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12-2011
THE ROLE OF TAK1 IN PANCREATIC CANCER DEVELOPMENT qianghua xia
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This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. THE ROLE OF TAK1 IN PANCREATIC CANCER DEVELOPMENT
AND THE MECHANISM OF TAK1 REGULATION
By Qianghua Xia, M.S.
APPROVED:
______Paul Chiao, Ph.D., Supervisory Professor
______Peng Huang, M.D., Ph.D.
______Wei Zhang, Ph.D.
______Shao-Cong Sun, Ph.D.
______Bingliang Fang, M.D., Ph.D.
APPROVED:
______Dean, The University of Texas Graduate School of Biomedical Sciences at Houston
THE ROLE OF TAK1 IN PANCREATIC CANCER DEVELOPMENT
AND THE MECHANISM OF TAK1 REGULATION
A
DISSERTATION
Presented to the Faculty of The University of Texas Health Science Center at Houston and The University of Texas M. D. Anderson Cancer Center Graduate School of Biomedical Sciences in Partial Fulfillment
of the Requirements
for the Degree of
DOCTOR OF PHILOSOPHY
------
By
Qianghua Xia, M.S.
Houston, Texas
August 2011
ACKNOWLEDGEMENTS
I would like to express my deepest gratitude to Dr. Paul Chiao for his support throughout my scientific endeavor, without which this project would never have reached fruition. I will always remember his charming personality, patience and kindness.
I would like to thank Dr. Peng Huang, Dr. Shao-Cong Sun, Dr. Bingliang Fang, Dr.
Kwong-Kwok Wong, Dr. Wei Zhang and Dr. Reuben Lotan for serving on my Ph.D. committee.
I would like to say special thanks to Dr. Yi Shi for his help with the MASS SPEC analysis, to Dr. Ruiying Zhao for her help with establishing cell lines. In addition, I want to express my appreciation to my colleagues in the laboratory: Dr. Zhongkui li, Dr. Jianghua
Lin, Dr. Zhe Chang, Dr. Yaan Kang, Ms. Yu Cao. It was a pleasure working with all of you.
Above all, I would like to thank my family for their endless support throughout my life.
iii
THE ROLE OF TAK1 IN PANCREATIC CANCER DEVELOPMENT AND THE MECHANISM OF TAK1 REGULATION
Publication Number: ______
Qianghua Xia, M.S.
Supervisory Professor: Paul Chiao, Ph.D.
Pancreatic cancer is the fourth leading cause of cancer-related mortality in the United
States and the fifth leading cause of cancer-related mortality worldwide. Pancreatic cancer
is a big challenge in large due to the lack of early symptoms. In addition, drug resistance is a major obstacle to the success of chemotherapy in pancreatic cancer. The underlying mechanism of drug resistance in human pancreatic cancers is not well understood. Better understanding of the mechanism of molecular pathways in human pancreatic cancers can help to identify the novel therapeutic target candidates, and develop the new preventive and clinic strategies to improve patient survival. We discovered that TAK1 is overexpressed in pancreatic cancer cell lines and patient tumor tissues. We demonstrated that the elevated activity of TAK1 is caused by its binding partner TAB1. Knocking down of TAK1 in pancreatic cancer cells with RNAi technique resulted in cell apoptosis and significantly reduces the size of tumors in mice and made a chemotherapy drug more potent. Targeting the kinase activity of TAK1 with the selective inhibitor LY2610956 strongly synergized in vitro with the antitumor activity of gemcitabine, oxaliplatin, or irinotecan
iv on pancreatic cancer cells. These findings highlighted that TAK1 could be a potential therapeutic target for pancreatic cancer.
We also demonstrated that TAK activity is regulated by its binding protein TAB1. We defined a minimum TAB1 sequence which is required and sufficient for TAK1 kinase activity. We created a recombinant TAK1-TAB1 C68 fusion form which has highly kinase activity. This active form could is used for screening TAK1 inhibitors.
In addition, several posttranslational modifications were identified in our study. The acetylation of lysine 158 on TAK1 is required for kinase activity. This site is conserved throughout all of kinase. Our findings may reveal a new mechanism by which kinase activity is regulated.
v
TABLE OF CONTENTS
Approval Page ...... i
Title Page ...... ii
Acknowledgements ...... iii
Abstract ...... iv
Table of Contents ...... vii
List of Illustrations ...... vi
List of Tables ...... vi
List of Abbreviations ...... xviii
CHAPTER 1. THE ROLE OF TAK1 IN PANCREATIC CANCER TUMORIGENESIS
1.1. Introduction...... 1
1.1.1. Overview of pancreas...... 1
1.1.1.1. Function and anatomy of Pancreas ...... 1
1.1.1.2. The origin of pancreatic cell lineage...... 2
1.1.2. Overview of pancreatic cancer...... 5
1.1.2.1. Causes and risk factors of pancreatic cancer ...... 5
1.1.2.2. The classification of ductal adenocarcinoma of the pancreas...... 5
1.1.2.2.1. Pancreatic intraepithelial neoplasia (PanIN) ...... 6
1.1.2.2.1.1. PanIN-1A...... 7
1.1.2.2.1.2. PanIN-1B...... 7
1.1.2.2.1.3. PanIN-2...... 7
vi
1.1.2.2.1.4. PanIN-3...... 9
1.1.2.2.1.5. Genetic alterations in pancreatic adenocarcinoma...... 9
1.1.2.2.1.5.1. K-RAS ...... 10
1.1.2.2.1.5.2. P21WAF1/CIP1 ...... 11
1.1.2.2.1.5.3. HER-2/neu ...... 11
1.1.2.2.1.5.4. Loss of p16 protein ...... 11
1.1.2.2.1.5.5. The p53 tumor suppressor ...... 12
1.1.2.2.1.5.6. Loss of SMAD4 ...... 12
1.1.2.2.1.5.7. BRCA2 mutation ...... 12
1.1.2.2.1.6. Other signaling pathways in PDAC...... 13
1.1.2.2.1.6.1. Growth factor pathways in pancreatic cancer...... 13
1.1.2.2.1.6.2. The serine/threonine kinase LKB1...... 12
1.1.2.2.1.6.3. NF-κB transcriptional factor in pancreatic cancer ...... 14
1.1.2.2.2. Mucinous cystic neoplasms (MCNs) ...... 15
1.1.2.2.3. Intraductal papillary mucinous neoplasms (IPMNs) ...... 16
1.1.2.3. Mouse models in pancreatic cancer studies...... 17
1.1.3. TAK1...... 18
1.1.3.1. TAK1 in C.elegans...... 18
1.1.3.2. TAK1 in Drosophila...... 20
1.1.3.3. TAK1 in zebrafish...... 21
1.1.3.4. TAK1 in Xenopus...... 22
1.1.3.5. TAK1 in mouse...... 23 vii
1.1.3.5.1. The role of TAK1 in cardiac ...... 23
1.1.3.5.2. The role of TAK1 in TGF-β signaling pathway...... 24
1.1.3.5.3. The role of TAK1 in the innate immunity ...... 24
1.1.3.5.4. TAK1 knockout mice ...... 26
1.1.3.6. TAK1 in human...... 28
1.1.3.7. TAK1 and cancer...... 30
1.1.3.8. Proposed model for the regulation of TAK1 in pancreatic cancer……………...33
1.2. Materials and method...... 35
1.2.1. Cell Lines, antibodies and Reagents...... 35
1.2.2. Plasmid and site-directed mutagenesis...... 36
1.2.3. Generating TAK1 Knockdown Cell Lines and TAK1 dominant negative mutant cell lines and TAB1 Knockdown Cell Lines...... 37
1.2.4. Western Blot Analysis...... 38
1.2.5. Electrophoretic Mobility Shift Assay (EMSA) ...... 39
1.2.6. NF-B reporter gene assay...... 40
1.2.7. Apoptosis...... 40
1.2.8. Cell Migration and Invasion Assay...... 40
1.2.9. RT PCR...... 42
1.2.10. SYBR green quantitative real-time PCR...... 42
1.2.11. MTT assay...... 43
1.2.12. Nude Mouse Orthotopic Xenograft Model...... 42
1.2.13. Immunohistochemistry and Tissue Mineral Analysis (TMA) ...... 45
viii
1.2.14. Statistical Analysis...... 48
1.3. Results...... 49
1.3.1. TAK1 splicing variants are differentially expressed in pancreatic cancer cells...49
1.3.2. TAK1 and TAB1 are overexpressed in pancreatic cancer cells; NF-B is activated in
pancreatic cancer cell lines...... 52
1.3.3. The expression levels of TAK1 and TAB1 are highly correlated in pancreatic cancer cell lines and human tumor tissues...... 54
1.3.4. TAK1 Silencing in Pancreatic Cancer Cells...... 56
1.3.5. Silencing TAK1 Suppresses NF-B DNA Binding Activitie ...... 58
1.3.6. Silencing TAK1 Induces a Potent in Vitro Chemosensitization and apoptosis of
Pancreatic Cancer cells...... 63
1.3.7. TAK is inactivated by the dominant negative mutant form...... 70
1.3.8. The inactivation of TAK1 suppresses pancreatic cancer cell migration and invasion..72
1.3.9. Targeting TAK1 kinase activity chemo sensitizes pancreatic cancer cells In Vitro...75
1.3.10. TAK1 and TAB1 stabilize each other in pancreatic cancer cell line...... 79
1.3.11. Silencing TAK1 or TAB1 reduced tumor size in Orthotopic Xenograft models...... 84
1.4. Discussion...... 87
1.4.1. Targeting TAK1 increased the sensitivity of pancreatic cells to chemotherapeutical drugs and blocks tumorigenesis...... 87
1.4.2. TAK1 activity is regulated in pancreatic cancer cells by TAB1...... 93
CHAPTER 2. THE REGULATORY MECHANISM OF TAK1 BY TAB1 AND
ACETYLATION AT LYSINE 158...... 95
ix
2.1. Introduction...... 95
2.1.1. TAK1 interaction proteins...... 95
2.1.1.1. TAB1...... 95
2.1.1.2. TAB2...... 97
2.1.1.3. TAB3...... 97
2.1.1.4. TAB4...... 98
2.1.2. Overview of Posttranslational modification (PTM) ...... 99
2.1.2.1. Phosphorylation...... 99
2.1.2.2. Methylation ...... 100
2.1.2.3. Ubiquitination...... 102
2.1.2.4.. Acetylation...... 104
2.2. Materials and methods...... 114
2.2.1. Primers and constructs...... 114
2.2.1.1. TAB1 primers and constructs...... 114
2.2.1.2. TAK1ΔN22 primer ...... 118
2.2.1.3. TAK1-TAB1 fusion primers...... 118
2.2.1.4. TAK1 site mutagenesis primers ...... 119
2.2.1.5. TAK1 primers for bacteria expression constructs...... 124
2.2.1.6. TAB1 primers for bacteria expression constructs...... 124
2.2.1.7. MKK6 primers for bacteria expression constructs...... 124
2.2.1.8. MEKK3 primers...... 127
2.2.2. Mass spectrometry ...... 128
x
2.2.3. Reagents and cell lines...... 128
2.2.4. Cycloheximide (CHX) chase assay...... 129
2.2.5. GST protein purification...... 130
2.2.6. His-tag protein purification...... 131
2.2.7. In Vitro Kinase assay...... 131
2.3. Results...... 132
2.3.1. The first 22 amino acids at the N-terminus did not stabilized TAK1...... 132
2.3.2. TAK1 is stabilized by TAB1...... 132
2.3.3. TAK1 Ubiquitination level is not affected by TAB1...... 136
2.3.4. TAB1 is indispensable for TAK1 kinase activity...... 138
2.3.5. C-terminus of TAB1 is required and sufficient to activate TAK1...... 144
2.3.6. All four transcriptional isoforms of human TAK1 have kinase activity dependent on
TAB1 but may be involved in different pathways...... 146
2.3.7. TAK1 activity is regulated by the acetylation at lysine 158...... 147
2.3.8. TAK1 is acetylated by CREB-binding Protein (CBP) ...... 171
xi
2.3.9. Lysine 158 of TAK1 is conserved throughout all kinases...... 175
2.4. Discussion...... 182
Reference...... 195
Vita...... 224
xii
List of Illustrations
CHAPTER 1
Figure 1: Anatomy of human pancreas...... 1
Figure 2 Cell differentiation signalling pathways in pancreatic development...... 4
Figure 3: Genetic progression model of pancreatic adenocarcinoma ...... 7
Figure 4: The central role of ubiquitin and TAK1 in multiple NF-κB signaling pathways.....25
Figure 5: Schematics of TAK1 mRNA splice variants and the protein isoforms...... 29
Figure 6: Proposed model for the regulation of TAK1 in pancreatic cancer ...... 34
Figure7: Different ratios of the 2 TAK1 mRNA variants expressed in normal pancreatic
tissues and pancreatic tumor tissues ...... 51
Figure 8: TAK1 and TAB1 are overexpressed in pancreatic cancer cells...... 55
Figure 9: TAK1 and TAB1 are correlated expression in and human pancreatic cancer
tissues...... 54
Figure 10: The effects of TAK1shRNA on pancreatic cancer cell lines ...... 57
Figure 11: TNF-κB activation is reduced in TAK1 knocking down cells...... 59
Figure 12: TAK1 kinase activity is reduced in TAK1 knock down cells ...... 60
Figure 13: P38 kinase activity is reduced in TAK1 knock down cells...... 61
Figure 14: TAK1 is required for IKKα/β phosphorylation and NF-κB activation in pancreatic
cancer cells ...... 62
Figure 15: Effects of TAK1 silencing on chemoresistance of pancreatic cancer
cells...... 64 xiii
Figure 16: Nuclear factor κB (NF-κB) activation after treatment with cytotoxic agents...... 67
Figure 17: Silencing TAK1 with the treatment of chemotherapeutical drugs induces proapoptosis phenotypes...... 68
Figure 18: TAK1 activity is inhibited by the dominant negative mutant K63A in pancreatic cancer cells...... 71
Figure 19: Blocking TAK1 inhibits pancreatic cancer cell migration ...... 73
Figure 20: Blocking TAK1 inhibits pancreatic cancer cell invasion...... 74
Figure 21: Activity of the TAK1 kinase–selective inhibitor LYTAK1 in vitro
...... 76
Figure 22: Inhibition of TAK1 activity and chemosensitization of human pancreatic cancer cells in vitro...... 78
Figure 23: TAK1 and TAB1 stabilize each other in pancreatic cancer cell line...... 81
Figure 24: The inactivation of TAK1 suppresses tumorigenicity in an orthotopic mouse model...... 85
Figure 25: The inactivation of TAB1 suppresses tumorigenicity in an orthotopic mouse model...... 81
CHAPTER 2
Figure 26: Biochemical mechanisms for protein-lysine and protein arginine methylation ..101
Figure 27: CHX chase assay for the full length TAK1 and TAK1ΔN22…...... 132
Figure 28: TAB1 interacts with TAK...... 134
xiv
Figure 29: TAK1 is stabilized by TAB1...... 135
Figure 30: Ubiquitination assay for TAK1...... 137
Figure 31: TAB1 is stabilized and phosphorylated by TAK1...... 139
Figure 32: TAK1 stabilization and autophosphorylation depend on TAB1...... 140
Figure 33: NF-κB activation is induced by TAK1 and TAB1...... 142
Figure 34: The phosphorylation of JNK is induced by TAK1 with TAB1...... 143
Figure 35: TAK1 is stabilized by the C-terminus of TAB1...... 145
Figure 36: EMSA Assay for NF-κB activation in TAK1 rescue cells...... 146
Figure 37: TAK1 is activated by TAB1...... 148
Figure 38: PTM sites on TAK1 identified by MASS SPEC ...... 149
Figure 39: The effects of phosphorylation at the new identified sites on TAK1...... 151
Figure 40: Maldi-MS identification of TAK1 lys 158 acetylation ...... 153
Figure 41: The structures of lysine, arginine, glutamine, asparagines and acetylated lysine154
CHAPTER 4
Figure 42: The effects of acetylation at the new identified sites on TAK1...... 155
Figure 43: Recombinant GST-TAK, GST-TAB1 and GST MKK6 purified from bacteria..157
Figure 44: TAK1 in vitro kinase assay...... 159
Figure 45: TAK1 in vitro kinase assay ...... 160
Figure 46: TAK1 and TAB1 interact with each other in vitro...... 161
Figure 47: TAK1 in vitro kinase assay...... 163
Figure 48: Light-microscopic phenotype in MEF cells expressing the various TAK1 forms or the empty vector as control ...... 165
xv
Figure 49: NF-κB activation induced by TNF-α treatment...... 166
Figure 50: The Expression of TAK1 Rescues the TNF-α Induced Apoptosis in TAK1Δ/Δ
MEFs, and TAK1-K158Q and TAK1-K158N Showed Partially Rescue Effect...... 168
Figure 51: Annexin V staining for apoptosis assay...... 169
Figure 52: Flow Cytometry analysis for apoptosis assay...... 170
Figure 53: TAK1 acetylation is induced by CBP...... 172
Figure 54: Acetylation level of TAK1 4R and TAK1 5R mutants is less than TAK1 wild type
...... 173
Figure 55: Acetylation level of TAK1 is increased by the treatment of deacetylase inhibitor nicotianamine...... 175
Figure 56: K 158 of TAK1 is highly conserved in most of kinases...... 177
Figure 57: Lysine 522 is required for MEKK3 kinase activity...... 179
Figure 58: MEKK31 acetylation is induced by CBP...... 181
xvi
List of Tables
Table 1: TAB1 constructs...... 117
Table 2: TAK1-TAB1 fusion constructs...... 118
Table 3: TAK1 site mutagenesis constructs...... 123
Table 4: Bacteria expression constructs...... 126
Table 5: MEKK3 constructs...... 128
xvii
List of Abbreviations
°C degrees celsius
µL microliter
µM micromolar
µg microgram
4E-BP1 eukaryotic initiation factor 4E binding protein
53BP1 p53-binding protein 1
ACC acetyl co-A carboxylase
ALS Amyotrophic lateral sclerosis disease
AMPK 5’ adenosine monophosphate-activated protein kinase
ATM ataxia telangiectasia mutated
ATP adenosine triphosphate
ATR ataxia telangiectasia and Rad3 related protein
C-terminus carboxyl terminus
CAG trinucleotide repeat consisting of cytosine, adenine and guanine
Chk2 checkpoint protein kinase 2
CKIP-1 casein kinase-2 interacting protein-1
DAPK death-associated protein kinase
DCFDA dichlorohydrofluorescein
DMSO dimethylsulfoxide
DNA deoxyribonucleic acid
DRAM damage-regulated autophagy modulator
xviii
DSB double strand breaks (in DNA) dsRNA double-stranded RNA (ribonucleic acid)
ERK extracellular signal-regulated kinase
FAT FRAP-ATM-TRRAP domain
FATC FAT-carboxyl-terminal domainxviii
FBS fetal bovine serum
GAP GTPase activating protein
GAPDH glyceraldehydes 3-phosphate dehydrogenase
GCN2 general control nonrepressed 2
GDP guanosine diphosphate
GFP green fluorescent protein
GSK glycogen synthase kinase
GTP guanosine triphosphate
Gy gray (unit of radiation exposure)
H2O2 hydrogen peroxide
H4K16 histone H4 acetylated at lysine 16
HCl hydrochloric acid
HDAC histone deacetylase
HEK 293 human embryonic kidney 293 cells
HER2 human epidermal growth factor receptor 2 hMOF human ortholog of MOF
HNSCC head and neck squamous cell carcinoma
xix
IKK IκB-kinase
IP immunoprecipitation
IR ionizing radiation
ISG20L1 interferon-stimulated 20kDa exonuclease-like
JNK c-Jun N-terminal kinase
LDH lactate dehydrogenase
LKB1 Liver kinase B1
M molar (or membrane fraction if in figure)
MAPK mitogen activated protein kinase
TGF-β Transforming growth factors beta
TAK1 TGF-β activation kinase 1
TAB1 TAK1 bind protein 1
PanIN-1A Pancreatic Intraepithelial Neoplasia 1-A
PTM post-translational modification
xx
CHAPTER 1. THE ROLE OF TAK1 IN PANCREATIC CANCER
TUMORIGENESIS
1.1. Introduction
1.1.1. Overview of pancreas
1.1.1.1 Function and anatomy of Pancreas: In humans, the pancreas is located
posterior to the stomach and associated with the duodenum. The pancreas is composed of
five regions: a head, neck, body, tail and uncinate process (Fig.1). The uncinate process of
the pancreas hooks the superior mesenteric artery and vein. The head is the rightmost portion
connected to the duodenum. The neck connects the head and the body portion. The body is
the main part of the pancreas that extends towards the tail. The tail of the pancreas is the
leftmost portion next to the spleen.
Figure 1. Anatomy of human pancreas.
http://pathology.jhu.edu/pancreas/BasicOverview2.php?area=ba. Reprinted by permission from Dr. Ralph Hruban
1
The pancreas includes exocrine glands and endocrine glands. The exocrine part consists of acinar cells that make and release digestive enzymes into the small intestine, which are required for the digestion of carbohydrates fats and proteins. The endocrine part consists of the islets of Langerhans which are surrounded by exocrine pancreatic acinar cells. The islets of Langerhans are composed of four different types of cells: α cells secret glucagon, β cells secret insulin, δ cells secret somatostatin and PP cells produce the pancreatic polypeptide[1].
1.1.1.2. The origin of pancreatic cell lineage: Many of the genes that control differentiation and proliferation in the normal developmental process are mutated in the progression of cancer. There is a well known idea that cancer is an aberrant recapitulation of embryogenesis because they share many of the same genes with each other. Therefore, the knowledge of the molecular signaling pathway involved in cell-fate determination of the progenitor cells during embryogenesis and organogenesis is also important for understanding cancer progression. We will briefly review how pancreatic cells originate and differentiate as below.
The pancreas arises from the fusion of the dorsal and ventral buds in the endoderm of the foregut at E9.5 in mice [2-4]. Several signaling molecules have been identified to determine the differentiation of pancreas (Fig.2). Sonic hedgehog (Shh) was expressed in stomach and duodenal endoderm at the stage E12.5 of mouse, but absent in pancreatic endoderm [5].
Ectopic expression of shh in the pancreatic epithelium lead to smooth muscle and interstitial cells [6-8]. The notochord secretes some signaling molecules including fibroblast growth
2
factor 2 (FGF2) and activin- βB that belong to the TGF-β during the early stage of pancreas
development. Both of them block the expression of Shh and the Shh receptor, Ptc1 and
therefore induce the expression of a variety of transcriptional factors including Pdx1,
Isl1, and Pax6 all of which play the important roles in the development of pancreas [9].
Notch signaling controls pancreatic cells determination at the different stage of pancreas
development. Activated Notch receptors bind to the DNA-binding protein RBP-Jk therefore
to lead to the expression of the negative basic helix–loop–helix (bHLH) HES genes which
block the expression of ngn genes [10-16]. Loss of Notch promotes ngn3 expression to
induce the pancreatic endocrine cell fate [10]. The exocrine markers such as amylase were
not detectable in the transgenic mice misexpressing activated Notch driven by PDX promoter.
Two independent labs demonstrated that Notch inhibited not only the endocrine but also
exocrine cell fate determination and thereby kept the progenitor cells at the undifferentiated
state [17, 18].
An elegant study using cre-loxp genetic system identified several specific transcription
factors [19]. In this study, they demonstrated that all three groups including islets, exocrine
and ductal cells originate from the same progenitors expressing the transcription factor pdx1.
All four types of islet cells including α, β, δ and PP cells are derived from the parent cells that express ngn3 transcription factors. However, ngn3 was not detected in the majority of acini or mature pancreatic duct cells. These studies strongly support that ngn3 transcriptional factor is highly specific for islet cells.
3
Embryonic Development
Foregut endoderm
Shh
Pdx1, Isl1, and Pax6 Stomach Pancreatic bud
Ngn3 Pdx1 Notch Islet cells Duct cells
Figure 2. Cell differentiation signalling pathways in pancreatic development. The genes that play an important role at the different stages of normal pancreatic development steps were showed. Adapted from Hezel A F et al. Genes Dev. 2006;20:1218-1249.
4
1.1.2. Overview of pancreatic cancer
The most of pancreatic cancers arise from the ductal exocrine glands and are called
adenocarcinoma of the pancreas [20, 21].Pancreatic cancer is the fourth leading cause of
cancer-related mortality in the United States and the fifth leading cause of cancer-related
mortality worldwide. Most people with pancreatic cancer in U.S.A. have developed
metastatic cancer by the time that pancreatic cancer is diagnosed. According to the estimation
of the American Cancer Society, approximately 43140 cases will be diagnosed with this
cancer and 36800 patients will die of it in 2010. These numbers indicate the difficulties in the
relative lack of curative approaches. The mechanism of invasion and metastasis in human
pancreatic cancers is not well understood. Better understanding the origin and biology of
invasive pancreatic cancer would therefore become an extreme important issue.
1.1.2.1. Causes and risk factors of pancreatic cancer: Epidemiology studies have
defined several risk factors related with pancreatic cancer. These factors include personal
habits and genetic background. A lot of reports have demonstrated heavy alcohol consumption and smoking are positively associated with pancreatic cancer [22-24].
1.1.2.2. The classification of ductal adenocarcinoma of the pancreas: Pancreatic ductal adenocarcinoma (PDAC) is the most cause and the most malignant type of pancreatic tumor cases. Important progress in the histopathologic and clinical studies has been made
5 that ductal adenocarcinoma primarily arise from premalignant precursor lesions. Three premalignant precursor lesions have been identified to progress to invasive cancer. These lesions have been classified into pancreatic intraepithelial neoplasia (PanIN), mucinous cystic neoplasms (MCNs), and intraductal papillary mucinous neoplasms (IPMNs) [25, 26].
We will review the classification of these lesions, including their distinguishing features and the molecular alterations associated with them below.
1.1.2.2.1. Pancreatic intraepithelial neoplasia (PanIN): PanIN is the most common non-invasive precursor lesion of pancreatic cancer. PanINs are defined as neoplastic epithelial proliferations (increased incidence of abnormal ductal structures) in the smaller caliber pancreatic ducts of patients with pancreatic adenocarcinoma. PanINs are graded into four groups according to the morphological criteria, cytological and architectural atypia:
PanIN-1A, PanIN-1B, PanIN-2 and PanIN-3 [27]. There is a lot of morphological difference between PanINs and normal ducts. Each grade of PanINs shows increasingly dysplastic growth (Fig.3).
6
Figure 3. Genetic progression model of pancreatic adenocarcinoma.
Nature Reviews Cancer 2, 897-909 (December 2002). Reprinted by permission from
Nature publishing group.
7
1.1.2.2.1.1. PanIN-1A: (Pancreatic Intraepithelial Neoplasia 1-A): Neoplastic PanIN-1A
represents the earliest step towards neoplasia. Grade of atypism is the lowest in PanIN-1A.
As described in detail elsewhere [27], PanIN-1A stage is characterized by the appearance of
a columnar, mucinous epithelium. These flat epithelia contain abundant supranuclear mucin
and basally located, round to ovoid nuclei with minimal atypia. The membrane of basement
is perpendicular to the nuclei if oval. There is much histological overlap between non- neoplastic flat hyperplastic lesions and flat neoplastic lesions without atypia. The neoplastic nature of PanIN-1A has not been well defined because they are also consistently found in histologically normal human pancreas in many cases [27].
1.1.2.2.1.2. PanIN-1B: (Pancreatic Intraepithelial Neoplasia 1-B): The epithelial cells in
PanIN-1B are similar to those in PanIN-1A, but in contrast to flat epithelial cells in PanIN-
1A, PanIN-1B lesions have increased crowding of tall columnar cells with papillary or micropapillary projections. These epithelial cells have pseudostratified architecture and are basally located [27].
1.1.2.2.1.3. PanIN-2: (Pancreatic Intraepithelial Neoplasia 2): The details of PanIN-2 have been described [27]. PanIN-2 lesions exhibit a flat or papillary organization. However, some mild cytological abnormalities are observed the mucinous epithelial lesions such as hyperchromatism and pseudo-stratification. In addition, nuclear crowding results in the enlarged nuclei. Cell polarity is lost. These nuclear abnormalities are less striking than those seen in PanIN-3. Rare mitoses characterized by non-atypical and non-luminal (not apical) are
8
observed in. PanIN-2 lesions show In contrast to PanIN-3, PanIN-2 lesions generally do not show dramatically cytologic abnormalities and true cribriforming luminal necrosis [27, 28].
1.1.2.2.1.4. PanIN-3: (Pancreatic Intraepithelial Neoplasia 3): The epithelial lesions are usually papillary or micropapillary in the architect, but in contrast to PanIN-2 lesions, they are rarely flat. PanIN-3 lesions are characterized by occurrence of severe cytologic abnormalities. Budding off into the lumen and true cribriforming are observed in the epithelial cells. Luminal necroses are observed in some cases. Nuclear polarity is significantly lost in PanIN-3 lesions. Mucinous cytoplasm display an orientation towards the basement membrane and dystrophic goblet cells display an orientation of nuclei towards the lumen. In addition, prominent (macro) nucleoli and abnormal or apical mitoses are detected.
PanIN-3 epithelial lesions are similar to carcinoma cells without invasion of the basal membrane, so they are referred to as “carcinoma in situ”. PanIN-3 is a late step in the course
of tumorigenesis [27, 28].
1.1.2.2.1.5. Genetic alterations in pancreatic adenocarcinoma: A dramatic shortening
of telomere length in PanIN-1A has been observed in PanIN-1A lesions. Telomere
shortening occurs in with a higher frequency than other genetic abnormalities. Telomere shortening has been one of the earliest demonstrable genetic aberrations in pancreatic precursor lesions and genetic events involved in pancreatic adenocarcinoma progression [29].
9
The molecular genetics PDAC has indicated that cancer genes are involved in PDAC progression. Genetic events and cancer signaling pathways have been linked the histological features of defined grade of PDAC progression. How these molecular events contribute to the characteristics of the neoplasms remains to be determined.
1.1.2.2.1.5.1. K-RAS oncogene is one of the earliest genetic abnormalities identified in the development of PDAC. The frequency of K-ras mutations reported in PDACs differed from study to study, but every study unambiguously showed that the frequency of KRAS mutations increases with the progression of PanIN from low-grade to high-grade with the mutation rate up to 100% for PanIN-3 lesions[30-32]. K-RAS is a GTPase which belongs to
GTP-binding protein family. The Ras GTPase-activating protein (GAP) is required for
GTPase hydrolyze to substrate. K-ras point mutation at codon 12 (substitution of GAT for
GGT) results in insensitivity to GAP and therefore attenuate the intrinsic rate of GTP hydrolysis [33, 34]. Therefore, this leads to the formation of constitutively active (GTP-
bound) protein which is independent of growth factor stimulation. Activated K-RAS has
been reported to function in several signaling pathways such as RAF-mitogen-activated kinase (MAPK) pathway, phosphoinositide-3-kinase/AKT signaling, and Ral GDS pathway
[35]. G12D K-ras mutation is related to resistance to TRAIL treatment in human pancreatic and lung cancer cell lines [36]. These downstream signaling elements are thought to contribute to PDAC progression.
MAP kinases are serine/threonine-specific protein kinases that control multiple cellular processes, such as gene expression, mitosis, differentiation, and cell survival/apoptosis[37].
10
The dominant negative forms of MEKK1, ERK1 and ERK2 inhibited the survival of colonies in pancreatic cancer cell lines [38]. Pharmacologic inhibitors of MEK induced cell cycle arrest in pancreatic cancer cell lines[39].A lot of studies by conditional knockout, xenograft and pharmacological inhibition have shown PI3K pathway plays a key role in PDAC progression [40-42]. RAL GTPases belong to the RAS subfamily. It has been found that
RAL A was activated in many PDAC cell lines. Tumorigenicity of RAS-transformed human
is inhibited by knockdown of RAL A using siRNA [43].
1.1.2.2.1.5.2. P21WAF1/CIP1 overexpression was found in 16% of patients with PanIN-1A
lesions and increased in frequency in PanIN-2 and PanlN-3 [44]. The activity of cyclin-
CDK2 and cyclin-CDK4 complexes is blocked by the binding of P21WAF1/CIP1 and thereby
and cell cycle progression is regulated.
1.1.2.2.1.5.3. HER-2/neu (epidermal growth factor receptor homologue) is
overexpressed in 82% (P = .008 vs. normal) of ducts with flat mucinous hyperplasia [45].
HER-2/neu oncogene encodes a transmembrane tyrosine kinase receptor that is highly
extensive homologous to the epidermal growth factor receptor. HER2/neu) play a key role in
the tumorigenesis.
1.1.2.2.1.5.4. Loss of p16 protein expression was observed in all PanIN lesions as early
as PanIN-1A [31, 46]. P16INK4A and p14ARF are two tumor suppressors which are encoded by
the CDKN2A/INK4A gene on chromosome 9p21. P16INK4A and p14ARF have distinct first
11
exons and alternative reading frames with shared downstream exons [47]. P16INK4A is a cell cycle checkpoint protein which blocks phosphorylation of RB, therefore inhibiting cell cycle entry into DNA synthesis phase. ARF can stabilize tumor suppressor p53 by preventing nucleo-cytoplasmic shuttling of Mdm2 [48]. INK4A is inactivated due to mutation, loss or promoter hypermethylation in up to 95% of human pancreatic cancers [31].
1.1.2.2.1.5.5. The p53 tumor suppressor mutations in the DNA-binding domain were identified in later-stage PanINs that are characterized by dysplasia [31, 49, 50]. The later loss of p53 and its functions decrease DNA repair checkpoint responses and leads to genomic instability that contribute to PDAC progression.
1.1.2.2.1.5.6. Loss of SMAD4 is identified in less than 50% of PanIn-3 lesions and considered as a late genetic event in PDAC [31]. Inactivation of smad4 Smad 4 is key modulators of TGF- β super family. SMAD4 can bind to receptor regulated SMADs (R-
SMADs), such as SMAD1 or SMAD2 to form the complexes. The complexes translocate into the nucleus and act as transcriptional factors. SMAD4 play a key role in differentiation, apoptosis, and the cell cycle.
1.1.2.2.1.5.7. BRCA2 mutation is found in familial pancreatic cancers [51]. BRCA2 plays a role in DNA repair and thereby maintains genomic stability. Loss of the wild-type
12
BRCA2 allele has been observed in those carriers of the BRCA2 germline heterozygous
mutations of BRCA2 in the late stage of PDAC.
1.1.2.2.1.6. Other signaling pathways in PDAC:
1.1.2.2.1.6.1. Growth factor pathways in pancreatic cancer: Grow factors are the
proteins that bind to their specific membrane receptors on the outside of the cell. Grow
factors promote cellular proliferation and differentiation. Multiple growth factor pathways
are dysregulated in pancreatic cancer cells. Transforming growth factors beta (TGF-β) is overexpressed in pancreatic cancer cells. As discussed before, the smad4, which is the member of TGF-β pathway cascade, is lost in pancreatic cancer cells. Epidermal growth factor (EGF), Vascular Endothelial Growth Factor (VEGF), insulin-like growth factor (IGF) fibroblast growth factor (FGF), hepatocyte growth factor (HGF) and their receptors are expressed at higher levels in pancreatic cancer cells than in normal pancreas[21].
1.1.2.2.1.6.2. The serine/threonine kinase LKB1 regulates cell polarity and energy sensing by phosphorylating AMPK [52]. Mutations or deletions in LKB1 lead to Peutz-
Jeghers syndrome [53]. LKB1 is also lost in 4%–6% of human pancreatic cancers [54], but the exact role of LKB1 loss in pancreatic cancers remains to be elucidated.
13
1.1.2.2.1.6.3. NF-κB transcriptional factor in pancreatic cancer: NF-κB is a family of
pleiotropic transcription factors that regulate the expression of a large number of genes
involved in inflammatory responses, apoptosis, oncogenesis, and metastasis. Mammalian
NF- B family consists of five members: NF-κB1 (p50 and its precursor, p105), NF-κB 2
(p52 and its precursor, p100), c-Rel, RelA (p65), and RelB. All of Rel/NF-κB proteins
contain a highly conserved Rel homology domain (RHD), which mediates DNA-binding,
inhibitor IB interaction, dimerization and nuclear localization[55][55][55][55][55]. NF-κB
family members can form heterodimers and homodimers with most other family members, to generate gene regulatory complexes with different properties. The classical pathway of NF-
κB is activated by a heterodimer of p50 and p65, which is usually the most abundant of the transactivating complexes [56].
The activity of NF-B is regulated by a shuttling mechanism between cytoplasm and nucleus. The interaction of NF-B with the inhibitors, known as IB, keeps inactive NF-
B/IB complexes in the cytoplasm. In response to cytokine stimulation, the inhibitor IB is
phosphorylated by IB kinase complex (IKK), and thereby NF-B translocates into nucleus
to induces expression of downstream target genes such as VEGF, IL-6, IL-8 and Bcl-xL [56,
57]. It has become increasingly evident that members of the NF-B and IB families plays a
key role in the development of cancer. Previous studies in our lab have showed NF-B is
constitutively activated in nearly 70% human pancreatic cancer tissues and most pancreatic
cancer cell lines[58, 59]. The dominant-negative mutant IB (S32, 36A) blocked
tumorigenesis and liver metastasis of pancreatic cancer cells [58, 59].
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1.1.2.2.2. Mucinous cystic neoplasms (MCNs) of the pancreas are uncommon tumors
compared with pancreatic intraepithelial neoplasia (PanIN). Basically, they are mucin-
producing (filled with mucinous fluid) cystic tumors, characterized by ovarian-type stroma
[60]. Most pancreatic MCNs are more frequently located in the body and the tail than in the
head of the pancreas. Most of MCNs are found in middle-aged females [61]. MCNs appeared
usually unilocular and occasionally multilocular. These cysts are lined by columnar mucin-
containing epithelial cells which are supported by ovarian-like cellular stroma composed of densely packed spindle cells. Generally, MCNs do not communicate with the pancreatic duct system[60].
Immunohistology has demonstrate that stroma of ovarian MCN was strongly immunopositive for α-smooth muscle actin (α-SMA) and vimentin and focally positive for desmin, and strong nuclear staining with estrogen and progesterone receptor [62].
The dysplasia of MCNs can be graded according to degree of cellular atypia. Low -grade dysplasia is characterized by uniform nuclei with minimal atypia, developing to middle grade dysplasia with moderate cytologic and architectural atypia, to high-grade dysplasia with significant architectural and cytologic atypia [60].
Our knowledge about genetic and epigenetic alterations in MCNs is still much less than that of ductal adenocarcinoma due to the very limit number neoplastic cells, however, MCNs have been shown some genetic alterations in common with those of PanINs as discussed
15
before. KRAS mutations have been found in 20% of mild dysplasia, 33% moderate dysplasia,
and 89% of carcinoma in situ and invasive carcinoma [63]. Immunohistochemical studies
have demonstrated overexpression of the tumor suppressor p53 in MCNs with the high-grade
dysplasia but not in MCNs with low-grade dysplasia [63, 64]. Loss of Smad4/Dpc4
expression has been found in advanced invasive MCNs [65]. In addition, Global analysis of gene expression has found a lot of over-expressed genes in MCNs including S100 calcium- binding protein P (S100P), Annexin 14 (ANX14), Prostate stem cell antigen (PSCA) [66].
1.1.2.2.3. Intraductal papillary mucinous neoplasms (IPMNs) are an uncommon type of pancreatic neoplasm characterized by non-invasive mucin producing, intraductal papillary or rarely flat epithelia. These epithelia arise from the main pancreatic duct or branch ducts, with ductal dilation and mucin retention in the pancreatic ducts [67].
As MCNs, IPMNs can also be graded according to degree of cytologic and architectural atypia, ranging from low-grade dysplasia (intraductal papillary mucinous adenoma), to IPMN with moderate dysplasia, to IPMN with high-grade dysplasia (intraductal papillary mucinous carcinoma). Most IPMN grow slower and are less aggressive than ductal adenocarcinoma.
One third of incidences of IPMNs are associated with invasive adenocarcinoma [68].
According architectural patterns, histology and mucin expression profile of the epithelium,
IPMNs can be group into four subtypes: the gastric type, intestinal type, pancreatobiliary type, and oncocytic type. The gastric-type express MUC5AC but are negative for MUC1 and
16
MUC2. The intestinal-type IPMN express MUC2 and MUC5AC but are negative for MUC1.
The pancreatobiliary-type IPMN is at least focally positive for MUC1 and consistently expresses MUC5AC but not MUC2. The oncocytic-type IPMN expresses MUC5AC consistently and MUC1 and MUC2 focally [68].
A lot of studies have demonstrated genetic alterations in IPMNs. The frequency of KRAS mutations increases with the progression of the grades of dysplasia in IPMNs [69]. In activation of tumor suppressor p53 is detected in high-grade dysplasia of IPMNs [70]. In contrast to PanINs, IPMNs showed the normal expression of SMAD4/DPC4 [68]. In addition, gene expression profiles in IPMNs have been identified by cDNA microarray technology. 62 up-regulated and 58 down-regulated genes have been found. Three members of the trefoil factor family (TFF1, TFF2, and TFF3) are identified as the most highly up- regulated genes in IPMNs. The mutations in the phosphoinositide-3-kinase catalytic-α
(PIK3CA) gene have been found at the rate of 11% [71].
1.1.2.3. Mouse models in pancreatic cancer studies: Molecular genetics and epidemiology of pancreatic cancer provide us the best evidence that pancreatic cancer arises from premalignant precursor lesions. Furthermore, the studies using mouse model of pancreatic cancer have demonstrate genetic mutations are responsible for progression of pancreatic cancer.
17
Activated KrasG12D mutation has been targeted the pancreatic epithelium by Cre
recombinase expression. The Pdx1 and Ptf1-p48 promoters are specific active in the
pancreatic cell types and use to delete LoxP-flanked Stopper Element to activate KrasG12D
expression. KrasG12D mutation mice show a gradual, age-dependent progression of lesions
which mimic human PanINs-I–III after a long latency [72]. KrasG12D mutation combined
with a conditional null allele of p53 [73] or a p53 R273H knock-in [74] allele resulted in
rapid progression of PDAC. Similarly, KrasG12D mutation combined with a conditional null
allele of p16 developed PDAC with shorter latency than KrasG12D alone [73]. These
evidences strongly support that pancreatic cancer arises from premalignant precursor lesions.
1.1.3. TAK1
Transforming growth factor-beta (TGF-beta) activation kinase (TAK1, MAP3K7) is a member of MAPKKK family. It was firstly identified in mouse by a yeast genetic screen using histidine as the selection marker. In the original study, TAK1 was showed to activate
Plasminogen Activator Inhibitor-1 (PAI-1) promoter after TGF-β or BMP-4 stimulation.
These experiments demonstrated that TAK1 may be a mediator of TGF-β signaling pathway
[75].
1.1.3.1. TAK1 in C.elegans. TAK1 homolog in C.elegans, MOM-4 was initially identified as an anterior-posterior (AP) polarity gene whose mutation resulted in the defects in polarity decisions in the early embryonic development. In the nematode Caenorhabditis
18 elegans, 4-cell stage embryonic cell, or blastomere, called EMS divides asymmetrically along the anterior-posterior axis [76]. The E produces blastomere endodermal cells, while the MS blastomere gives rise primarily to mesodermal cells [77, 78].
POP-1 is a member of a transcriptional factor family which contains the high mobility group (HMG or HMGB) domain controlled by Wnt signaling. POP-1 is a maternal gene which is asymmetrically expressed at lower levels in the posterior daughter,
E blastomere than the anterior daughter, MS blastomere in EMS. Genetic analysis demonstrated that mom-4 may act as the upstream of pop-1 and synergize with other mom mutant belonging to Wnt/WG components [76, 79]. MOM-4 is required for the WRM-1/LIT-
1-dependent phosphorylation of POP-1 which in turn down-regulate POP-1 activity [76,
80]. Mom-4 is homologous to vertebrate TAK1. Lit-1 is homologous to nemo-like kinase
(NLK) which is also shares 45% identical amino acids with human p38 MAP kinase and 43% identical amino acids with human ERK1 within the kinase domain [76]. These data support that a MAP kinase may contribute to AP cell determination during the early development of in C.elegans.
A binding protein called TAB1 is required for TAK1 kinase activity. TAP-1 encodes the homolog of vertebrate TAB1. Co-expression of MOM-4 and TAP-1 stimulate MOM-4
MAP3K activity, which support that the function of TAK1 is highly conserved between
C.elegans and mammals [76].
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1.1.3.2. TAK1 in Drosophila. Drosophila TAK1 was identified a Drosophila genomic library using mouse TAK1 as the probe [81]. A variety of developmental defects in different tissue were caused by expressing mTAK1 transgenic flies. A rough eye phenotype was observed in UAS-mTAK1/elav-GAL4 flies, whereas the antenna and distal parts of legs were missing in mTAK1/Dll-GAL4 flies. The activated form of mTAK1 in which the N-terminus is truncated caused early embryonic lethality. The size of the compound decreases by 30% in eye specific transgenic line compared with wild type flies. The active form of mouse TAK1 had more severe effects on eye development in the transgenic lines. Eye size was reduced to less than 30% and ommatidial units were almost totally lost in the transgenic lines expressing mTAK1ΔN. These results demonstrated that mTAK1ΔN has more severe effect than full length mTAK1 in transgenic flies [81]. Coexpression of mTAK1 with hTAB1 resulted in much smaller eye than mTAK1 alone.
TAK1 play a very important role in the control of JNK activity in Drosophila. TAK1 can activate the expression of JNK downstream targets including pp and puc. TAK1 induced the phosphorylation of the Bsk protein and activate Hep-Bsk cascade. Loss-of-function of bsk and hep suppressed the phenotype caused by the ectopic expression of TAK1. Both gain
of function and loss of function experiments support that TAK1-JNK pathway induced
apoptosis phenotype in Drosophila [81]. However, Genetic interaction analysis demonstrated
that TAK1 may not be involved in p38 or TGF-β/Dpp signaling. The function of TAK1 may
be specific for JNK pathway in Drosophila [81].
20
TAK1 also play an essential role in NF-κB pathway of Drosophila. Genetic approach identified that TAK1 acts as upstream of the IKK complex and downstream of Imd. TAK1 is required for Imd activation stimulated by natural Gram-negative bacterial infection and controls antibacterial peptide gene expression in Drosophila. The expression of the
Drosomycin-GFP reporter gene induced by Gram-negative bacteria strain E. carotovora 15 is significantly reduced in dTAK11 and DreddB118 mutants [82].
In Drosophila, downstream signaling of TAK1 diverges into two separate pathways.
Both the IKK/Relish branches and JNK are mediated by TAK1 and play a role in innate immunity. The IKK/Relish regulated genes reflect a sustained response while the JNK regulated genes exhibit a transient effect. Relish induces the proteasome-mediated degradation of TAK, therefore to negatively regulate JNK signaling [83].
1.1.3.3. TAK1 in zebrafish. The limited study of TAK1 in zebrafish was focus on its function in development. TAK1 homolog in zebrafish shares 71% identical amino acids with mice. Alk1 is TGFβ type I receptor. The mutations of the zebrafish Alk1 ortholog violet
Beauregard (vbg) resulted in cranial dilation which lead to the defect in blood circulation.
The experiments using the injection of antisense morpholinos against TAK1 and Alk1 demonstrated that they acted in the same pathway as they displayed a synergized effect on vascular development. The genetic interaction analysis put TAK1 in the downstream of
ALK1 for vascular development as the overexpression of TAK1 rescued the defects caused by ALK1 mutation [84].
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1.1.3.4. TAK1 in Xenopus. Not much is known about the function of TAK1 in Xenopus.
Limited studies have shown that TAK1 play an important role in early embryonic development of Xenopus. Injection of TAK1 mRNA into four-cell stage embryos leads to apoptosis and embryos. Co-injection of human bcl-2 mRNA reversed apoptosis caused by
TAK1 and induced the formation of normal embryos. These results support that overexpression of TAK1 results in apoptosis in Xenopus. The injection of TAK1∆N mRNA encoding the N-terminal truncated TAK1 resulted in apoptosis at lower doses than the full length TAK1. Although co-injection of xTAK1∆N mRNA and human bcl-2 mRNA into the ventral side did not exhibit phenotypes, co-injection of both the dorsal marginal zone of four- cell embryos strongly induced ventralization of the embryos. These results demonstrated that TAK1∆N specifically induces the formation of ventral mesoderm, which is same as the phenotype induced by BMP-4. These results support that TAK1 and BMP-4 may act in the same signaling pathway [85].
TAK1 acts as the downstream of BMP signaling at the early developmental stage of
Xenopus. Injection of TAK1 kinase dead form (TAK1DN) mRNA blocked the ventralization of Xenopus embryos induced by the injection of the mRNA encoding the constitutively active form of the BMP-2/4 type I receptor. However, TAK1 kinase dead form did not repress the expression of a pan-mesoderm marker (Xbra) stimulated by fibroblast growth factor (FGF). Injection of TAK1KN mRNA also did not block the expression of dorsalization marker molecules induced by activin, the constitutively active activin type IB receptor or
Smad2. These results indicate that TAK1 appears to be the specific downstream of BMP, smad pathway, but not activin [85].
22
Nemo-like kinase (NLK) is a downstream effector of TAK1. The injection of NLK
specific translational inhibitor, morpholino-antisense oligonucleotide (MO) inhibited
mesoderm induction at four-cell-stage. The whole-mount immunostaining by using a specific
phosphorylation antibody demonstrate that STAT3 is phosphorylated at Ser 728 during the
gastrula stage. Moreover, the injection of TAK1 MO greatly lowered STAT3 serine phosphorylation and the expression of Xbra, α-actin and Xnot induced by BMP, which are the mesodermal markers. These results demonstrate that TAK1, NLK and STAT3 cascade play a role in the early stage of Xenopus development [86].
1.1.3.5. TAK1 in mouse. The functional role of mouse TAK1 in multiple signaling pathways has been intensively studied. The following review will be focused on the role of
TAK1 in the innate immunity pathways and development.
1.1.3.5.1. The role of TAK1 in cardiac development. P19CL6 cell line originated from
P19 mouse embryonic teratocarcinoma cells. P19CL6 cell line has been used as an in vitro cardiomyocyte differentiation system. The treatment with 1% dimethyl sulfoxide induced most of P19CL6 cells to produce beating cardiomyocytes. However, the expression of contractile protein genes and cardiac transcription factors is lost in the progeny cells,
P19CL6noggin which constitutively expresses the BMP antagonist noggin. Therefore,
P19CL6noggin cell no longer differentiate into beating cardiomyocytes. The differentiation of P19CL6 cells into beating cardiomyocytes was reduced by TAK1 dominant negative mutant while WT TAK1 rescued the ability of P19CL6noggin cell to generate
23 cardiomyocytes. These results indicate that TAK1 play an important role in the control of cardiac development [87].
1.1.3.5.2. The role of TAK1 in TGF-β signaling pathway. TAK1 was originally identified as a transforming growth factor (TGF)-β-activated kinase. TAK1 activated transcription of the reporter gene driven by the PAI-1 gene promoter. Dominant negative
TAK1 mutant blocked this reporter gene activity induced by TGF-β stimulation [75]. These results demonstrated that TAK1 may be involved in the TGF-β signaling pathway.
Subsequent studies demonstrated that TAK1 was also required for NF-κB activation induced by TNF-α and IL-1β.
1.1.3.5.3. The role of TAK1 in the innate immunity. It was well established that TRAF family members including TRAF2, TRAF5, TRAF6 are required for NF-κB activation induced by TNF-α and IL-1β [88]. However, the mechanism how IKKβ is activated was unclear. Both IKK and JNK activation were inhibited by TAK1 specific inhibitor, [89]. The activation of IKK and JNK by TNF-α and IL-1β was reduced by the knock down of TAK1
[90]. These results strongly supported that TAK1 is involved in NF-κB signaling pathway activated by TNF-α and IL-1β.
A kinase complex consisting of three proteins TAK1, TAB1 and TAB2 was purified and identified by the biochemical approach. In vitro reconstitution revealed that IKK and MKK6
24 were phosphorylated by TAK1 in an E1, E2 (Ubc13-Uev1A) and E3 (TRAF6) dependent manner. Moreover, K63-linked polyubiquitination of TRAF6 was required and sufficient for
TAK1 activation [91].
Figure 4. The central role of ubiquitin and TAK1 in multiple NF-κB signaling pathways.
Cell Death and differentiation (2006) 13,687-692. Reprinted by permission from Nature publishing group.
25
1.1.3.5.4. TAK1 knockout mice. Several groups knocked out TAK1 independently and the genetic evidence demonstrated that TAK1 is necessary for the signaling pathway induced by TNF-α and IL-1β. Sato and colleagues used cre-loxP system to delete exon 2 of mouse TAK1, which encodes the ATP binding domain (K63). Deletion of TAK1 in the germ line resulted in embryonic death at E10.5. NF-κB, JNK and p38 activation by TNF-α and IL-
1β null MEF cells was impaired. IL-6 production by IL-1β stimulation was reduced and cell death was induced by TNF-α treatment. IκBα protein degradation and NF-κB DNA binding activity were also impaired in TAK1 knock out MEF cells. B cell–specific TAK1 deletion caused the defect in the development of B-1 B cells. Cell cycles into S phase in response to
LPS and/or CpG DNA were compromised in TAK1 null B cells. B cell antigen receptor
(BCR) was reduced. However, JNK, but not NF-κB, p38 or Erk was affected by BCR stimulation. Therefore, TAK1 is required for JNK signaling pathway induced by different stimuli while NF-κB activation by TAK1 depends on the specific stimuli. TAK1 is also required for adaptive immunity response. The production of antigen-specific IgG1 is significantly reduced in TAK1 heterozygous mice [92].
The second group used gene trap approach to disrupt TAK1 function in mice. The gene trap construct contains 2 genes: a fusion protein (βgeo), which is composed of β- galactosidase and neomycin phosphotransferase, and human placental alkaline phosphatase
(PLAP). This construct was inserted into the first intron to disrupt TAK1 expression. The mutant embryos exhibited the developmental defects in head and neural tube at E10 and
E10.5 and were dead at E11.5. NF-κB and AP-1 responsive promoter activity, IκBα degradation, JNK phosphorylation, C-jun phosphorylation and nuclear translocation and NF-
26
κB DNA binding activity all severe impaired In TAK1 mutant cells stimulated with TNF-α and IL-1. TNF-α treatment induced apoptosis in TAK1 mutant cells, as shown by Annexin-
V and PI staining, and pro Caspase 8 cleavage. In vitro kinase assay showed that TAK1 is essential for IKK activity induced by TNF-α and IL-1. However, TAK1 was not involved in the alternative NF-κB pathway activated by LT-β, BAFF, and CD40. Luciferase reporter gene assay also indicated that TAK1 is required for JNK phosphorylation, NF-κB and AP-1 activation induced by TGF-β [93].
Another group used the exact same ES cells containing the insertion in the first intron of the TAK1 gene to produce TAK1 mutant mouse. All TAK1 mutant embryos were dead at
E11.5 as described previously. In addition, the defect in heart development was identified in
TAK1 mutant mice, which was omitted by the above report. The TAK1 mutant embryos showed defective angiogenesis and the abnormal vascular architecture [84].
The fourth group used a different knockout strategy to abolish TAK1. Both sides of the first exon were flanked by LoxP site. Subsequently, TAK1 flox/flox mice were crossed with the Lck-Cre transgenic mice line in which the Cre recombinase was driven by the T cell- specific Lck to generate the deletion of TAK1 in T cells. T cells percentage in the peripheral lymphoid organs was significantly reduced up to 25%. CD4+ or CD8+ single positive thymocytes were much less in TAK1 conditional knockout mice than the flox mice. These result demonstrated that TAK1 is required for T cell development and maturation. Anti-CD3ε stimulation induced the significant increase in apoptosis of CD4+ and CD8+SP thymocytes
27
lacking of TAK1. The degradation of IκBα protein, IKK kinase activity, NF-κB DNA- binding activity and JNK phosphorylation were severely blocked in thymocytes of TAK1 conditional knockout mice. These data indicated that TAK1 is essential for NF-κB and JNK activation in thymocytes [94]. TAK1 knockout also resulted in the loss of the phosphorylation of AMPK at T172, which blocked AMPK activity [95]. In BMP signaling pathway, TAK1 was recruited to X-linked inhibitor of apoptosis protein (XIAP) mediated by
TAB1 upon BMP2/7 stimulation and thereby regulated Smad1/5/8 phosphorylation [96] .
1.1.3.6. TAK1 in human. The four splicing variants of TAK1 have been identified in human. TAK1b is the full length form which contains 17 exons. TAK1a, which was studied most intensively, lacks exon 12. TAK1 c contains no exon 16, which resulted in a reading frame shift and therefore produced a shorter protein with the earlier stop codon at the c- terminal. TAK1 d contains neither exon 12 nor exon16. These four different isoforms exhibited the tissue-specific expressing pattern [97, 98].
28
E2 E17
Figure 5. Schematics of TAK1 mRNA splice variants and the protein isoforms. TAK1a and TAK1d lack exon 12; TAK1c and TAK1d lack exon 16. The reading frame shift occurs at the 3’ end of the coding sequences of TAK1c and TAK1d due to the lack of exon 16.
Biochimica et Biophysica Acta 1517 (2000) 46-52. Reprinted by permission from Elsevier.
29
Most of studies about TAK1 function in human were carried out by transfection experiments. It has been demonstrated that TAK1 is required for the activation of SAPK/JNK induced by ceramide in COS7 cells [99]. TAK1 also mediated JNK activity stimulated by
TGF-β in the transient transfections of 293T cells[100]. Cotransfection of TAK1 and TAB1 stimulated IKK activity and NF-κB activations in HeLa cells [101]. TAK1 mediated also NF-
κB activation induced by Nontypeable Haemophilus influenzae (NTHi) in epithelial cells
[102]. TAK1 is required for protection from apoptosis induced by TNF-α and interleukin-1b converting enzyme (ICE; caspase 1) [103].
1.1.3.7. TAK1 and cancer. TAK1 play a key role in cancer development of mice. It has been reported that TAK1 regulate phosphorylation of JNK and p38 and therefore induced pulmonary metastasis in murine cancer colon cells 26 [104]. The inhibition of TAK1 with siRNA and the selective inhibitor 5Z-7-oxozeaenol increased in apoptosis induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) in HeLa cells and human lung adenocarcinoma A549 cells [105].
TNF-induced JNK activation mediated by TAK1 played a role in antiapoptotic effect in hepatoma [106]. However, the role of TAK1 in survival is controversial. It has been reported that transfection of TAK1 into human prostate cancer PC-3U resulted in apoptosis [107].
TAK was essential for adhesion of the metastatic human breast carcinoma cells (MDA-MB-
435) to the extracellular matrix [108]. It was found that TAK interacted with Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) and therefore activate JNK pathway required
30
for LMP1 oncogenic activity [109]. NF-κB activation in head and neck squamous cell
carcinoma (HNSCC) was blocked by TAK1 dominant mutant [110]. TAK1 plays a role in
tumor invasion and metastasis. The inhibition of TAK1 by siRNA or the dominant negative
mutant repressed the expression of matrix metalloproteinase-9 in the metastatic breast cancer
cell line and thereby reduced tumor angiogenesis and metastasis in orthotopic xenograft
model [111]. The oncoprotein induced constitutively NF-κB activation in HCT116 colon
cancer, HeLa cervical cancer cells, human ZR-75-1, MCF-7 breast cancer cells and MCF-
10A cells. Phosphorylation of IKK and IκBα induced by TNF-α is blocked by TAK1 siRNA
in MCF-10A cells [112]. Introduction of dominant negative mutant into MCF10A-CA1a cells reduced proliferation and invasion [113]. TAK1 is also required for AMPK activation and autophagy pathway induced by TRAIL-R2 in MCF10A cells [114].
TAK1 functions not only in NF-κB signaling pathway, but also in EGFR signaling
pathway. It has been reported that TAK1 is necessary for TNF-α-induced phosphorylation of
EGFR and internalization and these results implicated that the antiapoptotic effects of EGFR
induced by TNF- α may be mediated by TAK1 [115].
The SnoN is an oncoprotein which inhibits the expression of TGF-β-responsive genes.
The SnoN is phosphorylated by TAK1 upon TGF-β stimulation and thereby degraded to
allow the expression of downstream genes through smad activation in HeLa S3 cells [116].
The Tax oncoprotein encoded by of human T-cell leukemia retrovirus type I (HTLV-
31
I) constitutively activate IKK and NF-κB through TAK1 and IKK association. The loss of
TAK1 blocks NF-κB activation induced by Tax in MEF cells [117].
However, the oncogenic effect of TAK1 is controversial. TAK1 was involved with the recruitment of histone deacetylase (HADC) to the transcriptional factor Sp1 and thereby impaired the expression of the human telomerase reverse transcriptase (hTERT) gene [118] in human lung adenocarcinoma cell line, A549 cells. TAK1-P38 is required for the phosphorylation and internalization of epidermal growth factor receptor (EGFR) EGFR induced by TNF-α. This modification repressed the intercellular tyrosine kinase activity of
EGFR induced by EGF ligand binding in HeLa cells [119].
Mutations of the receptor tyrosine kinase Ror2 are associated with Robinow Syndrome.
The c-terminal domain of the tyrosine kinase receptor Ror2 was phosphorylated by TAK1 in the presence of an extracellular wnt ligand binding and thereby regulated Wnt-β-catenin-
LEF/TCF pathway [120]. TAK1 also controlled the expression of cytokines IL-8, TNF-α,
MIP-1α, and MIP-1β. The cytokine production was significantly reduced by TAK1 selective inhibitor 5z-7-oxozeaenol in human neutrophils [121]. The inhibition of TAK1 using RNAi repressed the expression of cyclooxigenase-2 (COX-2) induced by collagen-II-dependent in primary human chondrocytes (PHCs). These results supported that TAK1 may be used as a therapeutic target for osteoarthritis [122]. Etoposide (VP16) is a DNA double-strand break
(DSB) agent which leads to NF-κB activation in an ATM-dependent manner. TAK1 is
32
required for IKK and NF-κB activation by DNA damage in response to genotoxic stimuli
[123].
As summary, transforming growth factor beta activated kinase 1 (TAK1), known as
Mitogen-activated protein kinase kinase kinase 7 (MEKK7), is a member of MAP 3 kinase
family. TAK1 regulates multiple cellular processes. The yeast two hybrid experiments have
identified TAK1 binding protein 1 and 2, named TAB1 and TAB2, which interact with
TAK1 [124]. TAK1 plays a key role in both TNFα and IL-1β pathway. Knock down of
TAK1 RNAi impaired the activation of IKK and JNK by TNFα and IL-1β [90, 125]. The
inhibitor of TAK1, 5Z-7-oxozeaenol suppress kinase activity of TAK1, resulting in inhibition of JNK/p38 MAPK, IκB kinases, and NF-κB [89]. The genetic evidence revealed that knock out of TAK1 is embryonic lethal. NF-κB activation induced by TNFα and IL-1β are severely impaired in TAK1 knock out MEF cells [92]. co-transfection of TAK1 with its activator
TAB1 leads to the enhanced migration to fibronectin in vitro and metastasis to the lung in vivo [104]. However, what role TAK1 plays in pancreatic cancer and how it is regulate remain to be determined.
1.1.3.8. Proposed model for the regulation of TAK1 in pancreatic cancer. NF-B is constitutively activated in the most pancreatic cancer cell lines and tissues. Inhibition of constitutive NF-B activity by Ibα mutant suppresses tumorigenesis [58, 59]. It has been demonstrated that the autocrine mechanism of IL-1α accounts for the constitutive activation of NF-B in metastatic human pancreatic cancer cell lines[126]. TAK1 acts as the upstream
33 of NF-B signaling pathway and it is required for NF-B activation. Therefore, it is reasonable to propose that TAK1 plays a role in pancreatic cancer tumorigenesis and metastasis (Fig.6).
TNFα TGFβ
extracellular
cytoplasma I P
II TRAF2/5 ? RIP
Survival, TAB2/3 RSMAD MEKK3 P antiapoptosis, TAK1 SMAD4 Drug resistance? TAB1 P P MKK4/7 IκB IκB
NF-κB NF-κB
JNK
IκB α nucleus P P A20 P IL-8 C-JUNC-JUN NF-κB Bcl-2 SMAD4 RSMAD IL-2 ….. p15
Figure 6. Proposed model for the regulation of TAK1 in pancreatic cancer. This model demonstrates that TAK1 may play a key role of survival, antiapoptosis and drug resistance in pancreatic cancer through NF-κB activation.
34
1.2. Materials and method
1.2.1. Cell Lines, antibodies and Reagents
AsPc-1, PANC-1, BxPc-3, and CFPAC-1 human pancreatic cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA). MDAPanc-28 and
Panc-48 cell lines were obtained from the laboratory of Drs. Marsha L. Fraizer and Douglas
B. Evans. The Colo357FG cell line was obtained from the laboratory of Dr. Isaiah J. Fidler.
Human papillomavirus type 16 early gene 6 and 7-immortalized/nontumorigenic human
pancreatic ductal epithelial (HPDE) cells have been described previously [127]. All cell lines used in this study were authenticated using DNA fingerprinting at the genomic core facility
at Wayne State University (2009) and maintained as described previously [128]. The 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis,
MO) assay was used to obtain relative variable cell numbers.
TAK1, TAB1, Phospho-TAK1 (Thr184/187), IKKβ, Phospho-IKKα/β and Caspase-3
antibodies were purchased from Cell Signaling Technology, Inc. (#4505, #3225, #4531,
#2684, #2687, and #9632); Histone H1 antibody was purchased from Santa Cruz
biotechnology, Inc. (sc-8030). Monoclonal ANTI-FLAG® M2-Peroxidase (HRP) antibody was purchased from sigma (A8592).
35
Gemcitabine (Gemzar, Eli Lilly) and oxaliplatin (Eloxatin, Sanofi-Aventis) were
purchased as lyophilized products, which were then dissolved in sterile saline or 5% dextrose solution, respectively. LYTAK1 is an orally active TAK1 kinase-selective inhibitor (TAK1
Ki = 13 nM; p38 Ki > 20 mM; IKK-β Ki > 20 mM) generously provided by (Cancer Cell
Biology, Lilly Research Laboratories, 893 South Delaware Street, 48A/4103, DC0546
Indianapolis, IN 46285). For in vitro assays, LYTAK1 was dissolved in 100% dimethyl sulfoxide (DMSO) at a stock concentration of 1 mM. The concentration of DMSO did not
exceed 0.1% in any assay.
1.2.2. Plasmid and site-directed mutagenesis
To generate TAK1 sh RNA plasmids, two human TAK1 shRNA target sequences were chosen: 5’GAGGAAAGCGTTTATTGTATT3’ and 5’CCCAATGGCTTATCTTACATT3’.
These sequences were cloned into an FG12 lentivirus vector (gift from Dr. Qin Xiaofeng,
The University of Texas M. D. Anderson Cancer Center, Houston, Texas) with Bam HI and
Hind III.
To make “plenti-xia” vector, the multicloning site within pBluescript-KS was dropped with Bam HI and XhoI and cloned into Plenti6/v5.. WT-TAK1 tagged with myc was dropped from PRK6-MYC-TAK1 (gift from Dr. Xin Lin, The University of Texas M. D. Anderson
Cancer Center, Houston, Texas) with CLA I and ECORI. The insert was blunted with
36
Klenow and cloned into plenti-xia vector opened with ECOR V. TAK1 kinase dead dominant
negative mutant tagged with Flag was dropped from PC DNA3.1 Flag-TAK1 (k63A) (gift
from Dr. Xin Lin, The University of Texas M. D. Anderson Cancer Center, Houston, Texas )
with Xba I and Pme I. The insert was blunted with Klenow and cloned into plenti-xia vector
opened with ECOR V, too.
1.2.3. Generating TAK1 Knockdown Cell Lines and TAK1 dominant negative mutant
cell lines and TAB1 Knockdown Cell Lines
Lentiviruses were produced with the third generation packaging system plasmids
pMDLg/pRRE, pRSV-Rev, and pMD2.G into 293T cells [129]. The virus-containing
supernatants were collected after 72 hours of transfection and filtered through a Mini-pore
0.45 μm filtered. The AsPc-1, PANC-1, and MDAPanc-28 cells were transduced by the
lentivirus in the presence of the polycation Polybrene, and the stably transduced cells were
then sorted by GFP activity. AsPc-1, PANC-1, and MDAPanc-28 cell lines expressing TAK1
K63A mutant were produced by the same method and selected with blasticidine.
TAK1 shRNA rescue experiments were performed stably co-expressing by lentiviral infection wild-type murine TAK1 and human TAK1 shRNA in AsPc-1 and MDAPanc-28 cells.
37
TAB1 knocking down constructs were obtained from Open Biosystems. pMD2.G and
psPAX2 system are used for packaging
1.2.4. Western Blot Analysis
Human pancreatic cancer cells and HPDE were washed twice with cold phosphate- buffered saline and lysed into cold radioimmunoprecipitation assay buffer (50 mM Tris HCl
[pH 8], 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium
dodecyl sulfate). Each lysate (20 µg of protein) was separated by 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and probed with a rabbit polyclonal antibody
against TAK1 (#4505, Cell Signaling Technology, Inc.) and TAB1 (#3225, Cell Signaling
Technology, Inc.). For immunoprecipitation experiments, whole-cell lysates were prepared
with lysis buffer (50mM Tris pH 7.9, 0.5mM EDTA, 150mM NaCl, 5% glycerol, 0.5% NP-
40, 1 mmol/L sodium orthovanadate, 1 mmol/L sodium fluoride, 1 mmol/L
phenylmethylsulfonyl fluoride, 1 mmol/L DTT, 10 μg/mL aprotinin, 10 μg/mL leupeptin). 2
mg cell lysates were immunoprecipitated with 10 μL rabbit anti-TAK1 antibody (Bethyl
Laboratories A301-915A) at 4°C under rotary agitation for 4 hours and then the
immunocomplexes were captured by 40 μL protein A/ G agarose/sepharose bead slurry
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4°C for 2 h. The agarose/sepharose
beads were collected by centrifugation and washed three times with cold lysis buffer. The
agarose/sepharose beads were boiled in 3x sample buffer for 5 minutes. Each lysate (20 µL
of protein) was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
38
and probed with a rabbit polyclonal antibody against phospho-TAK1 (#4531, Cell Signaling
Technology, Inc.). Immunoreactive proteins were visualized with Lumi-Light Western
blotting substrate (Roche, Indianapolis, IN) according to the manufacturer’s instructions.
1.2.5. Electrophoretic Mobility Shift Assay (EMSA)
The nuclear extracts were prepared according to the method description before [130].
Briefly, cells were washed with cold PBS and lysed into cold buffer A (10 mM HEPES–
KOH pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM
phenylmethylsulfonyl fluoride) in the first step. The crude cellular lysate was centrifuged,
and the supernatant was taken of as the cytosolic fraction. The pellet was incubated the cold
high salt buffer C (20 mM HEPES–KOH pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride) to
extract nuclear fraction.
DNA binding assays for NF-B proteins were carried out using 10 μg of nuclear extracts
as described by Chiao et al [131]. The wild-type double-stranded oligonucleotides containing
the κB site (5'-AGT TGA GGG GAC TTT CCC AGG C-3') and mutant site (5'-AGT TGA
GGG GAC TTT CCC AGG C-3' ) were obtained from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA) and labeled with 32P to be used as probes. The reactions were resolved on
4% polyacrylamide gels with 0.25 X TBE (Tris/Borate/EDTA) running buffer.
39
1.2.6. NF-B reporter gene assay
The reporter gene luciferase assay has been described previously [132]. Briefly, one microgram each of the wild-type B, reporter plasmids containing the firefly luciferase reporter gene and the pRL-TK plasmid, containing the Renilla luciferase gene under the control of the herpes simplex virus thymidine kinase promoter as an internal control, was cotransfected into cells in triplicate by the lipotransfection method (FuGENE 6; Roche,
Indianapolis, Ind.) following the manufacturer's instruction. The activities of both firefly and
Renilla luciferases were determined 48 h after transfection with the dual luciferase reporter assay system (Promega, Madison, Wis.). The luciferase activities were normalized to the
Renilla luciferase activity of the internal control.
1.2.7. Apoptosis
The extent of apoptosis was analyzed by DNA fragmentation as previously described in
[133].
1.2.8. Cell Migration and Invasion Assay
40
Invasion assay was performed in 24-well plates by using a BD Biocoat growth factor-
reduced Matrigel invasion chamber (BD Biosciences) with an 8.0-μm pore size PET
membrane. Each membrane had a thin layer of GFR Matrigel Basement Membrane Matrix,
which serves as a reconstituted basement membrane in vitro. The inserts were rehydrated by
adding 0.5 ml of warm culture medium at 37 °C to the inserts for 2 h. Invasion of pancreatic
cancer cells expression TAK1 shRNA or TAK1 dominant negative mutant was determined
and pancreatic cancer cells expression scramble shRNA or empty vectors were used as the
control. These cells, kept in SFM without growth factors for 24 h, were seeded (5 × 104 cells
in 0.5 ml of SFM) to the invasion chambers with or without KGF (100 ng/ml) and heparin
(29.4 μg/ml), and 750 μl of complete keratinocyte-SFM was added to the lower well of the
invasion plate. Cells were incubated at 37 °C in a humidified atmosphere of 95% air and 5%
CO2 for 48 h. Noninvading cells from the interior of the inserts were removed by using
cotton-tipped swabs. Invading cells on the bottom side of the membrane were fixed by 10%
formaldehyde for 10 min, and then stained with 1% crystal violet, and at least three random
fields per insert were counted under an Olympus microscope. A representative field of each experiment was photographed with 4× lens and 2.5× magnification. Results are shown as the means ± S.E. of three independent experiments.
Migration assay was performed in 24-well plates by using Falcon cell culture inserts, which have a PET membrane with 1 × 105 8.0-μm pores per cm2 (BD Biosciences
Labware). Before the assay was run, HPDE cells were starved for 24 h, and 700 μl of complete keratinocyte-SFM was added to the lower well of the migration plate. The cells (5
× 104 cells/0.3 ml) were seeded to the insert in keratinocyte-SFM with or without KGF (100
41
ng/ml) and heparin (29 μg/ml). The incubation, staining, counting, and photographing procedures were the same as for the invasion assay. The representative fields of each experiment are shown. Results are shown as the means ± S.E. of three independent experiments.
1.2.9. RT PCR
The PCR was performed as follows: 940C for 5 min, 35 cycles at 940C for 30 s, 550C for
1 min, and 720C for 1min, and extension at 720C for 7 min. GAPDH was used as the internal control. To distinguish TAK1 isoform A and B, the forward primer 5’:
ACCTCTGAGGGCAAG AGGA3’ and the reverse primer 5’GCTTTTCTGAGGTTGGTCC
3’ are used to amplify the fragment of TAK1 covering exon 12.
1.2.10. SYBR green quantitative real-time PCR
PCR primers were synthesized by Sigma. The SYBR green real-time PCR was performed using Stratagenen Mx4000TM multiplex quantitative PCR system (La Jolla, CA) with
Brilliant SYBR green QPCR Master mix (Stratagene, La Jolla, CA) according the
manufacturer’s instructions. Real-time PCR was performed in a total volume of 25 μl using 1
μl of the first-strand cDNA synthesis mixture as the template. 2.5 pmol primer was used for
each reaction. The PCR was performed as follows: 940C for 5 min, 35 cycles at 940C for 30
42
s, 550C for 1 min, and 720C for 1min, and extension at 720C for 7 min. GAPDH was used as
the internal control to normalize the expression level. The relative quantification of gene
expression level was determined by the comparative CT method (2−ΔΔCT).
1.2.11. MTT assay
1,000 cells per well were plated on 96 well plate in triplicate wells, At day1, day2, day3,
day4, day5, the MTT assay was carried out by adding 10µl of 5 mg/ml solution of MTT
(Sigma Chemical Co., St. Louis, MO) to wells and incubating for 4 hours at 370C. The supernatant was removed and the blue MTT formazan precipitates was then dissolved in 100
µl/well of demethyl sulfoxide. The plates were placed on a shaker for 10 minutes at room temperature in the dark and absorbance was measured on a plate reader at 570nm. Data were shown as the mean value ± standard error of three independent experiments. Growth curves were generated by Excel.
1.2.12. Nude Mouse Orthotopic Xenograft Model
Six- to 8-week-old female athymic nude mice (NCI-nu) were purchased from the Animal
Production Area of the National Cancer Institute—Frederick Cancer Research Facility
(Frederick, MD). All mice were housed and treated in accordance with the guidelines of The
University of Texas M. D. Anderson Cancer Center’s animal care and use committee, and the
43
mice were maintained in specific pathogen-free conditions. The facilities were approved by
the Association for Assessment and Accreditation of Laboratory Animal Care and met all
current regulations and standards of the U.S. Departments of Agriculture and Health and
Human Services and the National Institutes of Health.
To produce pancreatic tumors, pancreatic cancer cells including AsPc-1, panc-1 and
panc-28 with or without TAK1 shRNA or TAK1 dominant negative mutant were harvested
from subconfluent cultures by brief exposure to 0.05% trypsin and 0.02% EDTA.
Trypsinization was quenched with medium containing 10% fetal bovine serum, and the cells were washed once in serum-free medium and resuspended in serum-free Hanks’ balanced salt solution. Only suspensions consisting of single cells with more than 90% viability were used for the injections. The orthotopic injection of pancreatic cancer cells was performed as described previously [128]. Briefly, the mice were anesthetized with a 1.5% isoflurane–air mixture. A small incision was made on the left side of abdominal flank, and then the spleen
was exteriorized. 1.0 × 106 tumor cells in 50 μL of Hanks’ balanced salt solution were
injected in a region of the pancreas just beneath the spleen. A 30-gauge needle, 1-mL
disposable syringe, and calibrated, push button–controlled dispensing device (Hamilton
Syringe, Reno, NV) were used to inject the tumor cell suspension. A successful subcapsular
intrapancreatic injection of tumor cells was identified by the appearance of a fluid bleb
without intraperitoneal (ip) leakage. To prevent leakage, a cotton swab was used to cover the injection site for 1 minute. One layer of the abdominal wound was closed with wound clips
(Auto-clip; Clay Adams, Parsippany, NJ). All the animals tolerated the surgical procedure well, and there were deaths due to anesthesia.
44
Eight weeks after injection, mice were dissected and the tumor was removed for photographing and measurement.
1.2.13. Immunohistochemistry and Tissue Mineral Analysis (TMA)
1. De-paraffin: Incubate the slides at 60°C for 1 hr.
2. Re-hydrated the tissue:
Put the slide in the following solution by order:
Xylene 4 min
Xylene 3 min
100%EtOH 1 min
100%EtOH 1 min
80%EtOH 1 min
30% EtOH 1 min
1X PBS 15 min
3. Target retrieves the antigen:
45
Turn on the steamer. Preheat the target retrieval solution (Antigen Unmasking H3300-
Vector) and steamer water in microwave oven, approx. Slides were incubated in hot target retrieval solution in the steamer for 15-20 min, and then cool down at RT for 20 min or more.
4. Immunohistochemistry assay:
(1) Wash the slides with PBS 2 X 5 min.
(2) Draw a circle along the tissue edge with ImmEdge pen (Vector) to keep the
solution on the surface of the tissue. Add the Biotin Block solution (Avidin Biotin
Blocking SP2001, Vector) on the surface of tissue and incubate at RT for 30 min.
(3) Wash the slides with PBS 2 X 5 min.
(4) Add the protein blocking solution (X0909-Dako) or 0.1 % BSA in PBS on the
surface of the tissue, incubate at RT for 30 min. Remove blocking solution.
(5) Incubate with primary antibody at 4C overnight. (Antibody concentration depends
on previous experiment or company information), antibody usually dilutes in 0.1% BSA
in PBS.
(6) Wash the slides with PBS 2 X 5 min.
(7) Incubate with biotinylated secondary antibody (Vectastain ABC-peroxidase Kit) at
RT for 30 min.
(8)Wash the slides with PBS 2 X 5 min.
(9) Incubate with ABC (from Vectastain ABC kit) reagent at RT for 30 min.
(10) Wash the slides with PBS 2 X 5 min.
46
(11) Use DAB substrate (Sigma #D4168) to develop the color about 2 to 5min, no
more 10 min.
(12) Rinse the slides in tap water.
(13) Counterstain the slides with Mayer’s Hematoxylin (Dako #S3309) solution for 10
min.
5. De-hydration of the tissue:
Put the slides in following solution by order:
30%EtOH 1 min
80%EtOH 1 min
100%EtOH 1 min
100%EtOH 1 min
Xylene 1 min
Xylene 2 min
Air dries the slides and add one drop of Permanent mount medium on the surface of the
tissue, cover them with the cover-slide.
IgG1 isotype was used as control.
47
1.2.14. Statistical Analysis
The results of in vitro proliferation are expressed as means (95% confidence intervals
[CIs]) for at least three independent experiments performed in triplicate. The statistical significance of differences in tumor growth was determined by one-way analysis of variance and Dunnett's test; differences in survival duration were determined using a log-rank test. All statistical tests were two-sided, and a P value less than 0.05 indicated statistical significance.
All statistical analyses were performed using GraphPad Prism software version 4.0c
(GraphPad Software, San Diego, CA).
48
1.3. Results
1.3.1. TAK1 splicing variants are differentially expressed in pancreatic cancer cells.
As stated above, TAK1 is an upstream kinase for IKK β which activates NF-B by phosphorylation of Ser32, 36 in IB. Four TAK1 alternative splicing variants have been identified, called TAK1a, TAK1b, TAK1c, and TAK1d. TAK1b is the longest transcript.
Compared with TAK1b, TAK1a lacks exon 12, TAK1c lacks exon 16 and TAK1d lacks both exon 12 and exon 16. The carboxyl-terminal domain in TAK1c and TAK1d is replaced by a truncated sequence generated by translation of the sequence downstream of the alternative splice site in another frame due to the lack of exon 16 (Fig.5, [98].
However, it was unclear which variant participated in TGF- pathways as it was originally discovered as a TGF- activating kinase, which TAK1 variants is required for activating NF-B and what function each TAK1 variant has. To determine the function of each TAK1 splicing variant, we reintroduced each of the four splicing variants of TAK1 back into TAK1-knockout MEF cells. We measured the NF-B activation using EMSA and found that only TAK1a and TAK1b, but not TAK1c and TAK1d, can restore cytokine-induced NF-
B activation (unpublished data). Because exon 16 is missing in TAK1c and TAK1d, these results implicate that each of the TAK1 variants may have the distinct role in regulation of
49
cytokine-induced NF-B activation. However, how exon 16 is involved in cytokine-induced
NF-B activation is still unknown.
To distinguish the function of TAK1 splicing variants in pancreatic cancer, we isolated
RNA from two normal pancreatic tissues and four pancreatic tumor tissues. We evaluated the expressing lever of the different TAK1 splicing variants in these tissues by RT-PCR. As shown in figure 7A, our result demonstrated that the ratio of TAK1a to TAK1a is much higher in most pancreatic tumor tissues than in normal pancreatic tissues. To confirm this finding, we tested twelve more tissues, which include six normal and six tumor tissues. Our result confirmed the previous finding (figure 7B). DNA sequence data confirmed that the lower band lacks the exact exon 12 (Data not shown). Taken together, TAK1 may be dysregulated by the alternative splicing in pancreatic cancer. However, what function of each
splicing variant of TAK1 in pancreatic cancer is still unclear.
50
AB
TAK1b TAK1a
GAPDH
Normal Tumor Normal Tumor
Figure 7. Different ratios of the 2 TAK1 mRNA variants expressed in normal pancreatic tissues and pancreatic tumor tissues. RT-PCR were carried out with RNAs isolated from six normal pancreatic tissues and six pancreatic tumor tissues. TAK1 primers are located outside of exon12. β-actin was used as control. Normal and tumor tissues were indicated.
51
1.3.2. TAK1 and TAB1 are overexpressed in pancreatic cancer cells; NF-B is activated in pancreatic cancer cell lines.
To study the role of TAK1 in pancreatic cancer chemoresistance, we initially evaluated the expression levels of TAK1 and TAK1 binding protein TAB1 in twelve different pancreatic cancer cell lines and in the immortalized and nontumorigenic E6E7 cell line, demonstrating the overexpression of TAK1 and TAB1 in most of the malignant cell lines compared with the nontumorigenic control (Fig.8A).
NF-B activity was analyzed by electrophoresis mobility shift assay. Consistent with the previous report [58, 59], NF-B is highly activated in most of pancreatic cancer cells
(Fig.8B). These data support that NF-B play a role in pancreatic cancer. More importantly,
NF-B activity is also highly correlated with the expression levels of TAK1 and TAB1. For example, the expression levels of TAK1 and TAB1 and NF-B activity are lower in BxPc-3 and Miapaca-2 than in most of other cell lines. This data support that TAK1 and TAB1 may contribute to NF-B activation in pancreatic cancer cell lines.
We also examined TAK1 expression in mouse pancreatic tumor tissue using immunohistochemistry staining with anti-TAK1. Our data demonstrated that the expression of TAK1 is also elevated in mice pancreatic tumor tissue (Fig.8C).
52
A. C. TAK1-IHC
TAK1
tumor TAB1
β-actin
B.
P65/p50 normal
Histone H1
Figure 8. TAK1 and TAB1 are overexpressed in pancreatic cancer. A.TAK1 and TAB1
are overexpressed in pancreatic cancer cell lines. Protein extracts were analyzed on
Western blot (50g/of protein extract/lane) and probed with the indicated antibodies.
Cytoplasm fraction extracted from the normal cell line (lane1) and twelve tumor cell lines
(lane2 through 13). E6E7 is immortalized but not transformed epithelial cells and was used
as a normal control. β-actin was used as the loading control. B. NF-κB activity is activated
in most of pancreatic cancer cell lines. EMSA was performed to determine the NF-κB activity in E7E7 and twelve pancreatic cancer cell lines as indicated. Histone H1 was determined as loading controls. C. TAK1 is overexpressed in human pancreatic tumor tissue. Immunohistochemistry staining was performed for mouse pancreatic tumor and normal tissues by TAK1 antibody.
53
1.3.3. The expression levels of TAK1 and TAB1 are highly correlated in pancreatic
cancer cell lines and human tumor tissues.
It has been demonstrated that TAB1 interacts with TAK1 and enhance TAK1 activity
[124]. Interestingly, the expression levels of TAK1 and TAB1 are highly correlated in
pancreatic cancer cell lines (Fig. 8A). For instance, both TAK1 and TAB1 are expressed at a
high level in most of cell lines such as AsPc-1, Capan-1, CFPAC-1, Hs766T, and L3.6 but at
a low level in the other cell lines such as BxPc-3 and Miapaca-2.
We also analyzed patient tumor tissue using immunohistochemistry staining with TAK1
and TAB1 antibodies. As shown in Fig. 9A, TAK1 and TAB1 were majorly expressed in
pancreatic ductal cells. More than 100 hundred tumor samples were analyzed with tissue
microarray. We classified TAK1 and TAB1 expression level into two levels for χ2 test. The statistics with a large χ2=180 demonstrated that TAK1 and TAB1 is highly associated with each other (Fig. 9B). These results strongly implicate that TAK1 may be regulated by TAB1 in pancreatic cancer.
54
A. TAK1-IHC B. TAB1-IHC
Human normal tissue Human normal tissue
Human tumor tissue Human tumor tissue
TAK1 TAB1 - + ++ +++ C. D. -101470
+ 33 152 23 0
++ 6 55 51 6
+++0707
Chi‐Square = 125.37, df=9, P<.0001
Figure 9. The expression of TAK1 and TAB1 are correlated in human pancreatic cancer tissue. A. Expression of TAK1 in human pancreatic cancer or normal tissue was analyzed by immunohistochemical staining. B. Expression of TAB1 in human pancreatic cancer or normal tissue was analyzed by immunohistochemical staining. C. TAK1 and TAB1 expression were classified into four levels. Tissue microarray analysis was summarized in the
4 X4 table. χ2 test was used to determine the association of TAK1 and TAB1. D. The association of TAK1 and TAB1 was demonstrated by the software R calculation.
55
1.3.4. TAK1 Silencing in Pancreatic Cancer Cells.
TAK1 plays a key role in the cellular processes of infection, inflammation. TAK1 is necessary for the activation of NF-κB in response to stimulation by proinflammatory cytokines such as TNF-α and IL-1β. NF-B activation is severely impaired in TAK1-null
MEF cells [92]. The study has shown that TAK1 acts as an upstream activating kinase for
IKKβ Phosphorylation of IKKβ leads to the activation of IKK. TAK1 can also phosphorylate
and activate MKK6 and MKK7, therefore activate p38 and JNK kinase pathways[91]. NF-B
is constitutively activated in most pancreatic cancer cells [58]. To determine the role of
TAK1 in pancreatic cancer, we knocked down the expression of TAK1 in pancreatic cancer
cell lines. Firstly, we tested effect of target sequences on RNAi efficiency for TAK1 in 293T
cells using siRNA. As shown in Fig. 10 A., both siRNAs have significant knock down of
TAK1 in 293T cells. Because the efficiency of transient transfection of siRNA into pancreatic cancer cells is very low, we generated constructs expressing shRNA of TAK1 using the lentivirus vector FG12 that contains the GFP selective marker. High infection efficiencies of the siRNA in AsPC-1, BxPc3, PANC-1, and PANC28 cells were yielded.
We used two pooled shRNA sequences to knock down the expression of TAK1 in three different pancreatic cancer cell lines, AsPc-1, PANC-1, and MDAPanc-28. These cell lines were transduced with lentiviruses expressing TAK1-specific shRNAs or a scramble sequence.
With this approach, we were able to significantly reduce the expression of TAK1 in the cell lines used (Fig.10 B). TAK1 shRNA rescue experiments were performed as control.
56
A
TAK1
β-actin
B
TAK1
β-actin
Figure 10. The effects of TAK1shRNA on pancreatic cancer cell lines. A. Testing TAK1 knocking down effects in 293T cells. Transfection of siRNA effectively knocked down the expression of TAK1 in 293T cells. B.TAK1 shRNA is infected into of TAK1 in AsPc-1,
PANC-1, and MDAPanc-28 pancreatic cancer cells by lentivirus. Protein extracts from these pancreatic cancer cell lines with or without infection were analyzed on Western blot (50g/of protein extract/lane) and probed with the indicated antibodies.
57
1.3.5. Silencing TAK1 Suppresses NF-B DNA Binding Activities.
TAK1 has been identified as an IKK kinase in TNF-α and IL-1 signaling pathway, which
contributes to of NF-B activation [91]. We analyzed NF-B activity in the pancreatic
cancer cell lines using different approaches. Luciferase assay demonstrated that whereas
AsPc-1 and MDAPanc-28 TAK1 knockdown cells had a very low NF-B activity, the same
cells stably expressing wild-type murine TAK1 demonstrated an NF-B activity similar to
the control cell lines (Fig. 11). Knocking down the expression of TAK1 suppressed the
autophosphorylation of residual TAK1 proteins (Fig.12), and reduced the phosphorylation of
p38, a downstream target of the TAK1-MKK6 intracellular pathway [102] (Fig.13).
To determine the effect of silencing TAK1 on the constitutive activation of NF-B, we
also evaluated IKK kinase activity and the DNA binding activity of NF-B transcriptional
factor in knockdown and control cell lines. We demonstrated that knocking down the
expression of TAK1 dramatically suppresses IKK phosphorylation (Fig.14 A) and the DNA-
binding activity of NF-B (Fig.14B) and in the pancreatic cancer cell lines compared with the respective controls.
Taken together, these data demonstrate that TAK1 is an essential mediator of the constitutive activation of NF-B in pancreatic cancer cells.
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Figure 11. TNF-κB activation is reduced in TAK1 knocking down cells. TAK1 shRNA rescue experiment. Nuclear factor κB (NF-κB) reporter gene assay in AsPc-1 and MDAPanc-
28 cells transduced with lentiviruses expressing TAK1-specific shRNA, a scramble sequence, or stably coexpressing human TAK1 shRNA and wild-type murine TAK1 sequences. The luciferase activities were normalized to the Renilla luciferase activity of the internal control. Error bars indicate 95% confidence intervals of two independent experiments performed in triplicate. Asterisks indicate statistical significance (P < .001) compared with scramble, as determined by two-sided one-way analysis of variance and Dunnett’s test.
59
Figure 12. TAK1 kinase activity is reduced in TAK1 knock down cells. Western blot analysis for the autophosphorylation of TAK1 in AsPc-1, PANC-1, and MDAPanc-28 pancreatic cancer cells transduced with lentiviruses expressing TAK1-specific shRNA or a scramble sequence as control.
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Figure 13. P38 kinase activity is reduced in TAK1 knock down cells. Western blot
analysis for the phosphorylation of p38 in AsPc-1, PANC-1, and MDAPanc-28 pancreatic
cancer cells transduced with lentiviruses expressing TAK1-specific shRNA or a scramble sequence as control.
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A
Phospho-IKKα/β
IKKα/β
B
P65/p50
histone H1
Figure 14. TAK1 is required for IKKα/β phosphorylation and NF-κB activation in pancreatic cancer cells. A. Western blot analysis for the phosphorylation of IKKα/β in
AsPc-1 and MDAPanc-28 pancreatic cancer cells transduced with lentiviruses expressing
TAK1-specific shRNA or a scramble sequence as control. Total IKKα/β was used as loading
control. EMSA was performed to determine the NF-κB activity in pancreatic cancer cell line
as indicated. Histone H1 was determined as loading controls.
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1.3.6. Silencing TAK1 Induces a Potent in Vitro Chemosensitization and apoptosis of
Pancreatic Cancer cells.
It is well known that pancreatic cancer is extremely resistance to chemotherapeutical drugs. To test our hypothesis that TAK1 would be responsible for the chemoresistance of pancreatic cancer, we determined the sensitivity of the TAK1 knockdown and control cell lines to three chemotherapeutic agents with different mechanisms of action that are either currently used or recently studied in combination with gemcitabine for pancreatic cancer treatment: DNA-intercalating agent oxaliplatin, topoisomerase I inhibitor SN-38, and nucleoside analog gemcitabine. AsPc-1, PANC-1, and MDAPanc-28 TAK1 knockdown cells demonstrated a significantly higher sensitivity to these chemotherapeutic agents than did their respective control cell lines. As indicated in Fig.15A, with the exception of the already gemcitabine-sensitive PANC-1 cells, after knocking down TAK1, we observed a significant increase in the sensitivity to gemcitabine for the moderately resistant AsPc-1 and the extremely gemcitabine-resistant MDAPanc-28 cells. All three knockdown cell lines were significantly more sensitive in the same extent to the irinotecan active metabolite SN-38. A dramatic chemosensitization to oxaliplatin was observed in all three knock down cell lines, showing differences in the IC50 between 40.2- and 72.7- fold (Fig.15B).
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Figure 15. Effects of TAK1 silencing on chemoresistance of pancreatic cancer cells. A.
Percent survival of AsPC-1, PANC-1, and MDAPanc-28 pancreatic cancer cells after treatment with increasing molar concentrations of gemcitabine, SN-38, and oxaliplatin.
Dimethyl sulfoxide–treated cells were assigned a value of 100% and designated as control.
Means and 95% confidence intervals of three independent experiments performed in quadruplicate are shown.
64
Figure 15. Effects of TAK1 silencing on chemoresistance of pancreatic cancer cells. B.
Percent survival of AsPC-1, PANC-1, and MDAPanc-28 pancreatic cancer cells after treatment with increasing molar concentrations of gemcitabine, SN-38, and oxaliplatin.
Dimethyl sulfoxide–treated cells were assigned a value of 100% and designated as control.
Means and 95% confidence intervals of three independent experiments performed in quadruplicate are shown.
65
Since several studies have shown that the proapoptotic stimuli elicited by the cytotoxic
agents activate the antiapoptotic function of NF-B [134], we determined the activation of
NF-B after treatment with IC50 doses of the cytotoxic agents. Whereas the control cells already demonstrated substantial activation of NF-B that could not be further augmented by
exposure to the cytotoxic agents, the TAK1 knockdown cells had much lower levels of NF-
B activation and were unable to increase NF-B activation, even if treated with IC50 doses
of the cytotoxic agents (Fig.16). In this regard, whereas the control pancreatic cancer cells
were protected from apoptosis, the TAK1 knockdown cells demonstrated a high induction of
apoptosis as demonstrated by an increased level of caspase-3 cleavage products (Fig. 17A )
and by the fragmentation of genomic DNA (Fig. 17B ). These data demonstrate that TAK1
is an important mediator of chemoresistance in pancreatic cancer.
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Figure 16. Nuclear factor κB (NF-κB) activation after treatment with cytotoxic agents.
AsPc-1, PANC-1, and MDAPanc-28 pancreatic cancer cells transduced with lentiviruses
expressing TGF-β-activated kinase-1 (TAK1)-specific small hairpin RNA (shRNA), or a
scramble sequence as control were treated for 24 hours with doses of gemcitabine, SN-38, or
oxaliplatin corresponding with the IC50for the control cells or saline as
control. Electrophoretic Mobility Shift Assay analysis was performed to determine the
DNA-binding activity of NF-κB p65/p50 and p50/p50 complexes.
67
SN-38 - -+ --+ A Oxaliplatin -+ --+ - TAK1 shRNA - - - + + +
32kD
Caspase-3 19kD AsPc-1 17kD
β-actin
32kD
Caspase-3 19kD Panc-1 17kD
β-actin
Figure 17. Silencing TAK1 with the treatment of chemotherapeutical drugs induces
proapoptosis phenotypes. A. Apoptosis of pancreatic cancer cell lines was measured by western blot analysis for caspase-3 cleavage after chemotherapeutical drugs treatment.
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WT TAK1 shRNA B - - - + - - - + gemcitabine SN-38 - - + - - - + - Oxaliplatin - + - - - + - -
AsPc-1
Panc-1
Panc-28
Figure 17. Silencing TAK1 with the treatment of chemotherapeutical drugs induces proapoptosis phenotypes. B. Apoptosis of pancreatic cancer cell lines was measured by
DNA fragmentation detected by agarose gel electrophoresis after chemotherapeutical drugs treatment.
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1.3.7. TAK is inactivated by the dominant negative mutant form.
To confirm the results from TAK1 shRNA knocking down, we also inactivate TAK1 function using the dominant negative mutant, which is the TAK1 kinase dead form in which
the ATP binding pocket was mutated from lysine to alanine at the amino acid residue 63.
Using lentivirus transduction, we were able establish the five stable cell lines which
expressed TAK1 K63A mutant. As shown in Fig. 18, IKKα/β phosphorylation was
significantly reduced and NF-B activation was blocked in pancreatic cancer cell line by the
kinase dead mutant K63A. These results furthermore support that TAK1 play an essential
role in the controlling NF-B activation in pancreatic cancer cells.
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TAK1 K63A
Flag-TAK1
β-actin
pIKKα/β
IKKα/β
P65/p50
Histone H1
Figure 18. TAK1 activity is inhibited by the dominant negative mutant K63A in pancreatic cancer cells. Lentivirus transduction was used to establish stable cell lines as indicated which expressed TAK1 kinase dead mutant form K63A. IKKα/β activity was analyzed by western blot and NF-κB activation was determined by EMSA. Total IKKα/β and histone H1 were used for loading control.
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1.3.8. The inactivation of TAK1 suppresses pancreatic cancer cell migration and invasion.
The metastatic property of pancreatic cancer is still a big challenge for patients.
Acquiring the potential of migration and invasion is a prerequisite for cancer metastasis. To study the role of TAK 1 in the controlling migration and invasion of pancreatic cancer cells, we performed Boyden chamber migration and Matrigel invasion for the TAK1 knocking down cells, TAK1 K63A mutant cells and control cells. Our results demonstrated that the inactivation of TAK1 with either shRNA or kinase dead mutant significantly reduced the migration and invasion of pancreatic cancer cells (Fig.19 and Fig.20).
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scramble shRNA TAK1 shRNA vector TAK1 K63A
AsPc-1
Panc28
160 35 140 30 120 25 100 20 80 15 60 10 40 5 20 0 0 scramble TAK1 Vector K63A scramble TAK1 Vector K63A shRNA shRNA shRNA shRNA
AsPc-1 migration cells Panc-28 migration cells
Figure 19. Blocking TAK1 inhibits pancreatic cancer cell migration. The Chamber migration assay was performed to determine the potentials of cell migration. The representative field of each cell line was shown. The migrated cells were quantified.
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scramble shRNA TAK1 shRNA vector TAK1 K63A
AsPc-1
Panc28
10 160 9 140 8 120 7
6 100
5 80 4 60 3 40 2
1 20
0 0 scramble shRNA TAK1 shRNA Vector K63A scramble shRNA TAK1 shRNA Vector K63A
AsPc-1 invasion cells Panc-28 invasion cells
Figure 20. Blocking TAK1 inhibits pancreatic cancer cell invasion. The Matrigel invasion was performed to determine the potentials of cell invasion. The representative field of each cell line was shown. The invaded cells were quantified.
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1.3.9. Targeting TAK1 kinase activity chemo sensitizes pancreatic cancer cells In Vitro
To establish TAK1 as a pharmacologically relevant target for the chemosensitization of
pancreatic cancer, we firstly tested the activity of the TAK1-kinase selective inhibitor
LYTAK1 in vitro. Four pancreatic cancer cell lines – AsPc-1, PANC-1, MDAPanc-28, and
Colo357FG – were exposed for 24 hours to increasing doses of LYTAK1. LYTAK1 had
potent in vitro cytotoxic activity, demonstrating an IC50 between 5 and 40 nM (Fig.21, A).
At this dose range, the kinase activity of TAK1 was markedly inhibited as indicated by the suppression of the autophosphorylation of TAK1 and the phosphorylation of p38 (Fig.21, B).
To demonstrate that the inhibition of the kinase activity of TAK1 would suppress the
activation of NF-B, we studied the effect of LYTAK1 on the DNA binding activity of this transcriptional factor. We observed that in the concentration range indicated, LYTAK1 was able to completely suppress the constitutive activation of NF-B in the pancreatic cancer cell lines (Fig.21, C).
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Figure 21. Activity of the TAK1 inhibitor LYTAK1 in vitro. A. Percent survival of AsPc-
1, PANC-1, MDAPanc-28, and COLO357FG pancreatic cancer cells after treatment with
increasing molar concentrations (log scale) of LYTAK1. Dimethyl sulfoxide (DMSO)–
treated cells were assigned a value of 100% and designated as control. Means and 95% confidence intervals of three independent experiments performed in quadruplicate are shown.
Curves were fitted by nonlinear regression analysis. B. Western blot analysis was performed to determine the expression and phosphorylation of TAK1 and p38, and C. Electrophoretic
Mobility Shift Assay analysis was performed to determine the DNA-binding activity of NF-
κB p65/p50 and p50/p50 complexes in AsPc-1, PANC-1, and MDAPanc-28 pancreatic cancer cells treated for 24 hours with increasing doses of LYTAK1 or DMSO as control.
Similar results were obtained with three independent experiments.
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To test our hypothesis that the pharmacologic inhibition of TAK1 would modulate the chemoresistance of pancreatic cancer, we determined whether the addition of LYTAK1 resulted in an increased in vitro antitumor activity when combined with classic chemotherapeutic agents. AsPc-1, PANC-1, and MDAPanc-28 pancreatic cancer cells, and
HPDE cells as control were treated with increasing doses of oxaliplatin, SN-38, or gemcitabine in combination with LYTAK1 at doses corresponding with the IC50 measured for each cell lines, or DMSO as control. The addition of LYTAK1 potentiated the cytotoxicity of these chemotherapeutic agents against the three different pancreatic cancer cell lines. No further significant sensitization was measured in the HPDE control cells
(Fig.22). The pharmacologic inhibition of TAK1 induced an increase in the sensitivity of the pancreatic cancer cell lines to chemotherapeutic agents to the same extent or, in the most resistant lines, considerably better than the sensitivity obtained with the genetic silencing of
TAK1.
Taken together, these results indicate that inhibiting TAK1 kinase by LYTAK1 effectively potentiates sensitivity of mechanistically diverse chemotherapeutic agents.
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Figure 22. Inhibition of TAK1 and chemosensitization of human pancreatic cancer cells in vitro. Percent survival of AsPC-1, PANC-1, and MDAPanc-28 pancreatic cancer cell lines, and human papillomavirus type 16 early gene 6- and 7-immortalized/nontumorigenic human pancreatic ductal epithelial (HPDE) control cells after treatment with increasing molar concentrations (log scale) of gemcitabine, SN-38, and oxaliplatin alone or in combination with LYTAK1 at doses corresponding with the IC50 measured for each cell line or dimethyl sulfoxide (DMSO) as control. DMSO-treated cells were assigned a value of 100% and designated as control. Means and 95% confidence intervals of three independent experiments performed in quadruplicate are shown.
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1.3.10. TAK1 and TAB1 stabilize each other in pancreatic cancer cell line.
As indicated above, TAK1 plays an important role in chemotherapeutic drug resistance in
pancreatic cancer cells. Block TAK1 activity can reverse pancreatic cancer cell resistance to chemotherapeutic drug. NF-B signaling pathway activates anti-apoptosis pathway and thereby mediates chemotherapeutic drug resistance. However, the underlying mechanism how TAK1 is activated in pancreatic cancer cells is still unknown. As shown above, the
expression levels of TAK1 and TAB1 are highly correlated in almost all of pancreatic cancer
cell lines and tumor tissues. To explore the regulatory mechanism of TAK1, we studied the role of TAK1 binding protein 1 (TAB1) in pancreatic cancer cell line. Firstly, we confirmed the existence of the interaction between TAK1 and TAB1 in pancreatic cancer cell lines. As shown Fig. 23A, TAK1 can coimunoprecipitate with TAB1 even without any stimulation.
A lot of experiments have demonstrated that TAK1 is regulated by TAB1 and TAB1 is required for TAK1 activity. However, however, the role of TAB1 in the control of TAK1 activity is still controversy. It was reported that IL-1 induced NF-κB activation was not
affected but IL-1 or TNF-α induced TAK1 activity was severely inhibited in TAB1 deficient
cells [135]. To clarify the role of TAB1 in pancreatic cancer cells, we silenced TAB1 in the
pancreatic cancer cell line Panc-1.
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We measure TAB1 level in TAK1 knocking down cells. Interestingly, TAB1 protein level was also decreased in TAK1 knocking down cells while the mRNA level of TAB1 did not change (Fig. 23B). These experiments demonstrated that TAB1 is stabilized by TAK1 at protein level but not increased by mRNA transactional level, determined by SYBR green quantitative real-time PCR. Conversely, TAK1 protein level was also decreased in TAB1 knocking down cells while the mRNA level of TAK1 did not change. TAK1 is stabilized by
TAK1 in Panc-1 cells (Fig. 23C). . This mechanism may be common in pancreatic cancer cell lines. We also analyzed the effect of TAB1 on NF-B activity. As indicated in Fig. 23D, silencing TAB1 inhibits NF-B activation in Panc-1 cells.
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AsPc‐1 Panc28 IP: anti‐TAB1 ‐ ++‐ WB: TAK1
TAK1
Figure 23. TAK1 and TAB1 stabilize each other in pancreatic cancer cell line. A. TAK1 and TAB1 interact with each other in pancreatic cancer cells. TAB1 antibody was used to precipitate the protein complex in TAK1 knocking down cells and the control cells as indicated and TAK1 level was analyzed by western blot.
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0.0006 AsPc‐1 The relative mRNA levels of TAK1 TAK1‐shRNA + 0.0005 The relative mRNA levels of TAB1 0.003 Scrambled 0.0004 + 0.0025 TAK1‐shRNA TAK1 0.0003 0.002 0.0002 0.0015 TAB1 0.001 0.0001 β‐actin 0.0005 0 0 AsPc‐1AsPc‐1+ AsPc‐1+TAK1 AsPc‐1AsPc‐1+ scramble AsPc‐1+TAK1 scramble shRNA shRNA shRNA shRNA Panc28 TAK1‐shRNA + The relative mRNA levels of TAB1 Scrambled The relative mRNA levels of TAK1 + 0.001 0.001 TAK1‐shRNA 0.0008 0.0008 TAK1 0.0006 0.0006
TAB1 0.0004 0.0004 β‐actin 0.0002 0.0002 0 0 Panc‐28 Panc‐28+ scramble Panc‐28+TAK1 Panc‐28 Panc‐28+ scramble Panc‐28+TAK1 shRNA shRNA shRNA shRNA
Figure 23. TAK1 and TAB1 stabilize each other in pancreatic cancer cell line. B. TAB1 is stabilized by TAK1 in pancreatic cancer cells. TAK1 and TAB1 protein levels were determined by western blot and mRNA level was determined by real time PCR.
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Panc‐1 TAB1‐shRNA + + The relative mRNA levels of TAB1 0.007 The Relative mRNA levels of TAK1 0.045 TAB1 0.04 0.006 0.035 0.005 0.03 0.004 TAK1 0.025 0.02 0.003 0.015 0.002 β‐actin 0.01 0.005 0.001 0 0 Panc‐1Panc‐1+ Panc‐1+TAB1 Panc‐1Panc‐‐1+ Panc‐1+TAK1 scramble shRNA shRNA scramble shRNA shRNA
Figure 23. TAK1 and TAB1 stabilize each other in pancreatic cancer cell line. C. TAK1 is stabilized by TAB1 in pancreatic cancer cells.
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1.3.11. Silencing TAK1 or TAB1 reduced tumor size in Orthotopic Xenograft models.
As indicated above, the inactivation of TAK1 suppresses pancreatic cancer cell migration
and invasion. To test our hypothesis that TAK1 play a role in pancreatic cancer progression,
we determined if targeting TAK1 or TAB1 could inhibit growth of human pancreatic cancer
cells in an orthotopic xenograft mouse model. Five mice for each group were orthotopically
injected with the different pancreatic cancer cell lines as indicated. Eight weeks after
injection, all mice were sacrificed, and the degrees of tumorigenesis were investigated.
AsPc-1 and Panc-28 are extremely malignant and aggressive cell lines. As shown in Fig.
24, the mice injected by the control cells (AsPc-1 or Panc-28) exhibited high tumor burden
and the inactivation of TAK1 by shRNA or kinase dead mutant significantly reduced tumor
size. Interestingly, tumor yield was also decreased in mice injected by TAB1 knocking down
cells (Fig. 25). These data support that TAK1 and TAB1 play an essential role in tumorigenesis of pancreatic cancer.
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average
3.5
3
2.5 AsPc-1 average 2
1.5 AsPc-1 scramble 1 Nude mice 0.5 0
le AsPc-1 ramb shRNA sc 1 K -1 AsPc-1 TAK1 shRNA TA sPc A AsPc-1
average Panc28
1.8 1.6 Panc28 scramble 1.4 Scid mice 1.2 1 average 0.8 0.6 Panc28 TAK1 shRNA+mouseTAK1 0.4 0.2 0
8 e . . 63A hRNA anc-2 se. s Panc28 TAK1 shRNA p rambl sc ou m -28 TAK1 28 -28 TAK1 K panc shRNA nc- anc p pa Panc28 TAK1 K63A TAK1 -28
panc
Figure 24. The inactivation of TAK1 suppresses tumorigenicity in an orthotopic mouse
model. One million viable cells for each cell line as indicated were suspended in 50 µl phosphate-buffered saline (PBS) and injected into the pancreatic parenchyma of female athymic BALB/c nude mice at 8 weeks of age (Charles River Laboratories, Inc. Wilmington,
MA, USA). Eight weeks after injection, all mice were sacrificed, and the degrees of tumorigenesis were investigated. The weights of tumors are shown in the graph.
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4.5 Panc-1 4 3.5 Nude mice 3 Panc-1 scramble 2.5 2 1.5 Panc-1 TAB1 shRNA 1 0.5 0
1 - le b Panc m RNA cra sh s B1 -1 A c n T a -1 P c n a P
Figure 25. The inactivation of TAB1 suppresses tumorigenicity in an orthotopic mouse model. One million viable cells for each cell line as indicated were suspended in 50 µl phosphate-buffered saline (PBS) and injected into the pancreatic parenchyma of female athymic BALB/c nude mice at 8 weeks of age (Charles River Laboratories, Inc. Wilmington,
MA, USA). Eight weeks after injection, all mice were sacrificed, and the degrees of tumorigenesis were investigated. The weights of tumors are shown in the graph.
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1.4. Discussion
1.4.1. Targeting TAK1 increased the sensitivity of pancreatic cells to chemotherapeutical drugs and blocks tumorigenesis.
As introduced before, NF-B is constitutively activated in the most pancreatic cancer cell
lines and tissues. Inhibition of constitutive NF-B activity by Ibα mutant suppresses
tumorigenesis [58, 59]. The genetic evidence revealed that NF-κB activation induced by
TNFα and IL-1β are severely impaired in TAK1 knock out MEF cells [92]. Therefore, we
rationalized that TAK1 plays a role in pancreatic cancer.
Pancreatic cancer is the fourth leading cause of cancer-related mortality in the United
States and the fifth leading cause of cancer-related mortality worldwide. According to the
estimation of the American Cancer Society, approximately 43140 will be diagnosed with this
cancer and 36800 will die of it in 2010. Pancreatic cancer is a big challenge in large due to
the lack of early symptoms. In addition, drug resistance is a major obstacle to the success of
chemotherapy in pancreatic cancer. The underlying mechanism of drug resistance in human
pancreatic cancers is not well understood. Better understanding of the mechanism of
molecular pathways in human pancreatic cancers can help to identify the novel therapeutic
target candidates, and develop the new preventive and clinic strategies to improve patient
87 survival. We found that TAK1 is overexpressed in most pancreatic cancer cell lines the protein plays an important role in the survival of the cells. The goal of this research project is to identify novel therapeutic agents that target transforming growth factor beta activated kinase 1 (TAK1, MEKK7) in human pancreatic cancer.
TGF- signaling pathway is frequently defective in various types of cancer such as pancreatic and colon cancers. TGF- legends and their receptors are overexpressed in pancreatic cancer cells. TGF- enhances invasion and the epithelial-to-mesenchymal transition (EMT). TAK1 is a key component in TGF- signaling pathway. In addition, TAK1 is required for TNF-α induced and IL-1β induced NF-κB activation. TAK1 plays a key role in pro-inflammatory signaling. Chronic or recurrent inflammation contributes to many types of cancer. Our lab previous demonstrated that nuclear factor-κB (NF-κB) is constitutively activated in most human pancreatic cancer cell lines but not in normal pancreatic tissues. The inhibition of the constitutive NF-κB activity in human pancreatic cancer cell lines blocked tumorigenesis of pancreatic cancer cells. NF-κB is a key mediator of pancreatic cancer cell survival. Since TAK1 acts as the upstream of NF-κB and it is required for NF-κB activation, we propose that TAK1 plays a key role of chemo-drug resistance in pancreatic cancer. In this study, we demonstrated a unique role for the MAP3K TAK1 in the chemoresistance of pancreatic cancer.
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TAK1 has recently emerged as a central regulator of diverse physiological processes including development, metabolism, and immune and stress responses leading to the activation of the transcription factors NF-B and AP-1 [136]. To demonstrate an important role for TAK1 in cancer, we silenced its expression or inhibited its kinase activity in pancreatic cancer cells. Our preliminary results demonstrated that TAK1 is overexpressed in most pancreatic cancer cell lines. Knocking down of TAK1 using RNAi technique blocked
NF-κB activity in pancreatic cancer cells and made chemotherapy drugs gemcitabine, oxaliplatin, or irinotecan more potent. Inhibition of TAK1 using either shRNA or TAK1 kinase dead mutant resulted in cell apoptosis, decreased pancreatic cancer cells invasion and significantly reduced the size of tumors in orthotopic mice model. These findings highlighted that TAK1 could be a potential therapeutic target candidate for pancreatic cancer.
To our knowledge, our present study is the first to demonstrate that silencing the expression of TAK1 leads to a proapoptotic phenotype in pancreatic cancer cells and in turn to their significantly higher sensitivity to the antitumor activity of chemotherapeutic drugs with different mechanisms of action. Considering the oxaliplatin in particular, whereas the
IC50s for this drug in control pancreatic cancer cells were higher than those achievable in patients [137], we found that silencing TAK1 decreased the IC50s for oxaliplatin into the range of plasma concentrations achievable in patients.
To confirm the results we obtained through genetic silencing of TAK1 expression, and to validate TAK1 kinase therapeutic target for treatment of pancreatic cancer patients, we 89
investigated the antitumor activity of the TAK1 kinase selective inhibitor LYTAK1 as a
single agent and in combination with gemcitabine, oxaliplatin, or irinotecan. In a different
study, screening for compounds that can inhibit TAK1 kinase activity resulted in the isolation
of one natural compound, 5Z-7-oxozeaenol, a resorcylic lactone of fungal origin with a
selective TAK1-inhibiting activity [89]. Together with anti-inflammatory activity, 5Z-7- oxozeaenol has recently demonstrated to potentiate in vitro the TRAIL- induced apoptosis in lung cancer cells [105]. To our knowledge, our current study is the first to demonstrate that targeting the kinase activity of TAK1 with the selective inhibitor LYTAK1 potentiate in vitro the antitumor activity of gemcitabine, oxaliplatin, or irinotecan against a panel of pancreatic cancer cells.
The potent in vitro antitumor activity of the pharmacological inhibition of TAK1 kinase activity in contrast to the milder proapoptotic phenotype demonstrated by the TAK1 knockdown pancreatic cancer cell lines could be due to different reasons. LYTAK1 is a potent and selective kinase inhibitor resulting in the almost complete inhibition of TAK1 activity, which in part may explain its cytotoxicity in vitro as single agent. On the other hand, shRNA did not completely silence the expression of TAK1, which may explain why TAK1 knockdown cells survived. Furthermore, our unpublished data from somatic cell (HCT116) knockout experiments indicated that complete ablation of TAK1 is lethal, consistently with the embryonic lethal phenotype noted in TAK1 knockout mice [92].
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In line with other murine models carrying a tissue- specific deletion of the TAK1 gene
[92, 94, 138], during the preparation of this manuscript two studies reported that TAK1 conditional ablation in liver parenchymal cells causes an early (6 weeks) spontaneous apoptosis of hepatocytes and cholangiocites [139, 140]. This leaded to chronic hepatitis, cholestasis, liver fibrosis, and to a subsequent compensatory liver cell proliferation that at later time point (16-33 weeks) drives carcinogenesis in the liver. The additional deletion of the receptor for TNF- was able to significantly suppress liver damage and inflammation
[139]. Whereas the spontaneous apoptosis of TAK1 knockout liver parenchymal cells is consistent with our observations in pancreatic cancer cells, these works could raise concerns on the potential hepatic toxicity of the pharmacologic inhibition of TAK1. As indicated before, in our work all regimens were well tolerated, and no weight loss or other signs of acute or delayed toxicity were observed. The low levels of TNF- in athymic nude mice kept under germ-free condition could explain the safety of LYTAK1 in comparison to the spontaneous apoptosis and inflammation observed in mice not kept under axenic conditions
[139, 140], and in particular the lack of activity in vivo of LYTAK1 as single agent treatment. In absence of other activator of apoptosis, the pharmacologic inhibition of TAK1 induced apoptosis of pancreatic cancer cells only when combined with proapoptotic chemotherapeutic agents.
Given its complex role in pancreatic cancer biology, targeting the TGF-β signaling pathways represents a challenging but important opportunity for the development of novel treatment for pancreatic cancer patients [141, 142]. In a different study, we investigated
91
whether targeting the TGF-β signaling though inhibition of TGF-β receptors I/II kinase
activity was a valid therapeutic approach to suppress growth and metastatic spread of pancreatic cancer [128]. Whereas we observed that targeting the kinase activity of the TGF-β receptor suppressed the Smad- dependent pathway and in turn significantly inhibited pancreatic cancer metastasis, we found that the combination of TGF-β receptor inhibition and gemcitabine resulted in a relatively minor impact on pancreatic tumor growth and mice survival. More recently, it has been demonstrated that the activation of TAK1 by TGF-β occurs in a TGF-β receptor kinase- independent manner [143]. These results suggest that targeting the kinase activity of TGF-β receptors I/II would not be likely to suppress the activation of TAK1, thereby explaining the weak potentiation of gemcitabine observed [128].
In the past two decades, the research for pancreatic cancer therapy has been focused on several molecules that are early players in the signal transduction cascade, with particular interest in membrane receptors such as the Epidermal Growth Factor Receptor (EGFR)
[144]. From the results of the clinical trials with inhibitors of this receptor in pancreatic [145]
and colorectal cancer [146], we have learned that the single mutation of K-Ras, the most
common genetic alteration in pancreatic cancer, is probably able to circumvent the antitumor
activity of anti-EGFR approaches. On the other hand, strategies for the direct inhibition of
the final players of the signal transduction cascade such the transcriptional factor NF-B still
face enormous challenges [134]. As a member of the MAP3K family, TAK1 is unique [136].
More than a single enzyme, it may be considered rather as the active component of a larger protein signaling complex made up of a variety of proteins such as TAK1 binding proteins
92
(TABs) and TRAF6, explaining its role as a key regulator of several signal transduction
cascades. We believe that targeting no redundant cytosolic mediators of the signal transduction cascade such as TAK1 could represent a better approach to inhibit key processes in tumor cells.
1.4.2. TAK1 activity is regulated in pancreatic cancer cells by TAB1.
TAB1 was initially identified as a TAK1 binding protein and many studies have
demonstrated that TAK1 activity I regulated by TAB1. However, NF-B is still activated in
TAB1 null MEF cells by TNF-α and IL-1. Our studies demonstrated that TAK1 and TAB1
are highly correlated in almost all of pancreatic cancer cells and they are stabilized by each other. Furthermore, silencing TAB1 suppressed NF-B activation. This result is conflicted
with that from MEF cells. One explanation is that TAB1 function may be cell type
dependent. In the context of TAK1 overexpression in Panc-1 cells, TAB1 may be involved
in NF-B activation through stabilizing TAK1 protein level. We tried to knock down TAB1 level in several different pancreatic cancer cell lines, but we only obtain the effective knocking down result in Panc-1 cells (data not shown). Thereby, we don’t know whether
NF-B is regulated in a common mechanism by TAB1.
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Importantly, TAB1 knocking down significantly reduced tumor burden in the Orthotopic
Xenograft models. As far as we know, this the first time to demonstrate TAB1 plays a role in pancreatic cancer tumorigenicity. Disruption of the interaction of TAK and TAB1 may reduce TAK1 level and therefore regulate TAK1 activity. Our results provide a new insight that the interaction of TAK1 and TAB1 may be used as a therapeutic target for pancreatic cancer.
In conclusion, this study is the first to demonstrate that the genetic silencing or the inhibition of the kinase activity of TAK1 is a valid approach to revert the intrinsic chemoresistance of pancreatic cancer. LYTAK1 is the first TAK1 selective inhibitor to be active in vivo and thus warrants further development for the treatment of pancreatic cancer patients.
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CHAPTER 2. THE REGULATORY MECHANISM OF TAK1 BY TAB1
AND ACETYLATION AT LYSINE 158
2.1. Introduction
2.1.1. TAK1 interaction proteins
A lot of studies have indicated that TAK1 is regulated by its binding proteins including
TAB1, TAB2, TAB3 and TAB4 and post-translational modification such as phosphorylation and Ubiquitination. We will briefly review several TAK1 interaction proteins and protein modifications.
2.1.1.1. TAB1
TAB1 was originally identified as a TAK1 interaction protein with yeast –two- hybrid.
TAB1 interacts with the N-terminus of TAK1 and the C-terminus of TAB1 is sufficient to activate TAK1 upon TGF-β stimulation in the overexpression experiments [124]. As introduced before, Co-expression of MOM-4 and TAP-1, which are the homologs of TAK1 and TAB1 in C.elegance respectively, stimulate MOM-4 MAP3K activity [76].
95
Coexpression of mTAK1 with hTAB1 resulted in reduced eye size in drosophila in transgenic flies [81]. TAK1 and TAB1 mediate BMP signaling pathway in xenopus at the early stage of development. TAB1 enhanced the function of TAK1 in inducing the formation of ventral mesoderm [85].
Target deletion of TAB1 in mice results in embryonic lethality E15.5 and E18.5. The development of heart and lung is largely impaired in TAB1 deficient embryos. TAB1 is required for TAK1 kinase activity which phosphorylates MKK6 in vitro and the reporter gene activity in response of TGF-β stimulation [147]. However, the role of TAB1 in the control of TAK1 activity is still controversy. It was reported that IL-1 induced NF-κB activation was not affected but IL-1 or TNF-α induced TAK1 activity was severely inhibited in TAB1 deficient cells [135]. These contradictory reports demonstrated that more detailed
analysis is required to dissect the function of TAB1 in regulating TAK1 activity.
It has been reported that the N-terminal domain of TAB1 is similar with protein phosphatase 2C (PP2C), however, the mutagenesis and structural analysis demonstrated that
TAB1 does not have the catalytic activity [148]. TAB1 Biochemical and structural analysis indicated that the N-terminal domain of TAB1interacts with the BIR1 domain of X-linked inhibitor of apoptosis (XIAP) and the interaction between them is required for NF-κB activation[149].
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2.1.1.2. TAB2
TAB1 was also isolated by yeast –two- hybrid. In contrast to TAB1, the C-terminal domain of TAB2TAB2 interacted with the C-terminal domain of TAK1 [124, 125] .
Biochemical experiments revealed that TAB1 acts as an adapt protein to mediate the interaction of TRAF6 and TAK1. The C-terminal domains of both TAK1 and TAB1 have the coiled-coil motif, which may provide the protein interaction between TAK1 and TAB1.
Cotransfection of TAB2 into 293 cells is not sufficient to induce TAK1 kinase activity that phosphorylates MKK6. However, TAB2 mediate NF-κB activation and JNK activation in a dose dependent manner followed by IL-1 stimulation and the C-terminal region of TAB2 acts as a dominant-negative mutant to block NF-κB activation and JNK activation in response of
IL-1 stimulation. However, the C-terminal region of TAB2 is unable to block NF-κB activation induced by TNF-α [125].
Targeted deletion of TAB2 in mice induced increased apoptosis of hepatoblasts and therefore resulted in embryonic lethality at day 12.5. However, NF-κB activation induced by
TNF-α and IL-1 is normal in TAB2 deficient cells. These data demonstrated that TAB2 nay not required for NF-κB activation in response of IL-1 and TNF-α [150].
2.1.1.3. TAB3
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TAB3 was initially identified in xenopus using cDNA microarrays [151]. TAB3 was
found from human and mice EST database according to the homolog of TAB2 and the full
length TAB3 was isolated from a human kidney cDNA library. Expression of TAB3 in 293T cells enhanced NF-κB activation. Cooverexpression of TAB3 increased the kinase activity of TAK1 and induced JNK activation. Either TAB2 or TAB3 siRNA alone did not block
NF-κB activation, however, combination of TAB2 with TAB3 significantly impaired block
NF-κB activation. These results implicated that TAB2 and TAB3 may have a redundancy function [152].
It was reported that both TAB2 and TAB3 contain a Zn finger (ZnF) domain which can bind the polyubiquitination chains. Once stimulated by IL-1 and TNF-α, the
polyubiquitination chains of RIP or TRAF6 interact with the ZnF domain of TAB2 and
TAB3, and therefore provided a scaffold to help TAK1 phosphorylate IKKβ [153].
2.1.1.4. TAB4
TAB4 was initially called type 2A-interacting protein (TIP), which firstly identified as
the homolog of the yeast Tip41 protein from a human T-cell lymphoma cDNA library.
Immunoprecipitation experiments demonstrated that TIP bound with PP2A family members
consisting of PP2Ac, PP4c and PP6c. TIP was shown to inhibit PP2A activity in the
ATM/ATR signaling pathway [154]. TAB4 transiently associated with TAK1 at 5 minute
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and disassociated at 15 min after IL-1β stimulation. Overexpression of TAB4 in 293T cells increased TAK1-TAB1 kinase activity and induced NF-κB activation [155].
2.1.2. Overview of Posttranslational modification (PTM)
Posttranslational modification (PTM) is a later step after protein translation. The common
modification includes phosphorylation, methylation, acetylation, ubiquitination and
glycosylation. Protein modification plays a very important role in the control of the functions
of a protein, for example, protein stability, protein interaction, protein-DNA interaction,
activating or inactivating an enzyme. We will give a brief review for several common
posttranslational modifications.
2.1.2.1. Phosphorylation: Phosphorylation is the most intensively studied
posttranslational protein modification. A phosphate (PO43-) group was added to Serine,
Threonine or Tyrosine during phosphorylation. Phosphorylation is catalyzed by kinase and
reversed by phosphatases. Phosphorylation introduces negative charges and therefore
changes protein conformation from hydrophobic to hydrophilic properties, which regulates
protein function in the multiple processes such as protein stability, protein interaction and
enzyme activity. For example, in canonical NF-κB signaling pathway, is caused by IKK-
induced phosphorylation [156]. It was reported that the interaction of MDM2 with p53 was
reduced by the phosphorylation at Thr-216 [157]. In MAP kinase pathway, the stress-
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activated protein kinases (SAPKs) activator (SEK1) is activated by MEKK1 induced
phosphorylation [158].
2.1.2.2. Methylation: Protein methylation is the addition of a methyl
group to arginine or lysine amino acid of a protein [159]. According to the structures of the guanidino nitrogens of arginine and the ε-amino group of lysine, arginine can be methylated at most twice and lysine can be methylated at most three times, respectively.
Dimethylation of arginine can be grouped into two different sub types: 1. asymmetric
NG,NG-dimethylarginine (aDMA) catalyzed by type I peptidylarginine methyltransferases (PRMTs) in which two methyl groups are added on the same one nitrogen and 2, symmetric dimethylated arginine catalyzed by type II PRMTs in which two methyl groups are added on both nitrogens. Several arginine methyltransferases including PRMT1 through 9 have been identified in mammalian and the substrate specificity of some PRMT enzymes have been determined [160].
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Figure 26. Biochemical mechanisms for protein-lysine and protein arginine methylation. Trends in Biochemical Sciences Volume 32, Issue 3, March 2007, Pages
146-152. Reprinted by permission from Elsevier.
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Protein-lysine methylation is most studied in histone. Multiple lysine sites in the H3 and
H4 histone subunits have been identified [161, 162]. Lysine methylation is reversible and
LSD1, which is a homolog of the amine oxidase homolog, has been identified as the first
demethylase [163]. Protein methylation is involved in multiple processes such as protein
stability, protein interaction, transcriptional regulation, enzyme activation and mRNA
splicing (Paik, Paik et al. 2007).
2.1.2.3. Ubiquitination
Ubiquitin is a small peptide consisting of 76 amino acids, which is highly conserved from
yeast to human. Ubiquitination is one of the most well known posttanslational modifications
in which one or several ubquitin molecules are added to the lysine of the targeted protein.
Ubiqutination was initially identified as the major mechanism of protein degradation. Three sequential steps are involved in protein ubiquitination and each step is catylazed by the corresponding enzyme [164].
In the first step, the C-terminal glycine of ubiquitin forms a high energy thiol-ester bond with the a cysteine of the ubiquitin-activating enzyme E1. This reaction is ATP dependent and the activated ubiquitin is produced during this step. In the following step, the activated ubiquitin from the high energy thiol-ester bond is passed on to the substrate by the ubiquitin- conjugating enzyme E2. In the final step, the substrate bound ubiquitin protein ligase E3 catalyzes the formation of a isopeptide bond between the ε-NH2 group of a lysine on the
102 substrate and the C-terminal glycine on a ubiquitin. The cycle consisting of the above three steps can be repeated to produce ubiquitin chains. The protein tagged with ubiquitin is targeted to the 26S proteosome for degradation. The ubiquitin molecules are removed from the proteins before degaration and the free ubiquitin molecules can be recycled. In most cases, the substrate sepecificity of ubiquitination is largely determined by E3 ligase and also depends in part on E2 enzyme. A lot of E2 and E3 enzymes have been identified [164] .
Ubiquitin contains seven lysine residues (Lys6, Lys11, Lys27, Lys29,
Lys33, Lys48 and Lys63) . A proteomics study using mass spectrometry demonstrated that polyubiquitin chain can be attached at any of these seven lysine sites [165]. The lysine 48 and lysine 63 are the most well known conjugating and the fuction of ubiquitination at other lysine site is still largely unknown. Historically the original studies were focused on the lysine 48 chains which is responsible for protein degradation. However, most recent studies have extended the role of ubiqutination from protein degradation to a variety of signaling pathways. The most well known sample is the role of lysine 63-linked ubiqutination in NF-
κB signaling pathway. It was reported that TAK1 interacted with the lysine 63-linked ubiquitination chain of TRAF6 through TAB2 or TAB3 upon IL- stimulation and furthermore activate the downsream IKK-IκBα - NF-κB cascade [136].
The recent studies have identified a new Ubiquitination during which a head-to-tail (Ub1
Gly76 to Ub2 Met1) linkage of polyubiquitination chain is produced. This polyubiquitination chain is conjugated to Lys 285 and Lys 309 in the coiled-coil 2 (CC2) and
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leucine zipper (LZ) domain of NF-κB essential modulator (NEMO, or called IKKγ).
Linear ubiquitin chain assembly complex (LUBAC), which contains two RING finger
components HOIL-1L and HOIP, induces the formation of the linear polyubiquitin chain and
is required for the canonical NF-κB activation [166, 167].
Like phosphorylation, methylation, ubiquitination is also a reversible process. The
cleavage of ubiquitin from substrates is catalyzed by deubiquitinating enzymes (DUBs). As
the reverse of Ubiquitination, deubiquitination plays an important role in multiple processes
such as kinase activation, protein interaction, protein degradation, endocytosis and regulation
of chromatin structure and transcription [136, 168-170]. Increasing evidence demonstrates
that deubiquitinating enzymes have high substrate specificity such as ubiquitin chain
specificity (Lys48 and Lys 63), the selection of ubiquitin and ubiquitin-like molecules
(NEDD8, ISG15, Sumo and Atg8 etal.), and DUB/E3 and interactions [170]. Approximate
95 genes encoding putative human deubiquitinating enzymes have been identified by
bioinformatics according to the DUBs catalytic domains composed of five
subgroups: ubiquitin-specific protease (USP), ubiquitin C-terminal hydrolase (UCH),
Otubain protease (OTU), and Machado-Joseph disease protease (MJD) and
JAB1/MPN/Mov34 metalloenzyme (JAMM) [170].
2.1.2.4.. Acetylation
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Acetylation is a very important post-translational modification in both prokaryotes and eukaryotes. Protein acetylation is grouped into two major classes: acetylation of N-terminal alpha-amine and acetylation of lysine.
N-terminal acetylation of proteins is one of the most well known protein post-translatioal modification with the addition of acetyl group onto N-terminal alpha-amine. More than 80% of proteins in eukaryotes are acetylated. This modification is irreversible and it is a very early and cotranslational event occuring when only about 25–50 amino acid residues of the nascent polypetide are extruding from the ribosome [171]. Little is known about the function of the acetylation of N-terminal alpha-amine. It was reported that the translocation of into endoplasmic reticulum was blocked by N-terminal acetylation [172]. Drosophila actin I is firstly translated as a precursor and followed by N-terminal acetylation, which plays a role in assembling microfilaments in vitro [173]. The role of N-terminal acetylation in stabilizing protein is controversial. The current view is N-terminal acetylation protects proteins from ubiquitination and therefore stablizes proteins. This view is supported by the experiments from several proteins such as cytochrome c, enolase, p16 and p14/p19ARF [174-177]. In
contrast to with the prevalent view, the most current study provides the evidence that N- terminal acetylation of Met acts as the N-terminal degradation signals ( called N-degrons) and N terminal-acetylated Ala, Val, Ser, Thr, and Cys residues are also recognized by the E3 ligase Doa10 of yeast. These two contradictory effects of N-terminal acetylation on protein stability may be determined by the folding of the N-terminal domain of the nascent proteins.
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The rapid folding protects the protein from degradation while the delayed or defective folding results in degradation [178].
Most studies of acetylation are focused on acetylation of lysine. The acetyl group was attached onto the ε-amino group of lysine during acetylation. The side chain of lysine containing the ε-amino group has a pKA value of approximate 10.53, therefore carrying positive charge at the pH of 7. Acetylation of lysine resulted in the removal of positive charge of the side chain and therefore generates two effects: (1) Reduce the affinity of the histone tail for negative charged DNA and produce a loose and relaxing of highly packed and order chromatin structure. Consequently, the transcriptional factors are able to bind DNA to activate transcription [179]; (2) Provide a docking site for the recruitment of transcriptional coactivators which contain bromodomains [180].
Histone acetylation is one of the most commonly studied modifications. The accumulating number of lysine sites has been identified as acetylation in histone H2A,
H2A.X, H2B, H3 and H4 (http://www.millipore.com/techpublications/tech1/pb1014en00).
Most of these sites are located in the N-terminus. As discussed above, acetylation resulted in the removal of positive charge. Neutralization of positive charge may change the interactions between histone and DNA or proteins. The alterations in these interactions could provide a more open and accessible conformation of chromatin for transcriptional factors and therefore modulate gene expression [181-183].
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Lysine acetylation is catalyzed by histone acetyltransferases (HATs). A lot of histone acetyltransferases which catalyze the transferring of the acetyl group onto the ε-amino group of lysine specific residues of histone have been identified by the different labs. Histone acetyltransferases are grouped into five families composed of p300/CBP HATs, GCN5- related N-terminal acetyltransferases (GNATs), MYST family (including MOZ, Ybf2/Sas3,
Sas2, and Tip60), the general transcription factor HATs (including the TAFII-250 family) and the nuclear hormone- related HATs ( including SRC1 and ACTR) [184].
P300/CBP is required for embryonic development in C.elegans, drosophila, mouse and human. RNAi experiments demonstrated that CBP-1, the C.elegans homolog of mammalian, is required for endoderm and mesoderm development [185]. Genetic screen and identification demonstrated that a deletion of CBP-1 results in embryonic lethality in c. elegans [186]. Drosophila CBP is involved in embryonic development such as establishment of dorsal-ventral polarity through the regulation of the transcriptional factors such as Cubitus interruptus twist and snail in Hedgehog, Wingless-Engrailed, TGF-β homolog
Decapentaplegic (Dpp) signaling pathway [187-189]. CBP and p300 are indispensable early embryonic development in mice. Loss of only one copy of either p300 or CBP gene is sufficient to result in embryonic lethality. These results indicate that the requirement of normal embryonic development for CBP and p300 is dose dependent [190]. In human, CBP is associated with Rubinstein-Taybi syndrome. Multiple mutations of CBP and p300 have been identified by DNA sequencing in patients with abnormal features including long eye
107 lashes, heavy and arched eyebrows, a prominent nose with a long hanging columella, a pouting lower lip, square distal fingertips, a shortened and broad thumb [191, 192].
Members of the GNAT family are shown to play an essential role in DNA repair, cell cycle progression, and embryonic development. Loss of GCN5 leads to a cell cycle blockage at the G2/M phase in yeast [193] . Target deletion of GCN512 in mice causes elevated apoptosis, development defect and embryonic lethality [194].
It was shown that p53 is regulated by Tip60 at two distinct levels: (1). Firstly it acts as the coactivator of p53 to induce transcription of the downstream target genes such as p21; (2) second, it is able to block E3 ligase Mdm2-induced ubiquitination and therefore protect p53 from degradation. Therefore it implicates that Tip60 is associate with DNA damage repair
[195]. The expression level of Tip60 is changed in human cancer. Tip60 expression is downregulated in colon and lung carcinomas [196]. However, upregulation of Tip60 protein is associated with the onset of prostate cancer through the acetylation of the androgen receptor (AR) [197]. These contradictory reports demonstrate that Tip60 play diverse roles in cancer progression and may not directly function as an oncogene or tumor suppressor gene
[198]. The other members of MYST family such as HBO1 (MYST2), MOZ and MORF are also involved in both tumor suppression and oncogenesis [199].
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TAFII-250 is one subunit of the transcription initiation factor TFIID. Drosophila TAFII-
230 is the homolog of human TAFII-250. Drosophila TAFII-230 has been identified to have
intrinsic HAT activity. Like hGCN5 and P/CAF, dTAFII230 displays the specific HAT activity for H3 and H4. TAFII-250 induced acetylation of nucleosomes could result in changes in conformation and therefore increase promoter exposure to transcription factors
[200].
Both steroid receptor coactivator-1 (SRC-1) and a novel cofactor (ACTR), which interact with nuclear hormone receptors, have been shown to have HAT activity. H3 and H4 can be
acetylated by recombinant SRC-1. ACTR increases the reporter gene activity induced by the
nuclear hormone receptor retinoids (RARs) [201, 202].
The function domains of different HATs have been identified. The catalytic mechanisms
of several HATs such as GCN5 and PCAF have been studied in detail by biochemical
approaches [183, 203-205]. The high resolution crystal structure and site mutagenisis
analysis of GCN5 have revealed the mechanism by which a conserved glutamate
residue(E173) plays an essential role in the substrate binding and catalysis [206]. However,
the selective HAT inhibitor of PCAF, composed of peptide/CoA conjugate, does not have
effect on the activity of p300. This implicates that the family of p300/CBP and the family of
GCN5/PCAF may employ distinct catalytic mechanism [207].
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A lot of studies have identified the HATs substrate specificities for histone. However, there is accumulating evidence that HATs also acetylate a variety of non-histone proteins
[208]. Several samples will be given as below.
(1). P53 is the most well known protein of non-histone substrates. It has been convincely shown that he DNA binding activity of p53 is largely regulated by p300/CBP mediated acetylation at several lysine residues of c-terminus [209, 210].
(2). Rel A subunit of NF-κB m is acetylates by p300/CBP at 218, 221 and 310.
Acetylation of 218 weakens the interaction with IκBα and inceases DNA binding activity.
Acetylation of 310 increases transcription activity [211, 212].
(3).The transcriptional factor smad7 is acetylated by p300 at 64 and 70. Acetylation protects smad7 from degradation by the ubiquitin E3 ligase and therefore enhances protein stability [213].
(4). The lysines 873 and 874 at the C terminus of the retinoblastoma (Rb) tumor suppressor is acetylated by p300 and PCAF. Acetylationof Rb is negatively corelated with the phosphorylation by E-Cdk2 and subsequently inhibits cell cycle progression. Rb
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acetylation also enhances binding affinity with MDM2 [214, 215]. The lysines 117, 120 and
125 in the N-terminal DNA binding domain of E2F1 are acetylated by PCAF. Acetylation of E2F2 increases DNA binding activity, protein atability and the interaction with p73 and
therefore contributes apoptosis [216-218].
(5). The transcription fator stat3 is acetylated at lysine 685 by p300/CBP. Acetylation
increases stat3 dimerization and thereby enhances DNA binding affinity and transactivation
[219, 220].
(6) Nuclear receptors such as androgen receptor (AR) and estrogen receptor are
acetylated by p300. Acetylation at the lysines 630, 632 and 633 is necessary for DNA
binding activity of AR [221, 222]. The lysines 299, 302 and 303 of the estrogen receptor α
(ERα) are acetylated by p300 [223]. It seems that acetylation of (ERα) produces opposite
effects on trancription based on the context: weakens hormone induced transcription but
enhancse the basal transcription [224].
(7). Yin Yang 1 (YY1) is a tarnscription factor which is involved in both transcription
activation and repression [225]. Transcriptional activity is regulated by the acetylation and
deacetylation of 170–200 of YY1 and DNA-binding activityis reduced by acetylation of
261–333 [226].
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(8). α-Tubulin is well knoawn as a cytoskeletal protein. Tubulin acetylation could be
involved in cell motility. Microtubules are classified into two subtypes: stable microtubules
with a half-life of hours and dynamic microtubules with a half-life of only 5–10 min. α-
tubulin is acetytlated atmuch higher level in table microtubules than in dynamic
microtubules. Deactylation of α-tubulin at lysine 40 is catalyzed by the histone
deacetylases, HDAC6 and SIRT2 [227-229]. Over-expression of HDAC6 reverses
acetylation of α-tubulin, results in depolymerization of microtubule and therefore
enhance cell motility [227].
Like other post-translational modifications, acetylation is also reversible. So far 18
histone deacetylases (HDAC) have been identified in human human and they are clsssified
into four groups according to their homology to yeast HDACs [230, 231]. The class I
HDACs are composed of four members (HDACs 1, 2, 3, and 8) , which are homologs of to
the yeast RPD3 protein. They are detected in nucleus fraction; the class II HDACs includes
six members (HDACs 4, 5, 6, 7, 9, and 10) , which are homologs of to the yeast HDA1
protein. They are located in both the nucleus and cytoplasm fractions; the class III HDACs
are composed of seven sirtuin proteins (SIRT1, 2, 3, 4, 5, 6 and 7), which are homologs of
the yeast protein sirtuin2 (Sir2). All of these seven sirtuin proteins share an evolutionary
conserved core domain composed of a NAD+ domain and a catalytic domain. The SIRT
family requires NAD+ for their activity; the class IV HDACs includes only one member
(HADC11). HDAC11 shares conserved residues with both class I and II HDAC enzymes
112 and therefore considered as a atypical group[232]. HDAC11 is found in both nucleus and cytoplasm fractions.
HDACs have been shown to be involved in a variety of signaling pathways. HDACs are opposite to HATs and therefore regulate gene expression by the removal of acetyl groups. It has been demonstrated that HDACs are associated with some diseases such as cancer. In acute myeloid leukaemia (AML), the recruitment of the N-CoR histone deacetylase complex to the fusion oncoproteins PML–RARA and PLZF–RARA leads to a repression of retinoic acid induced target genes [233]. The NAD+-dependent deacetylase SIRT1 has been shown to negatively regulate Notch signaling in endothelial cells. Deacetylation counteracts the stability of the Notch1 intracellular domain (NICD) by acetylation. Inhibition of SIRT1 activity by siRNA or nicotinamide (NAM) promotes the stability of NICD and thereby increases the activity of Notch target genes such as Nrarp, Hey2 and Lfng upon Notch ligand
DLL4 stimulation [234]. The treatment for acute myeloid leukemia cell line MLL-AF9 with the HDAC inhibitor (HDACi) valproic acid (VPA) induces G1 cell-cycle arrest and apoptosis in a p21 dependent manner [235].
Many deacetylase inhibitors have been discovered and the most well known samples are nicotinamide (NAM) and trichostatin A (TSA). The sirtuin Class III HDACs including
SIRT1, 2, 3, 4, 5, 6 and 7 contain a conserved NAD+ domain and thereby are inhibited by the
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binding of nicotinamide [236]. The Class I and II HDACs including HADC1 to HADC10
are inhibited by trichostatin A.
2.2. Materials and methods
2.2.1. Primers and constructs:
2.2.1.1. TAB1 primers and constructs:
5’ primer: (BamHI): CCC GGATCC GCC GCC ACCATGGCGGCGC AGAGGAGGAG C
3’ primer :( EcoRI+ HA tag): CCC GAA TTC TCA AGC GTA GTC CGG AAC GTC GTA
CGG GTA CGG TGCTGT CAC CAC GCT CTG C
3’ primer for N-terminal 418 AA: (EcoRI + HA tag): CCC GAA TTC TCA AGC GTA GTC
CGG AAC GTC GTA CGG GTA GAC CAT CTG GCC CTG GGA GGG
5’ primer for C-terminal 68 AA: (BamHI): CCC GGA TCC GCC GCC ACC ATGCA
AAGCCCGACC TTAACCCTGC
5’ primer for C-terminal 58 AA: (BamHI): CCC GGA TCC GCC GCC ACC ATG AA
CACGCACACG CAGAGCAGCA GC
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5’ primer for C-terminal 48 AA: (BamHI): CCC GGA TCC GCC GCC ACC ATG TC
TGACGGAGGC CTCTTCCGCT CC
5’ primer for C-terminal 38 AA: (BamHI): CCC GGA TCC GCC GCC ACC ATG GC
CCACTCGCTC CCGCCTGGCG
5’ primer for C-terminal 28 AA: (BamHI): CCC GGA TCC GCC GCC ACC ATG CG
TGTTGAGCCC TATGTGGACT
5’ primer for C-terminal 18 AA: (BamHI): CCC GGA TCC GCC GCC ACC ATG TT
TTACCGCCTC TGGAGCGTGG
5’ primer for C-terminal 8 AA: (BamHI): CCC GGA TCC GCC GCC ACC ATG GA
GCAGAGCGTG GTGACAGCAC
5’ primer for C-terminal 25 AA: (BamHI): CCC GGA TCC GCC GCC ACC ATG CCC
TATGTGGACT TTGCTGAG
3’ primer (1479-1462) (EcoRI -HA-tab1 1479-1462) (named 3’ c-11, last 11 aa deleted):
CCC GAA TTC CTA AGC GTA GTC CGG AAC GTC GTA CGG GTA CAC GCT
CCA GAG GCG GTA
3’ primer (1476-1459) (EcoRI -HA-tab1 1476-1459) (named 3’ c-12, last 12 aa deleted):
CCC GAA TTC CTA AGC GTA GTC CGG AAC GTC GTA CGG GTA GCT CCA
GAG GCG GTA AAA
3’ primer (1473-1456) ( EcoRI -HA-tab1 1473-1456) (named 3’ c-13, last 13 aa deleted):
CCC GAA TTC CTA AGC GTA GTC CGG AAC GTC GTA CGG GTA CCA GAG
GCG GTA AAA CTC 3’ primer (1470-1453) (EcoRI -HA-tab1 1470-1453) (named 3’ c-14,
115 last 14 aa deleted): CCC GAA TTC CTA AGC GTA GTC CGG AAC GTC GTA CGG
GTA GAG GCG GTA AAA CTC AGC
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Construct Cloning sites vector
1 Full length BamHI+EcoRI pcDNA3.1
2 Full length with HA BamHI+EcoRI pcDNA3.1
3 N418 with HA BamHI+EcoRI pcDNA3.1
4 C68 with HA BamHI+EcoRI pcDNA3.1
5 C58 with HA BamHI+EcoRI pcDNA3.1
6 C48 with HA BamHI+EcoRI pcDNA3.1
7 C38 with HA BamHI+EcoRI pcDNA3.1
8 C28 with HA BamHI+EcoRI pcDNA3.1
9 C18 with HA BamHI+EcoRI pcDNA3.1
10 C08 with HA BamHI+EcoRI pcDNA3.1
11 C24 with HA BamHI+EcoRI pcDNA3.1
12 C22 with HA BamHI+EcoRI pcDNA3.1
13 C20 with HA BamHI+EcoRI pcDNA3.1
14 C25 with HA BamHI+EcoRI pcDNA3.1
15 C15-68 with HA BamHI+EcoRI pcDNA3.1
16 C10-68 with HA BamHI+EcoRI pcDNA3.1
17 C05-68 with HA BamHI+EcoRI pcDNA3.1
18 C10-26 with HA BamHI+EcoRI pcDNA3.1
19 C11-26 with HA BamHI+EcoRI pcDNA3.1
20 C12-26 with HA BamHI+EcoRI pcDNA3.1
21 C13-26 with HA BamHI+EcoRI pcDNA3.1
Table 1. TAB1 constructs
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2.2.1.2. TAK1ΔN22 primer: CCC GGA TCC GCC GCC ACC ATG TCGC
AGGTCCTGAA CTTCGAAGAG
2.2.1.3. TAK1-TAB1 fusion primers:
5’ flag-Human tak1 primer: ( BamHI + Kazak +Flag+ Linker +TAK 5’)(F1): CCC GGA
TCC GCC GCC ACC ATG GAT TAC AAG GAT GAC GAC GAT AAG GCC CGG GCG
TCT ACA GCC TCT GCC GCC
3’-Human TAK1 (EcoRI+TAK1 3’) (F5): CCC GAA TTC TGA AGT GCC TTG TCG TTT
CTG
5’ Linker TAB1 C-68 5’ (F7): CCC GAA TTC GGA GGA GGA GGA GGA CAA AGC
CCG ACC TTA ACC CTG
3’ Human TAB1 –HA (XhoI) (F4): CCC CTC GAG TCA AGC GTA GTC CGG AAC GTC
GTA CGG GTA CGG TGCTGT CAC CAC GCT CTG C
Construct Cloning sites vector
Flag Full length TAK1 with TAB1 C68 1 BamHI+XhoI pcDNA3.1 HA
2 Flag N303 TAK1 with TAB1 C68 HA BamHI+XhoI pcDNA3.1
Table 2. TAK1-TAB1 fusion constructs
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2.2.1.4. TAK1 site mutagenesis primers:
S389A for: ACCTCTG AGGGCAAGAG GATG GCC GCT GACATGTCTG
AAATAGAAGC TA
S389A rev: TA GCTTCTATTT CAGACATGTC AGC GGC CATC CTCTTGCCCT
CAGAGGT
T440A for: CCCA GTGTCAGAAT GATTACT GCC TCAGGACCAA CCTCAGAAAA
GCC
T440A rev: GGC TTTTCTGAGG TTGGTCC TGA GGC AGTAATC ATTCTGACAC
TGGG
S441A for: CCCA GTGTCAGAAT GATTACT ACC GCC GGACCAA CCTCAGAAAA
GCC
S441A rev: GGC TTTTCTGAGG TTGGTCC GGC GGT AGTAATC ATTCTGACAC
TGGG
T440A, S441A for: CCCA GTGTCAGAAT GATTACT GCC GCC GGACCAA
CCTCAGAAAA GCC
T440A, S441A rev: GGC TTTTCTGAGG TTGGTCC GGC GGC AGTAATC
ATTCTGACAC TGGG
K72R for: AAATAGAAAG TGAATCTGAG AGG AGA GCGT TTATTGTAGA
GCTTCGGCAG
119
K72R rev: CTGCCGAAGC TCTACAATAA ACGC TCT CCT CTCAGATTCA
CTTTCTATTT
K72Q for: AAATAGAAAG TGAATCTGAG AGG CAA GCGT TTATTGTAGA
GCTTCGGCAG
K72Q rev: CTGCCGAAGC TCTACAATAA ACGC TTG CCT CTCAGATTCA
CTTTCTATTT
K158R for: GCGCTAATTC ACAGGGACCT G AGA CCACCA AACTTACTGC
TGGTTGCAGG
K158R rev: CCTGCAACCA GCAGTAAGTT TGGTGG TCT C AGGTCCCTGT
GAATTAGCGC
K158Q for: GCGCTAATTC ACAGGGACCT G CAA CCACCA AACTTACTGC
TGGTTGCAGG
K158Q rev: CCTGCAACCA GCAGTAAGTT TGGTGG TTG C AGGTCCCTGT
GAATTAGCGC
K227R for: GAAGTGATAA CGCGTCGG AGA CCCTTTGAT GAGATTGGTG
K227R rev: CACCAATCTC ATCAAAGGG TCT CCGACGCG TTATCACTTC
K227Q for: GAAGTGATAA CGCGTCGG CAA CCCTTTGAT GAGATTGGTG
K227Q rev: CACCAATCTC ATCAAAGGG TTG CCGACGCG TTATCACTTC
K520R for: CATTGTTATT ACAGAGA AGA CAAGAACTAG TTGCAGAACT
120
K520R rev: AGTTCTGCAA CTAGTTCTTG TCT TCTCTGT AATAACAATG
K520Q for: CATTGTTATT ACAGAGA CAA CAAGAACTAG TTGCAGAACT
K520Q rev: AGTTCTGCAA CTAGTTCTTG TTG TCTCTGT AATAACAATG
K562R for: TTTCTACTTA CTACCAGCAA TGC AGA AAA C AACTAGAGG
T CATCAGAAGT
K562R rev: ACTTCTGATG ACCTCTAGTT G TTT TCT GCA TTGCTGGTA
G TAAGTAGAAA
K562Q for: TTTCTACTTA CTACCAGCAA TGC CAA AAA C AACTAGAGGT
CATCAGAAGT
K562Q rev: ACTTCTGATG ACCTCTAGTT G TTT TTG GCA TTGCTGGTAG
TAAGTAGAAA
K563R for: TTTCTACTTA CTACCAGCAA TGC AAA AGA C AACTAGAGGT
CATCAGAAGT
K563R rev: ACTTCTGATG ACCTCTAGTT G TCT TTT GCA TTGCTGGTAG
TAAGTAGAAA
K563Q for: TTTCTACTTA CTACCAGCAA TGC AAA CAA C AACTAGAGGT
CATCAGAAGT
K563Q rev: ACTTCTGATG ACCTCTAGTT G TTG TTT GCA TTGCTGGTAG
TAAGTAGAAA
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K158N for: GCGCTAATTC ACAGGGACCT G AAC CCACCA AACTTACTGC
TGGTTGCAGG
K158N rev: CCTGCAACCA GCAGTAAGTT TGGTGG GTT C AGGTCCCTGT
GAATTAGCGC
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Construct vector
1 S389A pCMV-tag2A
2 T440A pCMV-tag2A
3 S441A pCMV-tag2A
4 T440A, S441A pCMV-tag2A
5 K72R pcDNA3.1
6 K72Q pcDNA3.1
7 K158R pcDNA3.1
8 K158Q pcDNA3.1
9 K227R pcDNA3.1
10 K227Q pcDNA3.1
11 K520R pcDNA3.1
12 K520Q pcDNA3.1
13 K562R pcDNA3.1
14 K562Q pcDNA3.1
15 K563R pcDNA3.1
16 K563Q pcDNA3.1
17 K158N pcDNA3.1
18 K72R, K227R pcDNA3.1
19 K563R, K72R pcDNA3.1
20 K563R. K227R pcDNA3.1
21 K563R. K520R pcDNA3.1
22 K72Q, K227Q pcDNA3.1
23 K563R, K72R, K227R (“3R”) pcDNA3.1
24 K563R, K72R, K227R, K520R (“4R”) pcDNA3.1
Table 3. TAK1 site mutagenesis constructs.
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2.2.1.5. TAK1 primers for bacteria expression constructs
TAK1 5’ (BamHI): CCC GGA TCC TCGACAG CCTCCGCCGC CTCC
TAK1 5' (EcoRI): CCC GAA TTC TCGACAG CCTCCGCCGC CTCC
TAK1 3' (XhoI): CCC CTC GAG TGAAGTG CCTTGTCGTT TCTG
K158amber for: GCGCTAATTC ACAGGGACCT G TAG CCACCA AACTTACTGC
TGGTTGCAGG
K158amber rev: CCTGCAACCA GCAGTAAGTT TGGTGG CTA C AGGTCCCTGT
GAATTAGCGC
2.2.1.6. TAB1 primers for bacteria expression constructs:
TAB1 N Pet28A: CCC GAA TTC GCGGCGC AGAGGAGGAG CTTGC
TAB1 C Pet28A: CCC CTC GAG CGGTGCTGTCACCACGCTCTGC
2.2.1.7. MKK6 primers for bacteria expression constructs
MKK6 was cloned from human 293T cell cDNA.
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MKK6 N PGEX 4T1 (and 6P1): CCC GCGGCCGC AT
TCTCAGTCGAAAGGCAAGAAGCG
MKK6 C PGEX 4T1 (and 6P1): CCC GCGGCCGC TC
GTCTCCAAGAATCAGTTTTAC
MKK6 K82A For: CC AGC GGG CA G ATC ATG GCA GTG GCG CGG A TC CGA
GCC AC A GTA AAT AGC
MKK6 K82A Rev: GCT ATT TAC TGT GGC TCG GAT CCG CGC CAC TGC CAT GAT
CTG CCC GCT GG
MKK6 S207A For: GGA ATC AGT GGC TAC T TG GTG GAC GCG GTT GCT AAA
ACA ATT GAT G CA GG
MKK6 S207A Rev: CC TGC ATC AAT TGT TTT AGC AAC CGC GTC CAC CAA GTA
GCC ACT GAT TCC
MKK6 T211A For: TAC T TG GTG GAC TC T GTT GCT AAA GCG ATT GAT G CA
GGT TGC AA A CCA TA
MKK6 T211A Rev: TA TGG TTT GCA ACC TGC ATC AAT CGC TTT AGC AAC AGA
GTC CAC CAA GTA
MKK6 S207A, T211A For: TAC T TG GTG GAC GCG GTT GCT AAA GCG ATT GAT
G CA GGT TGC AA A CCA TA
MKK6 S207A, T211A Rev: TA TGG TTT GCA ACC TGC ATC AAT CGC TTT AGC
AAC CGC GTC CAC CAA GTA
125
Construct vector
1 WT PET 28A
2 WT PGEX 4T1
3 WT PGEX 6P1
4 K63A PGEX 6P1
5 K158R PGEX 6P1 TAK1 6 K158Q PGEX 6P1
7 K158N PGEX 6P1
8 K158 amber PGEX 6P1
9 K563R, K72R, K227R (“3R”) PGEX 6P1
10 K563R, K72R, K227R, K520R (“4R”) PGEX 6P1
11 WT PGEX 6P1
12 K63A PGEX 6P1
13 K158R PGEX 6P1
14 K158Q PGEX 6P1 TAK1TAB1C68 fusion 15 K158N PGEX 6P1
16 K158 amber PGEX 6P1
17 K563R, K72R, K227R (“3R”) PGEX 6P1
18 K563R, K72R, K227R, K520R (“4R”) PGEX 6P1
19 WT PGEX 6P1
20 K82A PGEX 6P1
21 MKK6 S207A PGEX 6P1
22 T211A PGEX 6P1
23 S207A, T211A PGEX 6P1
24 WT PET 28A
25 TAB1 WT PGEX 4T1
26 WT PGEX 6P1
Table 4. Bacteria expression constructs
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2.2.1.8. MEKK3 primers:
K522R For: ACATGATTGTTCACCGGGACATTAGGGGAGCCAACATCCT
CCGAGACTCT
K522R Rev: AGAGTCTCGG AGGATGTTGG CTCC CCT AAT GTCCCGGTGA
ACAATCATGT
K522Q For: ACATGATTGTTCACCGGGACATTCAGGGAG CCAACATCC
T CCGAGACTCT
K522Q Rev: AGAGTCTCGG AGGATGTTGG CTCC CTG AAT GTCCCG
GTGA ACAATCATGT
K522N For: ACATGATTGT TCACCGGGAC ATT AAC GGAG
CCAACATCCT CCGAGACTCT
K522N Rev: AGAGTCTCGG AGGATGTTGG CTCC GTT AAT GTCCCGGTGA
ACAATCATGT
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Construct vector
1 WT pcDNA3.1
2 Kinase dead pcDNA3.1
3 K522R pcDNA3.1
4 K522Q pcDNA3.1
5 K522N pcDNA3.1
Table5. MEKK3 constructs.
All constructs above were confirmed by DNA sequencing.
2.2.2. Mass spectrometry
Mass spec was performed at the Proteomics Core of Baylor College of Medicine.
2.2.3. Reagents and cell lines:
Phospho-TAK1 (Thr184/187) Antibody, Phospho-MKK3 (Ser189)/MKK6 (Ser207)
Antibody, Phospho-p38 MAPK (Thr180/Tyr182) (12F8) Rabbit mAb were purchased from cell signaling (#4531, #9231, #4631). Mouse Anti-MEKK3 Monoclonal antibody was purchased from BD Biosciences (#611102). Human wildtype MEKK3, kinase dead MEKK3
128
and Phospho-MEKK3 are the generous gifts from Dr. Jianghua Yang (Baylor college of
Medicine, Houston, Texas). CBP and p300 plasmids are the generous gifts from Dr. Mien-
Chie Hung (The University of Texas M. D. Anderson Cancer Center, Houston, Texas).
GCN5 and PCAF plasmids are the generous gifts from Dr. Sharon Dent (The University of
Texas M. D. Anderson Cancer Center, Houston, Texas). Tip 60 plasmid is the generous gift
from Dr. Junjie Chen (The University of Texas M. D. Anderson Cancer Center, Houston,
Texas). HA-ubiquitin plasmid is the generous gift from Dr. Shao-cong Sun (The University of Texas M. D. Anderson Cancer Center, Houston, Texas).
TAK1 rescue cell lines (vector control, WT, K158R, K158Q, and K158N) were established by Dr. Ruiying Zhao. The plasmids were transfected into TAK1 null MEF cells and selected by zeocine.
2.2.4.. Cycloheximide (CHX) chase assay
TAK1 and TAB1 plasmids were transfected into 293T cells. AT 48 hours after transfection, cells were treated with 100 μg/mL CHX (Sigma) to inhibit protein synthesis and were harvested at deferent time points for western blot.
129
2.2.5. GST protein purification:
1) Pick a single colon from a fresh agar plate and grow overnight at 37˚C in 4 ml LB with the selection in a 10ml tube on a shaker.
2) Dilute 1:100 in 400ml LB with the selection and grow 3-4 hours at 37˚C in 1L bottle on a shaker until OD 0.6-0.8 is reached.
3) Induce with IPTG (1mM final) for 4 hours at 30˚C. Spin the culture, remove the supernatant and collect pellets. Wash the pellets twice with cold PBS.
4) Resuspend the bacterial pellet in 15 ml lysis buffer (50 mM Tris-Cl, PH7.6, 150 mM
NaCl, 10% glycerol, 0.5 mM EDTA, 250 µg/ml lysozyme and protease inhibitors) briefly sonicate the cells on ice for 6 x 10 second bursts (with 10 second intervals between each burst) using a Misonix XL 2020 sonicator fitted with a microtip probe and set at power setting 5.
5) Spin down the lysed cells by high speed centrifugation at 12k, 30min at 4C and collect the soluble protein extracts (avoid the precipitations)
6) While spinning, wash the 400 µl GSH beads (GE Healthcare, 17-0756-01) twice with lysis buffer.
7) Transfer the GSH beads into the supernatant lysate and rotate at 4˚C for 3 hr. Spin down the beads at 4000g X 5 min. Wash three times with lysis buffer.
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8) Add 400 glutathione buffer (10 mM Glutathione in 50 mM Tris pH 8.0, 10% glycerol )
elute for 1 hour at room temperature. Repeat elution once and combine the eluates. Aliquot
the eluates into eppendorf tubes and store them at -80˚C
2.2.6. His-tag protein purification:
Ni-NTA affinity resin was purchase from QIAGEN. Binding and Wash buffer: 50mM
sodium phosphate, 300mM NaCl, 10mM imidazole (pH 8.0). Elution buffer: 50mM sodium phosphate, 300mM NaCl, 250mM imidazole (pH 8.0). The other steps are same as GST purification.
2.2.7. In Vitro Kinase assay:
To analyze TAK1 kinase activity, GST purified TAK1 (or TAK1-TAB1c68 fusion protein) was incubated with the recombinant 1 µM MKK6( K82A or S207AT211A) , [γ-32
P]ATP in the kinase buffer (50 mM Tris-HCl, pH 7.5, 0.5 mM DTT, 5 mM MgCl2, 50 µM
ATP) at 30 °C for 1 h and then analyzed by SDS–PAGE.
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2.3. Results
2.3.1. The first 22 amino acids at the N-terminus did not stabilized TAK1.
It has been reported that that N-terminus 1 may play a negative role in regulation TAK1 activity. To test if the N-terminus increased TAK1 stability, we make an N-terminus truncated form in which the first 22 amino acids were deleted. The full-length and the truncated TAK1 were transfected into 293T cells. After 48 hours, cycloheximide
(CHX) chase assay was performed to determine the protein stability. As shown in Fig. 27, the deletion of N-terminus did not increase the stability of TAK1. There was not much deference in kinetics of the stability between the full-length and the TAK1 ΔN22.
TAK1 TAK1ΔN
CHX (hr) 0122 4 6 8 012264 8
Flag-TAK1
β-actin
Figure 27. CHX chase assay for the full length TAK1 and TAK1ΔN22. 293T cells were
transfected with the full length TAK1 and TAK1 TAK1ΔN22 plasmid. After 48 hours, cells
were treated with 100 μg/mL CHX (Sigma) to inhibit protein synthesis and were harvested at
0, 2,4,6,8 and 12 hours for western blot. β-actin was used as loading control.
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2.3.2. TAK1 is stabilized by TAB1.
TAB1 was shown to augment the level of TAK1 [124]. To demonstrate the role of TAB1 in regulating TAK1 activity, we firstly verified the existence of the interaction of TAK and
TAB1 in 293T system. The different Flag-TAK isoforms were cotransfected with HA-TAB1 into 293T cells. At 48 hours after transfection, cells were harvested and the extracts were immunoprecipitated by Flag antibody. The immunoprecipitates were analyzed by western blot with HA antibody. As shown in Fig. 28, all of four splicing forms, TAK1k63A and
TAK1 ΔN22 were shown to interact with TAB1. We measured TAK1 level with or without
TAB1 in 293T cell system. CHX chase assay was performed to measure TAK1 protein kinetics of the stability. As indicated in Fig. 29, cotransfection of TAB1 significantly increased TAK1 protein level where did not change TAK1 mRNA level. TAK1 degraded very quickly after CHX treatment. CHX chase assay at several time points demonstrated that the half-life time TAK1 alone is less than 8 hours but TAK1 level did not change even 16 hours after CHX treatment when TAK1 was cotransfected with TAB1.
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TAB1
HA-TAB1
Input
N.S.
Figure 28. TAB1 interacts with TAK1. The differnent Flag-TAK1 forms were tarnsfected with HA-TAB1 into 293T cells. The extracts were immunoprecipitated with Flag antibody followed by western blot with HA antibody.
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TAK1A TAK1A+TAB1
CHX 0162164 8 0 2 4 8
Flag-TAK1
β-actin
400 CHX chase assay
300 1.2 1 0.8 200 TAK1A 0.6 TAK1A+TAB1 TAK1A 0.4 Protein level 100 0.2 0 024816 0 CHX (hr) TAK1A TAK1A+TAB1
Quantification of TAK1 mRNA level Normalized by β-actin and the starting points
Figure 29. TAK1 is stabilized by TAB1. Real time PCR was used to quantify TAK1 mRNA level and CHX was used to determine TAK1 protein level after transfection into
293T cells.
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2.3.3. TAK1 Ubiquitination level is not affected by TAB1.
Since TAK1 stability is regulated by TAB1, we proposed that TAK1 ubiquitination is
regulated by TAB1. To test this hypothesis, the standard ubiquitination assay was performed in 293Tcells. Flag-TAK1 plus HA-ubiquitin plasmids with or without TAB1 were transfected into 293T cells. At 48 hours post-transfection 293T cells were treated with proteasome inhibitor MG132 at the final concentration of 50 μM for 6 h and then harvested for immunoprecipitation followed by western blot. As shown Fig.30, Coomassie blue staining or western blot demonstrated that TAK1 level was not affected by TAB1. TNF-α
treatment also did not change TAK1 ubiquitination.
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+ +++ MG132 + +++ + +++
Ubiquitin-HA + +++ + +++ + +++
+ TAK1-Flag + + + + + +++ +++
TAB1 - - + + -+- + -+- + TNFα - ++- - + - + - + - +
72KD input
IP: Flag IP: Flag
Coomassie Blue staining IB: HA
Figure 30. Ubiquitination assay for TAK1. Flag-TAK1 plus HA-ubiquitin with or without were transfected in to 293T cells. At 48 hours after transfection, cells were treated with proteasome inhibitor MG132 for 6 hours. Then cells with or without TNF-α treatment for 30 minutes were harvested for immunoprecipitation using flag antibody. The precipitates were analyzed by Coomassie blue staining or western blot.
137
2.3.4. TAB1 is indispensable for TAK1 kinase activity.
To test TAK1 kinase activity, we performed a series of experiments to analyze TAK1 downstream targets and substrates. Overexpression in 293T cells demonstrated that TAB1 is phosphorylated by TAK1. As in Fig. 31 A, the kinase activity of all the different TAK1 forms including the splicing isoform a, b, c and d depends on TAB1. TAB1 is also required for the kinase activity of the terminal truncated form TAK1 ΔN22. TAB1 was phosphorylated by the different TAK1 splicing isoforms a, b, c, d and TAK1 ΔN22 but not the kinase dead form TAK1K63A. The phosphorylated TAB1 is reduced by phosphatase treatment (Fig. 31B). TAB1 is phosphorylated by TAK1a and TAK1a in a dose dependent manner since TAB1 phosphorylation is increased with the amount of transfected plasmid
TAK1a and TAK1c as indicated in Fig. 31C. TAK1 k63A serves as a dominant mutant as it reduces TAB1 phosphorylation in a dose dependent manner (Fig. 31D).
138
TAB1 TAB1
A B
HA-TAB1
β-actin
D C TAK1a+TAB1 TAK1c+TAB1 TAK1K63A+TAB1 TAK1c+TAB1
HA-TAB1
β-actin
Figure 31. TAB1 is stabilized and phosphorylated by TAK1. A. TAB1 is phosphorylated by TAK1 splicing variants. B. TAB1 is dephosphorylated by phosphatase.
C. TAB1 is phosphorylated by TAK1 in a dose-dependent. D. TAK1 K63A acts as a dominant negative control.
139
TAK1 stabilization and phosphorylation also requires TAB1. To demonstrate that effects of
TAB1 on the stabilization of TAK1, we used phosphatase to treat extracts and made a bunch
of different dilutions for the extracts from TAK1-TAB1 cotransfection. As shown in Fig. 32,
cotransfection of TAB1 increases TAK1 level up to 25 fold.
25µg 25µg 5µg 1µg 25µg 1µg
Phosphotase --++ -+-+-+ - +
TAK1-Flag +++ + + + ++ + + + +
TAB1 --+++ + ++ - + + +
TAK1-Flag
β-actin
Figure 32. TAK1 stabilization and autophosphorylation depend on TAB1. TAK1 and
TAB1 were transfected into 293T cells and TAK1 level was analyzed by western blot.
Extracts were treated by phosphatase.
140
As introduced before, TAK1 is required for NF-κB activation. We measure NF-κB activity induced by TAK1. TAK1 with or without TAB1 were transfected into 293T cells. Nucleus extracts were fractionated and used for EMSA. As shown in Fig. 33, NF-κB activation was induced by TAK1 combined with TAB1 but TAK1 alone does not have much effect on NF-
κB activation. The splicing variants TAK1 c or TAK1 d were not shown to inhibit on NF-κB activation induced by TAK1a with TAB1. Although TAK1a or TAK1c alone did have effects on JNK phosphorylation, TAK1a plus TAB1 induced the phosphorylation of JNK (Fig.34).
The isoform TAK1c with TAB1 induced the phosphorylation of JNK in a dose dependent manner. The kinase dead form TAK1K63A served as a dominant negative mutant to inhibit
JNK phosphorylation.
141
TAK1a - + - + + + + + + +
TAK1c - - - - + + + - - -
TAK1d ------+++
TAB1 - - + + + + + + + +
P65/p65
Figure 33. NF-κB activation is induced by TAK1 and TAB1. TAK1a with or without
TAB1 plus the different amount of TAK1c or TAk1d plasmids were transfected into 293T cells. EMSA was performed using the nucleus fraction.
142
TAB1
Phospho-JNK
JNK
Figure 34. The phosphorylation of JNK is induced by TAK1 together with TAB1.
TAK1 with or without TAB1 plasmids were transfected into 293T cells. JNK phosphorylation was analyzed by western blot.
143
2.3.5. C-terminus of TAB1 is required and sufficient to activate TAK1.
To map the functional domain of TAB1 which stabilizes TAK1, we made a series of deleted TAB1 forms. These TAB1 deletion forms with Flag-TAK1 were transfected into
293T cells. At 48 hours after transfection, cells were harvested for western blot. As shown in
Fig. 35, our data confirmed that the N-terminal 418 could not stabilize and activate TAK
[237]. The C-terminal region containing 68 amino acids is sufficient to stabilize and activate
TAK. We mapped the shortest peptide from TAB1 which is sufficient and required for
TAK1 activation. This peptide contains only 17 amino acids (EPYVDFAEFYRLWSVD) between the C terminal site 10 and site 26. The deletion of Glutamic acid (E) at the C26 or
Valine (V) and Aspartic acid (D) resulted in the loss of function of TAB1.
144
TAB1 N-terminus C-terminus 1 418 68 1
26 10
TAB1
EPYVDFAEFYRLWSVDHGEQSVVTAP
Flag-TAK1 ++++++++++++ +++++ Flag-TAK1
β-actin
Flag-TAK1 ++++++ Flag-TAK1
β-actin
0.35
0.3
0.25
0.2
0.15
0.1
0.05 Relative Relative activity luciferase
0
Vector TAB1- TAB1C10-TAB1C11-TAB1C12-TAB1C13- C26-HA 26-HA 26-HA 26-HA 26-HA
Figure 35. TAK1 is stabilized by the C-terminus of TAB1. Flag-TAK1 with the different HA-TAB forms was transfected into 293T cells. TAK1 levels were analyzed by western blot with Flag antibody.
145
2.3.6. All four transcriptional isoforms of human TAK1 have kinase activity dependent
on TAB1 but may be involved in different pathways.
As introduced before, four transcriptional variants have been identified in human [97, 98].
To distinguish their functions, the four splicing isoforms of TAK1 were put back into TAK1
null MEF cells using retrovirus infection. The stable cell lines which express each splicing
form were established by hygromicin selection. The stable cell lines were treated with TNF-α
30 minutes. The nuleus proteins were isolated for EMSA. As shown in Fig. 36, NFκB activation was induced by TNF-α treatment in the cell lines expressing TAK1a and TAK1b.
These results demonstrated that TAK1a nad TAK1b successfully rescued the phnotypes.
However, TAK1c and TAK1d failed to induce NFκB activation.
TNF-α
P65/p50
Figure 36. EMSA Assay for NF-κB activation in TAK1 rescue cells. Four splicing
variants of TAK1 were put back into TAK1 null MEF cells. NF-κB activity induced by TNF-
α was analyzed by EMSA. 146
2.3.7. TAK1 activity is regulated by the acetylation at lysine 158.
Several reports have shown that TAK1 is regulated by post-translational modification such as phosphorylation and Ubiquitination [155, 238]. However, how TAK1 is stabilized and activated by TAB1 is still unknown. To answer this question, we made the TAK1-
TABC68 fusion and TAK1N303-TAB1C68 fusion constructs which expressed high active kinase (Fig.37A). We tried to map the new PTM site on TAK1 using these two fusions constructs. TAK1 with or without TAB, TAK1-TABC68 fusion and TAK1N303-TAB1C68 fusion constructs were transfected into 293T cells. At 48 hours after transfection, the cells were collected and the extracts were immunoprecipitated by Flag antibody. The precipitates were resolved in SDS-PAGE gel and followed by Coomassie blue staining. As shown in
Fig.37 B, TAK1 was successfully overexpressed in 293T cells and the interesting bands were excised from the gel and used for MASS SPEC analysis to identify the new post-translational modification. MASS spectra were summarized in the appendix. The identified amino acid sites were labeled in TAK1 sequence (Fig.38). Seven phosphorylation sites including Ser367,
Ser375, Ser192, Ser389, Ser412, Thr440, and Ser441 were identified. Among the
phosphorylation sites, Ser 192 and Ser412 were previously known and the other
five are newly discovered phosphorylation sites. More important, five acetylation sites at 72,
158,227,520 and 563 were identified.
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A
Phospho- p38
Phospho MKK3/6
β-actin
Flag Ab IP 3 hours Flag Ab IP overnight
B
Flag-TAK1TAB1C68 Flag-TAK1 HA-TAB1
Flag-TAK1N303TAB1C68
Figure 37. TAK1 is activated by TAB1. A. Flag-TAK1, Flag-TAK1TAB1C68, Flag-
TAK1N303TAB1C68 with or without TAB1 were transfected into 293T cells.
Phosphorylation of p38 and MKK3/6 were analyzed by western blot. B. Flag-TAK1 with or without TAB1, Flag-TAK1TAB1C68, Flag-TAK1N303TAB1C68 were transfected into
293T cells. The cells were harvested for immunoprecipitation using flag antibody. The precipitates were analyzed by Coomassie blue staining and the interesting bands were excised from the gel for MASS SPEC analysis.
148
1 MSTASAASSS SSSSAGEMIE APSQVLNFEE IDYKEIEVEE VVGRGAFGVV
51 CKAKWRAKDV AIKQIESESE RKAFIVELRQ LSRVNHPNIV KLYGACLNPV
101 CLVMEYAEGG SLYNVLHGAE PLPYYTAAHA MSWCLQCSQG VAYLHSMQPK
151 ALIHRDLKPP NLLLVAGGTV LKICDFGTAC DIQTHMTNNK GSAAWMAPEV
201 FEGSNYSEKC DVFSWGIILW EVITRRKPFD EIGGPAFRIM WAVHNGTRPP
251 LIKNLPKPIE SLMTRCWSKD PSQRPSMEEI VKIMTHLMRY FPGADEPLQY
301 PCQYSDEGQS NSATSTGSFM DIASTNTSNK SDTNMEQVPA TNDTIKRLES
351 KLLKNQAKQQ SESGRLSLGA SRGSSVESLP PTSEGKRMSA DMSEIEARIA
401 ATTGNGQPRR RSIQDLTVTG TEPGQVSSRS SSPSVRMITT SGPTSEKPTR
451 SHPWTPDDST DTNGSDNSIP MAYLTLDHQL QPLAPCPNSK ESMAVFEQHC
501 KMAQEYMKVQ TEIALLLQRK QELVAELDQD EKDQQNTSRL VQEHKKLLDE
551 NKSLSTYYQQ CKKQLEVIRS QQQKRQGTS
Phos: Ser367, Ser375, Ser192, Ser389, Ser412, thr440, ser441
Acetyl: Lys72, Lys158, Lys227, Lys520, Lys563
Figure 38. PTM sites on TAK1 identified by MASS SPEC. Seven phosphorylation sites
and five acetylation sites were identified.
149
To study the roles of phosphorylation at these sites in regulating TAK1 activity, Ser 389,
Thr440 and Ser 441 were changed into alanine to abolish phosphorylation with the site mutagenesis. The constructs containing the mutation at these sites were transfected with HA-
TAB1 into 293T cells. Three assays were used to evaluate TAK1 activity: TAK1 stability,
TAB1 phosphorylation and κB promoter activity. As indicated in Fig. 39, the TAK1 mutants
S389A, T440A, S441A and the double mutants still display the same activity as the wild type
TAK1.
150
HA TAB1
Flag TAK1
HA TAB1
N.S.
Figure 39. The effects of phosphorylation at the new identified sites on TAK1. Ser
389, Thr440 and Ser441 were changed into alanine. Western blot was used to analyze TAK1 stability, TAB1 phosphorylation and luciferase assay was performed to measure κB promoter activity.
151
It is a well established approach that arginine (R) is used as the mimics of unacetylated lysine and glutamine (Q) or asparagine (N) are used as the mimics of acetylated lysine due to the structural senilities (Fig. 41). Taking this advantage, we change the lysine at
72,158,227,520 and 563 into arginine or glutamine using the site mutagenesis approach. We also change lysine 158 into asparagines. These TAK1 mutants were transfected with HA-
TAB1 into 293T cells. TAK1 activity was evaluated by TAK1 stability, TAK1 phosphorylation, TAB1 phosphorylation and MKK6 phosphorylation. As shown in Fig. 42A, the changes from lysine to arginine or glutamine at sites 72, 227,520 and 563 did not have many effects on TAK1 activity based on the above assays. TAK1 kinase dead form K63A was used as the negative control for TAK1 activity. The different combinations of these mutations such as K72RK227R, K563RK72R, K563RK227R, K563RK520R, K72QK227Q,
K72RK227RK563R (3R), K72RK227RK520RK563R (4R) also kept the same activity as the wild type. However, the change from lysine to arginine at 158 (TAK1 K158R and TAK1 5R mutant) abolished TAK1 activity Fig. 42B. More important, the change from lysine to asparagine at 158 partially rescued TAK1 activity as indicated by TAK1 stability, TAK1 phosphorylation, TAB1 phosphorylation and MKK6 phosphorylation.
152
Fig. 40 Maldi-MS identification of TAK1 lys 158 acetylation.
153
Fig. 41 The structures of lysine, arginine, glutamine, asparagines and acetylated lysine.
154
HA-TAB1 A
Flag-TAK1
Phos-TAK1
HA-TAB1
β-actin
3R: K72R, K227R, and K563R 4R: K72R, K227R, K520R and K563R 5R: K72R, K227R, K520R and K563R plus K158R
HA-TAB1 B
Flag-TAK1
Phos-TAK1
HA-TAB1
Phos-MKK3/6
β-actin
Figure 42. The effects of acetylation at the new identified sites on TAK1. A. Screening
for the functional lysine sites on TAK1. Lys72, lys158, lys227, lys520 and lys563 were changed into arginine, glutamine or asparagine. Western blot was used to analyze TAK1
stability, TAK1 phosphorylation and TAB1 phosphorylation. B. The effect of acetylation at
lys158 was verified. Asparagine partially rescues lysine phenotype at 158.
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We also performed in vitro kinase assay to study how TAK1 activity is regulated by
TAB1. TAK1, TAB1 and MKK6 were cloned into PET 28 (His tag) or PGEX (GST tag)
systems. WT, K63A, K158R, K158Q, K158N. 4R, 5R and the deferent TAK1-TAB1 C68
fusion bacterial expression constructs were transformed into BL-21 cells and the bacterial cultures were induced by IPTG. The recombinant His-tag proteins were purified by Ni-NTA
affinity resin and GST-tag proteins were purified by GSH beads. We also use site
mutagenesis to produce MKK6 K82A kinase dead form in which ATP bind pocket is
disrupted. As the substrate, MKK6 K82A rules out the possibility of autophosphorylation in the following experiments. MKK6 S207AT211A was used as the negative control in which the phosphorylation sites were abolished. As shown in Fig. 43, we successfully produced the
GST tagged recombinant various TAK1 forms, TAB1 and the substrate MKK6 various forms were purified from bacteria expression system. Coomassie blue staining was performed to quantify the concentrations of the purified recombinant proteins.
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GST-TAK1 GST-TAK1-TAB1C68 fusion
A
GST-TAK1-TAB1C68 fusion GST-TAK1
4R: K72R, K227R, K520R and K563R
5R: K72R, K227R, K520R and K563R plus K158R
GST-MKK6
B
GST-TAB1
GST-MKK6
Figure 43. Recombinant GST-TAK, GST-TAB1 and GST MKK6 purified from bacterial. A. The different GST TAK1 forms and GST-TAK1-TAB1C68 fusion forms. B.
GST-TAB1 and the different GST MKK6 forms.
157
The recombinant proteins were used for in vitro kinase assay. As indicated in Fig. 44 and
Fig. 44, our data clearly demonstrated that only the recombinant TAK1-TAB1 C68 fusion protein could phosphorylate itself and MKK6 K82A while TAK1 alone does not have any kinase activity MKK6 are phosphorylated specifically at Ser 201 and T 211A since
S207AT211A form could not be phosphorylated by the fusion protein and thereby (Fig. 45).
Surprisingly, we did not detect the TAK1 kinase activity in the reaction using the combination of TAK1 and TAB1 which were separated purified from bacteria (Fig. 44).
Using His-tag or GST-tag TAK1, we demonstrated that TAK1 could be immunoprecipitated by TAB1 antibody (Fig. 46A) and conversely TAB1 could be immunoprecipitated by TAK1 antibody (Fig. 46B and Fig. 46C).
158
GST-MKK6K82A
GST-TAB1
GST-TAK1
WT-TAB1C68 Fusion
GST-MKK6K82A
Figure 44. TAK1 in vitro kinase assay. TAK1 mutants at lysine 158 were produced by site mutagenesis. TAK1 variants, TAB1 and MKK6K82A were purified from bacteria and used for in vitro kinase assay labeled with [γ-32 P] ATP. TAK1-TAB1 C68 fusion protein was used for the positive control.
159
GST-MKK6K82A GST-MKK6S207A, T211A
GST-TAK1-TAB1C68 Fusion
GST-TAK1-TAB1C68 Fusion
GST-MKK6K82A
Figure 45. TAK1 in vitro kinase assay. TAK1 mutants at lysine 158 were produced by site mutagenesis. TAK1 variants, TAB1, MKK6K82A and MKK6 S207AT211A were purified from bacteria and used for in vitro kinase assay labeled with [γ-32 P] ATP.
160
ABC
GST-TAK+GST-TAB1 GST-TAK+GST-TAB1
IP: IP:
GST-TAK1 GST-TAB1 GST-TAB1
IB: TAK1 IB: TAB1 IP: TAK1 IB: TAB1
Figure 46. TAK1 and TAB1 interact with each other in vitro. A. Binding reaction was performed by GST-TAK1 and GST-TAB1. Immunoprecipitation was performed with TAK1 or TAB1 antibody and immunoblotting was performed with TAK1 antibody. B. Binding reaction was performed by GST-TAK1 and GST-TAB1. Immunoprecipitation was performed with TAK1 or TAB1 antibody and immunoblotting was performed with TAB1 antibody. B. Binding reaction was performed by His-TAK1 and GST-TAB1.
Immunoprecipitation was performed with TAK1 and immunoblotting was performed with
TAB1 antibody.
161
We also generated TAK1 kinase from 293T cells. The different Flag-TAK1 forms with
HA-TAB1 were transfected into 293T cells. At 48 hours post transfection, the cells were harvested and the TAK-TAB1 complexes were immunoprecipitated by flag antibody. The precipitates were used for in vitro kinase assay. The recombinant GST-MKK6 kinase dead form was used for the kinase reaction. As shown in Fig. 47, TAK1 kinase activity was significantly reduced by the change from lysine to arginine at 158. More importantly, TAK1
K158N mutant partially recue the TAK1 kinase activity which consentient with the above results of 293T transfection in vivo.
162
HA-TAB1+GST-MKK6K82A
Flag-TAK1 GST-MKK6 HA-TAB1
Figure 47. TAK1 in vitro kinase assay. The different Flag-TAK1 forms with HA-TAB1 were transfected into 293T cells. At 48 hours post transfection, the cells were harvested and the TAK-TAB1 complexes were immunoprecipitated by flag antibody. The precipitates were used for in vitro kinase assay.
163
To demonstrate the important role of TAK1 acetylation at lysine 158, we performed a series experiments in MEF cells. Firstly, the TAK1 forms including TAK1 wild type, TAK1
K158R, TAK1 K158Q, TAK1 K158N and the empty vector were put back into TAK1 null
MEF cells and the stable cell lines expressing the various forms were established by the selection of zeocine. TAK1 expression was verified by western blot. The cells were treated by TNF-α for different times and several experiments were performed to investigate the role of lysine 158 in regulating TAK1 activity.
Firstly we tracked the morphological changes of the cells expressing the different TAK1 forms after TNF-α treatment under microscope. As indicated in Fig. 48, a lot of TAK1 null cells and the empty vectors cells were dead at 4 hours after TNF-α treatment. No phenotypic changes were observed in WT rescue cells. TAK1 K158R cells displayed a partially rescue phenotype but K158Q and K158N cell had a better rescue phenotype than TAK1 K158R. At
24 hours after TNF-α treatment, almost all of TAK1 null cells and the empty vectors cells clogged, floated and underwent apoptosis. Most of K158R cells were also dead but survival cells were a little more than TAK1 null cells and the empty vectors cells. This was still some
K158 N cells alive. It is not surprised that WT TAK1 had the most survival cells. As summary, the rescue effects followed the order: WT > K158N > K158Q > K158 N > K158 R> the empty vector, TAK1 null cells.
164
TNF-α (hr) 0 4 8 24
-/-
-/- + vector
-/- + WT
-/- + K158R
-/- + K158Q
-/- + K158N
Figure 48. Light-microscopic phenotype in MEF cells expressing the various TAK1 forms or the empty vector as control. The cells were treated with TNF-α and the photographs were taken at the different tines. Scale bars = 100 μm.
165
We also performed EMSA to evaluate NF-κB activation. As shown in Fig 49, only WT
rescue cells displayed the early NF-κB induction by TNF-α treatment at 30 minutes. TAK1
K158R, TAK1 K158Q and TAK1K158 N exhibited a later NF-κB induction at 8 hours and displayed different pattern from TAK1 null cell or the empty vectors which showed a slower decreasing process.
TNF-α (hr) 00.54812
-/-
-/- + vector
-/- + WT
-/- + K158R
-/- + K158Q
-/- + K158N
Figure 49. NF-κB activation induced by TNF-α treatment. Nuclear extracts were isolated from the different cell lines at several time points as indicated and used for EMSA to measure NF-κB activation.
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The expression of TAK1 rescues the TNF-α induced apoptosis in TAK1Δ/Δ MEFs, and
TAK1 K158Q and TAK1 K158N showed partially rescue effect. At 4 hours after TNF-α treatment, although K158 Q, K158N and K158 R showed less PARP cleavage than the
TAK1 null cells and the empty vector cells, TAK1 K158Q and TAK1 K158N cells had a better rescue effect than K158R. TAK1 K158Q and TAK1 K158N cells also had a stronger the induction of MKK6 phosphorylation than K158R. The phosphorylation of MKK6 level was close to the TAK1 null cells and the empty vector cells. Interestingly, the basal level of phosphor-ERK without TNF-α treatment displayed a decreasing order: WT < K158N <
K158Q < K158 N < K158 R< the empty vector, TAK1 null cells. It seemed that phosphor-
JNK and phosphor-ERK levels were lowered at 4 hours after TNF-α treatment (Fig.50).
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No TNF-α + TNF-α 4Hr
TAK1
PARP Cleaved-PARP
Phospho- MKK6
Phospho- JNK
Phospho- ERK (p42/p44)
β-Actin
Figure 50. The Expression of TAK1 Rescues the TNF-α Induced Apoptosis in TAK1Δ/Δ
MEFs, and TAK1-K158Q and TAK1-K158N Showed Partially Rescue Effect. The cells were harvested at 4 hours after TNF-α treatment followed by western blot using the different antibody as indicated. β- actin served as the loading control.
168
We also evaluated apoptosis cells using Annexin-V staining. As shown in Fig. 51, TAK1 wild type rescue cells, K158 N cells and K 158 Q cells displayed much slower apoptosis kinetics than TAK1 K158R cells, the empty vector cells and TAK1 null cells. At 24 hours after TNF-α treatment, more than 50% of TAK1 K158R cells, the amply vector cells and
TAK1 null cells showed Annexin-V positive, however, less than 30% of TAK1 wild type
rescue cells, K158 N cells and K 158 Q cells were Annexin-V positive. Once again, the
rescue effects followed the order: WT > K158N > K158Q > K158 N > K158 R> the empty
vector, TAK1 null cells.
Figure 51. Annexin V staining for apoptosis assay. The cells were collected at the different time points after TNF-α treatment and followed by Annexin V Staining. Annexin V positive cells were indicated with percentage.
169
Flow Cytometry Analysis was also performed to evaluate apoptosis. As indicated in Fig.
52, at 24 hours after TNF-α treatment, most of TAK1 null cells and the empty vector cells underwent apoptosis. TAK1 K158R cells showed a partially rescue effect while WT TAK1 rescue cells, TAK1 K158N cells and TAK1 K158Q cells displayed better rescue effects.
Consistent with the results from PARP cleavage, annexin-V staining, the rescue effects followed the exact same order: WT > K158N > K158Q > K158 N > K158 R> the empty vector, TAK1 null cells.
TAK1Δ/Δ MEFs TAK1Δ/Δ MEFs TAK1Δ/Δ MEFs Vector Flag‐TAK1‐WT + TNF‐α 24Hr + TNF‐α 24Hr + TNF‐α 24Hr
TAK1Δ/Δ MEFs TAK1Δ/Δ MEFs TAK1Δ/Δ MEFs Flag‐TAK1‐K158Q Flag‐TAK1‐K158N Flag‐TAK1‐K158R + TNF‐α 24Hr + TNF‐α 24Hr + TNF‐α 24Hr
Figure 52. Flow Cytometry analysis for apoptosis assay. The different cell lines were treated with TNF-α. After 24 hours, cells were collected for flow cytometry
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2.3.8. TAK1 is acetylated by CREB-binding Protein (CBP)
To screen for the acetyltransferase, we transfected Flag-TAK1 with or without TAB1 and
the several acetyltransferase constructs into 293T cells. TAB1 was used to increased TAK1
level. The cells were harvested at 48 hours after transfection and the extracts were
immunoprecipitated by Flag antibody followed by western blot with acetyl lysine antibody.
Our results demonstrated that the acetylation of TAK1 was significantly increased by CBP but not p300, GCN5 and PCAF (Fig. 53). We also examined the acetylation level of TAK1
4R and TAK1 5R mutants. As shown in Fig. 56, the acetylation level of these two mutants
was deduced compared with wild type TAK1 and TAK1 K158R. It seemed that TNF-α
treatment did not have much effect on the acetylation of TAK1.
171
Flag-TAK1 Flag-TAK1+ HA-TAB1
Acetylated-TAK1 IP: Flag IB: Acetyl-K
Total TAK1
IB: Flag+HA
N.S.
Figure 53. TAK1 acetylation is induced by CBP. Flag-TAK1 and the acetyltransferase
constructs CBP, p300, GCN5 and PCAF were transfected into 293T cells. The cells were harvested at 48 hours after transfection and the extracts were immunoprecipitated by Flag antibody. Acetylated-TAK1 was demonstrated by western blot with acetyl lysine antibody.
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CBP
TNF-α
TAK1
Acetyl-TAK1
N.S.
4R: K72R, K227R, K520R and K563R IP: Flag IB: Acetyl-K 5R: K72R, K227R, K520R and K563R plus K158R
Figure 54. Acetylation level of TAK1 4R and TAK1 5R mutants is less than TAK1 wild type. Flag-TAK1 and the acetyltransferase constructs CBP were transfected into 293T cells.
The cells were harvested at 48 hours after transfection and the extracts were immunoprecipitated by Flag antibody. Acetylated-TAK1 was demonstrated by western blot with acetyl lysine antibody.
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We also tested the effects of two deacetylase inhibitors trichostain A and nicotianamine.
As shown in Fig. 55, nicotianamine but not trichostain A treatment significantly increased the acetylation level of TAK1 compared with untreated control. The combined treatment had a stronger effect although we did not observe the effect of trichostain A.
174
Flag-TAK1
trichostatin A - -++ nicotianamine - + - +
Acetyl-TAK1
input
IP: Flag IB: Acetyl‐K
Figure 55. Acetylation level of TAK1 is increased by the treatment of deacetylase inhibitor nicotianamine. The 293 cells were transfected with Flag-TAK1 and treated with deacetylase inhibitors trichostain A and nicotianamine. Acetylated TAK1 was analyzed by western blot with acetyl lysine antibody.
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2.3.9. Lysine 158 of TAK1 is conserved throughout all kinases
We firstly aligned the kinase domain of different kinases using the software MEGA4 followed by a manual adjustment. As shown in Fig. 56, lysine 158 of TAK1 is conserved from c. elegans to human. It is also invariable in all of MAP3K members.
176
C.elegans_MOM-4 WMFQLSSAVDFFHSNS---QVHRDLKLQNMLLSDRYRT
Drosophila_tak1 WARQCAEGLAYLHAMTPKPLIHRDVKPLNLLLTNKGRN zebrafish_tak1 WCLQCSQGVSYLHGMKPKALIHRDLKPPNLLLVAGGTV
Xenopus_tak1 WCLQCAQGVAYLHSMKPKALIHRDLKPPNLLLVAGGTV mouse_tak1 WCLQCSQGVAYLHSMQPKALIHRDLKPPNLLLVAGGTV rat_tak1 WCLQCSQGVAYLHSMQPKALIHRDLKPPNLLLVAGGTV human_tak1 WCLQCSQGVAYLHSMQPKALIHRDLKPPNLLLVAGGTV
A-raf MVQLIDVARQTAQGMDYLH---AKNIIHRDLKSNNIFLHEG--
B-raf MIKLIDIARQTAQGMDYLH---AKSIIHRDLKSNNIFLHED-- map3k1 ESVVINYTEQLLRGLSYLH---ENQIIHRDVKGANLLIDST--- map3k2 ENVTRKYTRQILEGVHYLH---SNMIVHRDIKGANILRDS---- map3k3 ESVTRKYTRQILEGMSYLH---SNMIVHRDIKGANILRDS---- map3k4 EHVIRLYSKQITIAINVLH---EHGIVHRDIKGANIFLTS---- map3k5 EQTIGFYTKQILEGLKYLH---DNQIVHRDIKGDNVLINTY--- map3k6 ESTISFYTRQILQGLGYLH---DNHIVHRDIKGDNVLINTF--- map3k7 AAHAMSWCLQCSQGVAYLHSMQPKALIHRDLKPPNLLLVAGG-- map3k8 EFEIIWVTKHVLKGLDFLH---SKKVIHHDIKPSNIVFMS---- map3k9 PDILVNWAVQIARGMNYLHDEAIVPIIHRDLKSSNILILQKVEN map3k10 PHVLVNWAVQVARGMNYLHNDAPVPIIHRDLKSINILILEAIEN map3k11 PHVLVNWAVQIARGMHYLHCEALVPVIHRDLKSNNILLLQPIES map3k12 PSLLVDWSMGIAGGMNYLH---LHKIIHRDLKSPNMLITYD--- map3k13 PRLLVDWSTGIASGMNYLH---LHKIIHRDLKSPNVLVTHT--- map3k14 EDRALYYLGQALEGLEYLH---SRRILHGDVKADNVLLSSD--- map3k15 EPTIKFYTKQILEGLKYLH---ENQIVHRDIKGDNVLVNTY--- raf1 MFQLIDIARQTAQGMDYLH---AKNIIHRDMKSNNIFLHEG---
ZAK MDHIMTWATDVAKGMHYLHMEAPVKVIHRDLKSRNVVIA----- Figure 56. K 158 of TAK1 is highly conserved in most of kinases. Alignment was performed with the software MEGA4 followed by a manual adjustment. The conserved lysine was highlighted by red color.
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Therefore, we tested the role of this lysine in controlling kinase activity of MEKK3. We made three MEKK3 mutants K522R, K522Q and K522N. Kinase activity was examined by phosphorylation antibody. As shown in Fig. 57, the substation of lysine with arginine, glutamine or asparagine at position 522 which is corresponding to lysine 158 of TAK1 abolished its kinase activity.
178
Flag
MEKK3
Phos-MEKK3
N.S.
Figure 57. Lysine 522 is required for MEKK3 kinase activity. Site mutagenesis was used to create MEKK3 K522R, K522Q and K522N mutants. The various MEKK3 constructs were transfected into 293T cells and MEKK3 kinase activity was analyzed by phosphorylation antibody.
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MEKK3 was also acetylated by CBP. HA-MEKK3 and the acetyltransferase constructs
CBP, p300, GCN5, PCAF and tip60 were cotransfected into 293T cells. The cells were harvested at 48 hours after transfection and the extracts were immunoprecipitated by HA antibody. Acetylated-MEKK3 was analyzed by western blot with acetyl lysine antibody. As shown in Fig. 58, MEKK3 was also acetylated by CBP, but not by p300, GCN5, PCAF and tip60.
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HA-MEKK3
Acetyl-MEKK3
input
IP: HA IB: Acetyl‐K
Figure 58. MEKK31 acetylation is induced by CBP. HA-MEKK3 and the acetyltransferase constructs CBP, p300, GCN5, PCAF and tip60 were transfected into 293T cells. The cells were harvested at 48 hours after transfection and the extracts were immunoprecipitated by HA antibody. Acetylated-MEKK3 was demonstrated by western blot with acetyl lysine antibody.
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2.4. Discussion
In the current study, we investigate the three regulatory mechanism3: 1. N-terminus of
TAK1; 2. TAK1 binding protein TAB1; and 3. Post-translational modification, especially acetylation at lysine 158.
Several independent studies have demonstrated that TAK1 with N-terminus truncated has more activity than full-length TAK1. Ectopic expression of mouse TAK1ΔN has more severe effect than that of full length mTAK1 in transgenic flies [81]. The injection of TAK1∆N
mRNA encoding the N-terminal truncated TAK1 induced apoptosis at lower doses than the
full length TAK1 [85]. TAK1ΔN stimulated TGF-β target gene PAI-1 expression [75]. These
results strongly implicated that TAK1 function is regulated by the N-terminus. We
compared the stability of TAK between full length and N-terminus truncated form using the
classic cycloheximide (CHX) chase analysis. Both full length TAK1 and TAK1ΔN were
degraded in a time-course experiment after CHX treatment. We did not see much difference
in dynamics between them. In our experiments, either full length TAK1 or TAK1ΔN alone
does not have kinase activity. Both full length TAK1 and TAK1ΔN were stabilized by the
co-expression of TAB1. It seems that at least human N-terminus of TAK1 is not related to
stability. The mechanism of TAK1 N-terminus regulation may be species-dependent.
Another explanation is that the N terminus may be involved in TAK1 function other than
stability. More experiments are needed to define how it is involved with TAK1 activity.
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The previous study showed that TAB1 increased the expression level of TAK1 [124].
Here, we provided new evidence to understand the underlying mechanism. Real time PCR result showed that TAB1 did not increase TAK1 transcription. Furthermore, using cycloheximide (CHX) chase analysis we demonstrated that most of TAK1 was degraded after 16 hours of CHX treatment. However, co-expression of TAB1 significantly protected
TAK1 from degrading in the presence of CHX. As far as we know, this is the first evidence that TAK1 is stabilized by TAB1 at the protein level. TAK1 alone is not stable. As discussed before, we also tested TAK1ΔN stability. Our experiments demonstrated that the deletion of
TAK1 did not augment stability and TAK1ΔN stability also required TAB1. So the mechanism in which TAB1 stabilizes TAK1 may be N-terminus independent. In the physiological condition, TAK1 constitutively interacts with TAB1. This interaction is very important and may play a necessary role in controlling TAK1 activity.
To understand the mechanism how TAK1 is regulated by TAB1, we analyze TAK1
Ubiquitination level and tried to locate TAK1 Ubiquitination sites using MASS-SPEC approach. However, we did not observe much difference in TAK1 Ubiquitination between with and without TAB1. We also could not detect any Ubiquitination peptides of TAK1 with MASS SPEC (data not known). Most of proteins are degraded by the attachment of ubiquitin. However, some proteins were degraded through an ubiquitin-independent mechanism [239-242]. Such mechanisms may be involved with TAK1 degradation.
However, we cannot rule out the possibly that Ubiquitination of TAK1 is a transient event and the approaches we used was not sensitive enough to detect TAK1 Ubiquitination.
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A lot of studies have proposed that TAK1 kinase activity depends on TAK1 [85, 124,
243-246]. Our data recapitulated the role of TAB in regulating TAK activity and gave more evidence that TAB1 plays an essential role in regulating TAK1 activity.
The several signaling pathways, including TAK1 autophosphorylation, TAB1
phosphorylation, MKK6 phosphorylation, NF-κB promoter activity were induced by
coexpression of TAK1 and TAB1. In our experiments, transfection of TAK1 alone, without
TAB1, did not have any function. However, transfection of TAK1 alone, without TAB1, did
not have any function in all of these experiments. These results strongly support that TAK1
activity requires TAB1.
In vitro kinase assay using bacterially expressed recombinant protein is a very simple but
powerful technique. This approach can rule out contamination by other kinases. To take the
advantage of this approach, we first revealed that GST-TAK1-TAB1 fusion protein purified
from bacterial has high kinase activity. Since the substrate MKK6 itself is a kinase, we made
a kinase dead mutant of MKK6 to overcome its autophosphorylation in kinase assay. As
demonstrated in fig, GST-TAK1-TAB1 phosphorylated itself and MKK6 kinase mutant.
Mutations Ser207 and Thr211 at these two site abolished MKK6 phosphorylation. These
results confirmed that these two sites of MKK6 are specifically phosphorylated by TAK1.
However, GST-TAK1 alone does not have any kinase activity. This experiment clearly
demonstrated the requirement of TAB1 for TAK1 activity.
184
Since we demonstrated before that TAK1 could play an oncogenic in pancreatic cancer development, TAK1 is an attractive drug candidate. TAK1 inhibitors may be used as a promising therapeutic approach. Unfortunately, TAK1 alone does not have any kinase activity and therefore it cannot directly be used for screening inhibitors. Our fusion protein, which has high kinase activity, may provide a new assay for the screen of TAK1 inhibitors.
We also purified TAK1 and TAB1 separately. However, combination of TAK1 and
TAB1 proteins did not produce kinase activity. This inactivation of kinase was not due to the loss of the interaction between TAK1 and TAB1 because we verified the interaction still existed using co immunoprecipitation experiment. In the kinase reaction, we demonstrated that TAK1 can be pulled down by TAB1. Furthermore, we showed that this interaction was not resulted from GST since we observed the interaction between TAK1 and TAB1 using
His-TAK1 purified from TAK1. These experiments demonstrated that the interaction of
TAK1 and TAB1 was not sufficient to induce kinase activity. Some additional mechanism besides TAK1-TAB1 interaction may be required for TAK1 activity. One possibility is that
TAB1 may be involved in TAK1 folding. In bacterial expression system, TAK1 could not form an active conformation and subsequently lost kinase activity. More experiments need to be carried out to delineate its
It was reported that C-terminus of TAB1 is required for TAK1 activation [124]. Our data confirmed the previous discovery and mapped a minimum peptide sequence at the c-terminus
185
of TAB1 which is necessary for TAK1 activation. We made a series of deletion mutants of
TAB1 and defined that the short peptide from the c-terminus of TAB1
“EPYVDFAEFYRLWSVD” is not only required but also sufficient for TAK1 activation.
The deletion of an amino acid glutamic acid (E) at the N-terminus of this peptide completely
abolished TAK1 kinase activity. Homolog blast reveled that this sequence was found only in
TAB1 protein. This result demonstrated that the function of this sequence is very specific.
Unlike most other kinases, TAK1 alone does not have kinase activity. Only when interacted with TAB1, does it have kinase activity. The requirement of TAB1 for TAK1 kinase activity is quite unique. However, why TAK1 is activated by such a short peptide is a challenge question. This peptide-kinase interaction may be very similar with the ligand- receptor binding. It should be noticed that a majority of amino acids containing this peptide are hydrophobic. The interaction of this peptide with TAK1 may help change its kinase conformation, for example, to increase ATP affinity. However, the evidence is still largely absent. The data from structure biology may be required to reveal the detailed mechanism.
As introduced before, four transcriptional variants have been identified in human. All of
these isoforms interact with TAB1. Since alternative splicing in TAK1 C and D results in a
reading frame shift and an earlier stop codon, the c-terminus of TAK1 is required for its
TAB2 binding. TAB1 was phosphorylated by TAK1 since overexpression of different
TAK1 transcriptional variants with TAB1 resulted in TAB1 phosphorylation and TAK1
186 kinase dead form (K63A) was unable to induce phosphorylation. Cooverexpression of
TAK1 and TAB1 also resulted in JNK phosphorylation and NF-κB activation. In fact, the kinase domain is complete in these four transcriptional variant and thereby it is not surprised that all of them have kinase activity.
However, this does not mean their functions are completely overlapping. TAK1 A,
TAK1 B and TAK1C, TAK1 D may have different functions. Using TAK1 null MEF cells as the parental cell line, we made the stable cell lines expressing human TAK1A, TAK B, TAK
C and TAK D. Interested, the rescue experiments in TAK1 null MEF cells demonstrated that only isoform TAK1 A and TAK1 B, but not TAK C and TAK1 D can restore NF-κB activity induced by IL-1 and TNF-α. It has been shown that TAK1 c-terminus [(TAK1 403-579)] was required and sufficient for TAB1 binding [124]. TAB2 is an adapt protein which mediate the interaction of TAK1 and TRAF6 and regulates NF-κB activation induced by IL-1 [125].
The alternative splicing in TAK1 C and D results in a reading frame shift and an earlier stop codon and thereby lost the binding to TAB2. Consequently, TAK C and TAK1 D can no longer bind TRAF6 and thereby NF-κB cannot be activated through TAK C and TAK1 D.
Our data showed that TAK C and TAK1 D may not be involved in NF-κB activation and they may play different roles in specific tissue or at different developmental stage. However, what’s the deference in roles between TAK1 A and TAK1 B, or between TAK1 C and TAK1
D is still unknown. These splicing variants may be development stage specific or tissue specific. The current research is limited in NF-κB signaling pathway. However, they could
187 be involved in the different signaling pathways in response to the deferent stimuli. More experiments will be necessary to distinguish their functions.
TAK1 function is also regulated by post-translational modification. It has been showed that autophosphorylation of Thr 178, Thr 184 , Thr 187 and Ser 192 in activation loop is required for TAK1 activation [247-249]. Mutations at these sites abolished TAK1 kinase activity.
As discussed before, TAK1 was largely stabilized by TAB1. Taking advantage of co- overexpression of TAK1 and TAB1 in 293T cells, we tried to identify some post- translational modification sites of TAK1. A total of 7 phosphorylation sites were identified in
TAK1 (Ser192, Ser367, Ser375, Ser389, Ser412, Thr440 and Ser441). Some of these phosphorylation sites (Ser192, Ser375 and Ser412) have been identified before [155] and the other sites were identified first in this study. To study the roles of phosphorylation sites at these sites, we uses site mutagenesis to change serine or threonine into alanine. Although mutation of serine or threonine at Ser389, Thr440 and Ser441 to alanine abolished phosphorylation at these sites, TAK1 autophosphorylation level or TAB1 phosphorylation was not affected. Therefore, phosphorylation of TAK1 at these sites is not required for activation. However, we cannot exclude the possibility that these phosphorylation events may contribute to a different function of TAK1 which is independent of TAK1 kinase activity.
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More important, several acetylation sites were identified in our study. As introduced
before, acetylation is a very important post-translational modification. Here we firstly
demonstrated that TAK1 is regulated by acetylation. A total of 5 acetylation sites were
identified in TAK1 (Lys72, Lys158, Lys227, Lys520 and Lys563). As far as we know, this
is the first time that lysine acetylation was identified in TAK1. As shown Fig. 61, both lysine
and arginine are positively charged and they have the similar side chain. However, arginine
cannot be acetylated at ε-amino group, so it is used as the mimics of unacetylated lysine in
study of protein biochemistry. The positive charge is removed in lysine by acetylation at ε- amino group. In addition, it is a well established approach that glutamine or asparagine is used as the mimics of acetylated lysine.
To understand the roles of acetylation sites at these sites, we uses site mutagenesis to change lysine into arginine or glutamine or asparagine. We used the following different assays to determine TAK1 activity.
(1) As discussed before, when co expressed with TAB1 in 293T cells, TAK1 is phosphorylated by itself. However, TAK1 kinase dead form K63A, in which ATP binding pocket was mutated, does not have kinase activity;
(2) TAK1is stabilized by TAB1 when co-expressed in 293T cells but the level of TAK1
K63A are not affect by TAB1;
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(3) TAK1, but not TAK1 K63A, can stabilize TAB1;
Our results showed TAK1 activity was not affected by mutations at Lys72, Lys227,
Lys520 and Lys563. To determine if these sites have redundant function, we made the different combined mutant at these four sites. As shown in fig, the double mutants
K72RK227R, K72RK563R, K227RK563R, K520RK563R, and K72QK227Q displayed the same activity as the wild type TAK1. Furthermore, the triple mutant K72RK227RK563R
(called TAK1 3KR) also had the same kinase activity as the wild type TAK1. Finally, even the quad mutant in which all of the four lysine sites were mutated to arginine (TAK1 4KR) still held its kinase activity. These data clearly demonstrated these four sites are not involved with TAK1 kinase activity. However, we cannot rule out the possibility that acetylation at
Lys72, Lys227, Lys520 and Lys563 is involved in other mechanism independent of TAK1 kinase activity.
Surprising, the change of lysine 158 resulted in the loss of TAK1 kinase activity according to above 4 assays. It is unexpected that glutamine or asparagine substitution mimicked acetylation at lysine 158 in our experiments. As mentioned before, we established a kinase active form in bacterial expression system, which is a fusion protein, composed of
TAK1 and C-terminus of TAB1. We changed the lysine 158 of the fusion protein into arginine or glutamine or asparagines. In vitro kinase assay clearly showed that TAK1 kinase activity was severely impaired in these mutants.
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More important, the lysine acetylation mimics glutamine or asparagine at 158 of TAK partially rescue TAK1 function in survival and anti-apoptosis pathway. NF-κB activation
induced the expression of antiapoptotic gene such as Bcl-2 and Bcl-x [250]. It is well known that the cells lacking NF-κB would experience apoptosis upon TNF-α stimulation [251]. To study role of lysine 158 in controlling TAK1 function, we resorted the expression of TAK1 including K158R, K158Q and K158N mutants in TAK1 deficient MEF cells. Morphological
and flow cytometry analysis demonstrated that TAK1 deficient cells and the cells
containing the empty vector only cells experienced massive death at four hours after TNF-α
treatment. It is not surprised that the cells were totally rescued from apoptosis by the expression of wild type TAK1 at different time points. More interesting, the cells were partially rescued by TAK1 K158R, K158Q and K158N. Most importantly, K158Q and
K158N mutants had even better effects of rescue than K158R. These results are consistent with Poly (ADP-ribose) Polymerase (PARP) cleavage analysis and annexin-V staining.
It should be noticed that although TAK1 K158R did not display any kinase activity in our kinase assay, it still displayed a partially rescue effect. One explanation is that TAK1 k158R still has residual kinase activity. This kind of residual kinase activity could not be sensitive enough for our detection approach but it could still partially rescue the phenotype. Another explanation is that TAK1 served as not only a kinase, but also a scaffold for the other IKK kinase such as MEKK3 and thereby complement for the loss of TAK1 kinase activity. In fact, IKK complex is composes of a lot of proteins. It is possible that TAK1 provide a scaffold for the other components. We need more evidence to test this possibility.
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It should also be noticed that although TAK1 K158N purified from 293T cells displayed a partially kinase activity, we did not detect kinase activity using GST-TAK1K158N
TAB1C68 fusion. One possibility is that the recombinant TAK1 kinase purified from bacterial lacks some modifications which are important for kinase activity. GST- WT
TAK1K TAB1C68 fusion alone has high activity and therefore does not requires these modifications.
Lysine can be methylated, ubiquitinated and acetylated. These modifications can be regulated by each other since they compete for the same site. The study of acetylation is focused on histone although the increasing evidence demonstrated that acetylation also plays an important role in regulating non-histone proteins. Our study provides a new mechanism in which kinase activity is regulated by acetylation.
We also identified CBP as the acetyltransferase of TAK1. The histone acetyltransferase (HAT) CBP was originally identified as a transcriptional coactivator involved in histone modification. It interacts with cAMP-response element-binding protein
(CREB) and subsequently regulates the transcription of target genes [252-256]. A lot of studies have showed that CBP also acetylated the non-histone proteins such as p53 CBP
[257-261]. To identify the acetyltransferase of TAK1, we expressed TAK1 with several candidates including CBP, P300, GCN5, PCAF and Tip60. Acetylation of TAK1 was significantly increased by the expression of CBP. Therefore, CBP is a potential candidate
192 which acetylates TAK1. We are still unable to rule out the possibility that TAK1 is the substrate of other acetyltransferases since our list is not complete. We also tested other kinase such as MEKK3, TAK1-TAB1 C68 fusion, PLK3 and found that these kinases also were acetylated by CBP. It may be a common mechanism that kinase activity is regulated by acetylation.
We firstly aligned the kinase domain of different kinases using the software MEGA4 followed by a manual adjustment. As shown in fig, lysine 158 of TAK1 is conserved from c. elegans to human. It is also invariable in all of MAP3K members. Moreover, it is conserved in all of kinase. Therefore, we tested the role of this lysine in controlling kinase activity of
MEKK3. As shown in fig, the substation of lysine with arginine, glutamine or asparagines at position 522 which is corresponding to lysine 158 of TAK1 abolished its kinase activity.
These data demonstrated that the conserved lysine is very important for kinase activity. This conserved domain has been identified before but its role in controlling kinase activity is unknown. Our discovery provide a new mechanism and highlight that acetylation at this conserved site may be a common regulatory mechanism of kinase activity. However, we did not observe kinase activity in the mutants MEKK3 K522Q or MEKK3 K522N. One explanation is that the kinase activity is very week so that the MEKK3 phosphorylation antibody is not sensitive enough for detecting the activity. We could rule out possible that although this site is conserved, the acetylation mechanism is still depended on the specific kinase. The acetylation of MEKK3 we observed may occur in other sites.
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There are still unanswered questions. Since acetylation is reversible process, the
deacetylase which counteracts CBP is still unknown. The future plan includes screening for
the deacetylase and understanding the underlying mechanism how acetylation at this site
regulates kinase activity is still unknown. The acetylation is normally low in vivo. The pan
acetyl lysine antibody cannot give us any information about the specific information. To get accurate and detailed knowledge about acetylation efficiently, the site specific antibody is necessary. The specific antibody would help us answer the above question.
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Vita
Qianghua Xia was born in Weining, Gui Zhou province, P. R. China. During his childhood, he showed wide interests in mathematics, biology, history and other disciplines under the influence of his father. In high school, he was especially good at mathematics, but he insisted choosing biology as his major in college, due to his fascination in biology. In
1996, he got his B.S. degree in Environmental Biology & Ecology from Peking University,
Beijing, and P.R. China. Then he joined the Institute of Genetics and Developmental
Biology, Chinese Academy of Sciences as a graduate student. He graduated with a M.S. degree in Molecular Developmental Biology in 1999 and came to US afterwards. He spent three years studying the…Drosophila genetics in the lab of Dr. Patrick Callaerts at the
University of Houston and got his MS degree in Biology in 2004. After Dr. Patrick Callaerts moved to Belgium, Qianghua was recruited to the University Of Texas Houston Health
Science Center Graduate School Of Biomedical Sciences. After three rotations, he chose to further study in Dr. Paul Chiao lab and to focus his research on the role of TAK1 in pancreatic cancer.
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