A Dissertation Entitled the Regulation of Mixed Lineage Kinase 3 By

A Dissertation

entitled

The Regulation of Mixed Lineage Kinase 3 by Extracellular Signal-Regulated Kinases 1 and 2 and Stress Stimuli in Colorectal and Ovarian Cancer Cells

April L. Schroyer

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Doctor of Philosophy Degree in Biology

______Dr. Deborah Chadee, Committee Chair

______Dr. William Taylor, Committee Member

______Dr. Malathi Krishnamurthy, Committee Member

______Dr. Ajith Karunarathne, Committee Member

______Dr. Zahoor Shah, Committee Member

______Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies

The University of Toledo

December 2017

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

The Regulation of Mixed Lineage Kinase 3 by Extracellular Signal-Regulated Kinases 1 and 2 and Stress Stimuli in Colorectal and Ovarian Cancer Cells by

April L. Schroyer

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology

The University of Toledo

December 2017

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K), which activates the extracellular signal-regulated kinases 1 and

2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 MAPK pathways. Interestingly,

MLK3 can activate the ERK1/2 pathway through either kinase-dependent or - independent mechanisms; in the latter, MLK3 serves as a scaffold for transactivation of v-Raf murine sarcoma viral oncogene homolog B (B-Raf) and v-Raf-1 murine leukemia viral oncogene homolog 1 (Raf-1). MLK3 can transform NIH 3T3 cells and functions in migration and/or invasion of several human cancers. Computational studies recently suggested both MLK3 and ERK2 as master regulators of colorectal cancer (CRC) invasion. Human colorectal tumors display increased levels of reactive oxygen species

(ROS) or oxidative stress, which activates both MLK3 and MAPK signaling pathways in specific cellular contexts and is important for carcinogenesis. Therefore, we examined whether MLK3 promoted a malignant phenotype in CRC cells under oxidative stress. We found a ROS- and ERK1/2-dependent phosphorylation of MLK3 in H2O2-treated human colorectal carcinoma (HCT116) cells as well as an interaction between endogenous

MLK3 and both endogenous ERK1/2 and B-Raf. Active ERK1 phosphorylated kinase

iii dead FLAG-MLK3 in vitro, whereas ERK1 phosphorylation of kinase dead FLAG-

MLK3-S705A-S758A was reduced. MLK3 siRNA knockdown as well as FLAG-MLK3-

S705A-S758A expression decreased both ERK1/2 activation and CRC cell invasion in

H2O2-treated cells. These results suggest oxidative stress stimulates an ERK1/2- dependent phosphorylation of MLK3 on Ser705 and Ser758, which promotes MLK3- dependent B-Raf, MEK1/2, and ERK1/2 activation; this positive feedback loop (PFL) enhances the invasion of colon cancer cells. We also explored mechanisms to control the amount of MLK3 protein in cancer cells and discovered geldanamycin (GA), heat shock, and osmotic stress all reduced the abundance of endogenous MLK3 protein in ovarian cancer cells.

For my family.

Acknowledgements

I acknowledge all of my teachers, professors, and mentors from Longfellow

Elementary School, Crawford AuSable School District, Clonlara School, Monroe County

Community College, the University of Michigan, and the University of Toledo including, but not limited to, Marilyn Stancil, Carl Hatfield, Tracy Rayl, Dr. David Waggoner, Chris

Perria, Dr. Sundeep Kalantry, Clair Harris, Dr. Deborah Chadee, Dr. John Gray, Dr. John

Belizzi, Dr. Lirim Shemshedini, Dr. William Taylor, Dr. Malathi Krishnamurthy, Dr.

Ajith Karunarathne, Dr. Zahoor Shah, and Dr. Richard Komuniecki.

I especially thank my Ph.D. advisor, Dr. Deborah Chadee, for the invaluable opportunity, education, and support necessary for both personal and academic growth as well as my manuscript co-authors Nicholas W. Stimes, Dr. Widian Abi Saab, Dr. Natalya

Blessing, Srimathi Kasturirangan, and Evan Zink.

Table of Contents

Abstract ...... iii

Acknowledgements ...... v

Table of Contents ...... vi

List of Figures ...... ix

List of Abbreviations ...... xi

List of Symbols ...... xvi

1 Introduction ...... 1

1.1 Mitogen-Activated Protein Kinases (MAPKs) ...... 1

1.2 Extracellular Signal-Regulated Kinases 1 and 2 (ERK1/2) ...... 3

1.2.1 ERK1/2 Signaling ...... 3

1.2.2 ERK1/2 Substrate Specificity ...... 6

1.2.3 Nuclear ERK1/2 ...... 7

1.3 Mixed Lineage Kinase 3 (MLK3) ...... 9

1.3.1 MLK3 Characterization ...... 9

1.3.2 MLK3 Activation ...... 12

1.3.3 MLK3 and Apoptosis ...... 15

1.3.4 MLK3 and ERK1/2 Signaling ...... 21

1.3.5 MLK3 and Cancer Migration and Invasion ...... 25

1.3.6 MLK3 and Colorectal Cancer ...... 29

1.3.7 MLK3 and Oxidative Stress ...... 31

1.4 Significance ...... 36

2 Materials and Methods ...... 37

2.1 Cell Culture ...... 37

2.2 Cell Treatments and Electrophoresis ...... 38

2.3 Immunoblotting...... 39

2.4 Phosphatase Assay ...... 41

2.5 Plasmid and siRNA Transfections ...... 41

2.6 Immunoprecipitations and GST Pull-Down ...... 43

2.7 Multiple Sequence Alignment ...... 45

2.8 Site-Directed Mutagenesis ...... 46

2.9 In Vitro Kinase Assay ...... 48

2.10 In Vitro FluoroBlok Invasion Assay ...... 48

2.11 Quantitative Real-Time PCR ...... 50

2.12 Quantification and Statistical Analysis ...... 51

3 Results ……...... 53

3.1 MEK1/2 and ERK1/2 are activated and MLK3 is phosphorylated in response

to oxidative stress ...... 53

3.2 The ROS-induced phosphorylation of MLK3 is dependent on active ERK1/2

and endogenous MLK3 and ERK1/2 associate in HCT116 cells ……...... 58

3.3 ERK1 phosphorylates Ser705 and Ser758 of MLK3 in vitro ……...... 60

vii

3.4 Oxidative stress promotes association of MLK3 and B-Raf: ROS-induced

MEK1/2 and ERK1/2 activation requires phosphorylation of MLK3 Ser705 and

Ser758 ..……...... 63

3.5 MLK3 promotes oxidative stress-induced invasion of colon cancer cells

through phosphorylation of Ser705 and Ser758 ……...... 68

3.6 Geldanamycin reduces the abundance of endogenous MLK3 protein in

colorectal and ovarian cancer cells ……...... 72

3.7 Heat shock or osmotic stress decreases the amount of endogenous MLK3

protein in ovarian cancer cells, while TNFa does not ……...... 77

4 Discussion ……...... 81

References ...... 93

A Multiple Sequence Alignment of Humans MLKs 1-4 ...... 122

viii

List of Figures

1-1 MAPK signaling modules ...... 2

1-2 ERK1/2 pathway ...... 4

1-3 MLK family ...... 10

1-4 MLK3 autoinhibition and activation ...... 13

1-5 Active MLK3 signaling in apoptosis ...... 16

1-6 MLK3 regulation of NF-kB signaling ...... 20

1-7 MLK3-dependent regulation of ERK1/2 signaling ...... 22

1-8 MLK3 signaling in cancer migration and invasion ...... 26

1-9 Activation of ASK1 and MLK3 by oxidative stress ...... 32

3-1 Effect of H2O2 on MLKs, MEK1/2, and ERK1/2 in HCT116 cells ...... 54

3-2 ROS-dependent shift of MLK3 and ERK1/2 activation ...... 55

3-3 MLK3 is phosphorylated in response to H2O2 in HCT116 cells ...... 55

3-4 Effect of MLK3 catalytic inhibition on ROS-induced MLK3 phosphorylation ....57

3-5 Effect of ERK1/2 catalytic inhibition on ROS-induced MLK3 phosphorylation ..58

3-6 Effect of ERK1/2 siRNA knockdown on ROS-induced MLK3 phosphorylation .59

3-7 MLK3 and ERK1/2 associate in H2O2-treated HCT116 cells ...... 60

3-8 Specific ERK1/2 phosphorylation sites of MLK3 ...... 61

3-9 ERK1 in vitro kinase assay with FLAG-MLK3 variants as substrates ...... 62

3-10 Wild-type and S705A-S758A-FLAG-MLK3 expression and JNK activation ...... 63 ix

3-11 MLK3 and B-Raf associate in H2O2-treated HCT116 cells ...... 64

3-12 Effect of MLK3 siRNA knockdown on ROS-induced ERK1/2 activation ...... 65

3-13 Expression of FLAG-MLK3 variants and MEK1/2 and ERK1/2 activation ...... 66

3-14 Association of GST-B-Raf and FLAG-MLK3 variants ...... 67

3-15 Effect of prolonged oxidative stress on HCT116 cell invasion and ERK1/2 ...... 69

3-16 MLK3 siRNA knockdown impairs invasion of H2O2-treated HCT116 cells ...... 70

3-17 Expression of FLAG-MLK3 variants and ROS-treated HCT116 cell invasion ....71

3-18 GA treatment decreases the amount of MLK3 protein in HCT116 cells ...... 72

3-19 Effect of GA on MLK3 protein and ERK1/2 activation in SKOV3 cells ...... 73

3-20 Effect of GA on MLK3/MAP3K11 mRNA ...... 74

3-21 Effect of GA on the half-life of MLK3 protein ...... 75

3-22 GA-induced decrease of MLK3 protein is proteasome-dependent ...... 76

3-23 Effect of heat shock and osmotic stress on MLK3 and ERK1/2 ...... 78

3-24 Effect of heat shock and osmotic stress on MLK3/MAP3K11 mRNA ...... 79

3-25 Both heat shock and osmotic stress reduce the half-life of MLK3 protein ...... 79

3-26 Effect of TNFa on MLK3 protein ...... 80

4-1 Model of MLK3-mediated oxidative-stress induced colon cancer invasion ...... 82

4-2 MLK3 as a molecular switch for ERK1/2 activation in response to ROS ...... 85

A-1 Multiple sequence alignment of human MLKs 1-4 ...... 122

List of Abbreviations

AM ...... acetomethoxy AMPK ...... AMP-activation protein kinase AP-1 ...... activating protein-1 APAP ...... acetaminophen APS ...... ammonium persulfate A-Raf ...... v-Raf murine sarcoma 3611 viral oncogene homolog 1 ASK1 ...... apoptosis signal-regulated kinase 1 ATCC ...... American Type Culture Collection ATF ...... activating transcription factor ATP ...... adenosine triphosphate au ...... arbitrary units

β-Me ...... β-mercaptoethanol B-Raf ...... v-Raf murine sarcoma viral oncogene homolog Bax ...... Bcl2-associated x Bcl-2 ...... B-cell CLL/lymphoma 2

Cdc42 ...... cell division cycle protein 42 CHIP ...... carboxyl terminus of Hsc70-interacting protein CIAP ...... calf intestinal alkaline phosphatase CRC ...... colorectal cancer CRIB ...... Cdc42/Rac1 interactive binding CRM1 ...... chromosomal maintenance 1 CTF ...... C-terminal fragment

DLK ...... dual leucine zipper bearing kinase DMEM ...... Dulbecco’s modified Eagle’s medium DMSO ...... dimethyl sulfoxide DNA ...... deoxyribonucleic acid dNTP ...... deoxynucleotide triphosphate

EGF ...... epidermal growth factor EGTA ...... ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid Em ...... emission ERK...... extracellular signal-regulated kinase EV ...... empty vector Ex ...... excitation

ERM ...... ezrin/radixin/moesin

FAK ...... focal adhesion kinase FasL...... Fas ligand FBS ...... fetal bovine serum FL ...... full length FOXO ...... forkhead box O

GA ...... geldanamycin GCLC ...... glutamate-cysteine ligase catalytic subunit GDP ...... guanosine diphosphate GEF ...... guanine nucleotide exchange protein GF ...... growth factor GluR6 ...... glutamate receptor 6 GPCR ...... G-protein-coupled receptor Grb2 ...... growth factor receptor-bound protein 2 GSH ...... glutathione GSK3b ...... glycogen synthase kinase 3 beta GSNO ...... S-nitrosoglutathione GST ...... glutathione S-transferase GTP ...... guanosine triphosphate

HBx ...... hepatitis B virus x protein HO-1 ...... heme oxygenase-1 HPK1 ...... hematopoietic progenitor kinase 1 HPLC ...... high performance liquid chromatography HRP ...... horseradish peroxidase Hsp ...... heat shock protein

IgG ...... immunoglobulin G IL-1R ...... interleukin-1 receptor IkB ...... inhibitor of kappa B IKK ...... IkB kinase IKKK ...... IKK kinase

JIP1 ...... JNK interacting protein 1 JNK ...... c-Jun N-terminal kinase

KD ...... kinase dead KID ...... kinase insert domain KSR ...... kinase suppressor of Ras

LB ...... Luria-Bertani lncRNA ...... long non-coding RNA

MAP2K ...... MAPK kinase xii

MAP3K ...... MAPK kinase kinase MAPK ...... mitogen-activated protein kinase MEK1/2...... MAPK/ERK kinase 1 and 2 MEKK ...... MEK kinase METH ...... methamphetamine miR ...... microRNA miRNA ...... microRNA MKK ...... mitogen-activated protein kinase kinase MKP ...... MAPK phosphatase MLK ...... mixed lineage kinase MM ...... metastatic melanoma MMP ...... matrix metalloproteinase MOM ...... mitochondrial outer membrane MP-1 ...... MEK partner-1 MYD88 ...... myeloid differentiation primary response 88 Myr-Akt ...... myristylated Akt

NAC ...... N-acetyl-L-cysteine NCBI ...... National Center for Biotechnology Information NES ...... nuclear export signal NF2 ...... neurofibromatosis 2 NF-kB ...... nuclear factor kappa B

NGF ...... nerve growth factor NLS ...... nuclear localization signal nNOS ...... neuronal nitric oxide synthase NO ...... nitric oxide NPC ...... nuclear pore complex NS ...... non-specific NTS ...... nuclear translocation signal NUP ...... nucleoporin

PAK1 ...... p21 (Rac1) activated kinase PBS ...... phosphate buffered saline PBST ...... PBS-Tween 20 PCR ...... polymerase chain reaction PET ...... polyethylene terephthalate PFL ...... positive feedback loop PI3K ...... phosphatidylinositol-3-kinase Pin1 ...... prolyl-isomerase PKC ...... protein kinase C PMA ...... phorbol 12 myristate 13-acetate PMSF ...... phenylmethanesulfonyl fluoride POSH ...... plenty of SH3s PP2A ...... protein phosphatase 2A PSD95 ...... post-synaptic density protein 95 xiii

PTB ...... phospho-tyrosine-binding PTK1 ...... protein-tyrosine-kinase 1 PVDF ...... polyvinylidene difluoride

Rac1 ...... Ras-related C3 botulinum toxin substrate 1 Raf-1 ...... v-Raf-1 murine leukemia viral oncogene homolog 1 RFU ...... relative fluorescence units RLF ...... RalGDS-like factor RNA ...... ribonucleic acid ROS ...... reactive oxygen species Rpm ...... revolutions per minute RT ...... reverse transcriptase or room temperature RTK...... receptor tyrosine kinase

SAPK ...... stress-activated protein kinase SD ...... standard deviation SDS-PAGE ...... sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM ...... standard error of the mean SFM ...... serum-free media SH ...... Src homology shRNA ...... short hairpin-RNA siRNA ...... small interfering-RNA SNHG12 ...... small nucleolar RNA host gene 12 SNP ...... single nucleotide polymorphism SOS ...... son of sevenless SPRK ...... SH3-domain-containing proline-rich kinase SRF ...... serum responsive factor

TAK1 ...... TGF-beta activated kinase 1 TEMED ...... tetramethylethylenediamine TF ...... transcription factor TGFb ...... transforming growth factor beta TLR ...... toll-like receptor TNBC ...... triple-negative breast cancer TNFR ...... TNF receptor TNFa ...... tumor necrosis factor alpha Tpl2 ...... tumor progression locus 2 TRAF ...... TNFR-associated factor TRAIL ...... TNF-related apoptosis-inducing ligand TRB3 ...... TRIBBLES homolog 3 Trx ...... thioredoxin u-PA ...... urokinase plasminogen activator Ub ...... ubiquitin UTR...... untranslated region

xiv

WT ...... wild-type

ZAK ...... zipper-sterile-a-motif kinase

List of Symbols

*...... asterisk, statistical significance, p-value < 0.05 ° ...... degrees

α ...... alpha β ...... beta g ...... gamma d ...... delta k ...... kappa µ ...... micro

Arg ...... argenine Asp ...... aspartate C ...... Celsius or carboxyl Cys ...... cysteine g...... gram Gln...... glutamine Glu...... glutamate h...... hour l ...... liter L ...... liter Leu ...... leucine Lys...... lysine m ...... milli M ...... molar min ...... minutes n...... nano N ...... amino P ...... proline pH ...... potential of hydrogen Phe...... phenylalanine Pro ...... proline S ...... serine sec ...... seconds Ser ...... serine T ...... threonine Thr ...... threonine

xvi

Trp ...... tryptophan Tyr ...... tyrosine V ...... volts X ...... any amino acid Y ...... tyrosine

xvii

Chapter 1

Introduction

1.1 Mitogen-Activated Protein Kinases (MAPKs)

Mitogen-activated protein kinase (MAPK) signaling cascades translate a diverse range of extracellular stimuli into intracellular signals regulating biological processes such as proliferation, differentiation, metabolism, inflammation, survival, apoptosis, migration, and invasion (Figure 1-1) (Cargnello et al., 2011; Cuevas et al., 2007;

Morrison, 2012; Qi et al., 2005; Raman et al., 2007; Ramos, 2008; Wagner et al., 2009).

The basic component of conventional MAPK pathways is a three-tiered kinase signaling module, conserved in eukaryotes from yeast to humans, in which a serine/threonine

MAPK kinase kinase (MAP3K), generally activated by interactions with small GTPases and/or phosphorylation by upstream protein kinases, phosphorylates and activates a dual- specificity MAPK kinase (MAP2K) which in turn phosphorylates and activates a proline- directed serine/threonine MAPK. The activated MAPK either phosphorylates and activates cytosolic substrates or translocates into the nucleus and phosphorylates and activates transcription factors leading to transcription of specific genes and the appropriate cellular response. In mammals, there are four conventional MAPK pathways: the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinases 1

(JNKs) 1-3 (also called stress-activated protein kinases (SAPKs) a, b and g), p38 kinases

(α, β, γ, and δ isoforms), and ERK5. Atypical MAPKs ERK3, ERK4, ERK7, and Nemo- like kinase (NLK) also exist (Cargnello & Roux, 2011). This dissertation focuses on the

ERK1/2 MAPKs as well as the MAP3K mixed lineage kinase 3 (MLK3).

Figure 1-1: MAPK signaling modules. The ERK1/2, JNK, p38, and ERK5 MAPK pathways consist of a three-tiered phosphorelay of protein kinases, which transmit extracellular stimuli into the corresponding biological response.

1.2 Extracellular Signal-Regulated Kinases 1 and 2 (ERK1/2)

1.2.1 ERK1/2 Signaling

The ERK1/2 pathway includes several proto-oncogenes and is deregulated in approximately 30% of all human cancers (Fang et al., 2005). In colorectal cancer,

ERK1/2 signaling is important for proliferation, survival, invasion, metastasis, and angiogenesis (Fang & Richardson, 2005; Slattery et al., 2012; Urosevic et al., 2014). The classic ERK1/2 module consists of the MAP3Ks v-Raf murine sarcoma 3611 viral oncogene homolog 1 (A-Raf), v-Raf murine sarcoma viral oncogene homolog B (B-Raf), and v-Raf-1 murine leukemia viral oncogene homolog 1 (Raf-1), the MAP2Ks

MAPK/ERK kinases 1 and 2 (MEK1/2), and the MAPKs ERK1 and ERK2 (Figure 1-1)

(Cargnello & Roux, 2011). The ERK1/2 signaling pathway is mainly activated by ligands for transmembrane receptor tyrosine kinases (RTKs) such as insulin and growth factors

(GFs) including epidermal growth factor (EGF) and nerve growth factor (NGF) (Figure

1-2) (Cargnello & Roux, 2011; Raman et al., 2007; Ramos, 2008). The structural organization of RTKs consists of an extracellular ligand binding domain, a single transmembrane domain, and a cytoplasmic conserved protein tyrosine kinase domain

(Schlessinger, 2002). Binding of cognate ligands to the extracellular ligand binding domain of RTKs induces receptor dimerization, which promotes receptor activation through intermolecular autophosphorylation of cytoplasmic tail tyrosine residues

(Cargnello & Roux, 2011; Ramos, 2008). Some of the phospho-tyrosine residues reside in the activation loop of the catalytic domain stimulating protein tyrosine kinase activity, while other phospho-tyrosine residues serve as binding sites for proteins containing Src homology 2 (SH2) or phospho-tyrosine-binding (PTB) domains (Schlessinger, 2002).

Figure 1-2: The ERK1/2 signaling pathway. Ligand-induced receptor dimerization results in activation of RTKs. Grb2 binds to phospho-tyrosine residues of RTKs and recruits the SOS GEF, which activates Ras. Ras-GTP interacts with Raf proteins displacing bound 14-3-3 dimers. Along with PAK1, either Raf-1 or B-Raf, fully activated by PP2A and kinases, phosphorylates and activates MEK1/2, which phosphorylate and activate ERK1/2. ERK1/2 then phosphorylate and activate cytosolic substrates or translocate into the nucleus to promote gene transcription by regulating TFs. Inactive

ERK1/2 is retained in the cytoplasm by either inactive MEK1/2, PEA-15, Akt, microtubules, or MKPs.

The adaptor protein growth factor receptor-bound protein 2 (Grb2) recruits the guanine nucleotide exchange factor (GEF) son of sevenless (SOS) from the cytosol to the plasma membrane through protein-protein interaction. SOS associates with the membrane localized small GTPase Ras and promotes its activation by stimulating guanosine triphosphate (GTP) loading. Ras-GTP interacts with the Raf MAP3Ks to promote translocation of Rafs to the plasma membrane and displacement of bound 14-3-3, which maintains Rafs in an inactive state (Ramos, 2008). 14-3-3 dimers maintain a closed, catalytically inactive conformation of Raf-1 through binding the phosphorylated N- and

C-terminal residues Ser259 and Ser621 of the MAP3K. 14-3-3 displacement exposes phospho-Ser259, which is then dephosphorylated by protein phosphatase 2A (PP2A)

(Jaumot et al., 2001; Ramos, 2008). Dephosphorylation of Ser259 results in the complete release of 14-3-3, an open conformation, and phosphorylation of Raf-1 at multiple sites including Ser338 and Tyr341 by kinases such as protein kinase C (PKC) and Src to further

activate the MAP3K (Mercer et al., 2003; Ramos, 2008). 14-3-3 binds to B-Raf at phospho-Ser364 and phospho-Ser728; however, cytoplasmic B-Raf exists in an open conformation in contrast to cytoplasmic Raf-1 (Mercer & Pritchard, 2003). After translocation of B-Raf to the plasma membrane, PP2A dephosphorylates Ser364, and activating kinases phosphorylate and fully activate B-Raf at Ser601 and Thr598. Once fully activated, Rafs, along with p21 (Rac1) activated kinase (PAK1), phosphorylate and activate MEK1/2 (Frost et al., 1997; Frost et al., 1996; Ramos, 2008). MEK1/2 then phosphorylates ERK1/2 at both the threonine and the tyrosine residues of the conserved

TEY motif in the phosphorylation loop (Ramos, 2008). Active ERK1/2 is then released from MEK1/2 and either acts on cytoplasmic or nuclear targets such as transcription factors (TFs).

1.2.2 ERK1/2 Substrate Specificity

Approximately 160 substrates for ERK1/2 have been identified (Yoon et al.,

2006). In addition to the ERK1/2 consensus phosphorylation motif of PXS/TP (Gonzalez et al., 1991), docking domains of ERK1/2 substrates, such as the D-domain and the DEF domain, also provide target specificity (Biondi et al., 2003; Cargnello & Roux, 2011).

The D-domain, also known as the DEJL motif, is characterized by the sequence Arg/Lys-

X-X-Arg/Lys-X1-6-Leu-X-Leu (Biondi & Nebreda, 2003; Cargnello & Roux, 2011;

Kallunki et al., 1994; Ramos, 2008). MEK1/2 and the TFs c-Jun and ETS contain D- domains. The DEF-domain, also known as the FxFP motif, consists of a Ser/Thr-Pro phosphorylation site next to the sequence Phe-X-Phe-Pro (FxFP). The transcription factor c-Fos and MAPK phosphatases (MKPs) contain DEF-domains, and the transcription

factor Elk-1 contains both a D-domain and a DEF-domain (Fantz et al., 2001; Yang et al.,

1998). Different regions of ERK1/2 bind independently to the docking domains (Dimitri et al., 2005). The CD domain residues 316 and 319 of ERK1/2 bind to the D-domain, while a region of ERK1/2 including residues 185-261 binds the DEF-domain (T. Lee et al., 2004; Tanoue et al., 2000).

Scaffold proteins which interact with two or more components of the ERK1/2 signaling module also provide substrate specificity (Ramos, 2008). Kinase suppressor of

Ras (KSR) is the most well-characterized scaffold of the ERK1/2 pathway and binds to

Raf (Roy et al., 2002), MEK1/2 (Denouel-Galy et al., 1998; Yu et al., 1998), ERK1/2

(Yu et al., 1998), and 14-3-3 (Xing et al., 1997). Additional scaffold proteins for the

ERK1/2 pathway include MEK partner-1 (MP-1), Morg-1, IQGAP, b-arrestin 1 and 2,

Sur-8, MEK kinase 1 (MEKK1), major vault protein, paxillin, PEA-15, and MLK3

(Chadee et al., 2006; Ramos, 2008). The function of MLK3 as a scaffold protein for the

ERK1/2 signaling module will be discussed in a later section.

1.2.3 Nuclear ERK1/2

Active ERK1/2 translocate into the nucleus (Cargnello & Roux, 2011; R. H. Chen et al., 1992; Lenormand et al., 1993) and phosphorylate and activate TFs such as activating protein-1 (AP-1) and ETS (Figure 1-2) (Hollenhorst, 2012). AP-1 is composed of homo- or heterodimers of Jun (v-Jun, c-Jun, JunB, JunD), Fos (v-Fos, c-Fos, FosB,

Fral, Fra2), or activating transcription factor (ATF) (ATF2, ATF3/LRF1, B-ATF) (Karin et al., 1997). The ERK1/2 pathway is a master regulator of the G1- to S-phase transition of the cell cycle (Meloche et al., 2007). Many genes activated by AP-1 and/or ETS TFs

encode cell cycle regulators involved in proliferation such as cyclin D1, cyclin A, cyclin

E, p53, p21Cip1, p16Ink4a, and p19ARF (Hess et al., 2004) or extracellular proteases involved in cellular migration, invasion, and metastasis such as matrix metalloproteinases

(MMPs) and urokinase plasminogen activator (u-PA) (Hollenhorst, 2012). ERK1/2 phosphorylate and activate the TF Elk-1, which is involved in expression of immediate- early genes, such as the c-Fos gene (Gille et al., 1995). ERK1/2 stabilizes c-Fos protein by direct phosphorylation of Ser374 and Ser362 (Murphy et al., 2002; Plotnikov et al.,

2011) thereby allowing c-Fos to associate with c-Jun and form transcriptionally active

AP-1 complexes (Whitmarsh et al., 1996). Transcription of Fra1 is c-Jun- and AP-1- dependent (Adiseshaiah et al., 2005). ERK1/2 phosphorylates Fra1 protein, which enhances the stability of the Fra1/c-Jun heterodimer (Basbous et al., 2007; Belguise et al.,

2012). The transcriptional activity of ETS is also regulated by JNK and p38 signaling

(Hollenhorst, 2012). Ras—ERK1/2 signaling can also upregulate Jun expression (Hess et al., 2004), and JNK phosphorylates c-Jun protein on Ser63 and Ser73 which increases c-

Jun transactivation and AP-1 transcriptional activity (Derijard et al., 1994; Hibi et al.,

1993).

While ERK5 contains two nuclear localization signals (NLS), ERK1/2 do not contain an NLS (Plotnikov et al., 2011); thus, nuclear import of ERK1/2 is NLS- independent and may involve direct interaction with nucleoporins (NUPs) of nuclear pore complexes (NPC), which facilitate nuclear-cytoplasmic molecular exchange

(Matsubayashi et al., 2001; Whitehurst et al., 2002) and phosphorylation of a novel nuclear translocation signal (NTS), Ser244-Pro245-Ser246, in the kinase insert domain (KID) of ERK1/2 (Chuderland et al., 2008; Zehorai et al., 2010). The first step of nuclear import

of macromolecules through NPCs is interaction with shuttling factors such as importins.

Importin 7 directly interacts with ERK1/2, and this interaction is enhanced when the

ERK1/2 NTS is phosphorylated. Nuclear export of ERK1/2 may involve MEK1/2. The

N-terminus of MEK1/2 contains a leucine-rich chromosomal maintenance 1 (CRM1)- dependent nuclear export signal (NES) (Fukuda et al., 1996). Nuclear inactive MEK1/2 may bind nuclear inactive ERK1/2 and the exportin CRM1 resulting in rapid export from the nucleus (Adachi et al., 2000). Inactive ERK1/2 then remains localized in the cytoplasm due to interactions with anchoring proteins such as microtubules (Reszka et al., 1995), MEK1 (Fukuda et al., 1996; Rubinfeld et al., 1999), phosphatases (Zuniga et al., 1999) and PEA-15 mediated by Akt (Gervais et al., 2006).

1.3 Mixed Lineage Kinase 3

1.3.1 MLK3 Characterization

The 847 amino acid sequence of MLK3, also called SH3-domain-containing proline-rich kinase (SPRK) or protein-tyrosine-kinase 1 (PTK1), was first elucidated from nucleotide sequences of thymus (Ing et al., 1994), leukemia (Gallo et al., 1994), and melanocyte (Ezoe et al., 1994) cDNA clones. MLK3 belongs to the MLK family of protein kinases, which consists of three subfamilies: the MLKs (1, 2/MST,

3/SPRK/PTK1, 4α, and 4b) the dual-leucine-zipper bearing kinases (DLKs:

DLK/MUK/ZPK and LZK), and the zipper-sterile-α-motif kinase (ZAK: ZAKα and

ZAKb) (Figure 1-3) (Gallo et al., 2002; Z. Xu et al., 2001). MLK subfamily members each contain an N-terminus SH3 domain, a kinase catalytic domain, a leucine zipper motif, a cell division cycle protein 42 (Cdc42)/Ras-related C3 botulinum toxin substrate 1

Figure 1-3: The MLK family. The MLK family consists of three subfamilies: the

MLKs, the DLKs, and ZAK. The MLKs 1-4 each contain an SH3, kinase, LZ, and CRIB domain/motif.

(Rac1)- interactive binding (CRIB) motif, and C-terminus rich in proline, serine, and threonine residues (Gallo & Johnson, 2002; Gallo et al., 1994; Rattanasinchai et al.,

2016). The kinase domain of protein kinases is approximately 250-300 amino acid residues with 12 conserved subdomains: I, II, III, IV, V, VIa, VIb, VII, VIII, IX, X, and

XI. (Hanks et al., 1995). MLK subdomains share sequence identity with both serine/threonine and tyrosine kinases (Dorow et al., 1993; Gallo & Johnson, 2002); hence, the origin of the name “mixed lineage kinase.” Specifically, subdomain VIb contains a lysine residue diagnostic of serine/threonine kinases; while subdomains IX and

XI contain two tryptophan residues and a motif (Cys-Trp-X-X-Asp/Glu-Pro-X-X-Arg-

Pro-X-Phe), respectively, both of which are conserved in tyrosine kinases. During initial characterization, epitope-tagged MLK3 autophosphorylated on serine and threonine residues in an in vitro kinase assay, which designated MLK3 a serine/threonine protein kinase (Gallo et al., 1994). MLK3 was first identified to function in the proliferation of normal human melanocytes as MLK3 antisense oligonucleotides inhibited cell growth

(Ezoe et al., 1994) and was the first SH3 domain-containing kinase to demonstrate serine/threonine specificity (Gallo et al., 1994). Furthermore, using fluorescence in situ hybridization, the gene encoding MLK3, MAP3K11, was mapped to human chromosome

11q13.1-13.3, a region amplified in human malignancies of the breast, lung, esophagus, bladder, head and neck and in melanomas (Ing et al., 1994). MLK3 is now a well characterized MAP3K (Gallo et al., 1994) activated by a diverse range of stimuli such as

GFs and cytokines (Chadee et al., 2004), T-cell costimulation (Hehner et al., 2000), ceramide (Sathyanarayana et al., 2002), sorbitol/osmotic stress and camptothecin (Z. Xu et al., 2005), oxidative stress (Hong et al., 2007), and NGF deprivation in neuronal cells

(Mota et al., 2001). MLK3 regulates biological processes such as proliferation, osteoblast differentiation (Zou et al., 2011), ulcer healing (Kovalenko et al., 2012), neutrophil

motility (Polesskaya et al., 2014), macrophage-driven inflammation (Ibrahim et al., 2016;

Tomita et al., 2017), Golgi structural reorganization (Cha et al., 2004), G2/M microtubule instability (Swenson et al., 2003), G2/M cell cycle progression, cyclin D1 stability, and centrosome amplification through phosphorylation of prolyl-isomerase (Pin1) on Ser138

(Rangasamy et al., 2012), neuronal cell apoptosis (Z. Xu et al., 2001), and migration and invasion of human cancer cells (Chadee, 2013).

1.3.2 MLK3 Activation

The SH3 domain of MLK3 binds to a region between the leucine zipper and the

CRIB motif to promote autoinhibition or a closed conformation of MLK3 (Figure 1-4)

(H. Zhang et al., 2001). Binding of the GTP-bound Rho-family GTPases Rac1 and Cdc42 to the CRIB motif of MLK3 (Bock et al., 2000; Teramoto et al., 1996) disrupts SH3- mediated autoinhibition (J. Chen et al., 2010) and triggers leucine zipper-mediated homodimerization (Leung et al., 1998), subsequent trans/autophosphorylation of Thr277 and Ser281 within the kinase domain activation loop (Du et al., 2005; Leung et al., 2001), and translocation of MLK3 from the Golgi area (Cha et al., 2004; Swenson et al., 2003) to the plasma membrane and other membrane compartments (Du et al., 2005).

Hematopoietic progenitor kinase 1 (HPKl), a serine/threonine kinase is an upstream activator of MLK3. HPK1 phosphorylates and activates MLK3 on Ser281 in the activation loop leading to mitogen-activated protein kinase kinase 4 (MKK4) and JNK activation

(Kiefer et al., 1996; Leung & Lassam, 2001).

MLK3 activates the JNK and p38 MAPK pathways by phosphorylation and

Figure 1-4: MLK3 autoinhibition and activation. Rac1/Cdc42 binding to MLK3 disrupts MLK3 autoinhibition and promotes MLK3 dimerization and autophosphorylation. Depending on the cellular context, active MLK3 can phosphorylate

and activate MEK1/2, MKK 4/7, or MKK3/6, which result in ERK1/2, JNK1-3, and p38a-d signaling, respectively. HPK1 is also an upstream activator of MLK3.

activation of the MAP2Ks MKK4/7 and MKK3/6, respectively (Rana et al., 1996;

Teramoto et al., 1996; Tibbles et al., 1996; Whitmarsh et al., 1998). Specifically, MLKs phosphorylate MKK7 on Ser206 and Thr210 and MKK4 on Ser254 and Thr258 (Kyriakis et al., 2001; Vacratsis et al., 2000). Active MKK4/7 then phosphorylate and activate JNKs on Thr183 and Tyr185 located in a TPY motif (Lawler et al., 1998). GTPase-induced leucine zipper-mediated homodimerization of MLK3 is required for activation of JNK as a monomeric MLK3 leucine zipper variant failed to phosphorylate Thr258 of MKK4

(Vacratsis & Gallo, 2000) and expression of a MLK3 leucine zipper polypeptide blocked epitope-tagged JNK activation by wild-type MLK3 in HEK293 cells (Leung & Lassam,

1998). However, GTPase-induced leucine zipper-mediated homodimerization of MLK3 is not required for MLK3 activation as constitutively active Cdc42 activates monomeric

MLK3 in terms of autophosphorylation, kinase activity towards histones, and in vivo phosphorylation as compared to wild-type MLK3.

In vivo labeling with 32P in HEK293 cells co-expressing MLK3 and active Cdc42

(Cdc42V12) followed by mass spectrometry identified 11 MLK3 in vivo phosphorylation sites: Ser524, Ser555, Ser556, Ser654, Ser705, Ser724, Ser727, Ser740, Ser758, Ser770, and Ser793

(Vacratsis et al., 2002). A proline residue immediately follows 7 of the 11 in vivo phosphorylation sites, suggesting MLK3 is a potential substrate for proline-directed kinases such as the downstream MAPKs. Indeed, JNK phosphorylates MLK3 in vitro and

in vivo (Fig. 1-5) (Schachter et al., 2006). JNK-phosphorylation of MLK3 is essential for sustained JNK signaling.

1.3.3 MLK3 and Apoptosis

MLK3 and JNK function in both the extrinsic and intrinsic apoptotic signaling pathways, initiated by death receptor ligands such as tumor necrosis factor alpha (TNFa),

TNF-related apoptosis-inducing ligand (TRAIL), and Fas ligand (FasL) and mitochondrial events resulting in the release of pro-apoptotic factors such as cytochrome c, respectively (Figure 1-5) (Dhanasekaran et al., 2008; Fulda et al., 2006). Both apoptotic pathways result in the activation of the proteolytic caspase enzymes.

MLK3 is required for optimal activation of JNK signaling by the inflammatory cytokine TNFa (Brancho et al., 2005; Chadee & Kyriakis, 2004; Sathyanarayana et al.,

2002). In response to inflammatory signals, TNFa binds to TNF receptor 1 (TNFR1)

(Sedger et al., 2014). The adaptor proteins TNFR-associated factors (TRAFs) are then recruited to the TNFR cytoplasmic tails (Bradley et al., 2001). TRAFs also interact with the cytoplasmic tails of the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR). In response to TNFa and the inflammatory cytokine IL-1b, MLK3 associates with TRAF2 and TRAF6, respectively, in SKOV3 ovarian cancer cells (Korchnak et al., 2009), and

TNFa also induces an association between MLK3 and TRAF2 in Jurkat T lymphocyte cells (Sondarva et al., 2010). RNAi studies (Korchnak et al., 2009) or TRAF2 wild-type or deficient mouse embryonic fibroblasts (Sondarva et al., 2010) revealed a requirement for TRAF2 and TRAF6 in both MLK3 and JNK activation by TNFa. TNFa also stimulated ubiquitination (Ub) of MLK3 in both HEK293 and SKOV3 cells

Figure 1-5: Active MLK3 signaling in apoptosis. MLK3 is activated in response to apoptotic stimuli such as TNFa, IL-1b, NGF deprivation, b-amyloid, and HBx. MYD88- and TRAF6-dependent K63-linked Ub of MLK3 displaces bound JIP1 resulting in dimerization and autophosphorylation of MLK3, subsequent JNK signaling, MOM permeabilization, cytochrome c release, caspase activation, and apoptosis. MLK3 stabilizes TRB3, which inhibits Akt-mediated degradation of MLK3 protein and inhibition of Bax. GSK3b and POSH positively regulate MLK3 signaling in apoptosis, which can upregulate FasL expression.

(Korchnak et al., 2009). IL-1b induced activation of MLK3 requires both myeloid differentiation primary response 88 (MYD88) and TRAF6-dependent Lys 63-linked Ub of MLK3 on Lys264 in pancreatic beta cells (Humphrey et al., 2013). Cytokine-induced

Lys 63-linked Ub of MLK3 causes dissociation of inactive MLK3 from the JNK- interacting protein 1 (JIP1) scaffold protein resulting in MLK3 dimerization and autophosphorylation, JNK activation, and conformational changes of the pro-apoptotic protein B-cell CLL/lymphoma 2 (Bcl-2)-associated X (Bax). Bax activation results in permeabilization of the mitochondrial outer membrane (MOM) and apoptosis (Chipuk et al., 2004). The JIP1 scaffold protein interacts with MLKs, MKK7,and JNK and mediates activation of MLKs and JNK (Whitmarsh et al., 1998); however, in the cellular context above JIP1 sequesters monomeric inactive MLK3 in unstimulated cells (Humphrey et al.,

2013). Cytokine-activated MLK3 compromises mitochondrial integrity and induces apoptosis of pancreatic beta cells via direct interaction with and stabilization of the pseudokinase TRIBBLES homolog 3 (TRB3), which inhibits the pro-survival kinase Akt

(Humphrey et al., 2010). Akt is a known inhibitor of Bax translocation and mitochondrial membrane permeabilization (Simonyan et al., 2016; Tsuruta et al., 2002; Yamaguchi et al., 2001). In response to insulin, Akt phosphorylates MLK3 on Thr477 triggering Lys 48- linked Ub of MLK3 on Lys274 resulting in proteasomal degradation of MLK3 protein, decreased JNK activation, and survival of pancreatic beta cells (Humphrey et al., 2014).

Therefore, TRB3 in turn positively regulates MLK3 via inhibition of Akt-mediated phosphorylation, Ub, and degradation of MLK3 protein, and the relative activities of

MLK3, TRB3, and Akt and whether or not Lys264 is conjugated with K48- or K63-linked

Ub are pivotal in determining pancreatic beta cell fate.

The MLK family proteins MLKs 1-3 and DLK, activated by Cdc42 and Rac1, mediate neuronal cell apoptosis in response to deprivation of nerve growth factor (NGF) via a downstream pathway which includes activation of JNK, MKK4/7, and c-Jun followed by cytochrome c release and activation of caspases (Mota et al., 2001; Z. Xu et al., 2001). Expression of MLK family members induced apoptosis of neuronal PC12 cells and rat superior cervical ganglion sympathetic neurons, which was partially blocked by co-expression of myristylated Akt (myr-Akt) (Z. Xu et al., 2001). Glycogen synthase kinase 3b (GSK3b) also regulates MLK3 activation and neuronal cell apoptosis during

NGF withdrawal (R. Mishra et al., 2007). NGF deprivation activates GSK3b, which then directly phosphorylates MLK3 on Ser789 and Ser793 resulting in MLK3 activation, subsequent JNK activation, and caspase 3 cleavage in neuronal PC-12 cells. b-amyloid, implicated in Alzheimer’s disease, induces apoptosis of neuronal PC12 cells, sympathetic neurons, and cortical neurons likely through a MKK4—JNK—c-Jun—cytochrome c release—caspase 2 and 3 signaling pathway (Bozyczko-Coyne et al., 2001; Troy et al.,

2001). The proteolytic enzyme Calpain may also function downstream of JNK in b- amyloid-induced apoptosis of cortical neurons (Bozyczko-Coyne et al., 2001).

MLK3 (He et al., 2016; Tang et al., 2012), JNK (Le-Niculescu et al., 1999), c-Jun

(Faris et al., 1998), and AP-1 (Kasibhatla et al., 1998) induce expression of FasL, which initiates the extrinsic apoptotic pathway, in response to either hepatitis B virus X protein

(HBx), trophic factor withdrawal, stress, or DNA damaging reagents. MLK3 upregulates

FasL expression in response to HBx through activation of MKK7 and JNK in human hepatocellular carcinoma (HepG2) cells (Tang et al., 2012) and in NRK-52E rat kidney epithelial cells (He et al., 2016). AP-1 also induces expression of TNFa (Dhanasekaran

& Reddy, 2008) providing positive feedback on cytokine-induced MLK3-dependent apoptosis.

Plenty of SH3s (POSH) is a scaffold protein for a multiprotein complex consisting of Rac1, MLKs, MKK4/7, and JNK1/2 activating the JNK pathway in neuronal cell death

(Z. Xu et al., 2003). POSH expression induces apoptosis of neuronal PC12 cells and sympathetic neurons in a manner dependent on the MLKs, MKK4/7, c-Jun, and caspase activation. Neuronal death induced by POSH expression was also suppressed by myr-

Akt. In response to apoptotic stimuli, POSH and MLK3 undergo mutual stabilization, which is dependent on MLKs, MKK, and JNK, providing positive feedback and sustained cell death (C. Wang et al., 2010; Z. Xu et al., 2005).

MLK3 also induces apoptosis by the negative regulation of the nuclear factor kappa B (NF-kB) signaling pathway (Cole et al., 2009). The TF NF-kB is retained in the cytoplasm and maintained in an inactive state by inhibitor of kappa B (IkB) binding

(Figure 1-6) (Hayden et al., 2004). In response to stimuli such as TNFa, IkB kinase

Figure 1-6: MLK3 regulation of NFkB signaling. In stimulated cells, IKKK activates

IKK, which phosphorylates IkB targeting the protein for degradation. NF-kB is then freed from IkB and translocates to the nucleus and activates transcription of anti- apoptotic and mitogenic genes. IKK activity is negatively regulated by MLK3 in a kinase-independent manner.

(IKK) is activated. IKK then phosphorylates IkB targeting the protein for Ub and degradation. NF-kB then translocates to the nucleus and activates transcription of anti- apoptotic and mitogenic genes. MLK3 limits IKK activity in a kinase-independent mechanism possibly through inhibition of an upstream IKK kinase (IKKK) or direct interaction with the catalytic subunits of IKK, thereby negatively regulating NF-kB signaling (Cole et al., 2009). In this study, both siRNA or shRNA knockdown of MLK3 protected HEK293 cells from etoposide-induced apoptosis. Interestingly, expression of wild-type or kinase-dead MLK3 promoted etoposide-induced apoptosis, suggesting

MLK3 can also induce apoptosis through a mechanism independent of its catalytic activity.

1.3.4 MLK3 and ERK1/2 Signaling

MLK3 also activates the ERK1/2 MAPK pathway through both kinase-dependent

(Kovalenko et al., 2012; Marusiak et al., 2014) and -independent mechanisms (Figures 1-

4 and 1-7) (Chadee & Kyriakis, 2004; Chadee et al., 2006). During intestinal epithelial cell sheet migration to heal induced wounds or ulcers, MLK3 catalytic activity is required for both activation of JNK and ERK1/2 in vitro in colon-carcinoma derived Caco-2 cells and in an in vivo mouse model (Kovalenko et al., 2012). MLK3, along with the other members of the MLK subfamily, promotes survival of V600E-positive melanoma cells in the presence of Raf inhibitors by reactivating the MEK1/2— ERK1/2 pathway, presumably by direct phosphorylation and activation of MEK1/2 (Marusiak et al., 2014).

MLKs expressed in HEK293T cells activated MEK1/2 and ERK1/2 in a kinase- dependent manner and phosphorylated kinase-inactive MEK1 in an in vitro kinase assay.

Figure 1-7: MLK3-dependent regulation of ERK1/2. Depending on the cellular context MLK3 can activate ERK1/2 signaling, by either direct phosphorylation and activation of MEK1/2, mediating Raf-1 and B-Raf transactivation by a catalytically- independent scaffold function, or by an unknown mechanism. MLK3-induced ERK1/2 activation may lead to cell transformation or proliferation, migration, or invasion of certain cancer cells. Active MLK3 can also suppress ERK1/2 signaling.

In the latter, HEK293T cells were treated with Raf inhibitors PLX4032 (vemurafenib) or

L779450. MLKs expressed in A375 and A2058 V600E-positive melanoma cell lines treated with vemurafenib also activated MEK1/2 and ERK1/2 and promoted survival.

In colon cancer cells, lung fibroblasts, and human schwannoma cells bearing a loss-of-function mutation in the neurofibromatosis 2 (NF2) gene, MLK3 activates the

ERK1/2 pathway in a kinase-independent manner by serving as a scaffold for B-Raf and

Raf-1 transactivation in response to EGF treatment (Chadee & Kyriakis, 2004; Chadee et al., 2006). MLK3 RNAi silencing decreased mitogen-induced activation of B-Raf,

MEK1/2, and ERK1/2 and suppressed the proliferation of HCT15 colon adenocarcinoma cells, ST88-14 malignant schwannoma cells bearing loss of function NF1 mutations, and

HEI 193 human NF2 schwannoma cells (Chadee & Kyriakis, 2004). MLK3 failed to phosphorylate GST-B-Raf variants in an in vitro kinase assay, but did phosphorylate and activate MEK1. However, an interaction between MLK3 and B-Raf was observed in

EGF-treated CCD-18Co cells, and MLK3 RNAi silencing in EGF-treated HEK293 cells decreased the activity of B-Raf towards MEK1 in an in vitro kinase assay; importantly, this activity was restored by expression of either wild-type or kinase dead (K144R)

FLAG-MLK3 (Chadee et al., 2006). Furthermore, MLK3 interacted with both B-Raf and

Raf-1 in cells, and an MLK3-dependent association between B-Raf and Raf-1 was also observed in cells.

Another study reported MEK1 phosphorylation and activation by MLK3. MLK3 directly phosphorylated and activated MEK1 in vitro and also induced MEK1/2 phosphorylation on its activation sites in COS-7 cells (Shen et al., 2003). However,

MLK3-dependent MEK1/2 phosphorylation and activation does not activate ERK1/2 in

EGF-stimulated COS-7 cells. Rather, expression of active MLK3 attenuates mitogen- induced ERK1/2 activation in COS-7 cells in a manner requiring the kinase activities of

MLK3, MKK4, and JNK and c-Jun-mediated transcription, but is independent of Raf activation, suggesting the negative regulation of ERK1/2 by active MLK3 and sustained

JNK activation occurs downstream from Raf by uncoupling ERK1/2 activation from

MEK1/2 in a manner requiring c-Jun-mediated gene transcription.

Active MLK3 transforms NIH 3T3 cells in a MEK1/2-dependent mechanism

(Hartkamp et al., 1999). Expression of wild-type FLAG-MLK3, but not kinase dead

K144A FLAG-MLK3, in NIH 3T3 cells resulted in foci formation, morphological changes similar to v-Raf-transformed NIH 3T3 cells (refractile and spindle-shaped), elevated transcription from an AP-1-/ETS-1-driven promoter, activated JNK and ERK1, and induced phosphorylation of His-MEK1 in an in vitro kinase assay. Furthermore, activation of ERK1/2 by MLK3 was independent of Raf. In ovarian cancer, active MLK3 promotes invasion by increasing both the expression and activity of MMPs through both

JNK and ERK1/2 activation of AP-1-mediated transcription (Zhan et al., 2012). ERK1/2 promotes the formation of the transcriptionally active AP-1 complex by direct phosphorylation and stabilization of c-Fos, allowing c-Fos to associate with c-Jun

(Cargnello & Roux, 2011).

Active MLK3 drives invasion and transendothelial migration in triple-negative breast cancer (TNBC) cells through JNK/ERK1/2—Fra1—MMP-1/9 signaling

(Rattanasinchai et al., 2017). Induced expression of MLK3 in estrogen receptor-positive breast cancer cells, but not MLK3 knockout in 4T1 cells, increased both Fra1 transcript and protein abundance, MMP-1 and -9 mRNA, and invasion. The increase in MMP-1

protein and cell invasion observed with induced expression of MLK3 was abrogated by

Fra1 gene silencing, indicating Fra1 functions downstream of MLK3 in these events.

Wild-type MLK3, but not kinase-dead MLK3, increased Fra1 protein abundance and both JNK and ERK1/2 activation in MCF7 cells. JNK and ERK1/2 inhibitors, SP600125 and U0126, decreased endogenous Fra1 transcript and protein abundance in TNBC cells.

MLK inhibitor treatment decreased both MMP-1 mRNA abundance in SUM-159 TNBC cells and secretion of MMP-9 and invasion of parental 4T1 cells. Fra1 and MMP-1 mRNA expression was increased in circulating tumor cells isolated from the blood of mice bearing 4T1 mammary tumors and associated metastases, and this increase was dependent on MLK activity. These findings suggest MLK3 facilitates TNBC metastasis possibly through both vascular intravasation and extravasation.

1.3.5 MLK3 and Cancer Migration and Invasion

The involvement of MLK3 in cancer cell migration and invasion has recently been reviewed (Chadee, 2013; Rattanasinchai & Gallo, 2016). In addition to ovarian and

TNBCs as described above, MLK3 is implicated in or is required for the migration and/or invasion of several human cancers including lung carcinoma (Swenson-Fields et al.,

2008), non-small cell lung (Chien et al., 2011), gastric (P. Mishra et al., 2010), breast (J.

Chen et al., 2012; J. Chen et al., 2010; Cronan et al., 2012), melanoma (J. Zhang et al.,

2014), glioblastoma (Misek et al., 2017), and hepatocellular carcinoma (Figure 1-8) (Lan et al., 2017).

In human lung carcinoma cells, MLK3 functions as a scaffold in limiting Rho activation, which promotes directed cell migration (Swenson-Fields et al., 2008). Upon

Figure 1-8: MLK3 signaling in cancer migration and invasion. MLK3 can promote migration and/or invasion of human cancer cells through ERK1/2, JNK, and p38 MAPK pathways as well as through negative regulation of the Rho GTPase, resulting in increased expression of invasion genes and focal adhesion turnover, respectively.

direct protein-protein interaction of MLK3 and the Rho activator p63RhoGEF, MLK3 inhibits p63RhoGEF activation by Gaq downstream of G-protein-coupled receptors

(GPCRs) resulting in cytoskeletal changes necessary for migration such as focal adhesion turnover. While two MLK3 activating events, Rac binding and MLK3 autophosphorylation, promote the interaction between MLK3 and p63RhoGEF, kinase- inactive FLAG-MLK3 interacts with GST-p63RhoGEF in vitro and inhibits both Gaq- stimulated serum responsive factor (SRF)-mediated transcription and Rho activation after stimulation of Gaq-coupled GPCR/p63RhoGEF. MLK3 was implicated in the migration and invasion of non-small cell lung cancer cells through MKK3/6—p38 signaling leading to increased expression of MMPs 2 and 9 and u-PA (Chien et al., 2011).

MLK3 is required for the migration of human gastric cancer cells following stimulation with the gastrointestinal peptide hormone gastrin through SEK1—JNK1—c-

Jun signaling to increase MMP-7 promoter activity (P. Mishra et al., 2010). MLK3 promotes the invasion of Mel IM and Mel Ju melanoma cells likely through the MKK7 and c-Jun signaling pathway (J. Zhang et al., 2014), and as mentioned above, in V600E- positive melanoma cell lines, MLKs 1-4 act as MEK1 kinases which reactivate the

MEK1/2/ERK1/2 pathway to mediate resistance to RAF inhibitors (Marusiak et al.,

2014).

In breast cancer cells, MLK3 promotes a malignant phenotype via multiple mechanisms. Active MLK3 promotes the migration and invasion of mammary epithelial and breast cancer cells likely through activation of JNK—c-Jun—AP-1 signaling, which induces the expression of the invasion genes c-Jun, Fra-1, and vimentin (J. Chen et al.,

2010). Active MLK3 is required for the chemokine CXCL12-mediated migration and invasion of highly invasive basal MDA-MB-231 breast cancer cells and drives lung metastasis (J. Chen & Gallo, 2012). In the context of CXCL12-mediated migration and invasion of breast cancer cells, MLK3 signaling leads to active JNK, which phosphorylates paxillin at Ser178. Ser178 phosphorylation of paxillin recruits focal adhesion kinase (FAK) to mediate Tyr118 phosphorylation of paxillin, which leads to decreased Rho activity, enhancing focal adhesion turnover. As mentioned previously, active MLK3 promotes invasion and migration in TNBC cells through JNK/ERK1/2—

Fra1—MMP-1/9 signaling (Rattanasinchai et al., 2017). Phosphorylated paxillin was increased in MCF7 cells expressing MLK3; however, this increase was not affected by

Fra-1-silencing, suggesting MLK3 regulation of paxillin is independent of Fra-1, and

MLK3 controls at least two distinct pathways in breast cancer cells which both lead to a malignant phenotype.

Interestingly, MLK3 also functions as an anti-apoptotic protein in breast cancer cells. The MAP3Ks B-Raf, Tpl2, MEKK1-3, TAK1, and MLK3 were selected for short hairpin-RNA (shRNA) knockdown and orthotopic xenograft analysis to determine the

MAP3Ks required for MDA-MB-231 cell tumor growth and metastasis (Cronan et al.,

2012). Of the seven MAP3Ks tested, the most dramatic phenotype was observed with

MLK3. MLK3 shRNA knockdown decreased in vitro proliferation of MDA-MB-231,

SUM159 and Hs578T cells and inhibited tumor growth and metastasis in mice. Increased apoptosis was observed with TUNEL assays performed with both MLK3 shRNA knockdown in MDA-MB-231 cells and MLK3 knockdown xenografts, suggesting MLK3 is anti-apoptotic in this cellular context.

A role for MLK3 in glioblastoma migration and invasion was also identified

(Misek et al., 2017). In glioblastoma cells, both wild-type EGFR activated by EGF and an oncogenic, constitutively active EGFR variant (EGFRvIII) signal to dedicator of cytokinesis 1 (DOCK1), also known as DOCK180, which is a Rac1-specific GEF which promotes human glioma cell invasion (Jarzynka et al., 2007). DOCK180 and its cofactor engulfment and cell motility 1 (ELMO1) activate Rac1. Rac1-GTP activates MLK3, which promotes migration and invasion through activation of JNK.

1.3.6 MLK3 and Colorectal Cancer

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the third leading cause of cancer-related death in both men and women in the United States

(Siegel et al., 2015). In population-based control studies of colon and rectal cancers, genetic variation in MAPK genes was evaluated (Slattery et al., 2012). Single nucleotide polymorphisms (SNPs) in genes for both MLK3 and ERK2, MAP3K11 rs1784223 and

MAPK1 rs11913721 respectively, were associated with rectal cancer, and these SNPs demonstrated an approximately 40–50% increased risk for the high-risk genotype.

Cigarette smoking, a lifestyle factor which influences inflammation and oxidative stress, had the greatest impact on risk of colon cancer among those with MAP3K11 rs1784223 and MAP3K11 rs7116712. MAP3K11 and MAPK1 were associated with increasing risk

of a microsatellite instability tumor, a specific colon cancer molecular phenotype, and

MAP3K11 was associated with p53-mutated rectal tumors. The homozygote variant genotype of MAP3K11 rs7116712 reduced risk of death after diagnosis with rectal cancer; however, MAP3K11 rs11227234 and rs1151488 increased risk of death after diagnosis with colon cancer.

MLK3 missense mutations were identified in microsatellite instability sporadic

CRC cases and CRC cell lines (Velho et al., 2010). P252H A352R and A356V are located in the MLK3 kinase domain, but these residues are not highly conserved functional amino acids of the kinase catalytic core. Therefore, P252H, A352R, and

A356V are predicted to affect the scaffold properties of the protein rather than its kinase activity. P252H exhibited transforming and tumorigenic potential in vitro and in vivo, respectively, suggesting a scaffold function of MLK3 may be important for the malignant phenotype (Velho et al., 2010). P252H MLK3 harbors oncogenic activity likely through deregulation of several signaling pathways fundamental in the development and progression of CRC such as WNT, MAPK, NOTCH, transforming growth factor-beta

(TGF-β), and p53 (Velho et al., 2014).

System biology approaches and computational methods were used to identify signature genes and their regulatory pathways whose activation may specifically affect invasive tumor growth in two murine CRC cell lines 1638N-T1 and CMT-93

(Wlochowitz et al., 2016). MLK3 was among the top three upstream master regulators of

1638N-T1-specific TFs reaching 28 out of 135 TFs identified, while ERK2 was among the top three upstream master regulators of CMT-93-specific TFs reaching 31 out of 117

TFs identified. One of the pathways overrepresented in 1638N-T1 cells involved the

activation of ERK2 via IL-8. IL-8 increases the migration in human CRC cells through the integrin alpha-V/beta-6 and chemokine receptors CXCR1/2 involving the ERK2 and

ETS-1 signaling pathway (Sun et al., 2014).

1.3.7 MLK3 and Oxidative Stress

Human colorectal tumors have increased endogenous reactive oxygen species

(ROS) such as H2O2, a marker of oxidative stress (Perse, 2013). ROS-induced oxidative modification of signaling proteins is important for carcinogenesis and may include alteration of amino acid side chains, induction of peptide cleavage, and S-Nitrosylation which is a post-translational modification involving the covalent attachment of nitric oxide (NO) to the thiol moiety of reactive cysteine residues (Perse, 2013). Oxidative modification of MAPK signaling pathway components initiates MAPK signaling via direct activation of RTKs, direct or indirect activation of MAP3Ks, or direct inactivation of MAPK phosphatases (MKPs) which negatively regulate MAPKs (Figure 1-9) (Son et al., 2011). For example, ROS, produced by TNFa stimulation, oxidizes thioredoxin (Trx) causing dissociation from the MAP3K apoptosis signal-regulating kinase 1 (ASK1).

TRAF2 then facilitates ASK1 homo-oligomerization and activation leading to JNK and p38 signaling through MKK4 and MKK3/6, respectively (Liu et al., 2000). In addition, the ERK1/2 pathway can also be activated by ROS via direct oxidative modification and activation of the Ras GTPase at Cys118, which prevents exchange of GTP for GDP

(Lander et al., 1997; Liou & Storz, 2010).

MLK3 is both directly and indirectly activated by ROS in neuronal, kidney, mouse fibroblasts and primary hepatocytes, prostate cancer, and lung adenocarcinoma

Figure 1-9: Activation of ASK1 and MLK3 by oxidative stress. Both ASK1 and

MLK3 are activated by oxidative stress leading to JNK- and p38- mediated apoptosis. All signaling events depicted are regulated by ROS. Activation of MLK3 by ROS is observed in neuronal, kidney, mouse fibroblasts and primary hepatocytes, prostate cancer, and lung adenocarcinoma cells.

cells (Figure 1-9). MLK3 expressed in HEK293 cells is S-nitrosylated on Cys688, forming

SNO-MLK3, via a reaction with S-nitrosoglutathione (GSNO), an NO donor (Hu et al.,

2012). SNO-MLK3 undergoes dimerization followed by autophosphorylation. SNO-

MLK3 also exists in vivo during the early stages of rat brain global ischemia/reperfusion and is facilitated by neuronal nitric oxide synthase (nNOS) and results in the activation of the MKK4/7—JNK3—c-Jun pathway, Bcl-2 phosphorylation, FasL expression, and cytochrome c release, and the activation of caspase-3 (Hu et al., 2012; Yin et al., 2017).

Both MLK3 activation and the following events associated with MLK3 signaling in global cerebral ischemia are also dependent on oxidative stress. During ischemia, MLK3 translocates from the cytosol to the plasma membrane and binds to the scaffold protein post-synaptic density protein 95 (PSD95) (Pei et al., 2005) and glutamate receptor 6

(GluR6) (Savinainen et al., 2001). MLK3 subsequently activates JNK3 via MKK4/7

(Tian et al., 2003a; Tian et al., 2003b; Q. Zhang et al., 2003; Q. G. Zhang, Tian, et al.,

2006). MLK3 also interacts with the JIP1 scaffold protein during ischemia. First, oxidative stress disrupts an association between Akt1 JIP1 (Pan et al., 2006). JIP1 then interacts with both MLK3 and JNK3 promoting phosphorylation and activation of JNK3.

HPK1 is also an upstream activator of MLK3—MKK7—JNK3 signaling following

cerebral ischemia in the rat hippocampus (Li, Han, et al., 2008), and HPK1 is activated by Src-induced tyrosine phosphorylation in this cellular context (Li, Yu, et al., 2008).

Heme oxygenase-1 (HO-1) is a negative regulator of MLK3 signaling during ischemia.

HO-1 protects spinal cord neurons from oxidative stress-induced (S. Wang et al., 2017) or spinal cord injury-induced (Lin et al., 2017) apoptosis and brain ischemic injury (Shah et al., 2011; Y. J. Song et al., 2012) via suppression of Cdc42—MLK3—MKK7—JNK3 signaling. b-amyloid-induced MLK3 activation in neurons as described earlier is also dependent on oxidative stress (Y. Xu et al., 2009).

Oxidative stress regulates the assembly of MLK3 onto the JIP1 scaffold in

HEK293 and NIH 3T3 cells as well. In response to H2O2, the small GTPase RALA is activated by the exchange factor RalGDS-like factor (RLF), which is in a complex with

JIP1 and JNK (van den Berg et al., 2013). Active RALA consequently regulates assembly and activation of MLK3, MKK4, and JNK onto the JIP1 scaffold. Active JNK then phosphorylates and activates the TF and tumor suppressor protein forkhead box O

(FOXO). Also in response to H2O2, MLK3 is proposed to phosphorylate and activate

AMP-activated protein kinase (AMPK) on Thr172 (Luo et al., 2015).

In genipin-treated human prostate cancer cells, MLK3 is activated in a NADPH oxidase- and ROS-dependent manner (Hong & Kim, 2007). Active MLK3 then promotes cytochrome c release, caspase 3 activity, and cell apoptosis likely through activation of

JNK and c-Jun. The sphingolipid degradation product trans-2-hexadecenal induces cytoskeletal reorganization and apoptosis through oxidative stress-dependent MAPK signaling in HEK293T, NIH 3T3 and HeLa cells (Kumar et al., 2011). Trans-2- hexadecenal stimulated a MLK3—MKK4/7—JNK—c-Jun signaling pathway leading to

cytochrome c release, Bax activation, Bid cleavage, and increased translocation of Bim into the mitochondria.

Interestingly, two studies reported generation of oxidative stress, specifically superoxide production, downstream of MLK3 signaling. In an in vitro human model of

Parkinson's disease, methamphetamine (METH)- and iron (FeCl2)-exposed differentiated human mesencephalic neuron-derived cells presenting with cytoplasmic dopamine accumulation, oxidative stress was induced and the MKK4—JNK—c-Jun pathway was activated leading to neurodegeneration and eventually apoptosis (Lotharius et al., 2005).

Cep1347 prevented FeCl2/METH-induced oxidative stress, JNK activation, neurodegeneration, and apoptosis independent of the phosphatidylinositol-3-kinase

(PI3K)/Akt pathway. Cep1347 enhanced cellular protective mechanisms including cellular antioxidant capacity through increased expression of the oxidative stress- response modulator, activating transcription factor 4 (ATF4), and subsequent transcription of the cystine/glutamate and glycine transporters, which are responsible for the cellular uptake of the three key glutathione (GSH), a redox buffer, substrates: glutamate, cysteine, and glycine, which was correlated to elevated cellular cystine uptake and increased GSH biosynthesis resulting in an enhanced cellular antioxidant capacity.

MLK3 activates JNK during acetaminophen (APAP)-induced hepatotoxicity through

MKK4 (Sharma et al., 2012; J. Zhang et al., 2017). MLK3 activation also increased ROS formation in this cellular context. MLK3 depleted GSH by regulating its biosynthesis through glutamate-cysteine ligase catalytic subunit (GCLC) expression.

1.4 Significance

MLK3 is a ubiquitously expressed mammalian serine/threonine MAP3K with the ability to transform NIH 3T3 cells (Hartkamp et al., 1999) and demonstrates both kinase- independent and kinase-dependent functions in the positive regulation of migration and/or invasion in several human cancers including lung carcinoma (Swenson-Fields et al., 2008), non-small cell lung (Chien et al., 2011), gastric (P. Mishra et al., 2010), breast

(J. Chen & Gallo, 2012; J. Chen et al., 2010; Cronan et al., 2012; Rattanasinchai et al.,

2017), ovarian (Zhan et al., 2012), melanoma (J. Zhang et al., 2014), glioblastoma (Misek et al., 2017), and hepatocellular carcinoma (Fig. 1-7) (Lan et al., 2017). MLK3 SNPs and mutations have been identified in CRC (Slattery et al., 2012; Velho et al., 2010; Velho et al., 2014), and computational studies have identified MLK3 and ERK2 as master regulators of CRC invasion (Wlochowitz et al., 2016); however, the mechanisms for which MLK3 confers a malignant phenotype in CRC have not been elucidated biochemically. Human colorectal tumors present with intrinsic oxidative stress (Perse,

2013), which activates MLK3 in neuronal, kidney, mouse fibroblasts, primary hepatocytes, prostate cancer, and lung adenocarcinoma cells (Hong & Kim, 2007; Hu et al., 2012; Kumar et al., 2011; Lotharius et al., 2005; Luo et al., 2015; Pan et al., 2006; Pei et al., 2005; Savinainen et al., 2001; Sharma et al., 2012; Tian et al., 2003b; van den Berg et al., 2013; S. Wang et al., 2017; Q. G. Zhang, Tian, et al., 2006). Therefore, we sought to determine whether MLK3 promotes a malignant phenotype in CRC cells under oxidative stress. We also explored mechanisms to control the amount of MLK3 protein in cancer cells.

Chapter 2

Materials and Methods

2.1 Cell Culture

Human colorectal carcinoma (HCT116) cells, human embryonic kidney

(HEK293) cells, and human ovarian epithelial adenocarcinoma (SKOV3 and TOV21G) cells were purchased from the American Type Culture Collection (ATCC) (Manassas,

VA, USA). HEY1B ovarian cancer cells were a gift from Douglas Leaman, Ph.D. (The

University of Toledo/Wright State University). HCT116, HEK293, SKOV3, and HEY1B cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Mediatech,

Herndon, VA, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone,

Logan, UT, USA). TOV21G cells were cultured in medium 199 (Mediatech, Inc.) with

10% MCDB 105 (Sigma-Aldrich, St. Louis, MO) and 15% FBS. Both DMEM and medium 199 were supplemented with 2 mM l-glutamine, 25 µg/ml streptomycin, and 25

IU penicillin (Mediatech). All cells were cultured in a humidified atmosphere with 5%

CO2 at 37°C.

2.2 Cell Treatments and Electrophoresis

Cells were either left untreated or treated with the vehicle dimethyl sulfoxide

(DMSO) vehicle (Fisher Scientific, Hampton, NH, USA), 2 mM or 250 µM H2O2 (Fisher

Scientific), 10 µM geldanamycin (InvivoGen, San Diego, CA), 250 mM sorbitol (where from), 50 µM cycloheximide (Thermo Fisher Scientific, Rockford, IL), or 20 ng/ml

TNFa (Life Technologies, Grand Island, NY) for the indicated time periods. For heat shock treatments, cells were cultured at 37°C (control) or 42°C for the indicated time periods. Cells were pretreated with 10 mM N-acetyl-L-cysteine (NAC) (Acros Organics,

New Jersey, USA), 100 or 500 nM Cep1347 (Tocris Bioscience, Bristol, United

Kingdom), 10 µM UO126 (Promega, Madison, WI, USA), or 10 µM MG132

(Selleckchem, Houston, TX). All pretreatments were for 1 h, except for the use of

MG132, which was 2 h. Media was aspirated immediately after treatments, and the cells were rinsed with 1-3 ml sterile 1X phosphate-buffered saline (PBS) (prepared from 10X

PBS: 137 mM NaCl, 27 mM KCl, 14 mM Na2HPO4 pH 7.4 and 43 mM KH2PO4) and harvested on ice. Whole-cell extracts were prepared with 300 (for 6 cm dishes) or 500 µl

(for 10 cm dishes) 6X SDS sample buffer, which consists of 250 mM Tris pH 6.8, 8%

SDS, 20% β-mercaptoethanol (β-Me), 40% glycerol, and 2 mg of bromophenol blue.

Samples were boiled at 95°C for 5 min and subjected to western blot analysis.

6 µl of either prestained protein molecular weight marker (Pierce, Thermo Fisher

Scientific) or precision plus protein dual color standards (BIO-RAD) and 30 µl of each sample were loaded into the wells of either a 10% SDS-PAGE gel to examine electrophoretic mobility of MLKs or 12 or 15% SDS-PAGE gel for all other proteins.

The total volume of the resolving gel solution was 20 ml (7.5, 8, or 10 ml of 30%

acrylamide, 5 ml of 4X Tris-HCl/SDS pH 8.8 (0.5 M Tris base, 13.87 mM SDS), 7.5, 7, or 5 ml of sterile H2O, 90 µl of 50% ammonium persulfate (APS), and 10 µl of tetramethylethylenediamine (TEMED). The total volume of the stacking gel was 20 ml

(1.3 ml of 30% acrylamide, 2.5 ml of 4X Tris-Cl/SDS, pH 6.8, 6.2 ml of sterile H2O, 35

µl of 50% APS, and 10 µl of TEMED). 15 and 20 min, respectively, was allowed for the resolving and stacking gels to polymerize. The loaded acrylamide gels were submerged in 5 L of 1X SDS-PAGE running buffer (25 mM Tris base, 192 mM glycine, and 6.94 mM SDS) and electrophoresed overnight at 60 V.

The resolved proteins were transferred from the SDS gel to an Immobilon-P

Polyvinylidene Difluoride (PVDF) membrane in a transfer apparatus containing 1X transfer buffer (25 mM Tris base, 192 mM glycine and 20% methanol) set to 60 V for 2 h and 15 min at 4°C. Post-transfer, the PVDF membranes were stained with a Coomassie

Blue solution (40% (v/v) methanol, 10% (v/v) acetic acid, and 0.4 g Brilliant Blue R250 in 500 ml), de-stained with 70% methanol, and rinsed in PBST (1X PBS plus 0.05 %

(v/v) Tween 20) to ensure sufficient protein transfer. The de-stained membranes were immersed in blocking solution (5% (w/v) non-fat dry milk in 1X PBS) and gently rotated at room temperature (RT) for 1 h prior to immunoblotting as described below.

2.3 Immunoblotting

Immunoblotting was performed with the following Santa Cruz Biotechnology

(Dallas, TX, USA) antibodies: MLK1 (N-20) sc-19120, MLK3 (C-20) sc-536, B-Raf (F-

3) sc-55522, MEK1/2 (12-B) sc-436, ERK2 (C-14) sc-154, JNK1 (C-17) sc-474, p38a/b

(A-12) sc-7972, GST (B-14) sc-138, β-actin (C-4) sc-47778, Hsp90 (H-114) sc-7947,

Raf-1 (C-20) sc-227, and goat anti-rat IgG-HRP sc-2032. Activation state antibodies from Cell Signaling Technology (Beverly, MA, USA) include: phosphorylated MEK1/2

(p-MEK) (Ser221) 166F8, phosphorylated ERK1/2 (p-ERK) (Thr202/Tyr204) #4370S, phosphorylated JNK (p-JNK) (Thr183/Tyr185) 9251L, phosphorylated MLK3 (p-MLK3)

(Thr277/Ser281) #2811, and phosphorylated p38 (p-p38) (Thr180/Tyr182) #9211. Other antibodies used in this study were MLK4 NBP1-41081 (Novus Biologicals, Littleton,

CO, USA), rat anti-DYKDDDDK (FLAG) 200474-21 (Agilent Technologies, Santa

Clara, CA, USA), anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP) conjugate W401B (Promega), Hsp70 (W27) MAI-90504 (Thermo Scientific, Freemont,

CA), and Immun-Star goat anti-mouse IgG-HRP conjugate 1705047 (Bio-Rad, Hercules,

California, USA).

The antibodies were diluted into either antibody buffer (5% (w/v) non-fat dry milk in PBST) or phospho-antibody buffer (5% (w/v) bovine serum albumin in PBST).

The recommended antibody dilutions on the data sheets provided by the manufacturers were used. The blocked membranes were sealed in plastic sleeves with the appropriate amount of dilute antibody solutions and rotated overnight at 4°C. On the following day, the immunoblots were removed from the bags, washed three times for 15 min each in

PBST RT under gentle rotation, sealed in plastic sleeves containing the appropriate secondary antibodies diluted 1:5000 in antibody buffer, gently rotated at RT for 2 h, and washed as described above. The immunoblots were then developed using with

Immobilon-P development solution (Millipore, Billerica, MA, USA).

2.4 Phosphatase Assay

After H2O2-treatment and immunoprecipitation of endogenous MLK3, samples were suspended in either 50 µl 1X calf intestinal alkaline phosphatase (CIAP) buffer alone, 48 µl CIAP buffer plus 2 µl CIAP enzyme (Promega), or 47 µl CIAP buffer and 2

µl CIAP enzyme plus 1 µl phosphatase inhibitor cocktail 3 (Sigma-Aldrich, St. Louis,

MO, USA), and incubated for 1 h at 37°C with 300 revolutions per minute (rpm) in a thermomixer. Phosphatase inhibitor cocktail 3 contains (–)-p-Bromolevamisole oxalate which inhibits L-isoforms of alkaline phosphatases. The reaction was stopped with 1X

SDS sample buffer and 95°C heat for 5 min. The samples were then subjected to western blot analysis with MLK3 antibody.

2.5 Plasmid and siRNA Transfections

In addition to the mutant plasmids described below, pRK5-FLAG-MLK3, pRK5-

K144R-FLAG-MLK3, and pEBG-GST-B-Raf mammalian expression constructs

(Addgene, Cambridge, MA) were used for expression of human MLK3 and B-Raf.

Transient plasmid DNA transfections of HEK293 and HCT116 cells were performed with PolyJet (SignaGen Laboratories, Rockville, MD, USA) and Lipofectamine 2000

(Invitrogen, Carlsbad, CA, USA), respectively. Small interfering RNA (siRNA) knockdown in HCT116 cells was also executed with Lipofectamine 2000. Non-specific

(NS) siRNA, SignalSilence® p44/42 MAPK (ERK1/2) siRNA #6560 (Cell Signaling

Technology, Danvers, MA, USA), and MLK3 sense 5’-

GGGCAGUGACGUCUGGAGUdTdT-3’ (nt 903-923) and antisense 5’-

ACUCCAGACGUCACUGCCCdTdT-3’ siRNA were obtained from Santa Cruz

Biotechnology, Cell Signaling Technology, and GE Dharmacon (Lafayette, CO, USA), respectively.

For siRNA transfections, cells were seeded into 6 cm culture dishes and grown to

50-60% confluence overnight. Media was aspirated from the culture dishes, cells were rinsed with 2 ml fresh serum-free media (SFM), and 1.7 ml fresh SFM was added to the cells 30-60 min prior to the transfection. siRNA was pipetted into a sterile 1 ml microcentrifuge tube containing 800 µl of SFM, and the solution was gently mixed. 7 µl of Lipofectamine 2000 was added to the dilute siRNA solution, which was then gently mixed and incubated at RT for 20 min to allow transfection complex formation.

Immediately after incubation, the solution containing the transfection complexes was added drop-wise to the cells, which were then gently swirled and cultured in a humidified atmosphere with 5% CO2 at 37°C for 4-6 h. 2.5 ml of 20% FBS DMEM was then added to the culture dishes containing the cells and 2.5 ml SFM, bringing the final concentration of FBS to 10%. Cells were further incubated at 37°C for the appropriate amount of time and subjected to the appropriate treatments and harvesting. ERK1/2 and MLK3 siRNA transfection was performed with a final concentration of 100 nM for 72 and 36 h, respectively. Cells were then treated with either 2 mM H2O2 for 30 min or 250 µM H2O2 for 12 h and subjected to western blot analysis or an in vitro FluoroBlok invasion assay as described below.

For plasmid DNA transfections, cells were seeded into 6 or 10 cm culture dishes and grown to 50-60% confluence overnight. Media was aspirated from the culture dishes, and 2.8 (for 6 cm dishes) or 5 ml (for 10 cm dishes) fresh 10% FBS DMEM was added to the cells 30-60 min prior to the transfection. Plasmid DNA was pipetted into a sterile 1

ml microcentrifuge tube containing 200 (for 6 cm dishes) or 500 µl (for 10 cm dishes) of

SFM at a final concentration of 2 (for 6 cm dishes) or 5 µg (for 10 cm dishes), and the solution was gently mixed. 7 (for 6 cm dishes) or 15 µl (for 10 cm dishes) of PolyJet was added to the dilute plasmid DNA solution, which was then gently mixed and incubated at

RT for 15 min to allow transfection complex formation. Immediately after incubation, the solution containing the transfection complexes was added drop-wise to the cells, which were then gently swirled and cultured in a humidified atmosphere with 5% CO2 at 37°C for 24-36 h and then subjected to the appropriate treatments and harvesting. Plasmid

DNA transfections for in vitro FluoroBlok invasion assays were executed for 36 h, while all other plasmid DNA transfections were executed for 24 h.

2.6 Immunoprecipitations and GST Pull-Down

Immunoprecipitation (IP) of endogenous MLK3 from HCT116 cells was executed with MLK3 antibody, while immunoprecipitation of epitope tagged proteins from

HEK293 was performed with FLAG antibody. After appropriate transfections and possibly treatments (depending on the experiment), media was aspirated, and cells were rinsed with 2 ml sterile 1X PBS and harvested in 1 ml IP lysis buffer (20 mM Tris-HCL pH 7.4, 2 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA),

10 mM MgCl2, 0.1% (v/v) β-Me, 1% (w/v) Triton X-100, 100 µM phenylmethanesulfonyl fluoride (PMSF), 1 µM aprotinin, 2 µM leupeptin, 2 µM pepstatin, 50 mM b-glycerophosphate, 1 mM Na3VO4, and 1:100 phosphatase inhibitor cocktail 3 as described above). Samples were centrifuged at 7267 x g for 10 min in 4°C, and the supernatant was collected. 900 µl of the supernatant was pipetted into a new tube

and mixed with 25 µl of either A/G PLUS Agarose (Santa Cruz Biotechnology) or protein G resin (GenScript, Piscataway, NJ, USA) and one of the following antibodies: rat anti-DYKDDDDK (FLAG) CAT# 200474-21 (Agilent Technologies, Santa Clara,

CA, USA), anti-MLK3 (D-11) sc-166639 (Santa Cruz Biotechnology), anti-ERK2 (C-14) sc-154 (Santa Cruz Biotechnology), mouse IgG sc-2025 (Santa Cruz Biotechnology), or rabbit IgG sc-2027 (Santa Cruz Biotechnology). The supernatant/antibody/bead solution was rotated at 4°C for 2 h. Twenty µl of 6X SDS sample buffer was added to the remaining 100 µl of the supernatant, which was then boiled at 95°C for 5 min and subjected to western blot analysis. Immediately after the 2 h rotation, samples were centrifuged at 7267 x g for 1 min in 4°C, and the supernatant was discarded. The beads with bound protein were washed three times with 1 ml IP lysis buffer, 1 ml high salt wash buffer (IP lysis buffer with 0.1% Triton X-100 and 1 M LiCl, but without PMSF, b- glycerophosphate, Na3VO4, and phosphatase inhibitor cocktail 3) and 1 ml wash buffer

(high salt wash buffer without LiCl), respectively. After centrifugation and discard of the wash buffer, 30 µl 6X SDS sample buffer was added to the tubes, and the samples were then boiled at 95°C for 5 min and subjected to western blot analysis or radioactive kinase assays.

For the glutathione S-transferase (GST) pull-down, HEK293 cells were transfected with appropriate combinations of empty vector (EV), wild-type FLAG-

MLK3, S705A-S758A-FLAG-MLK3, and GST-B-Raf and treated with 2 mM H2O2 for 5 min 24 post transfection. Media was immediately aspirated, and cells were rinsed with 2 ml sterile 1X PBS and harvested in 1 ml IP lysis buffer (described above). Samples were centrifuged at 10,000 rpm for 10 min in 4°C, and the supernatant was collected. 900 µl of

the supernatant was pipetted into a new tube and mixed with 25 µl of immobilized glutathione (Thermo Scientific, Waltham, MA, USA). The supernatant/glutathione resin solution was rotated at 4°C for 2 h. Twenty µl of 6X SDS sample buffer was added to the remaining 100 µl of the supernatant, which was then boiled at 95°C for 5 min and subjected to western blot analysis. Immediately after the 2 h rotation, samples were centrifuged at 10,000 rpm for 1 min in 4°C, and the supernatant was discarded. The resin with bound protein was washed three times with 1 ml IP lysis buffer, 1 ml high salt wash buffer (described above) and 1 ml wash buffer (described above), respectively. After centrifugation and discard of the wash buffer, 30 µl 6X SDS sample buffer was added to the tubes, and the samples were then boiled at 95°C for 5 min and subjected to western blot analysis.

For both the co-immunoprecipitation of endogenous B-Raf with endogenous

MLK3 and the GST pull-down, Restore Western Blot Stripping Buffer (Thermo

Scientific) was used. After western blot analysis of endogenous B-Raf or FLAG-MLK3, the blots were washed with PBST for 5 min at RT, immersed in stripping buffer, incubated for 15 min at RT, washed with PBST for 5 min, blocked as described above for

30 min, and then probed with endogenous MLK3 or GST antibodies.

2.7 Multiple Sequence Alignment

Multiple sequence alignment of human MLKs was generated with T-Coffee and the following National Center for Biotechnology Information (NCBI) reference sequences: NP_149132.2 (MLK1), NP_002437.2 (MLK2), NP_002410.1 (MLK3), and

NP_115811.2 (MLK4). The alignment was formatted with Boxshade.

2.8 Site-Directed Mutagenesis

Site-directed mutagenesis of human MLK3 was performed with a QuikChange

Lightning Site-directed Mutagenesis kit from Agilent Technologies. The wild-type and kinase dead (K144R) MLK3 S705A mutant cDNA were generated with the sense oligonucleotide 5’-CTCAAGACGCCCGACGCCCCGCCCACTCCTGCA-3’ and the antisense oligonucleotide 5’- TGCAGGAGTGGGCGGGGCGTCGGGCGTCTTGAG-3’

(mismatch with the wild-type MLK3 template is underlined) using either pRK5-wild-type or pRK5-K144R MLK3 as templates, respectively. The MLK3 kinase dead S758A, wild- type S705A-S758A, and kinase dead S705A-S758A mutant cDNA were generated with the sense oligonucleotide 5’-CCAGGCACCCCACGTGCACCACCCCTGGGCCTC-3’ and the antisense oligonucleotide 5’-

GAGGCCCAGGGGTGGTGCACGTGGGGTGCCTGG-3’ using K144R MLK3, S705A

MLK3, and K144R S705A MLK3 as templates.

For all mutants, two complimentary oligonucleotide primers containing the point mutation of interest were designed using EnzymeX DNA sequence editor software, synthesized, and purified via high performance liquid chromatography (HPLC). 2.5 µl

10X reaction buffer, 6.75 µl double-distilled H2O, 1 µl 125 ng oligonucleotide primer #1,

1 µl oligonucleotide primer #2, 0.5 µl dNTP mix, 1 µl (50 ng) of dsDNA template, 0.75

µl QuikSolution reagent, and 0.5 µl QuikChange Lightning Enzyme were pipetted into a microcentrifuge tube on ice and then centrifuged briefly. Each reaction was cycled in a thermal cycler using the following parameters: 1 cycle of 95°C for 2 min, 18 cycles of

95°C for 20 seconds/60°C for 10 seconds/68°C for 3.5 min (30 seconds per kb of plasmid length…4661 bp pRK5-FLAG + 2541 bp MLK3 coding DNA sequence = 7202 bp =

7.202 kb), and 1 cycle of 68°C for 5 min. Each amplification reaction was then transferred to a microcentrifuge tube, and 1 µl of Dpn I restriction enzyme, which recognizes and cuts the palindromic sequence 5’-GAm6TC-3’, was pipetted into the tube on ice. The reaction mixture was gently mixed, briefly centrifuged, and incubated at 37°C for 5 min in a thermomixer. m6 refers to methylation of the nucleobase at the nitrogen-6 position. XL-10-Gold ultracompetent cells were gently thawed on ice, and 45 µl of the cells were pipetted into a prechilled 14 ml BD Falcon polypropylene round-bottom tube.

2 µl of b-ME mix were added to the ultracompetent cells, and the contents were swirled gently and incubated on ice for 2 min. 2 µl of the Dpn I-treated DNA was then added to the ultracompetent cells, and the transformation reaction was gently mixed and incubated on ice for 30 min. The tube containing the transformation reaction was then heat-pulsed in a 42°C water bath for 30 seconds, incubated on ice for 2 min, mixed with 0.5 ml of

NZY+ broth preheated to 42°C, and then incubated at 37°C for 1 h with 225 rpm shaking.

550 µl of the transformation reaction was streaked equally among 3 Luria-Bertani (LB)- ampicillin (50 µg/ml) agar plates. The plates were then incubated at 37°C for 16 h. 10 µl pipette tips were used to scrape single colonies and inoculate a starter culture of 5 ml LB broth containing 50 µg/ml ampicillin. The inoculated starter culture was incubated overnight at 37°C with 225 rpm shaking. Plasmid DNA was purified using the QIAGEN plasmid mini kit following the June 2005 QIAGEN Plasmid Purification Handbook, and

DNA concentration and purity was measured using a NanoDrop spectrophotometer.

Mutants were verified by DNA sequencing (Genewiz, South Plainfield, NJ, USA) with the following primer: MLK3 reverse 5’-TCAAGGCCCCGCTTCCGGCAC-3’.

Expression of the FLAG epitope tag was examined via transient transfection into

HEK293 cells and western blot analysis. Plasmid DNA was then purified using the

QIAGEN plasmid maxi kit following the QIAGEN handbook as above, and DNA concentration and purity was analyzed via NanoDrop technology.

2.9 In Vitro Kinase Assay

EV, wild-type FLAG-MLK3, K144R-FLAG MLK3, K144R-S705A-FLAG-

MLK3, K144R-S758A-FLAG-MLK3, and K144R-S705A-S758A-FLAG MLK3 were transiently transfected into and immunoprecipitated from HEK293 cells as described above. FLAG immunoprecipitates were suspended in kinase assay buffer (50 mM Tris-

HCl [pH 7.5], 2 mM EGTA, 10 mM MgCl2, 0.1 mM DTT, and 0.1 % Triton X-100) with

32 100 µM unlabeled adenosine triphosphate (ATP), 10 mM MgCl2, and 5 µCi γ- P-ATP

(PerkinElmer Health Sciences, Boston, MA, USA or MP Biomedicals, Solon, OH, USA).

0.01 µg purified full-length recombinant human active ERK1 (Boulton et al., 1991)

(SignalChem, Richmond, BC, CA) was added to appropriate samples. The assay was executed for 30 min at 30°C and stopped with 1X SDS sample buffer and 95°C heat for 5 min. Samples were centrifuged, subjected to SDS-PAGE, and transferred to PVDF membranes. Detection of radioactive materials was executed via autoradiography.

Membranes were then blocked, probed with FLAG and ERK1/2 primary antibodies, the appropriate secondary antibodies, and developed as described above.

2.10 In Vitro FluoroBlok Invasion Assay

HCT116 cells were either transfected with NS or MLK3 siRNA or EV, wild-type

FLAG-MLK3, or S705A-S758A-FLAG-MLK3 as described above, treated with vehicle

or 250 µm H2O2 for 12 h, immediately trypsinized, and transferred to 15 ml conical tubes.

The in vitro FluoroBlok invasion assay was performed as in Partridge and Flaherty, 2009

(Partridge et al., 2009) with slight modifications. 24 h prior to cell harvesting, 10 µl of 10 mg/ml BD Matrigel matrix (BD Biosciences, San Jose, CA, USA), thawed on ice, was diluted into 90 µl of cold SFM for each sample. 100 µl of diluted Matrigel was added to

FluoroBlok 24 well transwell inserts with 8.0 µm colored polyethylene terephthalate

(PET) membrane (Corning, Corning, NY, USA) in 24 well plates and allowed to solidify throughout a 24 h incubation at 37°C. Trypsinized cells were centrifuged, washed with 2 ml SFM twice, and counted in 2 ml fresh SFM with a hemocytometer under a light microscope. 300,000 cells in 500 µl SFM were gently pipetted into each apical chamber containing the Matrigel matrix, and 750 µl of 5% FBS in DMEM chemoattractant was added to each basal chamber, the bottom of the 24 well plate. The cells were incubated for 20-22 h at 37°C. The remaining cells were centrifuged, SFM was discarded, and 1 ml

PBS was added to the tubes. Cells were then transferred to microcentrifuge tubes and centrifuged. The PBS was discarded, and an appropriate amount of 6X SDS sample buffer (depending on the cell count) was added to each tube. Samples were sheared with needles, boiled at 95°C for 5 min, and subjected to western blot analysis with either

FLAG or MLK3 antibodies and b-actin antibody. Following incubation of the invasion assay, SFM was removed from the apical chambers by gently emptying the contents into a waste container all the while exerting care not to touch the bottom surface of the transwell insert. The transwells were placed into a second 24 well plate containing 500

µl/well of 4 µg/ml Calcein acetomethoxy (AM) fluorescent dye (Corning) in DMEM supplemented with 5% FBS, and incubated for 1 hour at 37°C. Fluorescence of invaded

cells (relative fluorescence units [RFUs]) was read at 494/517 nm (Ex/Em) wavelengths on a SpectraMax M5 bottom-reading fluorescent plate reader with SoftMax Pro 5 microplate data acquisition and analysis software. Photomicrographs of invaded cells were taken with an Olympus 1X81 inverted fluorescence microscope using Olympus cellSens microscope imaging software.

2.11 Quantitative Real-Time PCR

Total RNA was isolated from SKOV3, HEY1B, or TOV21G cells using TRIzol reagent (Invitrogen). Cells were cultured in 6 cm dishes to 90% confluence and then either treated with 10 µM GA or 250 mM sorbitol or cultured at 42°C for the indicated time periods. Media was aspirated, and cells were washed with 1 ml PBS. 1 ml of TRIzol reagent was added directly to the cells, which were then pipetted up and down several times, transferred to microcentrifuge tubes, and incubated at RT for 3 min. 200 µl of chloroform was added to each tube, which were then capped tightly, shaken vigorously by hand for 15 seconds, incubated at RT for 3 min, and centrifuged at 9400 rpm for 15 min at 4°C. During centrifugation, the mixtures separated into a lower red phenol- chloroform phase, an interphase, and a colorless upper aqueous phase which contained the RNA; hence, the aqueous phase was pipetted out of each tube, angled at 45°, and transferred to new microcentrifuge tubes. 0.5 ml of 100% isopropanol was added to each aqueous phase. The tubes were inverted several times, incubated at RT for 10 min, and centrifuged at 9400 rpm for 10 min. at 4°C. The supernatants were carefully discarded, and the remaining RNA pellets were washed with 1 ml of 75% ethanol. The samples were briefly vortexed and then centrifuged at 9400 rpm for 5 min at 4°C. The washes

were discarded, and the remaining pellets were air dried for 5-10 min, resuspended in 50

µl RNase-free water, and stored at -80°C until the next step.

Total RNA was reverse transcribed into cDNA. Total RNA concentration was measured using a NanoDrop spectrophotometer. For each sample, 2 µg of total RNA and

0.5 µg of random hexamer primer were diluted into water to 7 µl in a microcentrifuge tube, incubated at 70°C for 10 min using a thermomixer, quickly chilled on ice, and centrifuged. 13 µl of a master mix (4 µl 5X reverse transcriptase (RT) buffer, 2 µl 5 mM dNTP mix, 0.5 µl RNasin, 0.5 µl M-MLV RT (Fisher Scientific), and 6 µl water) was added to the sample, which was immediately incubated at 42°C for 1 h and then 70°C for

10 min. The RT product, cDNA, was verified with agarose gel electrophoresis and stored at -20°C until the next step.

For each sample, a standard polymerase chain reaction (PCR) reaction was performed with 1 µl cDNA, 100 ng of each forward and reverse primers (see below), 10

µl of 1X Ssofast EvaGreen Supermix (Bio-Rad Laboratories, Hercules, CA), and

RNase/DNase-free water to 20 µl. The primer sequences (human) were as follows: β-

Actin forward 5’- GGACTTCGAGCAAGAGATGG-3’ and reverse 5’-

AGCACTGTGTTGGCGTACAG-3’ and MLK3 forward 5’-

GTCATGGAATGGCAGTGG-3’ and reverse 5’- CACGGTCACCCTTCCTCA-3’.

2.12 Quantification and Statistical Analysis

For every data panel, immunoblots are representative of three independent experiments (n=3), and quantification and statistical analyses were performed on three independent experiments. Each experiment shown was replicated a minimum of three

5 1

times in the laboratory. Densitometric analysis of immunoblot or autoradiography bands was executed using Image J software (National Institutes of Health). The signal for a phosphorylated protein was normalized to the total abundance of the protein of interest.

Statistical analysis for comparison of two samples was performed with Student's t test

(two-tailed). For comparison of three plus samples, Kruskal-Wallis one-way analysis of variance followed by the Conover-Inman post hoc test was performed. All samples were included in the analysis, and P-values were not adjusted. Bars depict means, error bars represent standard deviations (SD) for RT-PCR data and standard error of the mean

(SEM) for all other data, asterisks indicate statistical significance (p-value < 0.05), and a.u.=arbitrary units.

Chapter 3

Results

3.1 MEK1/2 and ERK1/2 are activated and MLK3 is phosphorylated in response to oxidative stress

Oxidative stress can influence MAPK signaling by affecting MAP3K function.

ASK1 is indirectly activated by oxidative-modification resulting in JNK and p38 signaling (Liu et al., 2000). MLK3 is catalytically activated in response to oxidative stress by either direct oxidative modification or other mechanisms in neuronal, kidney, mouse fibroblasts and primary hepatocytes, prostate cancer, and lung adenocarcinoma cells (Hong & Kim, 2007; Hu et al., 2012; Kumar et al., 2011; Lotharius et al., 2005; Luo et al., 2015; Pan et al., 2006; Pei et al., 2005; Savinainen et al., 2001; Sharma et al., 2012;

Tian et al., 2003b; van den Berg et al., 2013; S. Wang et al., 2017; Q. G. Zhang, Tian, et al., 2006). To understand whether MLKs 1, 3, and 4 are regulated by ROS in colorectal cancer cells, HCT116 cells were treated with 2 mM H2O2 for 30 min (Figure 3-1).

MLK3, but not MLKs 1 or 4, exhibited reduced electrophoretic mobility (shift) by approximately 7 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE), and MEK1/2 and ERK1/2 were significantly activated. Pretreatment with

10 mM of the ROS scavenger N-acetyl-L-cysteine (NAC) abrogated the MLK3 mobility 53

Figure 3-1: MEK1/2 and ERK1/2 are activated and MLK3 electrophoretic mobility is retarded in response to H2O2 treatment. Western blot analysis of indicated proteins from HCT116 cell lysates treated with 2 mM H2O2 for 30 min. n=3 and a.u. stands for arbitrary units.

shift and significantly decreased the activation of ERK1/2 in H2O2-treated cells (Figure 3-

2), suggesting the MLK3 mobility shift and ERK1/2 activation are ROS-dependent. To examine if a post-translational modification such as a phosphorylation produced the shift,

HCT116 cells were either untreated or treated with 2 mM H2O2 for 30 min. Endogenous

MLK3 was immunoprecipitated and incubated with calf intestinal alkaline phosphatase

(CIAP) buffer alone, buffer plus CIAP, or buffer/CIAP/and a phosphatase inhibitor cocktail containing the alkaline phosphatase inhibitor (-)-p-bromolevamisole oxalate

(Figure 3-3). The H2O2-induced shift of MLK3 was not observed in the CIAP-treated

MLK3 immunoprecipitates, but was evident in the MLK3 immunoprecipitates treated with CIAP and phosphatase inhibitor. These results indicate the ROS-induced

Figure 3-2: The mobility shift of MLK3 and ERK1/2 activation in response to H2O2 is ROS-dependent. Western blot analysis of indicated proteins from HCT116 cell lysates pretreated with 10 mM NAC for 1 h followed by 2 mM H2O2 for 30 min. n=3.

Experiment performed by both April Schroyer and Nicholas Stimes.

Figure 3-3: MLK3 is phosphorylated in response to H2O2 treatment in HCT116 cells. Western blot analysis of endogenous MLK3 immunoprecipitated from HCT116 cells treated with 2 mM H2O2 for 30 min and then subjected to phosphatase treatment or treatment with phosphatase plus phosphatase inhibitor. n=3. 55

shift is due to phosphorylation. MLK3 activation involves autophosphorylation on Thr277 and Ser281 and leads to phosphorylation and activation of the downstream MAPK JNK

(Leung & Lassam, 2001). JNK phosphorylates MLK3 in vitro and in vivo giving rise to positive feedback (Schachter et al., 2006). To test if the ROS-induced phosphorylation of

MLK3 corresponds to JNK feedback or autophosphorylation of Thr277 and Ser281 and thus

MLK3 activation, HCT116 cells were pretreated with 100 nM (Figure 3-4A) or 500 nM

(Figure 3-4B) Cep1347, a small molecule competitive inhibitor of MLK3 kinase activity

(Maroney et al., 2001), for 1 h and then treated with 2 mM H2O2 for 30 min. Both 100 nM and 500 nM Cep1347 significantly reduced the activation of JNK in response to

H2O2, indicating MLK3 activation was blocked. Inhibition of MLK3 kinase activity did not prevent the mobility shift of MLK3 or the activation of ERK1/2, which suggests the

ROS-induced phosphorylation of MLK3 is separate from both JNK feedback onto MLK3 and MLK3 autophosphorylation and activation. Collectively, we observed ROS-induced activation of MEK1/2 and ERK1/2 as well as phosphorylation of MLK3, which is independent of MLK3 activation and JNK feedback.

Figure 3-4: The ROS-dependent phosphorylation of MLK3 is independent of both

MLK3 activation and JNK feedback. Western blot analysis of indicated proteins from

HCT116 cell lysates pretreated with either 100 nM (A) or 500 nM (B) Cep1347 for 1 h followed by 2 mM H2O2 for 30 min. n=3.

3.2 The ROS-induced phosphorylation of MLK3 is dependent on active ERK1/2, and endogenous ERK1/2 and MLK3 associate in HCT116 cells

Our Cep1347 results (Figure 3-4) suggested a kinase other than MLK3 or JNK phosphorylates MLK3 in response to ROS. Significant ERK1/2 activation was observed in H2O2-treated cells (Figures 3-1, 3-2, 3-4A, and 3-4B); therefore, we investigated whether ERK1/2 could promote the ROS-induced phosphorylation of MLK3. HCT116 cells were either untreated, treated with 2 mM H2O2 for 30 min, or pretreated with 10 µM of the MEK1/2 inhibitor UO126 and then treated with 2 mM H2O2 for 30 min (Figure 3-

5). The H2O2-induced mobility shift of MLK3 was not observed in cells with significant

ERK1/2 inhibition, suggesting the ROS-induced phosphorylation of MLK3 is ERK1/2-

Figure 3-5: The ROS-dependent phosphorylation of MLK3 is blocked by inhibition of ERK1/2 kinase activity. Western blot analysis of indicated proteins from HCT116 cell lysates pretreated with 10 µM UO126 for 1 h followed by 2 mM H2O2 for 30 min.

Lanes 1 and 2 are non-contiguous. n=3.

dependent. To confirm the UO126 observations, HCT116 cells were transfected with either non-specific (NS) or ERK1/2 siRNA and then treated with 2 mM H2O2 for 30 min

(Figure 3-6). ERK1/2 siRNA knockdown significantly reduced ERK1/2 activation and blocked the MLK3 mobility shift in H2O2-treated cells, indicating the ROS-induced phosphorylation of MLK3 is an ERK1/2-specific event. To determine whether ERK1/2

Figure 3-6: The ROS-dependent phosphorylation of MLK3 is prevented by ERK1/2 siRNA knockdown. Western blot analysis of indicated proteins from HCT116 cells transfected with non-specific (NS) or ERK1/2 siRNA and then treated with 2 mM H2O2 for 30 min. n=3.

interacts with MLK3, endogenous ERK1/2 was immunoprecipitated from HCT116 cells which were either left untreated or treated with 2 mM H2O2 for 30 min (Figure 3-7).

Endogenous MLK3 co-immunoprecipitated with endogenous ERK1/2, suggesting

endogenous ERK1/2 and MLK3 interact in HCT116 cells. This interaction was also observed in H2O2-treated cells.

Figure 3-7: MLK3 and ERK1/2 associate in H2O2-treated HCT116 cells. Western blot analysis of indicated proteins from HCT116 immunoprecipitates or cell lysates treated with 2 mM H2O2 for 30 min. n=3.

3.3 ERK1 phosphorylates Ser705 and Ser758 of MLK3 in vitro

We observed a ROS- and ERK1/2-dependent phosphorylation of MLK3 (Figures

3-2, 3-3, 3-5, and 3-6); thus, we hypothesized active ERK1/2 directly phosphorylates

MLK3. Six MLK3 residues conform to the ERK1/2 phosphorylation consensus sequence of PXS/TP (Gonzalez et al., 1991): Ser570, Thr605, Thr677, Ser705, Thr752, and Ser758 (Figure

3-8A). Both Ser705 and Ser758 are confirmed MLK3 in vivo phosphorylation sites

(Vacratsis et al., 2002). The MLKs share 75% sequence identity within the kinase catalytic domains and approximately 65% sequence identity from the SH3 domains to the

CRIB motifs; however, the MLKs carboxyl termini sequences are divergent and might have different regulatory functions (Gallo & Johnson, 2002). Ser705 and Ser758 are located within the C-terminal sequence unique to MLK3. MLKs 1 and 4 did not exhibit slower

mobility on SDS-PAGE in response to oxidative stress (Figure 3-1); thus, the potential

ERK1/2 phosphorylation sites should not be present in these kinases. We performed a multiple sequence alignment of the human MLKs and found the residues of MLKs 1-2 and 4 which correspond to Ser705 and Ser758 of MLK3 did not conform to the preferred

ERK1/2 phosphorylation consensus sequence of PXS/TP (Figure 3-8B and A-1). To

Figure 3-8: Ser705 and Ser758 of MLK3 are potential ERK1/2 phosphorylation sites specific to MLK3. (A) Residues in the MLK3 amino acid sequence which conform to the ERK1/2 phosphorylation consensus sequence of PXS/TP. (B) Results of a T-Coffee multiple sequence alignment of the human MLKs showing sequences of MLKs 1-2 and 4 homologous to Ser705 and Ser758 of MLK3.

investigate whether ERK1 could phosphorylate MLK3 in vitro on Ser705 and Ser758, these amino acids of kinase dead (K144R) FLAG-MLK3 were mutated to non- phosphorylatable alanine residues. Empty vector (EV), wild-type-, K144R-, K144R-

S705A-, K144R-S758A-, and K144R-S705A-S758A-FLAG-MLK3 were expressed in human embryonic kidney (HEK293) cells, immunoprecipitated, and used as substrate in 61

an ERK1 kinase assay (Figure 3-9). ERK1 phosphorylated wild-type- and K144R-FLAG-

MLK3 to a significantly greater extent than K144R-S705A- and K144R-S758A-FLAG

MLK3. ERK1 phosphorylation of K144R-S705A-S758A-FLAG-MLK3 was the least of all three mutants. K144R-FLAG-MLK3 phosphorylation was significantly less than wild- type-FLAG-MLK3, which is due to the inability of kinase dead K144R-FLAG-MLK3 to autophosphorylate on Thr277 and Ser281. When quantified relative to phospho-K144R-

FLAG-MLK3 (set at 1.0), the single mutants, phospho-K144R-S705A-FLAG-MLK3 and phospho-K144R-S758A-FLAG-MLK3, and the double mutant, phospho-K144R-S705A-

S758A-FLAG-MLK3, were 0.63, 0.83, and 0.31, respectively, suggesting that ERK1 phosphorylates MLK3 in vitro on Ser705 and Ser758. To verify the mutation of Ser705 and

Figure 3-9: ERK1 phosphorylates K144R-FLAG-MLK3 in vitro, but not K144R-

S705A-S758A-FLAG-MLK3. Autoradiography and western blot analysis of ERK1 kinase assays with EV and FLAG-MLK3 (wild-type, K144R, K144R-S705A, K144R-

S758A, or K144R-S705A-S758A) substrates. KD=kinase dead=K144R. Different capital letters over the bars indicate statistical significance, while same letters do not. n=3.

Ser758 to alanine did not affect MLK3 kinase function, wild-type FLAG-MLK3 and

S705A-S758A-FLAG-MLK3 were expressed in HEK293 cells, and the amount of phosphorylated, activated JNK (p-JNK) was assessed (Figure 3-10). There was no significant difference between wild-type- and S705A-S758A-FLAG-MLK3 in the ability to promote JNK activation.

Figure 3-10: S705A-S758A-FLAG-MLK3 expression in HEK293 cells activates JNK comparable to wild-type FLAG-MLK3 expression. Western blot analysis of indicated proteins from HEK293 cell lysates expressing EV, wild-type-FLAG-MLK3, and S705A-

S758A-FLAG-MLK3. n=3.

3.4 Oxidative stress promotes association of MLK3 and B-Raf: ROS-induced

MEK1/2 and ERK1/2 activation requires MLK3 Ser705 and Ser758 phosphorylation

MLK3, independent of its kinase activity, functions as a scaffold for B-Raf and

Raf-1 transactivation leading to ERK1/2 activation in EGF-treated colon cancer cells

(Chadee & Kyriakis, 2004; Chadee et al., 2006); therefore, we investigated whether this scaffold function of MLK3 is important for ERK1/2 activation in H2O2-treated colon cancer cells. To determine if endogenous MLK3 and B-Raf interact in response to 63

oxidative stress, endogenous MLK3 was immunoprecipitated from HCT116 cells either untreated or treated with 2 mM H2O2 for 5 min (Figure 3-11). Co-immunoprecipitation of endogenous B-Raf with endogenous MLK3 was significantly increased in response to

ROS. If oxidative stress enhances the MLK3 scaffold function associated with B-Raf

Figure 3-11: MLK3 and B-Raf associate in H2O2-treated HCT116 cells. Western blot analysis of indicated proteins from HCT116 immunoprecipitates or cell lysates treated with 2 mM H2O2 for 5 min. n=3.

activation, ERK1/2 activation should be dependent, at least in part, on MLK3. To test this hypothesis, HCT116 cells were transfected with either NS or MLK3 siRNA and then treated with 2 mM H2O2 for 30 min (Figure 3-12). MLK3 siRNA knockdown significantly reduced the ROS-dependent activation of ERK1/2. To examine if phosphorylation of Ser705 and Ser758 promotes MLK3-dependent ERK1/2 activation, wild-type FLAG-MLK3 and S705A-S758A-FLAG-MLK3 were expressed in HEK293 cells, which were then treated with 2 mM H2O2 for 30 min (Figure 3-13). No significant difference in JNK activation was detected between wild-type- and S705A-S758A-FLAG-

Figure 3-12: MLK3 siRNA knockdown abrogates ERK1/2 activation in response to

H2O2 treatment. Western blot analysis of indicated proteins from HCT116 cell lysates expressing either NS or MLK3 siRNA and treated with 2 mM H2O2 for 30 min. n=3.

MLK3 transfected cells; however, significantly less MEK1/2 and ERK1/2 activation was observed in cells expressing S705A-S758A-FLAG-MLK3 as compared to wild-type

FLAG-MLK3. These results indicate phosphorylation of Ser705 and Ser758 are critical for

MLK3-dependent MEK1/2 and ERK1/2 activation in H2O2-treated cells. To determine if

MLK3 and B-Raf interaction is dependent on phosphorylation of Ser705 and Ser758,

HEK293 cells were transiently co-transfected with appropriate combinations of EV, wild- type-FLAG-MLK3, S705A-S758A-FLAG-MLK3, and GST-B-Raf. Cells were treated with 2 mM H2O2 for 5 min and a GST pull-down was performed (Figure 3-14). An interaction between wild-type-FLAG-MLK3 and GST-B-Raf was observed in H2O2- treated cells confirming our results from Figure 3-11. The association of S705A-S758A-

FLAG-MLK3 and GST-B-Raf was significantly higher than wild-type-FLAG-MLK3, which suggests the phosphorylation of these residues regulates the binding of MLK3 to

B-Raf. Collectively, the data indicates oxidative stress promotes an interaction between

Figure 3-13: S705A-S758A-FLAG-MLK3 expression in H2O2-treated HEK293 cells decreases activation of MEK1/2 and ERK1/2 as compared to wild-type MLK3 expression. Western blot analysis of indicated proteins from HEK293 cell lysates expressing EV, wild-type-FLAG-MLK3, and S705A-S758A-FLAG-MLK3 and treated with 2 mM H2O2 for 30 min. n=3.

MLK3 and B-Raf, and the ERK1/2-mediated phosphorylation of MLK3 function in the activation of the B-Raf—MEK1/2—ERK1/2 signaling module.

Figure 3-14: The association between wild-type FLAG-MLK3 and GST-B-Raf in

H2O2-treated HEK293 cells is increased upon MLK3 S705A-S758A mutation.

Western blot analysis of indicated proteins from HEK293 cells co-expressing different combinations of EV, wild-type-FLAG-MLK3, S705A-S758A-FLAG-MLK3, or GST-B-

Raf, treated with 2 mM H2O2 for 5 min, and subjected to a GST pull-down. The amount of pull-down (GST-B-Raf) and bound (FLAG-MLK3) proteins was quantified. Different capital letters over the bars indicate statistical significance, while same letters do not. n=3.

3.5 MLK3 promotes oxidative stress-induced invasion of colon cancer cells through a mechanism which requires phosphorylation of Ser705 and Ser758

MLK3 promotes the migration and/or invasion of several human cancers (J. Chen

& Gallo, 2012; J. Chen et al., 2010; Chien et al., 2011; Cronan et al., 2012; Lan et al.,

2017; Misek et al., 2017; P. Mishra et al., 2010; Rattanasinchai et al., 2017; Swenson-

Fields et al., 2008; Zhan et al., 2012; J. Zhang et al., 2014), and system biology approaches and computational studies suggest both MLK3 and ERK2 are master regulators of CRC cell invasion (Wlochowitz et al., 2016). To investigate whether MLK3 is important for colon cancer cell invasion in a ROS-, ERK1/2-, and phosphorylation of

705 758 Ser and Ser -dependent mechanism, we first examined if prolonged H2O2 treatment affected colon cancer cell invasion and ERK1/2 activation. HCT116 cells were either untreated or treated with 250 µM H2O2 for 12 h and then invasion was analyzed by an in vitro FluoroBlok tumor invasion assay with Matrigel (Figure 3-15A). In comparison to untreated cells, H2O2 treatment significantly increased cell invasion. Significant ERK1/2 activation was observed in HCT116 cells treated with 250 µM H2O2 for 12 h as compared to untreated cells (Figure 3-15B). Next, we investigated whether MLK3 is required for oxidative stress-induced invasion of colon cancer cells. HCT116 cells were transfected with either 100 nM NS or MLK3 siRNA and treated with 250 µM H2O2 for 12 h. In

H2O2-treated cells, MLK3 siRNA knockdown significantly reduced cell invasion in comparison to cells transfected with NS siRNA (Figure 3-16). To determine if the phosphorylation of Ser705 and Ser758 is required for MLK3-dependent oxidative stress- induced colon cancer cell invasion, wild-type- and S705A-S758A-FLAG-MLK3 were expressed in HCT116 cells, and cells were treated with 250 µM H2O2 for 12 h followed

Figure 3-15: Prolonged oxidative stress increases invasion and activates ERK1/2 in

HCT116 cells. (A) Cell invasion, assessed by the FluoroBlok invasion assay and fluorescence microscopy, in HCT116 cells treated with 250 µM H2O2 for 12 h.

RFUs=relative fluorescence units. Scale bars represent 200 µm. n=3. (B) Western blot analysis of indicated proteins from HCT116 cell lysates treated with 250 µM H2O2 for 0 or 12 h. Lanes 1 and 2 are non-contiguous. n=3.

by cell invasion analysis. Wild-type-FLAG-MLK3 significantly increased colon cancer cell invasion in comparison to the EV control, while S705A-S758A-FLAG-MLK3 did not (Figure 3-17). Taken together, these results indicate MLK3 is required for the oxidative stress-induced invasion of colon cancer cells, and phosphorylation of Ser705 and

Ser758 are critical for this process.

Figure 3-16: MLK3 siRNA knockdown impairs invasion of H2O2-treated HCT116 cells. Cell invasion assessed as in 3-15A and western blot analysis in HCT116 cells expressing NS or MLK3 siRNA and treated with 250 µM H2O2 for 12 h. Lanes 1 and 2 of are non-contiguous. Scale bars represent 100 µm. n=3.

Figure 3-17: S705A-S758A-FLAG-MLK3 expression decreases invasion of HCT116 cells under prolonged oxidative stress as compared to wild-type MLK3 expression.

Cell invasion assessed as in 3-15A and western blot analysis in HCT116 cells expressing

EV, wild-type-FLAG-MLK3, or S705A-S758A-FLAG-MLK3 and treated with 250 µM

H2O2 for 12 h. Scale bars represent 100 µm. n=3.

3.6 GA reduces the abundance of endogenous MLK3 protein in colorectal and ovarian cancer cells

MLK3 interacts with the molecular chaperone heat shock protein 90 (Hsp90) and the co-chaperone p50cdc37, which function in the folding and stabilization of proteins and are required for MLK3 signaling (Whitesell et al., 2005; H. Zhang et al., 2004).

Geldanamycin (GA) is an Hsp90 ATPase inhibitor, which triggers the dissociation of

Hsp90 from its client proteins (Haupt et al., 2012; Roe et al., 1999; Stebbins et al., 1997;

H. Zhang et al., 2004). To determine the effect of Hsp90 on endogenous MLK3 protein in colorectal cancer cells, HCT116 cells were treated with either vehicle or 10 µM GA for 8 h (Figure 3-18). GA induced a decline in the amount of MLK3 protein as compared to the vehicle control. To examine if GA treatment also results in a decline of MLK3 protein in

Figure 3-18: GA treatment decreases the amount of MLK3 protein in HCT116 cells.

Western blot analysis of indicated proteins from HCT116 cells treated with vehicle or 10

µM geldanamycin (GA) for 8 h. n=3. Experiment performed by both April Schroyer and

Nicholas Stimes.

ovarian cancer cells, SKOV3 cells were treated with GA for either 0, 4, 6, 8, 10, or 12 h

(Figure 3-19). In SKOV3 cells treated with GA for 6 h, the amount of MLK3 protein was reduced to approximately 50% of the amount of MLK3 protein in the untreated 0 h cells.

Only 15% of MLK3 protein remained after 12 h GA treatment compared to the control.

GA treatment also reduced the activation of ERK1/2 by 4 h and prevented any activation of ERK1/2 throughout the 12 h time-course.

Figure 3-19: GA treatment reduces the abundance of MLK3 protein and ERK1/2 activation in SKOV3 cells. Western blot analysis of indicated proteins from SKOV3 cells treated with 10 µM GA for either 0, 4, 6, 8, 10, or 12 h (Blessing et al., 2014).

To determine the effect of GA treatment on the amount of MLK3/MAP3K11 mRNA, RT-

PCR was performed with MLK3 primers on cDNA prepared from total RNA isolated

from SKOV3 (Figure 3-20A) , HEY1B (Figure 3-20B), and TOV21G (Figure 3-20C) ovarian cancer cells were treated with GA for 0, 4, 8, and 12 h. GA treatment had no effect on the amount of MLK3/MAP3K11 mRNA at any time-point compared to the 0 h control, suggesting the GA-induced decrease in MLK3 protein is not correlated with a reduction in MLK3/MAP3K11 gene transcription. To examine if GA affects the half-life

Figure 3-20: GA treatment does not affect MLK3/MAP3K11 mRNA concentration in several ovarian cancer cells. (A) RT-PCR results of MLK3/MAP3K11 mRNA, normalized to b-actin, from SKOV3 cells treated with 10 µM GA for the indicated time periods. n=3. (B) RT-PCR results of MLK3/MAP3K11 mRNA, normalized to b-actin, from HEY1B cells treated with 10 µM GA for the indicated time periods. n=3. (C) RT-

PCR results of MLK3/MAP3K11 mRNA, normalized to b-actin, from TOV21G cells treated with 10 µM GA for the indicated time periods. n=3. (Blessing et al., 2014).

of MLK3 protein, SKOV3 cells were treated with either 50 µM of the protein synthesis inhibitor cycloheximide (CHX) alone or CHX followed by GA treatment 1 h later for either 0, 4, 6, 8, 10, or 12 h (Figure 3-21). The amount of MLK3 protein in the 12 h CHX sample was reduced to 39% of the amount in the untreated 0 h cells, while the amount of 74

MLK3 protein in the 12 h CHX+GA sample was reduced to 3% of the amount in the control cells, suggesting GA decreases the half-life of MLK3 protein in SKOV3 cells. To

Figure 3-21: GA treatment decreases the half-life of MLK3 protein in SKOV3 cells.

Western blot analysis of indicated proteins from SKOV3 cells pretreated with 50 µM cycloheximide (CHX) followed by either vehicle (left panel) or 10 µM GA (right panel) for either 0, 4, 6, 8, 10, or 12 h (Blessing et al., 2014).

determine whether the decline in MLK3 protein in response to GA is due to proteasomal degradation, SKOV3, HEY1B, and TOV21G cells were pretreated with 10 µM of the proteasome inhibitor MG132 for 1 h followed by vehicle or GA for 6 h (Figure 3-22).

MG132 prevented the GA-induced reduction in MLK3 protein. Taken together, the data suggests Hsp90 is required for MLK3 protein stability in colorectal and ovarian cancer cells by protection from proteasomal degradation.

Figure 3-22: GA-induced decrease in MLK3 protein is proteasome-dependent. (A)

Western blot analysis of indicated proteins from SKOV3 cells pretreated with vehicle or

10 µM MG132 for 2 h followed by vehicle or 10 µM GA for 6 h. (B) Western blot analysis of indicated proteins from HEY1B cells pretreated with vehicle or 10 µM

MG132 for 2 h followed by vehicle or 10 µM GA for 6 h. (C) Western blot analysis of indicated proteins from TOV21G cells pretreated with vehicle or 10 µM MG132 for 2 h followed by vehicle or 10 µM GA for 6 h. (Blessing et al., 2014).

3.7 Heat shock or osmotic stress decreases the amount of endogenous MLK3 protein in ovarian cancer cells, while TNFa does not

To determine whether stress stimuli also reduced the abundance of endogenous

MLK3 protein, SKOV3 cells were exposed to either heat shock (42°C) or osmotic stress

(250 mM sorbitol) for the indicated time periods. The amount of MLK3 protein in the 10 h heat shock sample was reduced to 1% of the amount of MLK3 protein in the 37°C 0 h control sample (Figure 3-23), while the amount of MLK3 protein in the 10 h osmotic stress sample was reduced to 13% of the amount of MLK3 protein in the untreated 0 h control sample. The results indicate both heat shock and osmotic stress respectively promote a decline in the abundance of MLK3 protein. Heat shock or osmotic stress also reduced the activation of ERK1/2. To determine the effect of heat shock or osmotic stress on the amount of MLK3/MAP3K11 mRNA, RT-PCR was performed with MLK3 primers on cDNA prepared from total RNA isolated from SKOV3 cells subjected to heat shock

(Figure 3-24A) or treated with 250 mM sorbitol (Figure 3-24B) for the indicated time periods. Both experimental conditions had no effect on the amount of MLK3/MAP3K11 mRNA at any time-point compared to the 0 h control, suggesting the heat shock- and osmotic stress-induced decrease in MLK3 protein is not correlated with a reduction in

MLK3/MAP3K11 gene transcription. To examine if either heat shock or osmotic stress affects the half-life of MLK3 protein, SKOV3 cells were treated with either 50 µM CHX alone or CHX followed by either heat shock or osmotic stress 1 h later for the indicated time periods (Figure 3-25) The amount of MLK3 protein in the 10 h CHX sample was reduced to 59% of the amount in the untreated 0 h cells, while the amount of MLK3 protein in the 10 h CHX + heat shock or sorbitol samples was reduced to 0% of the

Figure 3-23: Both heat shock and osmotic stress induce a decline in MLK3 protein and ERK1/2 activation. (A) Western blot analysis of indicated proteins from SKOV3 cells exposed to 42°C heat shock for either 0, 2, 4, 6, 8, or 10 h. (B) Western blot analysis of indicated proteins from SKOV3 cells treated with 0.25 M sorbitol for either 0, 4, 6, 8, or 10 h. (Blessing et al., 2014).

amount in the control cells, suggesting heat shock and osmotic stress both decrease the half-life of MLK3 protein in SKOV3 cells. To elucidate if the pro-inflammatory cytokine

TNFa could elicit a decline in MLK3 protein abundance, SKOV3 cells were treated with

20ng/ml TNFa for the indicated time periods (Figure 3-26). TNFa treatment did not cause a reduction in MLK3 protein, rather the amount of MLK3 protein increased compared to the 0 h control.

Figure 3-24: Both heat hock and osmotic stress do not affect MLK3/MAP3K11 mRNA concentration. (A) RT-PCR results of MLK3/MAP3K11 mRNA, normalized to b-actin, from SKOV3 cells exposed to 42°C heat shock for the indicated time periods. n=3. (B) RT-PCR results of MLK3/MAP3K11 mRNA, normalized to b-actin, from

SKOV3 cells treated with 0.25 M sorbitol for the indicated time periods. n=3. (Blessing et al., 2014).

Figure 3-25: Both heat shock and osmotic stress reduce the half-life of MLK3 protein. Western blot analysis of indicated proteins from SKOV3 cells pretreated with 50

µM cycloheximide (CHX) followed by either vehicle (left panel), 42°C heat shock

(middle panel), or 0.25 M sorbitol (right panel) for the indicated time periods (Blessing et al., 2014).

Figure 3-26: TNFa does not induce a decline in MLK3 protein in SKOV3 cells.

Western blot analysis of indicated proteins from SKOV3 treated with 20 ng/ml TNFa for either 0, 2, 4, 6, 8, 10, or 12 h (Blessing et al., 2014).

Chapter 4

Discussion

We propose a model (Figure 4-1) in which ERK1/2 activated by oxidative stress phosphorylates MLK3 on Ser705 and Ser758. Phosphorylated MLK3 binds B-Raf and promotes B-Raf, MEK1/2, and ERK1/2 activation, leading to maintenance of the

ERK1/2-phosphorylated MLK3 and heightened activation of ERK1/2 signaling. This positive feedback loop (PFL) increases invasion of colon cancer cells in response to oxidative stress. Collectively, our data support the hypothesis of an ERK1/2—MLK3—

B-Raf—MEK1/2—ERK1/2 PFL. We observed significant MEK1/2 (Figure 3-1) and

ERK1/2 (Figures 3-1, 3-2, 3-4A, 3-4B, 3-5, 3-6, 3-12, and 3-15B) activation in H2O2- treated HCT116 cells, indicating activation of the B-Raf—MEK1/2—ERK1/2 MAPK pathway. NAC pretreatment and MLK3 siRNA knockdown revealed ERK1/2 activation to be both ROS- and MLK3-dependent (Figures 3-2 and 3-12). In V600E-positive melanoma cell lines A375 and A2058, MLKs 1-4 act as MEK1 kinases that reactivate the

MEK1/2—ERK1/2 pathway mediating resistance to RAF inhibitors (Marusiak et al.,

2014). Inhibition of MLK3 kinase activity did not decrease ERK1/2 activation in response to oxidative stress (Figures 3-4A and 3-4B), suggesting the MLK3-dependent

ERK1/2 activation in HCT116 cells under oxidative stress occurs through a kinase- 81

Figure 4-1: Proposed model for MLK3-mediated invasion of colon cancer cells under oxidative stress. Oxidative stress activates ERK1/2, which then phosphorylates

MLK3 on Ser705 and Ser758. Phosphorylated MLK3 interacts with and promotes B-Raf activation in a kinase-independent manner, and MLK3 induces further ERK1/2 activation and invasion of colon cancer cells in a positive feedback loop.

independent mechanism. As previously mentioned, MLK3 functions as a scaffold for B-

Raf and Raf-1 transactivation leading to ERK1/2 activation (Chadee & Kyriakis, 2004;

Chadee et al., 2006). We detected an interaction between wild-type-FLAG-MLK3 and

GST-B-Raf as well as an interaction between endogenous MLK3 and both endogenous

B-Raf and ERK1/2 in cells exposed to oxidative stress (Figures 3-14, 3-11, and 3-7). We identified a ROS- and ERK1/2-dependent phosphorylation of MLK3 on Ser705 and Ser758, which was independent of MLK3 activation (Figures 3-2, 3-3, 3-4A, 3-4B, 3-5, 3-6, and

3-9). Furthermore, mutation of Ser705 and Ser758 to alanine significantly decreased the capacity of FLAG-MLK3 to activate both MEK1/2 and ERK1/2, but did not significantly affect FLAG-MLK3-dependent activation of JNK (Figure 3-13), which indicates phosphorylation of these residues are crucial for the MLK3 kinase-independent activation of B-Raf rather than MLK3 kinase activity. We observed a significant increase in the interaction between S705A-S758A-FLAG-MLK3 and GST-B-Raf compared to the interaction between WT-FLAG-MLK3 and GST-B-Raf (Figure 3-14), which suggests the

ERK1/2-mediated phosphorylation of MLK3 limits the interaction of MLK3 with B-Raf; however, we still detect a significant interaction between WT-MLK3 and B-Raf in H2O2- treated cells (Figure 3-11). The ERK1/2-mediated phosphorylation of MLK3 is required for MLK3-dependent activation of MEK1/2 and ERK1/2 providing positive feedback onto the ERK1/2 signaling pathway, which is the mechanism reported in our model

(Figure 4-1). We propose the decrease in MEK1/2 and ERK1/2 activation observed with

S705A-S758A-FLAG-MLK3 is due to its inability to activate B-Raf perhaps through influencing B-Raf homodimerization and/or heterodimerization with Raf-1.

Mathematical modeling of MAPK networks suggested the presence of an additional PFL involving MLK3 in response to ROS (Figure 4-2) (H. S. Lee et al., 2014).

An MKK4/7—JNK—POSH—active MLK3—MKK4/7 PFL is proposed to mediate the activating phosphorylations of ERK1/2 and JNKs by different ROS concentrations. JNK activation by this PFL promotes c-Jun-dependent expression of MKPs, which negatively regulate ERK1/2. Active JNK can cause direct positive feedback onto MLK3 through phosphorylation (Schachter et al., 2006) and along with the MLKs through stabilization of POSH protein (C. Wang et al., 2010; Z. Xu et al., 2005). POSH can in turn stabilize the MLKs and act as a scaffold promoting their activation, thus inducing JNK (Z. Xu et al., 2003). It is essential to note ERK1/2 is not a component of the PFL reported in Lee et al., 2014, rather ERK1/2 activation is attenuated as a consequence of the PFL (H. S. Lee et al., 2014). In addition, the MKK4/7—JNK—POSH—active MLK3—MKK4/7 PFL is dependent on MLK3 kinase activity. Therefore, our proposed PFL is mechanistically distinct from the PFL described in Lee et al., 2014 as it involves ERK1/2 activation and does not require MLK3 kinase activity. We propose MLK3, depending on its activation state, could act as a molecular switch to increase or decrease ERK1/2 activation in response to ROS. Inactive MLK3 could increase ERK1/2 activation through its scaffold function in activating B-Raf, while active MLK3 may decrease ERK1/2 activation through an active JNK—c-Jun—MKP pathway. In the latter, inhibition of MLK3 kinase activity would result in a decrease in MKP expression and therefore an increase in

Figure 4-2: MLK3 as a molecular switch for ERK1/2 activation in response to ROS.

MLK3, depending on its activation state, increases or decreases ERK1/2 activation in response to ROS. Active MLK3, along with POSH, MKK4/7, JNK, and c-Jun, promotes the transcription of MKPs, which negatively regulate ERK1/2. Inactive MLK3 functions as a scaffold in B-Raf and Raf-1 transactivation enhancing ERK1/2 signaling.

ERK1/2 activation. In our experiments, Cep1347 did not increase ERK1/2 activation in response to ROS (Figures 3-4A and 3-4B); hence, we propose the MKK4/7—JNK—

POSH—active MLK3—MKK4/7 PFL is not stimulated by oxidative stress in HCT116 cells.

Prolonged H2O2 treatment significantly increased both the activation of ERK1/2

(Figure 3-15B) and invasion of colon cancer cells (Figure 3-15A). MLK3 siRNA knockdown significantly decreased the oxidative stress-induced invasion of HCT116 cells (Figure 3-16). Significantly reduced invasion was also observed with S705A-

S758A-FLAG-MLK3 as compared to wild-type-FLAG-MLK3 in H2O2-treated HCT116 cells (Figure 3-17). Therefore, we propose MLK3 is required for oxidative stress-induced colon cancer cell invasion through a mechanism dependent on phosphorylation of Ser705 and Ser758. In ovarian cancer cells, MLK3 is required for ERK1/2-dependent activation of matrix metalloproteinases (MMPs) and induction of AP-1-dependent MMP expression leading to an invasive phenotype (Zhan et al., 2012). Possibly, the ERK1/2—MLK3—B-

Raf—MEK1/2—ERK1/2 PFL in colon cancer cells exacerbates invasion through enhanced ERK1/2—AP-1—MMP signaling. The mobility shift of MLK3 was not detected in Figure 3-15B; however, a slight mobility shift of MLK3 in response to 12 h

H2O2 treatment was observed in Figure 3-16. The electrophoretic shift of MLK3 is not as evident with the lower concentration of H2O2 as compared to the higher concentration, in which the entire pool of MLK3 protein is phosphorylated and ERK1/2 activation is more robust. It is also possible the ERK1/2 phosphorylation of MLK3 at high ROS concentrations is stable due to low MLK3 phosphatase expression and/or activity, whereas the ERK1/2 phosphorylation of MLK3 at low ROS concentrations is less stable

due to high MLK3 phosphatase expression and/or activity; however, a MLK3 phosphatase has not been identified.

While we are reporting a mechanism for positive regulation of MLK3 by ERK1/2,

ERK1/2 is implicated in the negative regulation of MLK3 as well (Liou, Zhang, et al.,

2010). When transiently expressed in untreated HEK293 cells or immortalized human T- lymphocyte (Jurkat TAg) cells stimulated with phorbol 12-myristate 13-acetate (PMA) or

TNFa, MLK3 undergoes a proteolytic event and generation of a stable C-terminal fragment (CTF). The MEK1/2 inhibitor UO126 prevented PMA- and TNFa-induced

MLK3 proteolysis, suggesting CTF generation is dependent on active ERK1/2. Cleavage of MLK3 occurs within the kinase domain between the amino acids Gln251 and Pro252; thus, the MLK3 CTF is 596 amino acids in length and lacks the N-terminal SH3 domain, but contains a truncated kinase domain and intact leucine-zippers and CRIB domain. The

MLK3 CTF negatively regulates full length (FL) MLK3 activation and signaling via two mechanisms: 1) MLK3 CTF heterodimerizes with FL MLK3. Since the CTF contains a truncated kinase domain, it cannot phosphorylate and activate the FL MLK3 within the heterodimer. 2) The CTF sequesters the active form of Cdc42 from FL MLK3 resulting in less activation of MLK3. The MLK3 protease and the exact role of ERK1/2 in MLK3 proteolysis remain to be identified as well as if endogenous MLK3 undergoes proteolysis and generates a CTF.

We have identified a kinase-independent function of MLK3 in the malignancy of colorectal cancer under oxidative stress; therefore, in this cellular context, therapeutic strategies must target the scaffold function of MLK3 or regulate the amount of total

MLK3 protein in cells. A reduction in the amount of MLK3 protein was observed in

response to GA in HCT116, SKOV3, HEY1B, and TOV21G cells (Figures 3-18, 3-19, 3-

21, 3-22A, 3-22B, 3-22C), suggesting Hsp90 is critical for MLK3 protein stability in colon and ovarian cancer cells. The decline in MLK3 protein induced by GA was dependent on the proteasome in SKOV3 cells (Figures 3-22A, 3-22B, and 3-22C), indicating GA stimulates the proteasomal degradation of MLK3. Both heat shock (Figure

3-23A) and sorbitol (Figure 3-23B), but not TNFa (Figure 3-26), also decreased the amount of MLK3 protein in SKOV3 cells. GA, heat shock, and osmotic stress also decreased the activation of ERK1/2 (Figures 3-19, 3-23A, and 3-23B). The decline in

MLK3 protein concentration in response to GA, heat shock, and osmotic stress in ovarian cancer cells was recently investigated (Blessing et al., 2014). Upon Hsp90 dissociation from MLK3 in response to GA, osmotic stress, and heat shock, Hsp70 recruits the E3 ubiquitin ligase carboxyl terminus of Hsc70-interacting protein (CHIP) to MLK3, and

CHIP mediates MLK3 Lys48-linked polyubiquitination and proteasome-dependent degradation leading to suppression of SKOV3 ovarian cancer cell invasion. Akt also triggers Lys48-linked ubiquitination and proteasome-dependent degradation of MLK3 through direct phosphorylation of MLK3 at Thr477 (Humphrey et al., 2014). In addition,

Akt1/2 negatively regulates POSH, Rac1, MLK3, MKK4/7, and JNKs 1-3 signaling pathway at various other tiers along the cascade (Figueroa et al., 2003; R. Wang et al.,

2006; Y. Xu et al., 2009; Yin et al., 2005; Q. G. Zhang, Han, et al., 2006; Q. G. Zhang,

Wang, et al., 2006). In some cases, Akt provides neuroprotection after global cerebral ischemia. Interestingly, GA provides neuroprotection in ischemic brain injury not only through suppression of MLK3 and ASK1 expression, but through an Akt-dependent mechanism as well (Wen et al., 2008; Yin et al., 2017).

Endogenous MLK4β protein is also degraded in response to GA, heat shock, and osmotic stress via a mechanism dependent on both Hsp70 and CHIP in ovarian cancer cells (Blessing et al., 2017). MLK4b is a negative regulator of MLK3 activation, MAPK signaling, and cell invasion (Abi Saab et al., 2012; Blessing et al., 2017). In either

SKOV3, HCT116, or HEY1B cells, MLK4b suppresses the activation of MLK3,

MKK3/6, JNK, p38, and/or ERK1/2 in response to a variety of stimuli (Abi Saab et al.,

2012; Blessing et al., 2017). Furthermore, epitope-tagged MLK4b and MLK3 interact in

HCT116 cells, and MLK4b blocks both MMP-9 gelatinase activity and cell invasion in

SKOV3 cells (Abi Saab et al., 2012).

Elevated levels of total MLK3 protein were observed in ovarian (Zhan et al.,

2012), breast (J. Chen et al., 2010), and melanoma (J. Zhang et al., 2014) cancer cells. In melanoma cell lines, both MLK3 mRNA and protein were significantly increased compared to a normal immortalized epidermal melanocyte (NHEM) cell line (J. Zhang et al., 2014). Bioinformatic tools uncovered a microRNA-125b (miR-125b) seed sequence binding site in the MLK3 3’ untranslated region (UTR). Expression of miR-125b, which negatively regulates MLK3 expression, is significantly decreased in metastatic melanomas (MM) accounting for the increase in both MLK3 mRNA and protein. miR-

125b is frequently down-regulated in hepatocellular carcinoma (Gong et al., 2013), cervical, breast, lung, ovarian cancers, and MM (Glud et al., 2011; Kappelmann et al.,

2013) possibly accounting for the increased levels of total MLK3 protein observed in ovarian and breast cancer. Up-regulation of MLK3/MAP3K11 mRNA, as well as MLKs 1,

2, and 4, was also observed in 9 of 21 melanoma patients and correlated with drug resistance in B-Raf V600E-positive patients (Marusiak et al., 2014). In this cellular

context, the MLKs acted as MEK1/2 kinases to reactivate ERK1/2 signaling after treatment with RAF kinase inhibitors vemurafenib or dabrafenib. Interestingly, miR-125a expression is upregulated via TGF-β signaling in human B-Raf V600E melanoma cells after the acquisition of resistance to B-Raf inhibitors. While miR-125a suppresses MLK3 expression, melanoma cells survive through upregulation of Akt (Koetz-Ploch et al.,

2017).

miR-199b-5p promotes proliferation of pancreatic beta cells by down-regulation of MLK3 expression during pancreatic regeneration in a partial pancreatectomy rodent model (Sato-Kunisada et al., 2016). Reduction of miR-199a-5p, which directly targets

MLK3/MAP3K11, contributes to the tumorigenesis of bladder urothelial carcinoma by regulating MLK3 (T. Song et al., 2015). miR-199a-5p is significantly down-regulated in bladder urothelial carcinoma. Expression of miR-199a-5p inhibited the tumorigenesis of bladder urothelial carcinoma in vitro and in vivo by inducing apoptosis. The expression level of miR-199a-5p was conversely correlated with MLK3, which promoted proliferation and interestingly inhibited apoptosis in bladder cancerous cells. Long non- coding RNA (lncRNA) small nucleolar RNA host gene 12 (SNHG12) expression was increased in hepatocellular carcinoma tissues compared to normal tissues. SNHG12 interacts with and acts as an endogenous sponge for miR-199a/b-5p leading to expression of MLK3 and promotes tumorigenesis and metastasis in hepatocellular carcinoma (Lan et al., 2017). Therefore, perhaps stresses such as GA, heat shock, and osmotic stress or proteins such as Hsp70, CHIP, MLK4b, or Akt, or miRNA may aid in combating oxidative-stress induced MLK3-dependent invasion of colon cancer cells.

Likely the most interesting candidate for interruption of the oxidative stress—

ERK1/2—MLK3—B-Raf—MEK1/2—ERK1/2—colon cancer cell invasion pathway is the Neurofibromatosis 2 (NF2) tumor suppressor protein Merlin. Merlin, the NF2 gene product, is an ezrin/radixin/moesin (ERM) family protein whose C-terminal region directly associates with MLK3 negatively regulating both its kinase-dependent and - independent functions respectively by disrupting the interactions between MLK3 and its upstream activator Cdc42 or those between MLK3 and either B-Raf or Raf-1 (Chadee et al., 2006; Zhan et al., 2010; Zhan et al., 2011). Merlin inhibits not only the catalytic activities of MLK3, B-Raf, and ERK1/2, but JNKs as well. Interestingly, MLK3 is required for the merlin-dependent suppression of T29 colon cancer proliferation and

SW10 mouse normal schwann cell invasion (Zhan et al., 2011).

Our findings indicate ERK1/2 is significantly activated by oxidative stress and promotes the phosphorylation of MLK3. In H2O2-treated HCT116 cells, MLK3 interacts with both B-Raf and ERK1/2 to promote further ERK1/2 activation in a manner that does not require MLK3 kinase activity, but is dependent on phosphorylation of Ser705 and

Ser758. We elucidated a requirement for MLK3 in the oxidative stress-induced invasion of colon cancer cells, which is also reliant on phosphorylation of Ser705 and Ser758. We propose a PFL involving MLK3 and ERK1/2 which promotes the MLK3 kinase- independent activation of B-Raf, MEK1/2, ERK1/2, and invasion of colon cancer cells.

This study identifies MLK3 as a direct ERK1/2 substrate and a positive regulator of both

ERK1/2 activation and oxidative stress-induced colon cancer cell invasion. In addition, our findings offer insight into the ill-defined kinase-independent functions of MLK3 and define the ERK1/2—MLK3—B-Raf—MEK1/2—ERK1/2 PFL as a molecular

mechanism by which MLK3 supports a malignant phenotype in CRC under oxidative stress, which may be combatted by certain stresses, GA, Hsp70 and CHIP, Akt, miRNA,

MLK4b, or Merlin.

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Appendix A

Multiple sequence alignment of human MLKs 1-4.

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MLK1 1 MEPSRALL--GCLASAAAAAPPGEDGAGAGAEEEEEEEEEAAAAVGPGELGCDAPLPYWT MLK2 1 MEEEEGAVAKE----W------GT-T------PAGPVWT MLK3 1 MEPLKSLFLKSPLGSWNGSG-SGGGGGGGGGRPEGSPK--AA-G------YANPVWT MLK4 1 MALRGAAG------ATDTPV-SSAGGAPGGSASSSSTSSGGSAS------AGAGLWA

MLK1 59 AVFEYEAAGEDELTLRLGDVVEVLSKDSQVSGDEGWWTGQL-NQRVGIFPSNYVTPRSAF MLK2 23 AVFDYEAAGDEELTLRRGDRVQVLSQDCAVSGDEGWWTGQLPSGRVGVFPSNYVAPGAP- MLK3 48 ALFDYEPSGQDELALRKGDRVEVLSRDAAISGDEGWWAGQV-GGQVGIFPSNYVSRGGG- MLK4 45 ALYDYEARGEDELSLRRGQLVEVLSQDAAVSGDEGWWAGQV-QRRLGIFPANYVAPCRPA

MLK1 118 SSRCQPGGEDPSCYPPIQLLEIDFAELTLEEIIGIGGFGKVYRAFWIGDEVAVKAARHDP MLK2 82 ------AAPA-GLQLPQEIPFHELQLEEIIGVGGFGKVYRALWRGEEVAVKAARLDP MLK3 106 ------PPPC------EVASFQELRLEEVIGIGGFGKVYRGSWRGELVAVKAARQDP MLK4 104 AS-----PAPPPS-RPSSPVHVAFERLELKELIGAGGFGQVYRATWQGQEVAVKAARQDP

MLK1 178 DEDISQTIENVRQEAKLFAMLKHPNIIALRGVCLKEPNLCLVMEFARGGPLNRVLS---- MLK2 132 EKDPAVTAEQVCQEARLFGALQHPNIIALRGACLNPPHLCLVMEYARGGALSRVLA---- MLK3 151 DEDISVTAESVRQEARLFAMLAHPNIIALKAVCLEEPNLCLVMEYAAGGPLSRALA---- MLK4 158 EQDAAAAAESVRREARLFAMLRHPNIIELRGVCLQQPHLCLVLEFARGGALNRALAAANA

MLK1 234 ------GKRIPPDILVNWAVQIARGMNYLHDEAIVPIIHRDLKSSNILILQKVEN MLK2 188 ------GRRVPPHVLVNWAVQVARGMNYLHNDAPVPIIHRDLKSINILILEAIEN MLK3 207 ------GRRVPPHVLVNWAVQIARGMHYLHCEALVPVIHRDLKSNNILLLQPIES MLK4 218 APDPRAPGPRRARRIPPHVLVNWAVQIARGMLYLHEEAFVPILHRDLKSSNILLLEKIEH

MLK1 283 GDLSNKILKITDFGLAREWHRTTKMSAAGTYAWMAPEVIRASMFSKGSDVWSYGVLLWEL MLK2 237 HNLADTVLKITDFGLAREWHKTTKMSAAGTYAWMAPEVIRLSLFSKSSDVWSFGVLLWEL MLK3 256 DDMEHKTLKITDFGLAREWHKTTQMSAAGTYAWMAPEVIKASTFSKGSDVWSFGVLLWEL MLK4 278 DDICNKTLKITDFGLAREWHRTTKMSTAGTYAWMAPEVIKSSLFSKGSDIWSYGVLLWEL

MLK1 343 LTGEVPFRGIDGLAVAYGVAMNKLALPIPSTCPEPFAKLMEDCWNPDPHSRPSFTNILDQ MLK2 297 LTGEVPYREIDALAVAYGVAMNKLTLPIPSTCPEPFARLLEECWDPDPHGRPDFGSILKR MLK3 316 LTGEVPYRGIDCLAVAYGVAVNKLTLPIPSTCPEPFAQLMADCWAQDPHRRPDFASILQQ MLK4 338 LTGEVPYRGIDGLAVAYGVAVNKLTLPIPSTCPEPFAKLMKECWQQDPHIRPSFALILEQ

MLK1 403 LTTIEESGFFEMPKDSFHCLQDNWKHEIQEMFDQLRAKEKELRTWEEELTRAALQQKNQE MLK2 357 LEVIEQSALFQMPLESFHSLQEDWKLEIQHMFDDLRTKEKELRSREEELLRAAQEQRFQE MLK3 376 LEALEAQVLREMPRDSFHSMQEGWKREIQGLFDELRAKEKELLSREEELTRAAREQRSQA MLK4 398 LTAIEGAVMTEMPQESFHSMQDDWKLEIQQMFDELRTKEKELRSREEELTRAALQQKSQE

MLK1 463 ELLRRREQELAEREIDILERELNIIIHQLCQEKPRVKKRKGKFRKSRL-KLKDG-NRISL MLK2 417 EQLRRREQELAEREMDIVERELHLLMCQLSQEKPRVRKRKGNFKRSRLLKLREGGSHISL MLK3 436 EQLRRREHLLAQWELEVFERELTLLLQQVDRERPHVRRRRGTFKRSKL-RARDGGERISM MLK4 458 ELLKRREQQLAEREIDVLERELNILIFQLNQEKPKVKKRKGKFKRSRL-KLKDG-HRISL

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MLK1 521 PSDFQHKFTVQASPTMDKRKSLINSRSSPPASPTIIPRLRAIQLTPGESS------MLK2 477 PSGFEHKITVQASPTLDKRKGSD--GASPPASPSIIPRLRAIRLTPVDCGGSSSGSSSGG MLK3 495 PLDFKHRITVQASPGLDRRRNVF--EVGPGDSPT-FPRFRAIQLEPAEPG------MLK4 516 PSDFQHKITVQASPNLDKRRSLNSSSSSPPSSPTMMPRLRAIQLTSDESN------

MLK1 571 -KTWGRSSVVPKEEGEEEEKRAPKKK-GRTWGPGTLGQKELASGDEGSPQRREKANGLST MLK2 535 SGTWSRGGPPKKEELV-----GGKKK-GRTWGPSSTLQKERVGGEE------MLK3 542 -QAWGRQSPRRLEDSS-----NGERRACWAWGPSSPKPGEAQNGRR------MLK4 566 -KTWGRNTVFRQEEFED-VKRNFKKK-GCTWGPNSIQMKDRTDCKE------

MLK1 629 PSESPHFHLGLKSLVDGYKQWSSSAPNLVKGPRSSPALPGFTSLM-EMALLAASWVVPID MLK2 575 ------RLKGLGEGSKQWSSSAPNLGKSPKHTPIAPGFASLN-EM------MLK3 582 ------RSR-MDEATWYLDSDDSSPLGSPSTPPALNG------MLK4 609 ------RIRPLSDGNSPWSTI---LIKNQKTMPLASLFVDQPGSC------

MLK1 688 IEEDEDS-EGPGSGESRLQHSPSQSYLCIPFPRGEDGDGPSSDGIHEEPTPVNSATSTPQ MLK2 613 -EEFAEAEDGGSSVPPSPYSTP--SYLSVPLPAEP---SPGARAP------WE MLK3 612 ------NPPRPSLEP--EEP------KR MLK4 645 -EEPKLSPDGLEHRKPKQIKLPSQAYIDLPLGKDAQRENPAEAESWEEAASANAATVSIE

MLK1 747 LTPTNSLKR--GGAHHRRCEVALLGCGAVLAATGLGFDLLEAGKCQLLPLEEPEPPAREE MLK2 654 PTPSAPPARWGH-GARRRCDLALLGCATLLGAVGLGADVAEARAAD------GEEQ MLK3 626 PVPAERGS--SS-GTPKLIQRALLRGTALLASLGLGRDLQPPGGP------GRER MLK4 704 MTPTNSLSR--S-PQRKKTESALYGCTVLLASVALGLDLRELHKAQAA--EEPL--PKEE

MLK1 805 KKRREGL-FQRSSRPRRSTSPPSRKLFKKEE--PMLLLGDPSASLTLLSLSSISECNSTR MLK2 703 RRWLDGLFFPRAGRFPRGLSPPARPHGRRED--VGPGL-GLAPSATLVSLSSVSDCNSTR MLK3 672 ------GESPTTPPTPT------MLK4 757 KKKREGI-FQRASKSRRSASPPTSLPSTCGEASSPPSL-PLSSALGILSTP----SFSTK

MLK1 862 SLLRSDSDEIVVYEMPVSP------VEAPPLSPCTHNPLVNVRVERFKRDPNQS MLK2 760 SLLRSDSDEAAP-AAPSP------PPSPPAPTPTPSP-STNPLVDLELESFKKDPRQS MLK3 683 ------PAPCP-TEPPPSPLICFSL-KTPDSP MLK4 811 CLLQMDSEDPLVDSAPVTCDSEMLTPDFCPTAPGSGREP-ALMPRLDTDCSVSRNLPSSF

MLK1 910 LTPTHVTL----TTPSQPSSHRRTPSDGALKPET--LLASRSPSSNGLSPSPGAGMLKTP MLK2 810 LTPTHVTA----A-CAVSRGHRRTPSDGALGQRG-----PPEPAGHG--PGP------MLK3 707 PTPAPLLL----D-LGIPVGQRSAKSP-----RR-----EEEPRGGTVSPPP------MLK4 870 LQQTCGNVPYCASSKHRPSHHRRTMSDGNPTPTGATIISATGASALPLCPSPAP------

MLK1 964 SPSRDPGEFPRLPDPNVVFPPTPRRWNTQQDSTLE--RPKTLEFLPRPRPSANRQRLDPW MLK2 850 ---RDLLDFPRLPDPQALFPA--RRR---PPEFPG--RPTTLTFAPRPRPAASRPRLDPW MLK3 744 ------G------T--SRS---APGTPGTPRSPPLGLISRPRPSPLRSRIDPW MLK4 924 ------HSHL---PR------EVSPK--KHSTVHIVPQRRPASLRSRSDLP

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MLK1 1022 W-FVSPSHARSTSPANSSSTE-TPSNLDSCFASSSSTVEERPGLPALLPFQA-GPL-PPT MLK2 900 K-LVSFGRTLTISPPSRP------DTPE------SP-GP--PSV MLK3 780 S-FVSAGPRPSPLPSPQPAPRRAPW---TLFPDS-DPFWDS---PPANPFQG-GP--QDC MLK4 958 QAYPQT--A------VSQLAQT-ACVVGRPGPHPTQ-FLAAKERTKSH

MLK1 1078 ERTLLDLDAEGQSQDSTVPLCRAELNTHRPAPYEIQQEFWS MLK2 928 QPTLLDMDMEGQNQDSTVPLCGAHGSH------MLK3 829 RAQTKDMGAQAP----WVPEAG----P------MLK4 996 VPSLLDADVEGQSRDYTVPLCRMRSKTSRPSIYELEKEFLS

Figure A-1. Multiple sequence alignment of human MLKs 1-4. Multiple sequence alignment of human MLKs 1-4 generated with T-Coffee and formatted with the

Boxshade program.

125