IDENTIFICATION OF NOVEL SUBSTRATES OF LRRK2,
A PARKINSON’S DISEASE ASSOCIATED KINASE
By
CAROLINE H. LEE
Submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy
Dissertation Adviser: Dr. Shu G. Chen
Department of Biochemistry
CASE WESTERN RESERVE UNIVERSITY
May 2014
Case Western Reserve University
School of Graduate Studies
We hereby approve the thesis/dissertation of
CAROLINE H. LEE
Candidate for the degree of Doctor of Philosophy
Committee Chair
Dr. Michael E. Harris
Committee Members
Dr. Shu G. Chen
Dr. Robert Petersen
Dr. Hung-ying Kao
Dr. Neena Singh
Date of Defense
December 18, 2013
*We also certify that written approval has been obtained for any proprietary
material contained therein.
1
Table of Contents
Table of Contents ...... 2 List of Tables ...... 4 List of Figures ...... 5 List of Abbreviations ...... 6 Acknowledgements ...... 7 Abstract ...... 10 Chapter 1: Review ...... 12 Parkinson’s Disease ...... 13 Clinical features of Parkinson’s disease and its prevalence ...... 13 Pathological features of Parkinson’s disease ...... 13 Genetics of Parkinson’s disease ...... 14 Leucinerich repeat kinase 2 (LRRK2) ...... 16 Prevalence of LRRK2 mutations in PD ...... 16 LRRK2-associated neuropathology ...... 17 Structure of LRRK2 ...... 18 Biological function of LRRK2 ...... 19 Animal models of LRRK2linked PD ...... 21 Identifying substrates of LRRK2 ...... 22 Chemical genetic approach ...... 22 Current findings on proposed substrates ...... 24 Chapter 2: Chemical Genetic Identification of LRRK2 Substrates ...... 28 Abstract ...... 29 Introduction ...... 29 Experimental Procedures ...... 32 Generation of analog-sensitive LRRK2 variants ...... 32 Immunoprecipitation of flag-tagged LRRK2 ...... 33 In vitro kinase assay ...... 33 Indirect competition assay ...... 34 Synthesis of N6-cPe-ATPγS using Ynk1p ...... 34 In vivo Substrate labeling using digitonin ...... 35 Immunoprecipitation of in vivo labeled substrates ...... 36 Mass spectrometry analysis ...... 36 Generation of flag-tagged eIF4G1 variants ...... 37 Results ...... 38 Characterization of analog-sensitive LRRK2 ...... 38 In vivo substrate phosphorylation by AS-G2019S...... 41 Global analysis of potential LRRK2 substrates ...... 43 Validation of eIF4G1 as a LRRK2 substrate ...... 44 Discussion ...... 45 Chapter 3: Future Directions ...... 51
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Tables ...... 58 Table 1. LRRK2 mutations and clinical phenotypes, as reported in various populations...... 59 Table 2. List of small molecule LRRK2 inhibitors. There are several LRRK2 inhibitors with different molecular structures...... 59 Table 3. List of proposed LRRK2 substrates...... 60 Figures ...... 61 Figure 1...... 62 Figure 2...... 62 Figure 3...... 63 Figure 4...... 64 Figure 5...... 65 Figure 6...... 66 Figure 7...... 67 Figure 8...... 68 Figure 9...... 70 Figure 10 ...... 71 Figure 11 ...... 72 Appendix ...... 73 Appendix I ...... 74 References ...... 81
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List of Tables
Table 1. LRRK2 mutations and clinical phenotypes…………………………………60
Table 2. List of small molecule LRRK2 inhibitors…………………………………...60
Table 3. List of proposed LRRK2 substrates…………………………………………61
4
List of Figures
Figure 1. Lewy bodies in PD brains………...... 63
Figure 2. Schematic diagram of LRRK2 functional domains, and disease-associated mutations in LRRK2 ...... 63
Figure 3. Generation of analog-sensitive versions of LRRK2...... 64
Figure 4. AS-LRRK2 shows specificity toward N6-cPe-ATPγS...... 65
Figure 5. AS-LRRK2 and AS-G2019S show similar sensitivity to N6-Bn-
ATPγS...... 66
Figure 6. The kinetics of AS-G2019S induced MBP thiophosphorylation...... 67
Figure 7. In vivo labeling of substrates using AS-G2019S...... 68
Figure 8. Global identification of AS-G2019S substrates using tandem mass
spectrometry...... 69
Figure 9. Tandem mass spectra...... 71
Figure 10. eIF4G1 is a direct substrate of LRRK2...... 72
Figure 11. Putative mechanism of LRRK2 regulation in protein
synthesis...... 73
5
List of Abbreviations
4E-BP1 Eukaryotic translation initiation factor 4E-binding protein 1
AS Analog-sensitive
Bn N6-benzyl-ATPγS
cPe N6-cyclopentyl-ATPγS eIF3 Eukaryotic translation initiation factor 3 eIF4E Eukaryotic translation initiation factor 4E eIF4F Eukaryotic translation initiation factor 4F eIF4G1 Eukaryotic translation initiation factor 4gamma 1
DA Dopaminergic
GFP Green fluorescent protein
HDAC2 Histone deacetylase 2
HEK293T Human embryonic kidney cell line 293 T
LRRK2 Leucine-rich repeat kinase 2
MBP Myelin basic protein
mTOR Mammalian target of rapamycin
NDPK Nucleoside diphosphokinase
PABP1 Polyadenylate-binding protein 1
PD Parkinson’s disease
Phe N6-phenyl-ATPγS
PhEt N6-phenyethyl-ATPγS
PP2A Protein phosphatase 2A
VPS35 Vacuolar protein sorting-associated protein 35
6
Acknowledgements
I would like to thank my thesis adviser Dr. Shu G. Chen, for all the support and advice he has provided during my graduate career. I joined his lab rather in later stage of my graduate career, and I needed to learn the entire field from scratch, yet he has shown me great amplitude of patience and support. He not only trained me to become a professional, but he also provided me a second chance to appreciate the joy of science. I will be forever grateful.
I would also like to thank all my thesis committee members, Dr. Michael Harris,
Dr. Robert Petersen, Dr. Neena Singh, and Dr. Hung-ying Kao, for their kind support and advices. I will always be grateful to Dr. Harris for always being there for me for the past
8 years. I will never forget Dr. Petersen’s warm smiles and hellos in the hallways. It made joining a new department so much more welcoming and easier. I learned so much, so fast in Dr. Singh’s class when I joined the new lab. It was one of the most memorable learning experiences, and without the class I would never have survived my new research. Last but not least, I am grateful for Dr. Kao always keeping me on edge. Your advices shaped me to become a better scientist, to strive for better results. I would also like to thank Drs. Chao Zhang and Kevan Shokat from UCSF for advice on chemical genetic design, and Dr. Janna Kiselar from Case Center for Proteomics and
Bioinformatics for mass spectrometry facility and data analysis.
I would like to thank everyone in the department of biochemistry, especially Dr.
William Merrick, for such overwhelming support and encouragement when I went through difficult times during my graduate career. I spent 10 years in the department of biochemistry, for both undergraduate and graduate school. The biochemistry department
7
is truly a second home for me. I would also like to thank the department of pathology for
administrative arrangements and support that enabled me to conduct the thesis research in the Chen lab.
I would like to thank everyone from my lab, Dr. Luxuan Guo who generated some reagents before I joined the lab, and Dr. Chen Yao, Wen Wang, and William Johnson who helped me tremendously when I needed to learn everything from the beginning.
Will, I am very grateful that you always have been keeping me in good spirit in the lab both scientifically and personally. I could not ask for a better co-worker and a friend, who knows how to tickle me in the brain. And always make me laugh.
I would like to thank all my friends who have been there for me in both good times and bad times. It made my life in Cleveland so much better, and without you all, I might have gone crazy. I wish our friendship will last forever, and I sincerely hope all your science or non-science careers soar and leave a dent in the universe. Cheers, y’all.
Lastly, I would like to thank my family. Judi and Tom Rogers, you have taken me in when I was in need, and treated me like a real family. Without your love and support, I would not be at Case, I would not even be in the States. I am in forever debt, and you will always be my second parents. Sungjoon, meeting you is the best thing that happened in my graduate career. You are the love of my life, and I wish our life to be happily ever after. My little brother, I am thankful that you put up with me when I was going through rough times. I cherish our times together in Cleveland, and in Indiana, and we will make more great memories in our lifetime. Finally, mom and dad, there is no word that I can describe how grateful I am for your unconditional love and support. I will do my best to
8 repay what you have been providing me and to love you with all my heart. 사랑합니다.
오래오래 사세요.
9
Identification of Novel Substrates of LRRK2, a Parkinson’s disease
Associated Kinase
by
CAROLINE H. LEE
Abstract
Parkinson’s disease (PD) is the second most common neurodegenerative disease
characterized neuropathologically by progressive loss of dopaminergic neurons, affecting
about 2% of the general population over the age of 65. Most cases of PD are considered
idiopathic with unknown etiology, although both genetic and environmental factors have
been implicated. Recent population studies have identified mutations in leucine-rich
repeat kinase 2 (LRRK2) as the most frequent known cause for both familial and sporadic
cases of PD. The biological function and substrates of LRRK2 are yet to be discovered.
LRRK2 is an active protein kinase, and common LRRK2 mutations are postulated to
enhance its kinase activity. Thus, it raises an intriguing question about how dysregulated
substrate phosphorylation by LRRK2 may cause PD. To understand the
pathophysiological role of LRRK2, we have identified its substrates using a chemical
genetic approach. In the chemical genetic approach, the ATP-binding pocket of LRRK2
was modified (via gatekeeper mutation) to accommodate not only ATP, but also ATP
analogs that carry bulky side groups. In this way, only the analog-sensitive version of
LRRK2, but not wild-type LRRK2, is able to phosphorylate substrates using the ATP
analogs. In order to distinguish phosphorylation from endogenous, abundant ATP in the
cell, we used various ATPγS analogs that are specific to analog-sensitive LRRK2
10 variants. Using this method, we have successfully identified many cellular proteins that
are specifically phosphorylated by analog-sensitive LRRK2 in a human cell line using
Western blot and tandem mass spectrometry. These putative LRRK2 substrates belong to
proteins involved in various cellular processes. We discovered that two of these proteins,
VPS35 and eIF4G1, are encoded by the corresponding genes linked to PD. Furthermore,
we verified eIF4G1 as a novel LRRK2 substrate, suggesting that LRRK2 may play an
important role in the regulation of protein translation. Our findings may provide useful insights into the pathogenic role of LRRK2 and facilitate the development of therapeutic strategies targeting LRRK2-linked signaling pathway for treatment of PD.
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Chapter 1: Review
12
Parkinson’s Disease Clinical features of Parkinson’s disease and its prevalence
Parkinson’s disease (PD) is a progressive neurodegenerative disorder that affects part of the nervous system that is responsible for movement. It is the most common neurodegenerative movement disorder, and it is the second most common neurodegenerative disorder, after Alzheimer’s disease. PD is characterized by progressive
loss of dopaminergic neurons in the substantia nigra, which results in motor
abnormalities. Clinical symptoms include bradykinesia, tremor, rigidity and postural
instability [1, 2]. However, these symptoms are not universal to all PD patients. For
example, patients who are affected by mutations in a particular gene, called SNCA (α-
synuclein), tend to develop distinctive clinical features, such as autonomic dysfunction,
speech problems, behavioral changes and cognitive decline, in addition to the more
common symptoms described above [3]. The underlying mechanism of how these
pathophysiological symptoms develop is yet unclear. Levodopa, the most commonly used
therapeutic treatment for PD thus far, can alleviate these symptoms in the early stage of
the disease. However, as the disease progresses, levodopa loses its effectiveness.
PD affects about 2% of individuals aged 65 years and the percentage increases
with age, affecting about 5% of individuals with age of 85 [4, 5]. Because of prolonged
lifespan and aging population, the number of individuals suffering from PD is expected to increase [6].
Pathological features of Parkinson’s disease
PD is best described by progressive loss of dopaminergic neurons in the
substantia nigra, which is responsible for locomotion and coordination. Additional
13 pathological hallmarks include formation of Lewy bodies, which is mainly composed of
α-synuclein, and other protein aggregates (Figure 1) [7-10]. Lewy bodies may form throughout various brain regions, such as brain stem, diencephalon, basal ganglia and neocortex. These pathological changes result in clinical symptoms that include
progressive dementia, such as visuospatial and executive dysfunction, hallucination and
fluctuation in cognition [11]. However, the formation of Lewy bodies is not universal to
all PD patients and unfortunately, there are no biomarkers available to detect either
dopaminergic neuronal loss or Lewy body formation. In addition, the mechanism of how
protein aggregates form is not yet completely understood.
Genetics of Parkinson’s disease
Most PD cases are thought to be idiopathic or sporadic, with unknown etiology.
However, about 10% of PD cases arise from genetic mutations. Recent population studies
have found that mutations in seven genes, SNCA (α-synuclein), PARK2 (parkin),
PARK7 (DJ-1), PINK1 (phosphatase and tensin homologue induced putative kinase 1),
VPS35 (vacuolar protein sorting 35), eIF4G1 (eukaryotic translation initiation factor
gamma 1), and LRRK2 (leucine-rich repeat kinase 2), are linked to inherited forms of PD
[12-14]. More specifically, SNCA, LRRK2, VPS35 and eIF4G1 are autosomal dominant
while parkin, DJ-1 and PINK1 are autosomal recessive for PD [15, 16]. Among the four
autosomal dominant PD genes, Mutations in LRRK2, VPS35, and eIF4G1 have been
shown to cause late-onset PD.
VPS35 is part of the retromer, which is responsible for mediating retrograde
transport of transmembrane proteins from endosomes to the trans-Golgi network [17]. It
is the largest subunit of a trimer complex that is responsible for recognizing cargo
14 proteins, which is comprised of VPS26, VPS29 and VPS35. VPS35 serves as a central platform in assembly of the trimer complex. Retromer has been shown to play roles in various pathways, including cargo sorting, Wnt signaling, cell polarity, and regulation of
amyloid precursor processing [18-20]. Recently, a mutation in VPS35, D620N, has been
identified as a cause of autosomal dominant PD [13, 21]. However, according to
population studies, this particular mutation has a low frequency from 0.1% to 1% in
familial cases, which suggests that the cause of PD by this mutation is rare [22].
Although the exact mechanism of how the mutation in VPS35 causes PD pathogenesis is
unknown, there are two proposed pathways in which it may be involved; one is the
involvement in Wnt signaling pathway, and the other is the trafficking of divalent metal
transporter, DMT1. As previously mentioned, retromer plays an important role in the
Wnt signaling pathway. Hence, it has been postulated that the mutation in VPS35 may
affect the assembly of the retromer complex, thus disrupting Wnt signaling pathway,
since VPS35 serves as a central platform of cargo recognition subcomplex [23, 24]. The
mutation in VPS35 may affect the sorting of DMT1 to LAMP2 (lysosomal-associated membrane protein 2), which may affect its function as an iron transporter [25].
Mutations in eIF4G1, A502V and R1205H, have been recently identified as a novel cause of autosomal dominant PD [14]. However, the frequency of these mutations is low in PD patients from the European, African and Indian decent, and China, which suggest that the mutations in eIF4G1 are a rare cause of PD [26-29]. eIF4G1 serves as a scaffold protein, which binds to eIF4E and eIF4A, to form eIF4F complex, which regulates translation of mRNAs with 5’ m7G-cap. Proper assembly of eIF4F is required in
order to initiate binding of the 40S ribosome to the mRNA, thus initiating translation.
15
The exact role of eIF4G1 in PD pathogenesis is not yet clear. However, overexpression of eIF4G1 and/or eIF4F complex has been reported in many types of cancers. For examples,
overexpression of eIF4G1 has been found in squamous lung carcinomas and
inflammatory breast cancer [30, 31]. Increased expression of eIF4E has been reported in
breast carcinoma, primary bladder cancer, and non-Hodgkin’s lymphoma [32]. Increased
expression of EIF4A has been found in human melanoma cells [33]. Taken together,
deregulation of translation initiation plays an important role in human diseases.
The discovery of mutations in LRRK2 has been one of the most significant
contributions in understanding PD pathogenesis. Mutations in LRRK2 are the most
frequent known cause for both familial and sporadic PD. Thus, there have been vigorous
efforts to understand the biological function of LRRK2 and its role in PD pathogenesis.
More specific details will be discussed in the next section.
Leucine-rich repeat kinase 2 (LRRK2) Prevalence of LRRK2 mutations in PD
Mutations in LRRK2 are associated with late-onset, autosomal-dominant
inherited PD [34-36], and five specific mutations in LRRK2, I1371V, R1441C/G,
Y1699C, G2019S and I2020T, have been shown to be pathogenic [37, 38]. Patients with
I2020T mutation have been mainly found in a large Japanese family [39], those with
Y1699C mutation in a large British family, those with R1441C in western Nebraska [34,
35], and those with R1441G in the Basque region of Spain [40-42]. Among these five
mutations, G2019S is the most common LRRK2 mutation accounting for up to 10% of
familial PD and 2% of sporadic PD cases in many populations in America and Europe,
40% of familial and sporadic PD in Arab patients from North Africa, and 30% of familial
16 and sporadic PD in Ashkenazi Jews [43-45]. Such a high prevalence highlights the
important role of LRRK2 and protein phosphorylation in both genetic and sporadic forms of PD.
LRRK2-associated neuropathology
As previously mentioned, all patients display loss of dopaminergic neurons even though the severity of loss may vary. The Lewy body formation and PD-associated dementia are also not universal. About 30 disease-associated mutations in LRRK2 have
been reported (Figure 2) [46]. The frequency of each mutation varies by population [47].
In parallel, patients with different mutations in LRRK2 showed variable disease
phenotypes. Patients with I2020T showed dopaminergic neuronal loss without Lewy
body formation [48]. Patients with Y1699C mutation showed dementia and protein
aggregates, in addition to the dopaminergic neuronal loss [35, 49]. Patients with R1441C
mutation showed rather scattered phenotype; some displayed formation of Lewy bodies,
some displayed Tau pathology, and some did not develop either [50]. Patients with the
G2019S mutation, the most common mutation in LRRK2, displayed no difference in the
age of disease onset and duration compared to sporadic PD [51]. In addition, the clinical
symptoms from the patients with this mutation did not show significant difference
compared to the patients with sporadic PD [52]. However, the frequency of Lewy body
formation was not uniform among the patients with this mutation [53]. This suggests that
LRRK2 may not exert its effect on one particular pathway, but rather it may be involved
in various cellular pathways (Table 1).
17
Structure of LRRK2
LRRK2 is a multi-domain protein of 2527 amino acids encoded by 51 exons [54].
It is part of a ROCO superfamily, which contains Roc (Ras of complex) domain,
followed by COR (C-terminal of Roc) domain. Usually, a kinase domain from MAPKKK
(mitogen-activated protein kinase kinase kinase) follows immediately after the Roc and
COR domains. In addition, LRRK2 has an N-terminal LRR (leucine-rich repeat) domain, and C-terminal WD40 repeats (Figure 2). LRR domain may play an important role in protein-protein interactions, as many other proteins with this motif do [55]. Roc domain functions as a GTPase [56-58], while COR domain has been postulated to be important in
LRRK2 dimerization [54, 59, 60]. It has been shown that LRRK2 normally exists as
dimer, and its dimeric state is required for its kinase activity [59, 61-63]. MAPKKK domain functions as a kinase, and the exact role of WD40 repeats of LRRK2 has not been elucidated. Pathogenic mutations I1371V, R1441C and R1441G are located in the
GTPase domain, and G2019S and I2020T are located in the kinase domain. Despite that there are many mutations throughout LRRK2, clusters of pathogenic mutations in the
GTPase domain and the kinase domain indicate that its intrinsic GTPase and kinase activities play important roles in PD pathogenesis.
Obtaining three-dimensional structure of LRRK2 could provide tremendous opportunity for potential therapeutics. Ever since the success of Gleevec, which inhibits the tyrosine kinase BCR-Abl from phosphorylating its targets, thus preventing further signaling cascade of cancer development in chronic myeloid leukemia, kinase inhibitors have been one of the highlights in drug discovery [64]. Although there are several experimental LRRK2 inhibitors available (Table 2) [65], it has been difficult to develop
18 therapeutic targets for LRRK2 signaling. Because the substrates of LRRK2 are largely unknown, it is challenging to determine the exact target of such LRRK2 inhibition, which may produce uncertain consequences. In addition, current LRRK2 inhibitors are non- selective in relation to wild type and mutant LRRK2, which means that these LRRK2 inhibitors would terminate all activities of LRRK2 regardless of wild type or mutant.
Therefore, it is crucial to determine the three-dimensional structure of LRRK2, in order
to accurately assess structural consequence of pathogenic mutations and to develop more
effective and safe treatment. There are predicted structures of GTPase domain, kinase domain, and WD40 repeats [36, 66]. Although the modeling of the GTPase and kinase domain of LRRK2 reveals resemblance to other known GTPases and kinases, and the structures of isolated Roco domain of LRRK2 [59, 60] or a Dictyostelium homologue have been reported [67], yet the three-dimensional structure of full-length human LRRK2 or its kinase domain must be solved for better structural insights into mammalian
LRRK2.
Biological function of LRRK2
LRRK2 is a functional kinase, which can catalyze authophosphorylation,
phosphorylation of a generic substrate myelin basic protein (MBP), and phosphorylation
of pseudosubstrate peptides LRRKtide and Nictide [68-70]. The pathogenic mutation
G2019S has been shown to increase its kinase activity in vitro [71]. However, little is
known about the cellular effect of LRRK2 autophosphorylation or its kinase activity.
LRRK2 is also a functional GTPase, which hydrolyzes GTP to GDP. Because two
functional enzymatic domains are present adjacent to each other, a potential regulatory
mechanism between the GTPase and kinase domains has been postulated. Initially, it was
19 thought that binding of GTP to LRRK2 enhances its kinase activity, but later it was
revealed that the kinase activity is dependent on the GTP binding capacity of LRRK2
[72, 73]. On the other hand, its GTPase activity does not require a functional kinase
domain. Moreover, autophosphorylation studies of LRRK2 showed that many of the sites
within the Roc domain, which possesses GTPase activity, are autophosphorylated. Using
tandem mass spectrometry of LRRK2 overexpressed in cells, T1343, S1403, T1404,
T1410, and T1491 were found to be autophosphorylated, which are located in the
GTPase domain of LRRK2. Moreover, T1967, T1969, T2031, S2032, and T2035 were
also autophosphorylated, which are located in the kinase domain of LRRK2 [74-76].
Taken together, autophosphorylation may serve a regulatory role in the activities of the
GTPase domain and kinase domain of LRRK2.
Using in vitro kinase assay of a peptide library, it was reported that LRRK2
phosphorylation favors peptide containing the F/Y-x-T-x-R/K motif [77]. Among the
identified LRRK2 autophosphorylation sites, T1410 specifically follows the suggested
peptide motif described above. However, this peptide motif has not been vigorously
validated as the site of phosphorylation in previously proposed LRRK2 substrates (Table
3).
Although cellular substrates of LRRK2 have not been identified, wide expression
of LRRK2 in various tissues and cell types indicates that LRRK2 is involved in various
cellular pathways. LRRK2 is mainly cytosolic but can also localize to endosomes,
lysosomes, multivesicular bodies, mitochondrial outer membrane, lipid rafts,
microtubule-associated vesicles, the Golgi complex, and the endoplasmic reticulum (ER)
[78, 79]. Surprisingly, mRNA and the protein levels of LRRK2 are relatively low in
20 dopaminergic neurons in substantia nigra, despite the link between LRRK2 and PD.
Overexpression of G2019S has been shown to be toxic to primary neurons and neuroblastoma cell lines, and induces cell death [57, 80]. In addition, overexpression of the G2019S mutant has been shown to cause reduction in neurite length, which suggests that LRRK2 may be involved in cytoskeletal organization and microtubule assembly [81,
82].
Animal models of LRRK2-linked PD
Humans have diverse cellular pathways that are not only complex, but many of the pathways are also intertwined. Such complexity presents significant challenges in
determining the exact role of a kinase and its consequences when a mutation occurs.
Hence, many studies have used simpler organisms to establish the fundamental function
of LRRK2. The consequences of LRRK2 mutations are much more dramatic in
invertebrate models. In transgenic Drosophila, expression of human G2019S has been
shown to affect locomotion, and cause visual impairment and dopaminergic neuron loss
[83, 84]. In transgenic C. elegans, overexpression of R1441C and G2019S has been
shown to cause age-dependent impairment in dopamine-dependent locomotive behavior
(such as basal slowing response or food-sensing), and dopaminergic neurodegeneration
[85, 86].
In transgenic mice, it has been more challenging to show robust Parkinsonism
phenotypes; it has been difficult to generate pathological features of PD in transgenic
mice. BAC (bacterial artificial chromosome) transgenic mice harboring R1441G
mutation developed motor deficits and hyperphosphorylated Tau proteins, but failed to
induce dopaminergic neuronal loss [87]. BAC transgenic G2019S mice showed defects in
21 dopamine neurotransmission but no apparent neurodegeneration [88]. Another transgenic case model that expression of G2019S mutant by platelet-derived growth factor-β promoter showed mild dopaminergic neuronal loss, yet they failed to develop protein aggregates [89]. Viral delivery of human G2019S to the brains of adult rats was shown to cause dopaminergic neuronal loss [90, 91]. It is possible that despite genetic
manipulations, its consequences may have been alleviated by other cellular pathways in
mice, causing milder phenotypes. Also, it is possible that proper disease phenotype or
pathological hallmark does not appear due to the difference in mouse lifespan compared
to humans; neurodegenerative diseases in humans may not appear until >60s, except
early onset cases. In addition, a comprehensive understanding of disease pathogenesis
and LRRK2 mechanism is still lacking, which hinders generation of a better mammalian
animal model.
Identifying substrates of LRRK2 Chemical genetic approach
A protein kinase is an enzyme that chemically adds phosphates to its target
proteins, resulting in changes of activities of the target proteins. This phosphorylation
typically turns on or off its substrates, thus altering their functions and activities.
Phosphorylation plays a pivotal role in regulation of various cellular pathways, especially
signaling cascades. Hence, disruption of proper kinase activity leads to various human
diseases, such as cancer, inflammatory and autoimmune diseases, neurodegenerative
diseases, and diabetes [92-96]. Therefore, establishing substrates for each kinase would
broaden our understanding of the role of the kinases, and facilitate development of future
therapeutics.
22
Unfortunately, identifying substrates of kinases has been extremely challenging; many of the kinases have overlapping substrates that can functionally compensate for each other. In addition, all kinases use ATP as a phosphodonor, which hinders the ability to distinguish phosphorylated proteins by a specific kinase [97, 98]. Furthermore, complex and versatile nature of cellular localization and protein-protein interactions provide another layer of in difficulty identifying kinase substrates by in vitro studies.
Numerous strategies have been developed to identify substrates, such as simultaneous measurement of multiple active kinase states using polychromatic flow cytometry, immunoprecipitation of relevant kinase complexes, mass spectrometry and functional proteomics, and modulation of divalent metal requirements in cell lysates [99-104].
Although the identified substrates may not directly recapitulate cellular events in vivo, they can still contribute to the understanding of underlying mechanism of kinases.
To address the technical difficulties of identifying direct substrates of a kinase, a chemical genetic approach has been developed, most prominently by the Shokat group
[105]. In this method, a kinase of interest is engineered to accept a non-natural phosphate donor, which is poorly accepted by wild-type protein kinase in the cell. More specifically, a functionally conserved residue within ATP-binding pocket of the kinase active site is altered to a smaller residue, such as alanine or glycine, thus creating a bigger active site.
This mutation has been designed in a way that the mutation must be functionally silent and must not alter kinase phosphoacceptor specificity [106]. Such a residue, which follows all the necessary criteria mentioned above, is called the “gatekeeper” residue, because it has been shown that this residue occupies an existing ATP-binding pocket in the kinase active site [107]. Hence, synthetic ATP analogs can easily access the enlarged
23 kinase active site, and effectively phosphorylate its substrates [108, 109]. Since wild-type
kinases cannot accommodate phosphates from synthetic ATP analogs with bulkier side chains, this method can be used to identify substrates that are specific to the engineered
analog-sensitive kinases. To date, this method has been used to identify direct substrates
of many different kinases, such as c-Jun N-terminal kinase (JNK), cyclin-dependent
kinase 2 (CDK2), proto-oncogene tyrosine-protein kinase (Src), and AMP-activated
protein kinase (AMPKα2), which suggests that the chemical genetic screening is a
powerful tool [109-112].
Current findings on proposed substrates
Phosphorylation of LRRK2 and identifying its substrates has been under intense investigation, in an effort to understand the biological function of LRRK2 and its role in
PD pathogenesis. Due to cellular complexity and overlapping substrates among many
kinases, most of the studies identifying substrates have been conducted in vitro. To date,
several substrates have been proposed as candidates of LRRK2 substrates (Table 3).
ArfGAP1, ADP-ribosylation factor GTPase-activating protein 1, has been
identified as a possible LRRK2 substrate and also a regulator of its GTPase activity as its
name suggests. It was shown that LRRK2 and ArfGAP1 co-localize in cytoplasm and the
Golgi network, and ArfGAP1 enhances GTP hydrolysis of LRRK2. In addition, it was shown that LRRK2 can phosphorylate ArfGAP1, which supports a potential regulatory mechanism between the GTPase domain and the kinase domain of LRRK2 [113].
Ezrin/radixin/moesin (ERM) proteins play an important role in bridging actin filaments and the plasma membrane. Phosphorylation of ERM is critical in regulation of its activity; phosphorylation in Thr558 activates ERM [114, 115]. They were proposed as
24
LRRK2 substrates, and it was reported that expression of G2019S mutation of LRRK2 in
neuronal filopodia impairs neurite outgrowth, while introducing exogenous polypeptide containing ERM phosphorylation site remedies the neurite defects [69, 116]. Such findings support the involvement of LRRK2 in cytoskeletal organization, yet more direct evidence or mechanistic studies are needed.
LRRK2 may play an important role not only in actin filament, but also microtubule assembly. It was shown that LRRK2 physically interacts with α- and β- tubulin, and phosphorylates brain-derived β-tubulin, thus enhancing microtubule stability
[117, 118]. However, in vivo evidence confirming direct phosphorylation of β-tubulin is still lacking. In addition to β-tubulin, Tau, a microtubule-associated protein, was suggested to be regulated by LRRK2 [119]. Tau is phosphorylated by many serine/threonine kinases, and phosphorylation of Tau facilitates binding to microtubule, thus increasing its stability [120]. Although the underlying mechanism of LRRK2- mediated regulation of Tau remains unknown, deregulated Tau, due to hyperphosphorylation and/or abnormal accumulation, has been reported in some transgenic mice models that harbor LRRK2 mutations, and brains from AD and PD patients [121, 122].
Drp1 is a small GTPase that regulate mitochondrial fission, and has been proposed to be phosphorylated by LRRK2 [123]. Recent evidence suggests a potential interaction between Drp1 and LRRK2, thus regulating mitochondrial function and morphology. It was shown that G2019S causes increased mitochondrial fragmentation resulting in neuronal toxicity. However, inhibition of Drp1 ameliorated the fragmented mitochondrial level [124, 125].
25
4E-BP1 is so far the most emphasized, yet controversial, substrate of LRRK2. 4E-
BP1, the eIF4E binding protein, acts as a translational repressor by competing with eIF4G1 for binding site of eIF4E [126]. This binding to eIF4E is regulated by 4E-BP1 phosphorylation, and it has been shown that hyperphosphorylation of 4E-BP1 abolishes this binding [127]. Initially, it was shown that LRRK2 directly phosphorylates 4E-BP1, and G2019S promotes hyperphosphorylation of 4E-BP1, thus resulting in dopaminergic neuronal loss in Drosophila [128]. Subsequently, it was shown that down-regulation of
LRRK2 causes a decrease in 4E-BP1 protein [129, 130]. Such findings have provoked great interest in the potential relationship between LRRK2 and translation initiation;
deregulation of protein synthesis may lead to loss of dopaminergic neurons, or protein
aggregate formations. However, other studies have shown that overexpression of human
LRRK2 causes only weak 4E-BP1 phosphorylation in HEK293T cells, and direct
phosphorylation of 4E-BP1 by LRRK2 could not be confirmed in mammalian cells [128,
131]. In addition, LRRK2 and 4E-BP1 showed minimal co-localization in mammalian
neurons, and no change in 4E-BP1 expressions was found in LRRK2 transgenic mouse
models [132].
In addition to the potential substrates described above, there is evidence of other
substrates for LRRK2. Phosphorylation of FOXO1, forkhead box transcription factor, by
LRRK2 has been linked to LRRK2-mediated apoptosis in dopaminergic neurons of
Drosophila [133]. Endophilin A (EndoA), a key player in synaptic vesicle endocytosis,
has been shown to be phosphorylated and regulated by LRRK2 [134]. There are also
suggestions of a relationship between α-synuclein and LRRK2, because some of Lewy
bodies contain co-localized LRRK2 and α-synuclein aggregates [135]. Other proposed
26 substrates include Prdx3 [136] and snapin [137]. All of this evidence further implicates
how many diverse cellular pathways LRRK2 is involved in, thus emphasizing the
significance of identifying LRRK2 direct substrates. We have developed an unbiased
method to search for LRRK2 substrates, which would not only be specific to LRRK2, but
is also a global approach. The previously proposed substrates have been based on in vitro
experiments, which monitored changes of activities of one potential candidate upon
LRRK2 overexpression or pathogenic mutation. Our method allows us to screen all the
proteins that are phosphorylated specifically by LRRK2, without disrupting cellular
organization. As a result, we can create a global profile of proteins that are involved in
various cellular pathways. Revealing LRRK2 substrates and its immediate downstream
signaling pathways could serve as a key in deciphering PD pathogenesis.
27
Chapter 2: Chemical Genetic Identification of LRRK2
Substrates
28
Abstract
Parkinson’s disease (PD) is the most common neurodegenerative movement disorder, characterized by progressive loss of dopaminergic neurons in the substantia nigra. Most PD cases are thought to be idiopathic with unknown etiology. About 10% of the PD cases are familial due to inheritance of genetic mutations. Mutations in LRRK2
(leucine-rich repeat kinase 2) is the most frequent known cause for the late-onset and autosomal-dominantly inherited PD. LRRK2 has been implicated in diverse molecular pathways and cellular functions, but many of its substrates are yet to be discovered. In this
study, we have used a chemical genetics approach, which allows a minimally modified
kinase to be sensitized to synthetic ATP analogs for phosphorylation of its direct substrates, to identify previously unidentified substrates that are specific to LRRK2. We
have applied this analog-sensitive strategy and have identified many putative cellular
substrates of LRRK2 in human cells using Western blot and tandem mass spectrometry.
We have discovered that two of these proteins, VPS35 and eIF4G1, are encoded by the
corresponding genes linked to PD. Furthermore, we have verified eIF4G1 as a novel
LRRK2 substrate, suggesting that LRRK2 may play an important role in the regulation of
protein translation.
Introduction
Parkinson’s disease (PD) is the most common neurodegenerative movement
disorder, which results in motoric abnormalities, such as bradykinesia, tremor, rigidity
and postural instability [1, 2]. PD is characterized by progressive loss of dopaminergic
29 neurons of the substantia nigra. In addition, its pathological hallmark includes the
presence of Lewy bodies, which is mainly composed of α-synuclein and other protein
aggregates [7-9]. PD affects about 2% of individuals aged 65 years and the percentage
increases with age, affecting about 5% of individuals with age of 85 [4, 5]. Because of
prolonged lifespan and aging population, the number of individuals suffering from PD is
expected to increase [6].
Most PD cases are thought to be idiopathic or sporadic, with unknown etiology.
However, about 10% of PD cases arise from genetic mutations. Recent population studies
have identified that mutations in seven genes, SNCA (α-synuclein), PARK2 (parkin),
PARK7 (DJ-1), PINK1 (phosphatase and tensin homologue induced putative kinase 1),
VPS35 (vacuolar protein sorting 35), eIF4G1 (eukaryotic translation initiation factor
gamma 1), and LRRK2 (leucine-rich repeat kinase 2), are associated inherited form of
PD [12-14]. Among these seven genes, mutations in LRRK2 are the most frequent known
cause for the late-onset and autosomal-dominantly inherited PD [34-36]. Several specific
mutations in LRRK2, I1371V, R1441C/G, Y1699C, G2019S and I2020T, are shown to
be pathogenic [37, 38]. Among them, G2019S is the most common LRRK2 mutation that
accounts for up to 10% of familial PD and 2% of sporadic PD cases in many populations
in America and Europe, 40% of familial and sporadic PD in Arab patients from North
Africa, and 30% of familial and sporadic PD in Ashkenazi Jews [43-45]. Such a high
prevalence highlights an important role of LRRK2 and protein phosphorylation in both
genetic and sporadic forms of PD.
LRRK2 has been implicated in diverse molecular pathways and cellular
functions, such as vesicular trafficking, neurite outgrowth, cytoskeletal regulation,
30 autophagy, mitochondrial function, and translational control [46, 118, 138]. More specifically, pathogenic mutation G2019S has been reported to cause cellular toxicity in
vitro, such as reduction in neurite length, altered cellular distribution and apoptosis [57,
81, 139], as well as visual impairment in Drosophila, and locomotive dysfunction in
Drosophila, C. elegans, and transgenic mice [84, 91, 140, 141]. However, the majority of
these studies are based on indirect evidences, and the exact mechanism of how LRRK2
mutations cause disease pathology is yet to be solved. Interestingly, G2019S has been
reported to enhance kinase activity by about 2-3 folds compared to that of LRRK2 wild
type, even though its intrinsic targets remain unclear [71]. Hence, it is critical to establish
a deeper understanding of how LRRK2 mechanistically regulates these cellular processes
and the role of LRRK2 in substrate phosphorylation and associated downstream signaling
pathways.
There have been numerous efforts to identify potential substrates for LRRK2,
and several putative substrates have been postulated, such as Tau, tubulin, Drp1,
endophillin A, 4E-BP1 [119, 125, 131, 134, 142]. Although these previous findings
provide useful insights about possible function of LRRK2, a generally applicable
approach remains to be developed for the identification of LRRK2 substrates in order to
elucidate the overall signaling pathways and biological function regulated by LRRK2.
One of the major challenges in identifying LRRK2 substrates is to distinguish cellular
targets that are specific to LRRK2. There is an enormous variety of endogenous kinases
present in cells, which makes it difficult to differentiate the substrates phosphorylated by
individual kinases. In this study, we used a chemical genetic approach, which allows a
minimally modified kinase to be sensitized to synthetic ATP analogs for phosphorylation
31 of its direct substrates [112, 143], to identify previously unidentified substrates that are specific to LRRK2. We applied this analog-sensitive (AS) strategy to identify novel cellular substrates of LRRK2 in human cells. We successfully generated AS-LRRK2 and
AS-G2019S, which displayed sensitivity to ATPγS analogs, and then specifically monitored LRRK2-induced thiophosphorylation in whole cells under relatively physiological conditions. We identified individual substrates using a combination of immunochemistry and mass spectrometry. Notably, we discovered that two of these proteins, VPS35 and eIF4G1, are encoded by the corresponding genes linked to PD.
Finally, we confirmed eIF4G1 as a direct target of LRRK2, suggesting a molecular mechanism underlying the role of LRRK2 in the regulation of protein translation.
Experimental Procedures Generation of analog-sensitive LRRK2 variants
FLAG-tagged analog-sensitive (M1947G) versions of LRRK2 were derived from the original constructs in pCEP4 vector [56] using site-directed mutagenesis with
QuikChange II XL kit (Agilent Technologies). After cloning, individual plasmids were confirmed by restriction endonuclease digestion, and by DNA sequencing. FLAG-tagged
LRRK2 wild type, G2019S, AS-LRRK2, and AS-G2019S were overexpressed in human embryonic kidney cells (HEK293T) via transient transfection. A mixture of 7.6 μg of
LRRK2 plasmid and 0.4 μg of a GFP plasmid was transfected to 100 mm plate using polyethylenimine (Sigma-Aldrich). Cells were collected 48 h post transfection in lysis buffer [1% Triton-X 100, 1mM EDTA, and the Complete protease inhibitor cocktail
32
(Roche), in Dulbecco’s Phosphate Buffered Saline (DPBS). The whole cell lysates were clarified by centrifugation at 16,000xg for 10 min at 4 oC.
Immunoprecipitation of flag-tagged LRRK2
Flag-tagged LRRK2 proteins were immunoprecipitated by the addition of anti-
FLAG M2 affinity gel (Sigma-Aldrich) to the clarified lysates, followed by gently
rotation for 3 h at 4°C. The beads were collected by low centrifugation at 1000xg for 1 minute. To wash the beads, wash buffer (1% TritonX-100, 1 mM EDTA, 0.5 M NaCl, and the Complete protease inhibitor cocktail in DPBS) was added. The mixture was gently rotated at room temperature for 5 minutes, and the beads were collected by low centrifugation. The wash step was repeated 5 times in total; 4 times with wash buffer and finally with DPBS. After the final wash, the beads were resuspended in 150 μl of DPBS.
In vitro kinase assay
A volume of 10 to 20 μl of immunoprecipitated LRRK2 variants were used per
reaction, depending on the amount of LRRK2 purified in each immunoprecipitation. In a
single reaction, 1 mM GTP, 3.64 g MBP, immunoprecipitated LRRK2, and 1mM
ATPγS (or ATPγS-analogs) were added to 1X kinase buffer (15 mM MgCl2, 5 mM
EGTA, and 20 mM β-glycerol phosphate in 20 mM HEPES, pH 7.4), and incubated at
30°C for 1 hour. After the incubation, thiophosphorylated MBP was alkylated by the
addition of 2.5 mM p-nitrobenzyl mesylate (PNBM, Abcam) and 25 mM EDTA and
incubation for 2 hour at room temperature. After the alkylation, the reaction mixture was diluted with an equal volume of 2xK buffer (6 % SDS, 4 mM EDTA, 20% glycerol, and
2.5% β-mercaptoethanol in 0.1M Tris, pH 7.0), boiled at 95°C for 5 min, and then loaded on to a SDS-PAGE gel for protein separation. Detection of thiophosphorylated MBP was
33 accomplished by Western blot with the thiophosphate-specific rabbit monoclonal antibody (α-thioP mAb 51-8, Abcam) and ECL kit (GE Health).
Indirect competition assay
Identical conditions of in vitro kinase assay were used as described above, except as noted below. About 10 ng of AS-LRRK2 or AS-G2019S was used to catalyze thiophosphorylation of MBP (30 M) using 0.5 mM ATPγS in the absence or presence of
3 mM ATP, N6-cyclopentyl-ATP (N6-cPe-ATP) or N6-phenylethyl-ATP (N6-PhEt-ATP)
at 30 oC for 60 min. The reaction was terminated by the addition of 20 mM EDTA and
the mixture was alkylated with PNBM for 2 hour at room temperature.
Synthesis of N6-cPe-ATPγS using Ynk1p
Recombinant Ynk1p construct with a C-terminal hexa histidine tag (a gift of D.
Pain, University of Medicine and Dentistry of New Jersey) was expressed in E. coli, according to the published method [144]. The his6-tagged Ynk1p was bound to Ni-NTA
agarose (Qiagen). The enzyme was extensively washed with buffer A (20% glycerol in
50mM Tris, pH 7.5). Then, the enzyme was collected by serial elution with buffer A
containing imidazole at concentrations between 100 mM and 500 mM. Each fractionation
was examined for the presence of purified enzyme using a SDS-PAGE gel and
Coomassie blue staining. The fractions with 200 mM and 300 mM imidazole were found
to contain the most amounts of purified enzyme. The eluted enzyme was dialyzed to
remove imidazole using Slide-A-Lyzer dialysis cassette (Thermo Scientific) and buffer
A. After the dialysis, the enzyme concentration was measured using a spectrophotometer.
6 For synthesis of N -cPe-ATPγS, a mixture of 20 μg of his6-tagged Ynk1p, and 4 mM
GTPγS in 20 mM Tris, pH 7.6 were incubated for 1 hour at 30°C. The reaction mixture
34 was added to 30μl Ni-NTA agarose for binding by his6-tagged Ynk1p for 15 minutes at
30°C. The beads were then washed twice with 20 mM Tris, pH 7.6. A solution containing
6 8 mM N -cPe-ADP (Axxora) in 1X reaction buffer (5 mM MgCl2 in 20 mM Tris, pH7.5)
was added to the beads, and incubated for 1 hour at 30°C. After the reaction, the beads
were precipitated by centrifugation. The supernatant, which contains synthesized N6-cPe-
ATPγS, was collected.
In vivo Substrate labeling using digitonin
Both G2019S and AS-G2019S were overexpressed in HEK293T cells via transient transfection, as described above. Cells were counted 48 hours post transfection using Trypan blue (HyClone). Cells at a density of 5× 106 cells/ml were washed with
serum-free DMEM, collected by low centrifugation, and then resuspended in
permeabilization buffer (25 mM Tris, pH 7.5, 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM sodium orthovandate, 10 mM MgCl2, Complete protease inhibitor cocktail,
PhosStop phosphatase inhibitor tablet in DPBS) containing 50μg/mL digitonin. The
resuspended cells were incubated on ice for 5 minutes, and then collected by
centrifugation. The cells were then resuspended in resuspension buffer (permeabilization
buffer without digitonin), followed by the addition of 100 μM N6-Bn-ATPγS and 1 mM
GTP. The mixture was incubated for 30 min at 30 °C. After the reaction, the cells were collected by low centrifugation. The cells were lysed in 250 μl lysis buffer, and incubated on ice for 15 min. Next, the lysates were clarified by centrifugation at 16,000xg for 10 minutes. Then, 2.5 mM PNBM was added to the clarified lysates and incubated for 2 h at room temperature to alkylate the proteins. After the alkylation, 5 μl was used for Western blot, and the rest was preceded to immunoprecipitation.
35
Immunoprecipitation of in vivo labeled substrates
PNBM is known to interfere with immunoprecipitation [143]. Hence, PNBM was removed by filtration with PD10 desalting columns (GE Healthcare Life Sciences). After removing PNBM and prior to immunoprecipitation, we pre-cleared the collected samples to eliminate unspecific protein binding to agarose beads. The PD10 column-filtered samples were incubated with Protein A/G Plus agarose (Santa Cruz Biotech) for 3 h at
4°C. Then, the agarose beads were discarded following centrifugation. To the supernatant, -thioP antibody was added, and the mixture was incubated overnight at
4°C. Protein A/G Plus agarose was then added to the sample, and incubated for 3 h at
4°C. The beads were collected by low centrifugation, and washed several times. Finally, the beads were diluted with 2xK buffer, boiled at 95°C for 5 min, and loaded onto a SDS-
PAGE gel. Once the proteins were separated, the gel was stained overnight with Sypro
Ruby (Bio-Rad), washed in water, and then further proceeded to mass spectrometric analysis.
Mass spectrometry analysis
The gel above the 50kD marker (to avoid abundant IgG), was cut into 6 gel slices.
The gel samples were then prepared for mass spectrometry using a standard in-gel
digestion procedure. The gel slices were reduced with DTT, and alkylated with
iodoacetamide. Then, the gel slices were digested with modified trypsin. Liquid
chromatography-mass spectrometry (LC-MS) data were acquired on a Waters nanoAcquity LC system (Waters, Taunton, MA) interfaced to an Orbitrap Elite mass
spectrometer (Thermo Electron, San Jose, CA). Peptides digested from each gel slice
were combined, concentrated, and loaded on column in a 9 μL injection. Peptides were
36 desalted on a trap column [180 μm × 20 mm packed with C18 Symmetry, 5μm, 100Å
(Waters, Taunton, MA)] and subsequently resolved on a reversed phase column [75μm x
250 mm nano column, packed with C18 BEH130, 1.7μm, 130Å (Waters, Taunton, MA)] using a gradient of 2 to 50% mobile phase B (0.1% formic acid and acetonitrile) over a period of 90 minutes at ambient temperature and a flow rate of 300 nl/min. Peptides eluting from the capillary tip were introduced into the nano-electrospray source with a capillary voltage of 2.4 kV. A full scan was obtained for eluted peptides in the range of
380–1800 m/z followed by twenty data dependent MS/MS scans. MS/MS spectra were generated for peptides with a minimum signal of 2000 by collision-induced dissociation of the peptide ions at normalized collision energy of 35%, an isolation width of 2.5, and an activation time of 30 msec to generate a series of b- and y-ions as major fragments.
The resulting MS/MS data were searched against human database using Mascot search engine. The data were searched for tryptic peptides of human proteins using accuracy values of 10 ppm and 0.8 Daltons for MS1 and MS2 scans respectively. Data files generated from Mascot search were then processed by Scaffold software, from
Proteome Software. We selected proteins above 90% protein identification threshold and
90% peptide identification threshold. Among these proteins, we selected the proteins that have 100% identity probability in AS-G2019S and 0% identity probability in G2019S.
Generation of flag-tagged eIF4G1 variants
The constructs of full-length eIF4G1 [145] (a gift of N. Sonenberg, McGill
University, Canada) and eIF4G1 isoform b [146] (a gift of M. Gromeier, Duke
University) were FLAG-tagged. The PD-linked mutation (R1205H) was introduced by
37 site-directed mutagenesis. All individual plasmids were confirmed by restriction
endonuclease digestion, and by DNA sequencing.
Results Characterization of analog-sensitive LRRK2
A challenge in the discovery of a kinase substrate is the fact that there are more
than 500 protein kinases in a cell, and all of them utilize ATP for phosphorylation. We employed an unbiased chemical genetic strategy [106] that will maximize the specificity of phosphorylation event to identify protein substrates that are unique to LRRK2. In chemical genetic approach, a large, hydrophobic amino acid in the conserved ATP binding pocket known as the “gatekeeper residue” is mutated to a small residue to generate an analog-sensitive (AS) kinase that can accept a synthetic ATP analog with a
N6-substituted bulky group due to shape complementarity (Figure 3A). In contrast, wild type kinases cannot use the bulky ATP analog for phosphorylation. However, it would be difficult to distinguish proteins that are phosphorylated with ATP analogs from the ones that are phosphorylated by naturally present ATP. Accordingly, ATPγS analogs are used to yield a thiophosphate moiety that is targeted for unique recognition by a monoclonal antibody, the -thiophosphate antibody [143]. Hence, this chemical genetic approach would allow specific phosphorylation and epitope-tagging of its direct substrate by AS- kinase using synthetic ATPγS analog without the interference by any other kinases. By sequence alignment with well-characterized kinases, the gatekeeper residue in LRRK2 is identified as methionine (M) at codon 1947 (Figure 3B). We generated analog-sensitive
38 version of LRRK2 (AS-LRRK2) by introducing the gatekeeper mutation M1947G. In
addition, we generated AS-G2019S, which is LRRK2 carrying the pathogenic G2019S
mutation, in addition to the gatekeeper mutation (M1947G+G2019S). We overexpressed
FLAG-tagged LRRK2 variants including LRRK2 wild type, G2019S, AS-LRRK2 and
AS-G2019S in human embryonic kidney cell line 293T (HEK293T). We immunopurified
these LRRK2 variants with anti-FLAG beads, and verified the proteins using silver
staining (Figure 3C) and on Western blot with an anti-LRRK2 antibody (Figure 3D).
To confirm whether AS-LRRK2 variants function as active kinases, we conducted
in vitro kinase assay using immunopurified LRRK2 variants, a generic substrate myelin
basic protein (MBP), and ATPγS, which serves as a thiophosphor donor. We found that
AS-LRRK2 readily utilized ATPγS to thiophosphorylate MBP (Figure 4A, first lane).
Then, we investigated whether AS-LRRK2 can accept ATP analogs by performing an
indirect competition assay. In this assay, various ATP analogs were added in addition to
ATPγS. Hence, if AS-LRRK2 could accept any of the ATP analogs, the level of
thiophosphorylated MBP would decrease, because the ATP analog and ATPγS would
compete for AS-LRRK2 kinase active site. We examined whether N6-phenylethyl-ATP
(PhEt), N6-phenyl-ATP (Phe), or N6-cyclopentyl-ATP (cPe) would be accepted by AS-
LRRK2. We observed that only N6-cPe-ATP resulted in decreased thiophosphorylation
of MBP, in addition to the ATP control (Figure 4A).
The data from the indirect competition assay suggested the possibility of N6-cPe-
ATP as an analog for AS-LRRK2. However, they did not prove whether AS-LRRK2 could actually utilize N6-cPe-ATP as a phosphor donor; it is possible that N6-cPe-ATP
may indirectly interfere with the access of ATPγS to the kinase active site. Thus, we
39 investigated whether N6-cPe-ATPγS could be used as a phosphor donor by AS-LRRK2
following the synthesis of N6-cPe-ATPγS that is not commercially available. We used recombinant Ynk1p, a yeast nucleoside diphosphokinase (NDPK) [144], to catalyze the transfer of γ-phosphate group from GTPγS to N6-cPe-ADP to generate N6-cPe-ATPγS
(Figure 4B). As a control for successful synthesis using Ynk1p, we also synthesized
ATPγS using GTPγS and ADP. We then examined whether AS-LRRK2 could directly
utilize these synthetic ATPγS variants as phosphor donors to thiophosphorylate MBP. As
expected, AS-LRRK2 could successfully thiophosphorylate MBP using both synthesized
ATPγS and N6-cPe-ATPγS, indicating that N6-cPe-ATPγS is a ATPS analog for kinase
reaction by AS-LRRK2 (Figure 4B). In addition, we examined whether AS-G2019S can
also accept N6-cPe-ATPγS as a phosphor donor. Interestingly, AS-G2019S did not
appreciably utilize N6-cPe-ATPγS for thiophosphorylation, as compared to the control
using Ynk1p-synthesized ATPγS (Figure 4C). We concluded that N6-cPe-ATPγS is
specific for AS-LRRK2, but not AS-G2019S.
It is intriguing that the G2019S mutation in AS-G2019S conferred diminished
sensitivity to N6-cPe-ATPγS. Therefore, AS-G2019S and AS-LRRK2 might have distinct
sensitivities to different ATPγS analogs. To identify ATPγS analogs that can be utilized
by AS-G2019S and their sensitivity to AS-LRRK2, we examined additional variants of
ATPγS analogs that are commercially available, including N6-phenyethyl-ATPγS (PhEt),
N6-furfurylated-ATPγS (Fur), and N6-benzyl-ATPγS (Bn). In addition, we tested the
parallel reactivity of wild type LRRK2 and G2019S as a control of specificity for the
corresponding AS-LRRK2 and AS-G2019S proteins. As expected, we observed that
LRRK2 wild type and G2019S were not able to accept any of the ATPγS-analogs tested
40
(Figure 5). AS-G2019S exhibited sensitivity to N6-Bn-ATPγS, and N6-PhEt-ATPγS but to a lesser extent. Interestingly, AS-LRRK2 also displayed sensitivity to N6-Bn-ATPγS
(Figure 5), in addition to N6-cPe-ATPγS. As a result, N6-Bn-ATPγS was chosen as the best phosphor donor, because it is highly specific to AS-LRRK2 and AS-G2019S, and is the most efficiently utilized ATPγS analog. We focused our subsequent studies on the
AS-G2019S and G2019S pair, since they contain the most common PD mutation that confers a gain of function in LRRK2 kinase activity [68, 80, 147].
We assessed whether AS-G2019S can achieve similar catalytic activity as
G2019S by performing a time course assay. We incubated either G2019S or AS-G2019S, with MBP and ATPγS in kinase reaction that was terminated at different time points, in the increment of 15 minutes (Figure 6A). We observed faster phosphorylation rate in
G2019S compared to AS-G2019S, yet the overall phosphorylation activities using either
G2019S or AS-G2019S was similar by 60 minutes (Figure 6B). This result suggests that the AS-G2019S is not impaired in overall kinase activity, consistent with the observations that many other kinases engineered to their AS versions retain active kinase activity [106].
In vivo substrate phosphorylation by AS-G2019S
Our ultimate goal is to identify substrates of LRRK2 in mammalian cells.
Initially, we used whole lysates of HEK293T cells to identify any proteins that would be specifically thiophosphorylated using N6-Bn-ATPγS by AS-G2019S, but not by G2019S.
We performed in vitro kinase assay with the addition of either G2019S or AS-G2019S, and N6-Bn- ATPγS to HEK293T whole lysates, followed by immunoprecipitation using
41
α-thiophosphate antibody. Surprisingly, we observed wide range of thiophosphorylated bands in both G2019S and AS-G2019S (data not shown). This could be due to high background reactivity associated with whole cell lysates in which cellular compartments are disrupted. Thus, to investigate whether AS-G2019S can specifically phosphorylate cellular proteins under relatively physiological conditions, we performed in vivo labeling of substrates in minimally treated HEK293T cells. To achieve this, we overexpressed AS-
G2019S or G2019S in HEK293T cells, and then incubated cells with N6-Bn-ATPγS in
the presence of digitonin, a gentle detergent that permeabilizes cell membrane to allow
the uptake of N6-Bn-ATPγS without overall disruption of cellular organization. Previous
studies have shown that these in vivo labeling conditions preserve endogenous kinase
signaling [112, 148]. Following labeling of digitonin-permeabilized cells using N6-Bn-
ATPγS, we immunoprecipitated thiophosphorylated proteins using -thiophosphate antibody for subsequently detection on Western blot. We observed that many more proteins contained thiophosphate moiety in cells expressing AS-G2019S than those expressing G2019S, indicating relatively specific phosphorylation of protein substrates by AS-G2019S (Figure 7A). Several protein bands were labeled to a minor extent in the cells expressing G2019S, which may represent possible low reactivity by some unidentified kinases toward N6-Bn-ATPγS for a few proteins or simply reflect minimal
cross-reactivity in detection by Western blot.
Recent human genetics studies have revealed several genes linked to PD [149].
Among these, mutations in LRRK2, eIF4G1, and VPS35 have been shown to cause
autosomal, late-onset PD. In order to explore possible biochemical mechanisms linking
these PD-associated proteins using our chemical genetic approach, we re-probed the
42 immunoprecipitated thiophosphorylated proteins for LRRK2, eIF4G1 and VPS35 (Figure
5B). We found that LRRK2, eIF4G1 and VPS35 were among the proteins specifically thiophosphorylated in the cells expressing AS-LRRK2. None of them were detected in the control cells expressing G2019S (Figure 7B). These results suggest that LRRK2 mediated phosphorylation of eIF4G1 and VPS35, as well as autophosphorylation that was previously reported to occur in vitro [76].
Global analysis of potential LRRK2 substrates
To identify all potential substrates of LRRK2, thiophosphorylated proteins labeled with N6-Bn-ATPγS were immunoprecipitated with α-thiophosphate antibody from either
the cells expressing AS-G2019S or the control cells expressing G2019S. They were
resolved by SDS-PAGE, and entire lanes of proteins with a molecular weight above 50
kDa (to avoid abundant IgG bands) were excised and digested with trypsin. The resulting
peptides were identified by tandem mass spectrometry. This unbiased global analysis
identified 10-fold more proteins in samples thiophosphorylated in the presence of AS-
G2019S than G2019S. We focused on those proteins that were only present in the AS-
G2019S sample, but not in the G2019S sample. As a result, a total of about 200 proteins
with a minimal of two uniquely identified peptides were detected specifically in the AS-
G2019S sample (Appendix I). They belong to many different groups associated with
diverse cellular functions (Figure 8A, Appendix I). Among them, proteins involved in
post-transcriptional regulation, protein translation, post-translational modification and
protein transport, are well represented. A few putative LRRK2 substrates are listed in
Figure 8B. LRRK2 and eIF4G1 were positively identified by tandem mass spectrometry
as potential substrates specific for AS-G2019S (Figure 8B, Figure 9), in good agreement
43 with the findings by Western blot (Figure 7). In addition, we identified other translation
factors, such as eIF3, elongation factor 1 and 2. Intriguingly, we also identified the
mammalian target of rapamycin, mTOR, which is an important serine/threonine kinase
that controls protein synthesis by regulating a translation repressor, translation initiation
factor 4E-binding protein (4E-BP1). Possible linkage between LRRK2 and some of these
proteins may suggest a pivotal role of LRRK2 in regulation of protein translation, with
relevance to PD pathogenesis (Figure 8B). We have also identified other proteins that
have been previously suggested as possible LRRK2 substrates such as tubulin [117],
which is also implicated as a protein dysregulated in PD.
Validation of eIF4G1 as a LRRK2 substrate
Our above data from the small-scale Western blot, and global screening of
substrates by mass spectrometry repeatedly identified eIF4G1 as a potential substrate of
AS-G2019S. Thus, we validated whether G2019S can directly target eIF4G1 for
phosphorylation. We generated FLAG-tagged eIF4G1 variants and then overexpressed
them in HEK293T cells. Unfortunately, the full-length eIF4G1 was not expressed to an
appreciable level as an intact protein (Figure 10A), likely due to its known difficulty in
expression or tendency to be degraded by proteolytic cleavage. However, it has been
shown that some eIF4G1 isoforms, such as eIF4G1 isoform b that results from the
alternative usage of a translation initiation codon 40 amino acids downstream of the full-
length eIF4G1 have higher stability in cells [145, 146]. We confirmed that FLAG-tagged
eIF4G1 isoform b (wild type and a PD-linked R1205H mutant) could be stably expressed
in HEK293T cells (Figure 10A). We immunopurified FLAG-tagged G2019S and eIF4G1
isoform b using anti-FLAG beads, and performed in vitro kinase assay using ATPγS. We
44 observed increase phosphorylation by ATPγS in the presence of both G2019S and eIF4G1 (Figure 10B), indicating that LRRK2 mediated autophosphorylation, as well as transphosphorylation of eIF4G1. Taken together, our data are consistent with the finding that eIF4G1 is a novel LRRK2 substrate.
Discussion
LRRK2 is a novel protein kinase implicated in the pathogenesis of PD, but its substrates and signaling pathways remain elusive. In this study, we employed an unbiased chemical genetic strategy to identify LRRK2 substrates in mammalian cells. Specifically, we have combined a PD-linked, gain-of-function mutation G2019S with the gatekeeper mutation M1947G to generate AS-G2019S mutant of LRRK2 that is active and sensitized to utilize a synthetic ATP analog N6-Bn-ATPγS to specifically label LRRK2 substrates with a thiophosphate epitope tag in mildly permeabilized cells. The chemical genetic screen of LRRK2 substrates presented here provided us an unprecedented opportunity to begin the reconstruction and validation of LRRK2-mediated signaling cascade that in turn may offer useful insights into the molecular pathways leading to PD pathogenesis.
We have identified many putative LRRK2 substrates. Among them, there is an enrichment of the proteins involved in transcription, post-transcriptional regulation, protein transport, and protein translation. Interestingly, we have discovered that LRRK2,
VPS35, and eIF4G1 are LRRK2 substrates phosphorylated by AS-G2019S, identified through immunoprecipitation with phospho-specific α-thioP antibody followed by
Western blot with protein-specific antibodies and/or unbiased mass spectrometric analysis. While our results have confirmed previous findings that LRRK2 is a substrate of
45 its own kinase activity both in vitro [76, 150] and in vivo [151], it is highly intriguing that
VPS35 and eIF4G1 have been recently linked to PD. Mutations in VPS35 [13, 21], a protein critical for membrane protein trafficking, and in eIF4G1 [14], a protein required for protein translation, cause autosomal-dominant, late-onset PD. Thus, to our knowledge, these findings represent the first study that putatively links VPS35 and
eIF4G1 to the LRRK2 kinase signaling pathways relevant to PD pathogenesis.
We have further confirmed eIF4G1 as a direct target of LRRK2 through in vitro kinase assay with G2019S instead of AS-G2019S. Interestingly, several proteins identified in our chemical genetic screen are also involved in the regulation of protein translation. These data have enabled us to provide a novel hypothesis about how LRRK2 might regulate protein translation through these putative substrates (Figure 11). One of the identified LRRK2 substrates is mTOR, a master regulator of protein synthesis that integrates nutrient sensing and energy metabolism. Identification of both mTOR and eIF4G1 as LRRK2 substrates suggests that LRRK2 may play an important role in protein translation (Figure 11). mTOR has been shown to directly phosphorylate 4E-BP1 in regulating cap-dependent translation [152]. 4E-BP1 readily binds to eIF4E, which is a component of eIF4F complex along with eIF4G1 and EIF4A. Proper assembly of eIF4F is required to recruit 40S ribosome, which in turn initiates scanning of amino acids and
the start codon. Binding of 4E-BP1 to eIF4E hinders eIF4F formation, thus repressing
translation initiation. Phosphorylation of 4E-BP1 by mTOR diminishes the 4E-BP1
binding to eIF4E [126, 127]. Subsequent eIF4G1 binding to eIF4E facilitates eIF4F
assembly, thus allowing translation initiation. It has been shown that eIF4G1 and 4E-BP1
binding to eIF4E is mutually exclusive, therefore maintaining their balance by the
46 activity of mTOR is critical for protein synthesis regulation [153]. In addition to eIF4G1, recruitment of 40S ribosome is thought to also involve eIF3 [154], and our mass spectrometric analysis also identifies eIF3 as a candidate substrate of LRRK2.
mTOR is known to be regulated by several other upstream proteins, including phosphoinositol dependent kinase 1 (PDK-1), phosphatidylinositol 3-kinase (PI3K), Akt and tuberous sclerosis complex proteins 1 and 2 (Tsc1/2) [155]. Our data revealed that
LRRK2 may serve as a novel regulator of mTOR but its functional consequence remains to be elucidated; whether such phosphorylation has further downstream effect, such as affecting phosphorylation of 4E-BP1. Phosphorylation of 4E-BP1 by LRRK2 independent of mTOR has also been reported to occur in Drosophila [128], but remains to be confirmed in a mammalian system [131]. Furthermore, the cellular consequence of
LRRK2 phosphorylation of eIF4G1, which serves an opposite function from 4E-BP1, needs to be deciphered; whether such phosphorylation modulates eIF4G1 binding to eIF4E. Interestingly, eIF4G1 has also been shown to be phosphorylated by mTOR at
S1108 [156]. However, the cellular effect of such a phosphorylation event has not been revealed. Therefore, identifying the site(s) of LRRK2-induced phosphorylation could uncover whether the phosphorylation by LRRK2 serves as an additive or opposing effect from mTOR. Taken together, LRRK2 may regulate protein translation by affecting 4E-
BP1 phosphorylation via mTOR, or by modulating eIF4G1 phosphorylation to impact eIF4F complex formation.
Another finding suggesting the involvement of LRRK2 in protein synthesis is the identification of PABP1 as one of the substrates. PABP1 binds to 3’ polyadenylated tail of mRNA, and plays an important role in translation initiation. PABP1 interacts with
47 eIF4G1 to circularize mRNA, thus allowing the unwinding of the proximal 5’ UTR of mRNA to facilitate translation initiation. However, PABP1 also plays another role in mRNA turnover, by recruiting deadenylation factors, thus promoting mRNA decay [157].
The balance between promoting protein synthesis and mRNA decay by PABP1 is not completely understood yet. However, phosphorylation of PABP1 by MAPKAP kinase 2
(MK2) seems to promote mRNA deadenylation [158]. Hence, phosphorylation of PABP1 by LRRK2 may promote mRNA decay via a similar mechanism. Alternatively, it may enhance binding of PABP1 to eIF4G1, thus promoting protein synthesis.
It is thus plausible that LRRK2 may be physiologically important in orchestrating fine control of protein synthesis through phosphorylation of eIF4G1, mTOR, and PABP1 that either positively or negatively impact protein translation. On the other hand, it is also possible that the observed substrate phosphorylation is due to the presence of pathogenic mutation G2019S in our AS-LRRK2 construct. G2019S mutation is thought to increase the LRRK2 kinase activity [68]. Hence, it is conceivable that G2019S may cause exaggerated phosphorylation of eIF4G1 and mTOR, leading to uncontrolled protein translation. Heightened protein synthesis has been implicated in neurodegenerative diseases including PD, and pharmacological inhibition of mTOR activity confers neuroprotective effects in experimental disease models [159]. Understanding the dynamic regulation of LRRK2-mediated phosphorylation of eIF4G1 and mTOR under both physiological and pathological conditions will likely provide unprecedented insights into the role of LRRK2 in protein translation and proteostasis, and more importantly into the molecular pathways leading to PD pathogenesis.
48
We have also identified several other substrates that may implicate LRRK2 in
novel cellular function. One of them is histone deacetylase 2 (HDAC 2). HDAC plays an
important role in regulation of gene expression; deacetylation, or removal of acetyl group
from lysine, causes histones to be more positively charged, thus increasing interaction between negatively charged DNA backbone. As a result, more tightly compacted chromatin is formed. Recently, HDAC inhibitors have been developed as therapeutic agents for many different diseases, including neurodegenerative diseases [160]. There are many different types of HDAC inhibitors, depending on which isoforms of HDAC is being targeted. For PD, some HDAC inhibitors have displayed neuroprotective effects by increasing dopamine uptakes and dopamine biosynthetic enzyme, tyrosine hydroxylase
[161-163]. However, how these HDAC inhibitors yield such effects is not completely understood. Hence, identifying HDAC 2 as one of the LRRK2 substrates may reveal
HDAC 2 as a downstream effector of LRRK2, leading to more fine-tuned therapeutic approach involving HDAC inhibitors.
Tubulin is among the identified substrates of LRRK2 that have also been previously proposed [118]. Another substrate that has been previously suggested but is absent in our dataset is the microtubule-associated protein Tau, which is largely neuron- specific. Interestingly, we have identified protein phosphatase 2A (PP2A). PP2A has been shown to dephosphorylate Tau at S396, which results in disassociation of Tau from microtubules [164, 165]. The activation of PP2A has been suggested to increase in age- dependent manner, and it also has been shown that the PP2A activity is increased in
Alzheimer’s disease brains [166-168]. Our results suggest that LRRK2 may regulate the activity of PP2A, which may in turn modulate the stability of microtubules.
49
Although more than 200 potential LRRK2 substrates were identified by mass
spectrometry, only a fraction of these have been previously reported. This may suggest
that signaling pathways regulated by LRRK2 are much more extensive than previously
realized. Similar chemical genetic screens of other AS-kinases have also yielded large
numbers of identified substrates as reported by other groups [112, 169, 170]. It is also
likely that not every protein that we have identified in this screen may serve as a direct
substrate of LRRK2, since a few proteins may be inadvertently bound to
thiophosphorylated substrates that are directly recognized by the α-thioP antibody.
Corroboration with results from covalent capture of thiophosphopetides will further enhance the specificity of the screen [170]. Another possibility is that the overexpression of the active mutant of LRRK2, G2019S, necessary for maximizing detection sensitivity in our assay may causes over-representation of phosphorylated substrates being identified. Conversely, some substrates may have evaded the detection by mass spectrometry (e.g. VPS35) due to low abundance, the effect of 50-kDa cut-off (to avoid
IgG contamination) or limited sites available for thiophosphate labeling. Improved strategies of enrichment and more sensitive detection of direct kinase substrates will likely overcome these hurdles.
Our analysis of identified LRRK2 substrates suggests that LRRK2 is involved in various cellular processes. Perturbations of LRRK2-mediated signaling pathways are
likely detrimental to neuronal function and may contribute to PD pathogenesis. While it
remains a formidable challenge to experimentally validate all individual LRRK2 substrates, such an endeavor will likely lead to identification of suitable drug targets for the development of therapeutic strategies for PD.
50
Chapter 3: Future Directions
51
In this study, we have identified substrates of LRRK2 using a chemical genetic approach; minimally modified analog-sensitive G2019S, which harbors the most common pathogenic mutation in LRRK2, specifically accepts synthetic N6-Bn-ATPγS as a phosphor donor to yield thiophosphorylated proteins, while endogenous LRRK2 is not able to accommodate such synthetic ATPγS-analogs. Using this strategy, we have performed global screening of all thiophosphorylated proteins in HEK293T cells, and have identified individual proteins by Western blot and mass spectrometry. The results indicate that LRRK2 may be involved in various cellular processes, such as regulation of transcription, translation and protein transport. Among these processes, we are particularly interested in the role of LRRK2 in regulation of protein translation, and how it affects PD pathogenesis. Toward this goal, we propose the following future studies.
Specific aim I. To investigate the effects of pathogenic mutations in LRRK2 and eIF4G1.
We have confirmed eIF4G1 as a LRRK2 substrate by investigating whether
LRRK2 can directly phosphorylate eIF4G1 using an in vitro kinase assay (Figure 10).
The data shown is a direct confirmation of our chemical genetic screening, which is a
phosphorylation event between G2019S of LRRK2 and wild-type eIF4G1. It has been
reported that a pathogenic mutation, R1205H, in eIF4G1 causes autosomal dominant,
late-onset PD [14]. Hence, it will be interesting to monitor the phosphorylation event
between G2019S and R1205H, to investigate whether two pathogenic mutations
responsible for PD impose additive effect; in other words, whether G2019S-induced
phosphorylation of R1205H is greater than that of wild-type eIF4G1, and whether
G2019S indeed increases phosphorylation of eIF4G1 compared to wild-type LRRK2. In
order to do so, we can perform in vitro kinase assay using wild-type LRRK2 and wild-
52 type eIF4G1, wild type LRRK2 and eIF4G1 R1205H, LRRK2 G2019S and wild type eIF4G1, and finally LRRK2 G2019S and eIF4G1 R1205H. All of these experiments can be performed using ATPγS. Then, we can quantitate the levels of thiophosphorylated
eIF4G1 variants for direct comparison.
Specific aim II. To identify phosphorylation site(s) of eIF4G1 specific to LRRK2.
eIF4G1 is a well-known phosphoprotein [171-173]. There are many different
kinases that can phosphorylate eIF4G1, such as mTOR, PI3K, which are from upstream
of translation initiation. Phosphorylation of eIF4G1 is thought to increase binding affinity
to eIF4E, which will increase cap-dependent translation initiation [146, 174]. Thus, it will
be important to assess whether LRRK2-induced phosphorylation of eIF4G1 reflects such
signaling cascade. In order to do so, LRRK2-induced phosphorylation site(s) must be
investigated. Then, we can generate eIF4G1 mutants with either constitutively active
phosphorylation site(s), or phosphorylation inactive site(s). All of these eIF4G1 mutants
can be generated with or without the pathogenic mutation R1205H. Using these mutants,
we can confirm whether LRRK2 recognizes these eIF4G1 mutants as substrates. In addition, we can overexpress these eIF4G1 mutants in HEK293T cells, and monitor
whether these phosphorylation site mutations cause any cell toxicity. In addition, we can
immunoprecipitate eIF4G1 mutants to investigate whether their binding affinity to eIF4E
has changed, by Western blot using eIF4E-specific antibody.
Specific aim III. To investigate transcripts that are specific to the LRRK2 G2019S.
If LRRK2 indeed plays an important role in regulation of protein synthesis, it will
be crucial to identify specific transcripts that are affected by LRRK2 mutations, more
specifically G2019S. In animal models, G2019S-induced phenotypes have not been
53 consistent. It is possible that true parkinsonism phenotypes could be difficult to generate,
because LRRK2 is involved in many different cellular pathways. In addition, there are
many proteins that are involved in translation and post-translational modification, along
with many chaperones, thus overall phenotypic effect may be minimal by LRRK2
mutations. However, we can still investigate whether particular transcripts are either up-
regulated or down-regulated when G2019S is present, which can pin-point specific
cellular pathway it may affect, and elucidate the biological function of LRRK2. Hence,
we can overexpress either wild-type LRRK2 or G2019S in HEK293T cells, isolate
mRNAs, and then analyze transcripts by microarrays. Moreover, we can compare the
changes of transcripts when eIF4G1 R1205H is present in addition to LRRK2 G2019S.
Specific aim IV. To investigate mTOR as a direct substrate of LRRK2.
In addition to eIF4G1, we have also identified mTOR as a LRRK2 substrate by
mass spectrometry. mTOR plays an important role in translation initiation by directly
phosphorylating 4E-BP1, which competes with eIF4G1 for binding to eIF4E.
Phosphorylation of 4E-BP1 has been shown to release itself from eIF4E, thus releasing eIF4E for eIF4G1 binding. As a result, eIF4F complex formation increases, thus promoting cap-dependent translation initiation. Moreover, LRRK2 has been postulated to target 4E-BP1 as a substrate in Drosophila, yet more direct evidence from mammalian cells is lacking. Our data suggest that LRRK2 may regulate phosphorylation of 4E-BP1 via mTOR, hence facilitating eIF4E availability for eIF4G1. To establish putative mechanism of how LRRK2 may regulate protein synthesis via eIF4G1 and mTOR, more mechanistic studies on LRRK2 and mTOR are required (Figure 9). Hence, we will
54 validate whether mTOR is indeed a direct substrate of LRRK2 by in vitro kinase assay using mTOR and G2019S, and in cell-based experiments.
Specific aim V. To investigate the relationship between mTOR and LRRK2, in their
kinase activity.
We have identified mTOR as a LRRK2 substrate, but mTOR is also a
serine/threonine kinase. Thus it is possible that mTOR may also phosphorylate LRRK2,
hence mTOR and LRRK2 may mutually regulate each other. In order to test such a
theory, we can investigate phosphorylation sites of these two proteins. LRRK2
autophosphorylation sites have already been investigated [75], and several
phosphorylation sites of mTOR by other kinases have also been identified [175-178].
Therefore, when we allow mTOR phosphorylation by LRRK2, we will also examine
phosphorylated LRRK2, which could be a result from autophosphorylation or
phosphorylation by mTOR. We can identify phosphorylation site(s) of each protein, and
then compare to phosphorylation sites that are already known. Identifying LRRK2-
specific phosphorylation site(s) of mTOR will further allow generation of
phosphorylation-inactive mutant of mTOR, which will be used to study the signaling
cascade.
Specific aim VI. To investigate the signaling cascade from LRRK2 to 4E-BP1.
As previously mentioned, mTOR regulates phosphorylation of 4E-BP1 and 4E-
BP1 has been postulated to be one of LRRK2 substrates. Since we identified mTOR as
one of LRRK2 substrates, but not 4E-BP1, we hypothesized that there is a putative
signaling cascade from LRRK2 to mTOR, and then to 4E-BP1. To confirm this
hypothesis, we can investigate phosphorylation events between these three proteins using
55 in vitro kinase assay. First, we need to evaluate whether LRRK2 can directly
phosphorylate 4E-BP1, as some have postulated, using only LRRK2 and 4E-BP1. Then,
we can compare phosphorylated 4E-BP1 when mTOR is also present, by using LRRK2,
mTOR and 4E-BP1. If phosphorylated 4E-BP1 when LRRK2 and mTOR are present is
much greater than the phosphorylated 4E-BP1 by LRRK2 alone, then the phosphorylated
4E-BP1 by LRRK2 can serve as a background level, if there is any. To address specific
signaling event between these three proteins, we can generate phosphorylation-inactive
mutant of mTOR from the previous study proposed, and then investigate whether
phosphorylation of 4E-BP1 is impaired, or falls to the background level.
Global chemical genetic screening has provided such a powerful to investigate a
broad range of substrates that are specific to a kinase. As a result, we have identified
about 200 proteins as putative substrates of LRRK2. However, it is also possible that some of the proteins are not direct substrates of LRRK2, because these proteins are
identified after immunoprecipitation. In other words, some of the proteins are identified
due to strong protein-protein interactions that are not necessarily specific for LRRK2.
Hence, additional replications of experiments could provide more fine-tuned list of
putative LRRK2 substrates by eliminating non-overlapping proteins. In addition, despite
the complete list of putative substrates illuminating the potential role of LRRK2, the
possibility of increased or aberrant phosphorylation must not be neglected, because we
have identified substrates by using a gain-of-function LRRK2 mutant G2019S. Hence, additional screening of LRRK2 substrates using wild type LRRK2 variants would provide another analysis of LRRK2 putative substrates, and the difference in putative
substrates between LRRK2 G2019S and wild type could elucidate possible mechanisms
56 leading to PD pathogenesis. Ideally, individual verification of each substrate, whether
LRRK2 can directly induce phosphorylation or not, should be performed. Unfortunately,
such verification requires enormous time and resources that it may not be feasible.
Despite these caveats, chemical genetic screening allows for investigation of a broad
range of substrates of a kinase in a rather unbiased way. Applying this method, we have
identified several putative substrates, such as eIF4G1, VPS35, and mTOR, which have
been implicated in PD pathogenesis in several different studies.
57
Tables
58
Table 1. LRRK2 mutations and clinical phenotypes, as reported in various populations. Mutation Phenotype Reference R144C/G Scattered phenotype between [50] Lewy body formation, Tau pathology, or neither. Y1699C Dementia, protein aggregates, DA [35, 49] neuronal loss G2019S Identical as sporadic PD, DA [51, 52] neuronal loss I2020T DA neuronal loss without Lewy [48] body formation
Table 2. List of small molecule LRRK2 inhibitors. There are several LRRK2 inhibitors with different molecular structures. Name Structure IC50 WT IC50 G2019S Reference (nM) (nM) LRRK2-IN-1 13 6 [179]
CZC-25146 4.8 6.9 [180]
CZC-54252 1.3 1.85 [180]
TAE684 7.8 6.1 [181]
59
HG10-102-01 20.3 9.8 [182]
GSK2578215A 10.9 8.9 [183]
Table 3. List of proposed LRRK2 substrates. Proposed Substrates Function Reference ArfGAP1 (ADP-ribosylation GTPase-activating protein [113] factor GTPase-activating protein 1) ERM (Ezrin/radixin/moesin) Crosslinking actin filaments with [116] plasma membranes β-tubulin Microtubule assembly [117] Tau Microtubule stability [119] Drp1 (Dynamin-related Mitochondrial fission [125] protein 1) 4E-BP1 (Eukaryotic Transcription repression [128, 130] translation initiation factor 4E- binding protein 1) FOXO 1 (Forkhead box Regulation of gluconeogenesis, [133] protein O1) glycolysis, adipogenesis EndoA (Endophilin A) Recruitment of endocytic proteins [134] Prdx3 (Thioredoxin-dependent Antioxidant [136] peroxide reductase) Snapin (SNARE-associated Vesicle docking [137] protein)
60
Figures
61
Figure 1. Lewy body formation, which is mainly composed of α-synuclein and other protein aggregates, is a pathological hallmark of Parkinson’s disease. Taken from [10]. Copyright permission obtained from John Wiley and Sons.
R1441C R1441G R793M I1122V R1441H G2019S E334K L1151T A1442P Q930R M1869T I2012T I2020T S1096C I1192V R1514Q L1795F A211V P755L S973N I1371V R1628P Y2006H T2031S
K544E R1067Q S1228T Y1699C R1941H T2356I G2385R E10K 1 LRR RocCOR MAPKKK WD40 2527 984 1278 1334 15121513 1878 1879 2138 2142 2498
Figure 2. Schematic diagram of LRRK2 functional domains, and disease-associated mutations in LRRK2. Mutations that have been identified as pathogenic are shown in red. Mutations that serve as a PD-risk factor are shown in green, and other mutations that have been identified in LRRK2 with unknown consequences are shown in blue. Taken from [46]. Copyright permission obtained from John Wiley and Sons.
62
A WT- kinase B
substrate substrate Kinase regions A*TPS c-Src AS -LRRK2 Lpl1 P-S v-erbB PKC substrate substrate CDC28 CaMKIIa A*TPS FUS3
X LRRK2 WT M L V M E L D Y G AS-LRRK2 M L V G E L D Y G -NH2 ATPS AS-G2019S M L V G E L D Y S X= 1947 2019 A*TPS
CD
Figure 3. Generation of analog-sensitive versions of LRRK2. A) Schematic diagram of the chemical genetics approach to LRRK2 substrate identification. A synthetic ATPγS analog (A*TPγS) contains a bulky group at the N6 position (X) cannot be utilized by wild type (WT) kinase LRRK2 for thiophosphorylation. When the “gatekeeper residue” in the ATP binding pocket of LRRK2 is mutated to accommodate a bulky A*TPγS, the analog- sensitive version of LRRK2 (AS-LRRK2) will catalyze thiophosphorylation of its direct substrates. The thiophosphate moiety on the substrates can be specifically recognized by a monoclonal antibody (α-thioP). B). Sequence alignment of amino acids in the ATP binding pocket of the conserved kinase regions showing the large gatekeeper residue (red) in different kinases. The gatekeeper residue of wild-type LRRK2 is identified as M1947, which is mutated to a smaller residue G in AS-LRRK2 and in mutant LRRK2 carrying PD-linked G2019S mutation (AS-G2019S). C). FLAG-tagged LRRK2 variants (WT, G2019S, AS-LRRK2, and AS-G2019S) are immunoprecipitated by anti-FLAG M2 agarose from transiently transfected HEK293T cells, and visualized by silver staining. D). The anti-FLAG immunoprecipitated LRRK2 variants are confirmed on Western blots with anti-LRRK2 antibody.
63
MBP + + + + + A ATPγS + + + + + ATP ‐ + ‐ ‐ ‐ PhEt‐ATP ‐‐+ ‐ ‐ Phe‐ATP ‐‐‐ + ‐ cPe‐ATP ‐‐‐ ‐+ Thio P‐MBP
AS‐LRRK2
B
MBP + ‐ + + + ‐ + + ATPγS ‐ + ‐ ‐ ‐ + ‐ ‐ Ynk1p‐ATPγS ‐‐+ ‐ ‐ ‐ + ‐ Ynk1p‐cPe‐ATPγS ‐‐‐+ ‐ ‐ ‐ + Thio P‐MBP Wild type AS‐LRRK2
MBP + + + + Ynk1p‐ATPγS + ‐ + ‐ C Ynk1p‐cPe‐ATPγS ‐ + ‐ + Thio P‐MBP G2019S AS‐G2019S
Figure 4. AS-LRRK2 shows specificity toward N6-cPe-ATPγS. A) Indirect competition assay using ATP-analogs. N6-cPe-ATP competes with ATPγS for AS-LRRK2 catalyzed thiophosphorylation of MBP. B) Synthesis of N6-cPe-ATPγS using yeast NDPK Ynk1p, which mediates the transfer of γ-phosphates to dinucleosides. In vitro kinase assay shows that AS-LRRK2, but not LRRK2 WT, can utilize N6-cPe-ATPγS as a phosphor donor. C) N6-cPe-ATPγS is not utilized by G2019S and AS-G2019S for thiophosphorylation of MBP.
64
MBP + + + + ATPγS + ‐ ‐ ‐ PhEt‐ATPγS ‐ + ‐ ‐ Fur‐ATPγS ‐‐+ ‐ Bn‐ATPγS ‐‐‐+
WT Thio P‐MBP
AS‐LRRK2 Thio P‐MBP
G2019S Thio P‐MBP
Thio P‐MBP AS‐G2019S
Figure 5. AS-LRRK2 and AS-G2019S show similar sensitivity to N6-Bn-ATPγS. Various commercially available ATPγS-analogs were examined for thiophosphorylation of MBP by AS-LRRK2 and AS-G2019S. Both AS-LRRK2 and AS-G2019S show sensitivity to N6-Bn-ATPγS. In addition, AS-G2019S shows sensitivity to N6-PhEt- ATPγS, but to the lesser extent than N6-Bn-ATPγS.
65
15 30 45 60 15 30 45 60 mins A WB: α-LRRK2
WB: α- thio-phosphate
Coomassie-stained MBP
G2019S AS-G2019S
B
Figure 6. The kinetics of AS-G2019S induced MBP thiophosphorylation. As compared to G2019S. AS-G2019S shows slower initial enzymatic catalysis, yet similar final level of MBP thiophosphorylation is achieved in 60 minutes. A) G2019S and AS-G2019S thio- phosphorylation was measured by time course assay. Four identical sets of in vitro kinase reaction are performed at the indicated time points. The signal intensity of MBP thiophosphorylation on Western blot (arbitrary unit) is shown. B) Quantitative representation of the time course assay. Thiophosphorylated-MBP level is normalized by the amount of LRRK2 corresponding to the time point. The scatter plot represents the ratio of thiophospho-MBP formation over the time course.
66
A B kD 250
WB: 150 kD α‐LRRK2 250
100 210 α‐eIF4G1 75 90 α‐VPS35
50 IgG
Figure 7. In vivo labeling of substrates using AS-G2019S reveals phosphorylation of many endogenous proteins in HEK293T cells. A) AS-G2019S thio-phosphorylates endogenous proteins present in HEK293T cells. After labeling of digitonin-permeabilized cells expressing G2019S or AS-G2019S with N6-Bn-ATPγS, thiophosphorylated proteins are immunoprecipitated with the thioP antibody, and detected on Western blot. Many cellular proteins are thiophosphorylated in the presence of AS-G2019S, while G2019S shows a minor level of background activity. B) AS-G2019S induces phosphorylation of several PD-associated proteins Probing the blot from A) using protein-specific antibodies shows that AS-G2019S phosphorylates eIF4G1 and VPS35, in addition to autophosphorylation.
67
Figure 8. Global identification of AS-G2019S substrates using tandem mass spectrometry. A) Putative LRRK2 substrates are identified as thiophosphorylated proteins present only in AS-G2019S-expressing cells but not in G2019S-expressing cells that are permeabilized with digitonin to allow uptake of N6-Bn-ATPγS. without disrupting overall cellular organization. A total of about 200 proteins are identified as putative AS-G2019S substrates. They belong to different groups with diverse cellular functions. About 50% of the proteins are involved in transcription, post-transcriptional modification, translation and protein transport. B) Putative LRRK2 substrates identified in this study. Several putative substrates may play an important role in protein translation and synthesis. In addition to LRRK2, identified candidate substrates include translation factors, such as eIF4G1, eIF4G2, elongation factor 1 and 2, and mTOR, a master regulator of protein synthesis, as well as tubulin, which has been previously proposed as a LRRK2 substrate.
68
69
Figure 9. Tandem Mass spectra representing each unique peptide identified in A) LRRK2 and B) eIF4G1.
70
Figure 10. eIF4G1 is a direct substrate of LRRK2. A) Flag-tagged G2019S, eIF4G1 isoform b and its pathogenic mutant R1205H are overexpressed, and then immunoprecipitated using anti-flag beads. eIF4G1 isoform b is more stably expressed than the full-length eIF4G1. B) The immunoprecipitated G2019S and eIF4G1 isoform b are used to perform in vitro kinase assay to assess phosphorylation activity. G2019S undergoes autophosphorylation and phosphorylation of its substrate, eIF4G1. Both autophosphorylation of LRRK2 and phosphorylation of eIF4G1 increase, when they are present together.
71
ADP ATP mTOR LRRK2 ATP ATP ADP ADP 4E‐BP1 eIF4G1
eIF4E
eIF4F (eIF4G ∙ eIF4E∙ eIF4A)
Cap‐dependent translation
Figure 11. Putative mechanism of LRRK2 regulation in protein synthesis. Phosphorylation of eIF4G1 by LRRK2 may increase its binding affinity to eIF4E, and then enhances the eIF4F complex formation. LRRK2 may regulate mTOR, which phosphorylates 4E-BP1. 4E-BP1 is tightly bound to eIF4E, but phosphorylation of 4E- BP1 by mTOR releases eIF4E, thus eIF4G1 can bind. All of these steps are important in progression to cap-dependent translation, and deregulation of any of these steps could result in cellular dysfunction.
72
Appendix
73
Appendix I. Complete list of putative LRRK2 substrates identified by tandem mass spectrometry. The columns represent the protein accession number, number of unique peptide identified by the tandem mass spectrometry, and its protein identity, respectively.
Accession Number of Protein Name Number peptides Identified IPI00296337 40 DNA-dependent protein kinase catalytic subunit IPI00844578 22 ATP-dependent RNA helicase A IPI00013452 15 Bifunctional aminoacyl-tRNA synthetase
IPI00783271 15 Leucine-rich PPR motif-containing protein, mitochondrial
IPI00953508 15 DNA topoisomerase 2 IPI00329719 13 Myosin-Id IPI00003865 11 Heat shock cognate 71 kDa protein
IPI00180675 9 Tubulin alpha-1A chain IPI00008557 9 Insulin-like growth factor 2 mRNA-binding protein 1 IPI00100160 9 Cullin-associated NEDD8-dissociated protein 1 IPI00411559 8 Structural maintenance of chromosomes protein 4 IPI00420014 8 U5 small nuclear ribonucleoprotein 200kD helicase IPI00829992 8 Myosin-1c IPI00553185 8 T-complex protein 1 subunit gamma IPI00006196 8 Nuclear mitotic apparatus protein 1 IPI00006482 7 Sodium/potassium-transporting ATPase subunit alpha-1 IPI00297211 7 SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 IPI00000690 7 Apoptosis-inducing factor 1, mitochondrial IPI00004233 7 Antigen KI-67 IPI00000846 7 Chromodomain-helicase-DNA-binding protein 4 IPI00022744 7 Exportin-2 IPI00306369 7 tRNA (cytosine-5-)methyltransferase NSUN2 IPI00017297 7 Matrin-3 IPI00219420 7 Structural maintenance of chromosome protein 3 IPI00873959 7 Myosin VA isoform 2 IPI00291939 6 Structural maintenance of chromosomes protein 1A IPI00651653 6 Probable ATP-dependent RNA helicase DDX17 IPI00640703 6 Exportin-5 IPI00300127 6 N-acetyltransferase 10 IPI00305068 6 Pre-mRNA-processing factor 6 IPI00028275 6 Cytoskeleton-associated protein 5 IPI00444452 6 Putative helicase MOV-10 IPI00175649 6 Leucine-rich repeat serine/threonine kinase 2 IPI00186290 6 Elongation factor 2 IPI00400922 5 Protein RRP5 homolog IPI00008524 5 Polyadenylate-binding protein 1 IPI00031519 5 DNA (cystosine-5)-methyltransferase 1 IPI00644127 5 Isoleucyl-tRNA synthetase, cytoplasmic
74
IPI00376344 5 Myosin-1b IPI00925477 5 Topoisomerase II beta IPI00394788 5 Probable alanyl-tRNA synthetase, mitochondrial IPI00004860 5 Arginyl-tRNA synthetase, cytoplasmic IPI00295857 5 Coatomer subunit alpha IPI00215743 5 Ribosome-binding protein 1 IPI00017283 5 Isoleucyl-tNRA synthetase, mitochondrial IPI00156374 5 Importin-4 IPI00015806 5 General transcription factor 3C polypeptide 3 IPI00414482 4 General transcription factor 3C polypeptide 1 IPI00024067 4 Clathrin heavy chain 1 IPI00397904 4 Nuclear pore complex protein Nup93 IPI00007188 4 ADP/ATP translocase 2 IPI00008603 4 Actin IPI00145593 4 Nucleolar MIF4G domain-containing protein 1 (NOM1) IPI00297779 4 T-complex protein 1 subunit beta IPI00302927 4 T-complex protein 1 subunit delta IPI00784090 4 T-complex protein 1 subunit theta IPI00101186 4 RRP-12-like protein IPI00022202 4 Phosphate carrier protein, mitochondrial IPI00025087 4 Cellular tumor antigen p53 IPI—384456 4 Isoform GTBP-N of DNA mismatch repair protein Msh6 IPI00470649 4 Nicalin IPI00006379 4 Nucleolar protein 58 IPI00411937 4 Nucleolar protein 56 IPI00793443 4 Importin-5 IPI00007401 4 Importin-7 IPI00604664 4 NADH-ubiquinone oxidoreductase 75kDa subunit IPI00940786 4 DNA replication licensing factor MCM3 IPI00456969 4 Cytoplamic dynein 1 heavy chain 1 IPI00003519 4 116kDa U5 small nuclear ribonucleoprotein component IPI00655631 4 DNA polymerase IPI00375358 4 Replication factor C subunit 1 IPI00654555 4 NOP2 protein IPI00396435 4 Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX15 IPI0041173 4 Putative ATP-dependent RNA helicase DHX30 IPI00027442 4 Alanyl-tRNA synthetase, cytoplasmic IPI00032374 4 Ribosomal RNA processing protein 1 homolog B IPI00157790 4 Proteasome-associated protein ECM29 homolog IPI00025057 4 Double-stranded RNA-specific adenosine deaminase IPI00027230 4 Endoplasmin IPI00007752 4 Tubulin beta-2C chain IPI00514053 4 Coatomer subunit delta IPI00300659 4 Parafibromin IPI00045946 4 ATP-dependent metalloprotease YME1L1 IPI00163085 4 Angiomotin IPI00242630 4 HEAT repeat-containing protein 2 IPI00024279 3 HEAT repeat-containing protein 1
75
IPI00550689 3 UPF0027 protein C22orf28 IPI00185146 3 Importin-9 IPI00001639 3 Importin subunit beta-1 IPI00002214 3 Importin subunit alpha-2 Karyopherin alpha 2 IPI00183462 3 Exosome complex exonuclease RRP44 IPI00783781 3 Nuclear pore complex protein Nup205 IPI00411886 3 Nucleolar complex protein 2 homolog IPI00171542 3 Nucleoporin 85 IPI00550021 3 60S ribosomal protein L3 IPI00003918 3 60S ribosomal protein L4 IPI00013485 3 40S ribosomal protein S2 IPI00020729 3 Insulin receptor substrate 4 IPI00745266 3 Eukaryotic translation initiation factor 3 subunit L IPI00956559 3 Eukaryotic translation initiation factor 4 gamma 1 IPI00337541 3 NAD(P) transhydrogenase, mitochondrial IPI00026089 3 Splicing factor 3B subunit 1 IPI00021187 3 RuvB-like 1 IPI00658000 3 Insulin-like growth factor 2 mRNA-binding protein 3 IPI00006197 3 Nuclear valosin-containing protein like IPI00941161 3 General vesicular transport factor p115 IPI00337385 3 Pre-mRNA-processing factor 40 homolog A IPI00018465 3 T-complex protein 1 subunit eta IPI00158615 3 THO complex subunit 2 IPI00644576 3 Filamin A, alpha IPI00219352 3 Cystathionine beta-synthase IPI00604527 3 Threonyl-tRNA synthetase, mitochondrial IPI00549566 3 Probable arginyl-tRNA synthetase, mitochondrial IPI00783726 3 Kinectin isoform b IPI00472160 3 Rho/rac guanine nucleotide exchange factor 2 IPI00017303 3 DNA mismatch repair protein Msh2 IPI00418797 3 DNA-directed RNA polymerase IPI00027808 3 DNA-directed RNA polymerase II subunit RPB2 IPI00015953 3 Nucleolar RNA helicase 2 IPI00001091 3 AFG3-like protein 2 IPI00026781 3 Fatty acid synthase IPI00034049 3 Regulator of nonsense transcripts 1 IPI00328306 3 Zinc finger CCCH domain-containing protein 11A IPI00374476 3 G patch domain-containing protein 4 isoform 1 IPI00641364 3 PPAN-P2RY11 protein IPI00006721 3 Dynamin-like 120kDa protein, mitochondrial IPI00879242 3 Ewing sarcoma breakpoint region 1 IPI00292953 3 Ankycorbin IPI00179057 3 Cullin-4B IPI00783302 3 Pentatricopeptide repeat-containing protein 3, mitochondrial IPI00008575 2 KH domain-containing, RNA-binding, signal transduction- associated protein 1 IPI00025874 2 Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 precursor (DDOST)
76
IPI00297492 2 Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit STT3A IPI00005675 2 NF-kappa-B-repressing factor IPI00102815 2 Nucleolar complex protein 3 homolog IPI00152890 2 Nucleolar protein 6 IPI00022613 2 Nucleolar protein 14 IPI00291200 2 Nuclear pore complex protein Nup133 IPI00748807 2 Nuclear pore complex protein Nup160 IPI00554777 2 Asparagine synthetase (glutamine-hydrolyzing) IPI00294834 2 Aspartyl/asparaginyl beta-hydroxylase IPI00011603 2 26S proteasome non-ATPase regulatory subunit 3 IPI00010720 2 T-complex protein 1 subunit epsilon IPI00178431 2 ATP-dependent DNA helicase Q1 IPI00032423 2 Probable ATP-dependent RNA helicase DDX52 IPI00879999 2 ATP-dependent RNA helicase DDX54 IPI00007084 2 Calcium-binding mitochondrial carrier protein Aralar2 IPI00844172 2 Myosin IPI00293426 2 Transcription activator BRG1 IPI00001159 2 Translational activator GCN1 IPI00410330 2 Transcriptional repressor p66-alpha IPI00644079 2 Heterogeneous nuclear ribonucleoprotein U IPI00916060 2 DNA-directed RNA polymerase IPI00153051 2 Poly (A) RNA polymerase, mitochondrial IPI00183626 2 Polypyrimidine tract-binding protein 1 isoform a IPI00020194 2 TATA-binding protein-associated factor 2N IPI00029324 2 Midline-1 IPI00069750 2 Poly(U)-binding-splicing factor PUF60 IPI00007928 2 Pre-mRNA-processing splicing factor 8 IPI00004968 2 Pre-mRNA-processing factor 19 IPI00010740 2 Splicing factor, proline- and glutamine-rich IPI00300371 2 Splicing factor 3B subunit 3 IPI00216113 2 Kinesin-like protein KIF2C IPI00175193 2 Chromosome-associated kinesin KIF4B IPI00414973 2 FAST kinase domain-containing protein 5 IPI00401959 2 Protein SON isoform F IPI00010590 2 Lymphoid-specific helicase IPI00926560 2 Leucyl-tRNA synthetase, mitochondrial IPI00925046 2 Glutaminyl-tRNA synthetase IPI00031410 2 Serine/threonine-protein kinase mTOR IPI00554737 2 Serine/threonine-protein phosphatase 2A 65kDa regulatory subunit A alpha isoform IPI00647720 2 Serine/arginine repetitive matrix protein 1 IPI00782992 2 Serine/arginine repetitive matrix protein 2 IPI00456919 2 E3 ubiquitin-protein ligase HUWE1 IPI00180305 2 E3 ubiquitin-protein ligase UBR4 IPI00289776 2 Probable E3 ubiquitin-protein ligase MYCBP2 IPI00645078 2 Ubiquitin-like modifier-activating enzyme 1 IPI00304187 2 RNA-binding protein 28 IPI00032355 2 Pumilio homolog 1 (Drosophila), isoform CRA_c
77
IPI00072534 2 Protein unc-45 homolog A IPI00217957 2 Lamin-B1 IPI00009771 2 Lamin-B2 IPI00337325 2 Hyaluronan mediated motility receptor IPI00334914 2 TRM1-like protein IPI00025447 2 Elongation factor 1-alpha IPI00782985 2 Eukaryotic translation initiation factor 4 gamma 2 IPI00844264 2 Cold shock domain-containing protein E1 isoform 3 IPI00430472 2 Activating signal cointegrator 1 complex subunit 3 IPI00299524 2 Condensin complex subunit 1 IPI00003886 2 Guanine nucleotide-binding protein-like 3 IPI00478657 2 G-rich sequence factor 1 IPI00465429 2 RUN and FYVE domain-containing protein 1 IPI00002372 2 ATP-binding cassette sub-family D member 3 IPI00005045 2 ATP-binding cassette sub-family F member 2 IPI00941747 2 Calnexin IPI00789740 2 Gem (Nuclear organelle) associated protein 4 IPI00107531 2 DNA repair protein RAD50 IPI00783872 2 Caprin-1 IPI00024364 2 Transportin-1 IPI00298961 2 Exportin-1 IPI00302458 2 Exportin-7 IPI00216694 2 Plastin-3 IPI00143753 2 U2-associated protein SR140 IPI00219160 2 60S ribosomal protein L34 IPI00385495 2 Lipase maturation factor 2 IPI00411614 2 WD repeat and HMG-box DNA-binding protein 1 IPI00006451 2 Vesicle-fusing ATPase IPI00289601 2 Histone deacetylase 2 IPI00641873 2 Staufen, RNA binding protein, homolog 1 IPI00939488 2 Anaphase-promoting complex subunit 7 IPI00009464 2 Exosome component 10 IPI00013830 2 SNW domain-containing protein 1 IPI00014310 2 Cullin-1 IPI00023409 2 Regulator of nonsense transcripts 3B IPI00024642 2 Coiled-coil domain-containing protein 47 IPI00024664 2 Ubiquitin carboxyl-terminal hydrolase 5 IPI00031397 2 Long-chain-fatty-acid-CoA ligase 3 IPI00075081 2 Fanconi anemia group D2 protein IPI00106495 2 Condensin complex subunit 3 IPI00219740 2 DNA replication licensing factor MCM7 IPI00477123 2 Protein TBRG4 IPI00413265 2 Structural maintenance of chromosomes protein 5 IPI00413895 2 Golgin subfamily A member 2 IPI00030275 2 Heat shock protein 75kDa, mitochondrial IPI00056499 2 Zinc finger protein 622 IPI00154283 2 Protein CIP2A IPI00644715 2 Chaperone, ABC1 activity of bc1 complex homolog IPI00783982 2 Coatomer subunit gamma
78
IPI00100106 1 Synembryn-A IPI00007927 1 Structural maintenance of chromosomes protein 2 IPI00642982 1 LONP1 protein IPI00167941 1 Midasin IPI00641719 1 Surfeit 4 IPI00010368 1 Kinesin-like protein KIF2A IPI00027834 1 Heterogeneous nuclear ribonucleoprotein L
IPI00012074 1 Heterogeneous nuclear ribonucleoprotein R
IPI00013070 1 Heterogeneous nuclear ribonucleoprotein U-like protein 1
IPI00655649 1 EXOC4 protein IPI00293655 1 ATP-dependent RNA helicase DDX1 IPI00003768 1 Pescadillo homolog IPI00479432 1 Methyltransferase-like protein 13 IPI00293859 1 Histone-lysine N-methyltransferase MLL2 IPI00844000 1 E3 UFM1-protein ligase 1 IPI00026320 1 E3 ubiquitin-protein ligase UBR5 IPI00328911 1 E3 ubiquitin-protein ligase HECTD1 IPI00054042 1 General transcription factor II IPI00339381 1 Helicase-like transcription factor IPI00166010 1 CCR4-NOT transcription complex subunit 1 IPI00299507 1 Condensin complex subunit 2 IPI00152377 1 Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit STT3B IPI00000279 1 Zinc finger CCCH domain-containing protein 15 IPI00000725 1 Zinc finger protein 451 IPI00030624 1 Serine/threonine-protein phosphatase 6 regulatory subunit 3 IPI00010080 1 Serine/threonine-protein kinase OSR1 IPI00647073 1 Cullin 2 IPI00183572 1 Dedicator of cytokinesis protein 7 IPI00010153 1 60S ribosomal protein L23 IPI00329389 1 60S ribosomal protein L6 IPI00299573 1 60S ribosomal protein L7a IPI00025273 1 Trifunctional purine biosynthetic adenosine-3 IPI00031820 1 Phenylalanyl-tRNA synthetase alpha chain IPI00163381 1 Prolyl 3-hydroxylase 1 IPI00644087 1 Progerin IPI00377261 1 Far upstream elemnt-binding protein 3 IPI00293464 1 DNA damage-binding protein 1 IPI00102864 1 Hexokinase-2 IPI00009790 1 6-phosphofructokinase type C IPI00219483 1 U1 small nuclear ribonucleoprotein 70kDa IPI00101532 1 Centrosomal protein of 55kDa IPI00100151 1 5’-3’ exoribonuclease 2 IPI00106502 1 Kelch-like ECH-associated protein 1 IPI00023344 1 Symplekin
79
IPI00028980 1 Unhealthy ribosome biogenesis protein 2 homolog IPI00020127 1 Replication protein A 70kDa DNA-binding protein subunit IPI00009379 1 DNA-binding protein SMUBP-2 IPI00873518 1 MAD1 mitotic arrest deficient-like 1 IPI00306048 1 ATPase family AAA domain-containing protein 3B IPI00328200 1 Glutamine-rich protein 1 IPI00409635 1 Extended synaptotagmin-2 IPI00217952 1 Glucosamine-fructoase-6-phosphate aminotransferase (isomerizing) 1 IPI00030774 1 Tubulin-specific chaperone D IPI00384176 1 Protein polybromo-1 IPI00431405 1 UPF0465 protein C5orf33 IPI00293260 1 DnaJ homolog subfamily C member 10 IPI00333913 1 Neuroblastoma-amplified sequence IPI00743121 1 Sphingomeylin phosphodiesterase 4 isoform 1 IPI00217240 1 WD repeat-containing protein 75 IPI00152441 1 Minor histocompatibility antigen H13 IPI00002335 1 Huntingtin
80
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