Education & Certifications
Post-doc, MRC Protein Phosphorylation Unit, Signal Transduction (2010)
PhD, Medical College of Wisconsin, kinases & molecular genetics (2006)
BS, Austin Peay State University, Biology (2000)
Senior Research Scientist, Stanford University (8/1/2018 - Present)
- Targeting LRRK2 mutations in Parkinson's disease. Future medicinal chemistry 2022
Discovery of 1H-Pyrazole Biaryl Sulfonamides as Novel G2019S-LRRK2 Kinase Inhibitors.
ACS medicinal chemistry letters
2022; 13 (6): 981-988
G2019S (GS) is the most prevalent mutation in the leucine rich repeat protein kinase 2 gene (LRRK2), a genetic predisposition that is common for Parkinson's disease, as well as for some forms of cancer, and is a shared risk allele for Crohn's disease. GS-LRRK2 has a hyperactive kinase, and although numerous drug discovery programs have targeted LRRK2 kinase, few have reached clinical development. We report the discovery and preliminary development of an entirely novel structural class of potent and selective GS-LRRK2 kinase inhibitors: biaryl-1H-pyrazoles.
View details for DOI 10.1021/acsmedchemlett.2c00116
View details for PubMedID 35707141
Evaluation of Current Methods to Detect Cellular Leucine-Rich Repeat Kinase 2 (LRRK2) Kinase Activity.
Journal of Parkinson's disease
BACKGROUND: Coding variation in the Leucine rich repeat kinase 2 gene linked to Parkinson's disease (PD) promotes enhanced activity of the encoded LRRK2 kinase, particularly with respect to autophosphorylation at S1292 and/or phosphorylation of the heterologous substrate RAB10.OBJECTIVE: To determine the inter-laboratory reliability of measurements of cellular LRRK2 kinase activity in the context of wildtype or mutant LRRK2 expression using published protocols.METHODS: Benchmark western blot assessments of phospho-LRRK2 and phospho-RAB10 were performed in parallel with in situ immunological approaches in HEK293T, mouse embryonic fibroblasts, and lymphoblastoid cell lines. Rat brain tissue, with or without adenovirus-mediated LRRK2 expression, and human brain tissues from subjects with or without PD, were also evaluated for LRRK2 kinase activity markers.RESULTS: Western blots were able to detect extracted LRRK2 activity in cells and tissue with pS1292-LRRK2 or pT73-RAB10 antibodies. However, while LRRK2 kinase signal could be detected at the cellular level with over-expressed mutant LRRK2 in cell lines, we were unable to demonstrate specific detection of endogenous cellular LRRK2 activity in cell culture models or tissues that we evaluated.CONCLUSION: Further development of reliable methods that can be deployed in multiple laboratories to measure endogenous LRRK2 activities are likely required, especially at cellular resolution.
View details for DOI 10.3233/JPD-213128
View details for PubMedID 35599495
The E3 ligase TRIM1 ubiquitinates LRRK2 and controls its localization, degradation, and toxicity.
The Journal of cell biology
2022; 221 (4)
Missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease (PD); however, pathways regulating LRRK2 subcellular localization, function, and turnover are not fully defined. We performed quantitative mass spectrometry-based interactome studies to identify 48 novel LRRK2 interactors, including the microtubule-associated E3 ubiquitin ligase TRIM1 (tripartite motif family 1). TRIM1 recruits LRRK2 to the microtubule cytoskeleton for ubiquitination and proteasomal degradation by binding LRRK2911-919, a nine amino acid segment within a flexible interdomain region (LRRK2853-981), which we designate the "regulatory loop" (RL). Phosphorylation of LRRK2 Ser910/Ser935 within LRRK2 RL influences LRRK2's association with cytoplasmic 14-3-3 versus microtubule-bound TRIM1. Association with TRIM1 modulates LRRK2's interaction with Rab29 and prevents upregulation of LRRK2 kinase activity by Rab29 in an E3-ligase-dependent manner. Finally, TRIM1 rescues neurite outgrowth deficits caused by PD-driving mutant LRRK2 G2019S. Our data suggest that TRIM1 is a critical regulator of LRRK2, controlling its degradation, localization, binding partners, kinase activity, and cytotoxicity.
View details for DOI 10.1083/jcb.202010065
View details for PubMedID 35266954
A Phosphosite Mutant Approach on LRRK2 Links Phosphorylation and Dephosphorylation to Protective and Deleterious Markers, Respectively.
2022; 11 (6)
The Leucine Rich Repeat Kinase 2 (LRRK2) gene is a major genetic determinant of Parkinson's disease (PD), encoding a homonymous multi-domain protein with two catalytic activities, GTPase and Kinase, involved in intracellular signaling and trafficking. LRRK2 is phosphorylated at multiple sites, including a cluster of autophosphorylation sites in the GTPase domain and a cluster of heterologous phosphorylation sites at residues 860 to 976. Phosphorylation at these latter sites is found to be modified in brains of PD patients, as well as for some disease mutant forms of LRRK2. The main aim of this study is to investigate the functional consequences of LRRK2 phosphorylation or dephosphorylation at LRRK2's heterologous phosphorylation sites. To this end, we generated LRRK2 phosphorylation site mutants and studied how these affected LRRK2 catalytic activity, neurite outgrowth and lysosomal physiology in cellular models. We show that phosphorylation of RAB8a and RAB10 substrates are reduced with phosphomimicking forms of LRRK2, while RAB29 induced activation of LRRK2 kinase activity is enhanced for phosphodead forms of LRRK2. Considering the hypothesis that PD pathology is associated to increased LRRK2 kinase activity, our results suggest that for its heterologous phosphorylation sites LRRK2 phosphorylation correlates to healthy phenotypes and LRRK2 dephosphorylation correlates to phenotypes associated to the PD pathological processes.
View details for DOI 10.3390/cells11061018
View details for PubMedID 35326469
The Roc domain of LRRK2 as a hub for protein-protein interactions: a focus on PAK6 and its impact on RAB phosphorylation.
Leucine-rich repeat kinase 2 (LRRK2) has taken center stage in Parkinson's disease (PD) research as mutations cause familial PD and more common variants increase lifetime risk for disease. One unique feature in LRRK2 is the coexistence of GTPase/Roc (Ras of complex) and kinase catalytic functions, bridged by a COR (C-terminal Of Roc) platform for dimerization. Multiple PD mutations are located within the Roc/GTPase domain and concomitantly lead to defective GTPase activity and augmented kinase activity in cells, supporting a crosstalk between GTPase and kinase domains. In addition, biochemical and structural data highlight the importance of Roc as a molecular switch modulating LRRK2 monomer-to-dimer equilibrium and building the interface for interaction with binding partners. Here we review the effects of PD Roc mutations on LRRK2 function and discuss the importance of Roc as a hub for multiple molecular interactions relevant for the regulation of cytoskeletal dynamics and intracellular trafficking pathways. Among the well-characterized Roc interactors, we focused on the cytoskeletal-related kinase p21-activated kinase 6 (PAK6). We report the affinity between LRRK2-Roc and PAK6 measured by microscale thermophoresis (MST). We further show that PAK6 can modulate LRRK2-mediated phosphorylation of RAB substrates in the presence of LRRK2 wild-type (WT) or the PD G2019S kinase mutant but not when the PD Roc mutation R1441G is expressed. These findings support a mechanism whereby mutations in Roc might affect LRRK2 activity through impaired protein-protein interaction in the cell.
View details for DOI 10.1016/j.brainres.2022.147781
View details for PubMedID 35016853
- Editorial: LRRK2-Fifteen Years From Cloning to the Clinic. Frontiers in neuroscience 2022; 16: 880914
Discovery of G2019S-Selective Leucine Rich Repeat Protein Kinase 2 inhibitors with invivo efficacy.
European journal of medicinal chemistry
1800; 229: 114080
Mutations in the Leucine Rich Repeat Protein Kinase 2 gene (LRRK2) are the most common genetic causes of Parkinson's Disease (PD). The G2019S mutation is the most common inherited LRRK2 mutation, occurs in the kinase domain, and results in increased kinase activity. We report the discovery and development of compound 38, an indazole-based, G2019S-selective (>2000-fold vs. WT) LRRK2 inhibitor capable of entering rodent brain (Kp=0.5) and selectively inhibiting G2019S-LRRK2. The compounds disclosed herein present a starting point for further development of brain penetrant G2019S selective inhibitors that hopefully reduce lung phenotype side-effects and pave the way to providing a precision medicine for people with PD who carry the G2019S mutation.
View details for DOI 10.1016/j.ejmech.2021.114080
View details for PubMedID 34992038
Protein phosphatase 2A holoenzymes regulate leucine-rich repeat kinase 2 phosphorylation and accumulation.
Neurobiology of disease
LRRK2 is a highly phosphorylated multidomain protein and mutations in the gene encoding LRRK2 are a major genetic determinant of Parkinson's disease (PD). Dephosphorylation at LRRK2's S910/S935/S955/S973 phosphosite cluster is observed in several conditions including in sporadic PD brain, in several disease mutant forms of LRRK2 and after pharmacological LRRK2 kinase inhibition. However, the mechanism of LRRK2 dephosphorylation is poorly understood. We performed a phosphatome-wide reverse genetics screen to identify phosphatases involved in the dephosphorylation of the LRRK2 phosphosite S935. Candidate phosphatases selected from the primary screen were tested in mammalian cells, Xenopus oocytes and in vitro. Effects of PP2A on endogenous LRRK2 phosphorylation were examined via expression modulation with CRISPR/dCas9. Our screening revealed LRRK2 phosphorylation regulators linked to the PP1 and PP2A holoenzyme complexes as well as CDC25 phosphatases. We showed that dephosphorylation induced by different kinase inhibitor triggered relocalisation of phosphatases PP1 and PP2A in LRRK2 subcellular compartments in HEK-293 T cells. We also demonstrated that LRRK2 is an authentic substrate of PP2A both in vitro and in Xenopus oocytes. We singled out the PP2A holoenzyme PPP2CA:PPP2R2 as a powerful phosphoregulator of pS935-LRRK2. Furthermore, we demonstrated that this specific PP2A holoenzyme induces LRRK2 relocalization and triggers LRRK2 ubiquitination, suggesting its involvement in LRRK2 clearance. The identification of the PPP2CA:PPP2R2 complex regulating LRRK2 S910/S935/S955/S973 phosphorylation paves the way for studies refining PD therapeutic strategies that impact LRRK2 phosphorylation.
View details for DOI 10.1016/j.nbd.2021.105426
View details for PubMedID 34144124
Genetic and Environmental Factors Influence the Pleomorphy of LRRK2 Parkinsonism.
International journal of molecular sciences
2021; 22 (3)
Missense mutations in the LRRK2 gene were first identified as a pathogenic cause of Parkinson's disease (PD) in 2004. Soon thereafter, a founder mutation in LRRK2, p.G2019S (rs34637584), was described, and it is now estimated that there are approximately 100,000 people worldwide carrying this risk variant. While the clinical presentation of LRRK2 parkinsonism has been largely indistinguishable from sporadic PD, disease penetrance and age at onset can be quite variable. In addition, its neuropathological features span a wide range from nigrostriatal loss with Lewy body pathology, lack thereof, or atypical neuropathology, including a large proportion of cases with concomitant Alzheimer's pathology, hailing LRRK2 parkinsonism as the "Rosetta stone" of parkinsonian disorders, which provides clues to an understanding of the different neuropathological trajectories. These differences may result from interactions between the LRRK2 mutant protein and other proteins or environmental factors that modify LRRK2 function and, thereby, influence pathobiology. This review explores how potential genetic and biochemical modifiers of LRRK2 function may contribute to the onset and clinical presentation of LRRK2 parkinsonism. We review which genetic modifiers of LRRK2 influence clinical symptoms, age at onset, and penetrance, what LRRK2 mutations are associated with pleomorphic LRRK2 neuropathology, and which environmental modifiers can augment LRRK2 mutant pathophysiology. Understanding how LRRK2 function is influenced and modulated by other interactors and environmental factors-either increasing toxicity or providing resilience-will inform targeted therapeutic development in the years to come. This will allow the development of disease-modifying therapies for PD- and LRRK2-related neurodegeneration.
View details for DOI 10.3390/ijms22031045
View details for PubMedID 33494262
Parkinson's disease-associated mutations in the GTPase domain of LRRK2 impair its nucleotide-dependent conformational dynamics.
The Journal of biological chemistry
Mutation in Leucine Rich Repeat Kinase 2 (LRRK2) is a common cause of familial Parkinson's disease (PD). Recently, we showed that a disease-associated mutation R1441H rendered the GTPase domain of LRRK2 catalytically less active and thereby trapping it in a more persistently "on" conformation. However, the mechanism involved and characteristics of this "on" conformation remained unknown. Here, we report that the ROC domain of LRRK2 exists in a dynamic dimer-monomer equilibrium that is oppositely driven by GDP and GTP binding. We also observed that the PD-associated mutations at residue 1441 impair this dynamic and shift the conformation of ROC to a GTP-bound-like monomeric conformation. Moreover, we show that residue Arg-1441 is critical for regulating the conformational dynamics of ROC. In summary, our results reveal that the PD-associated substitutions at Arg-1441 of LRRK2 alter monomer-dimer dynamics and thereby trap its GTPase domain in an activated state.
View details for DOI 10.1074/jbc.RA119.007631
View details for PubMedID 30796162
LRRK2-mediated Rab10 phosphorylation in immune cells from Parkinson's disease patients.
Movement disorders : official journal of the Movement Disorder Society
BACKGROUND: Leucine-rich repeat kinase 2 is a potential therapeutic target for the treatment of Parkinson's disease, and clinical trials of leucine-rich repeat kinase 2 inhibitors are in development. The objective of this study was to evaluate phosphorylation of a new leucine-rich repeat kinase 2 substrate, Rab10, for potential use as a target engagement biomarker and/or patient enrichment biomarker for leucine-rich repeat kinase 2 inhibitor clinical trials.METHODS: Peripheral blood mononuclear cells and neutrophils were isolated from Parkinson's disease patients and matched controls, and treated ex vivo with a leucine-rich repeat kinase 2 inhibitor. Immunoblotting was used to measure levels of leucine-rich repeat kinase 2 and Rab10 and their phosphorylation. Plasma inflammatory cytokines were measured by multiplex enzyme-linked immunosorbent assay.RESULTS: Mononuclear cells and neutrophils of both controls and Parkinson's disease patients responded the same to leucine-rich repeat kinase 2 inhibitor treatment. Leucine-rich repeat kinase 2 levels in mononuclear cells were the same in controls and Parkinson's disease patients, whereas leucine-rich repeat kinase 2 was significantly increased in Parkinson's disease neutrophils. Rab10 T73 phosphorylation levels were similar in controls and Parkinson's disease patients and did not correlate with leucine-rich repeat kinase 2 levels. Immune-cell levels of leucine-rich repeat kinase 2 and Rab10 T73 phosphorylation were associated with plasma inflammatory cytokine levels.CONCLUSIONS: Rab10 T73 phosphorylation appears to be a valid target engagement biomarker for potential use in leucine-rich repeat kinase 2 inhibitor clinical trials. However, a lack of association between leucine-rich repeat kinase 2 and Rab10 phosphorylation complicates the potential use of Rab10 phosphorylation as a patient enrichment biomarker. Although replication is required, increased leucine-rich repeat kinase 2 levels in neutrophils from Parkinson's disease patients may have the potential for patient stratification. leucine-rich repeat kinase 2 activity in peripheral immune cells may contribute to an inflammatory phenotype. © 2018 International Parkinson and Movement Disorder Society.
View details for DOI 10.1002/mds.27601
View details for PubMedID 30597610
P62/SQSTM1 is a novel leucine-rich repeat kinase 2 (LRRK2) substrate that enhances neuronal toxicity.
The Biochemical journal
2018; 475 (7): 1271-1293
Autosomal-dominant, missense mutations in the leucine-rich repeat protein kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson's disease (PD). LRRK2 kinase activity is increased in several pathogenic mutations (N1437H, R1441C/G/H, Y1699C, G2019S), implicating hyperphosphorylation of a substrate in the pathogenesis of the disease. Identification of the downstream targets of LRRK2 is a crucial endeavor in the field to understand LRRK2 pathway dysfunction in the disease. We have identified the signaling adapter protein p62/SQSTM1 as a novel endogenous interacting partner and a substrate of LRRK2. Using mass spectrometry and phospho-specific antibodies, we found that LRRK2 phosphorylates p62 on Thr138 in vitro and in cells. We found that the pathogenic LRRK2 PD-associated mutations (N1437H, R1441C/G/H, Y1699C, G2019S) increase phosphorylation of p62 similar to previously reported substrate Rab proteins. Notably, we found that the pathogenic I2020T mutation and the risk factor mutation G2385R displayed decreased phosphorylation of p62. p62 phosphorylation by LRRK2 is blocked by treatment with selective LRRK2 inhibitors in cells. We also found that the amino-terminus of LRRK2 is crucial for optimal phosphorylation of Rab7L1 and p62 in cells. LRRK2 phosphorylation of Thr138 is dependent on a p62 functional ubiquitin-binding domain at its carboxy-terminus. Co-expression of p62 with LRRK2 G2019S increases the neurotoxicity of this mutation in a manner dependent on Thr138. p62 is an additional novel substrate of LRRK2 that regulates its toxic biology, reveals novel signaling nodes and can be used as a pharmacodynamic marker for LRRK2 kinase activity.
View details for DOI 10.1042/BCJ20170699
View details for PubMedID 29519959
Reduced LRRK2 in association with retromer dysfunction in post-mortem brain tissue from LRRK2 mutation carriers.
Brain : a journal of neurology
2018; 141 (2): 486-495
Missense mutations in leucine-rich repeat kinase 2 (LRRK2) are pathogenic for familial Parkinson's disease. However, it is unknown whether levels of LRRK2 protein in the brain are altered in patients with LRRK2-associated Parkinson's disease. Because LRRK2 mutations are relatively rare, accounting for approximately 1% of all Parkinson's disease, we accessioned cases from five international brain banks to investigate levels of the LRRK2 protein, and other genetically associated Parkinson's disease proteins. Brain tissue was obtained from 17 LRRK2 mutation carriers (12 with the G2019S mutation and five with the I2020T mutation) and assayed by immunoblot. Compared to matched controls and idiopathic Parkinson's disease cases, we found levels of LRRK2 protein were reduced in the LRRK2 mutation cases. We also measured a decrease in two other proteins genetically implicated in Parkinson's disease, the core retromer component, vacuolar protein sorting associated protein 35 (VPS35), and the lysosomal hydrolase, glucocerebrosidase (GBA). Moreover, the classical retromer cargo protein, cation-independent mannose-6-phosphate receptor (MPR300, encoded by IGF2R), was also reduced in the LRRK2 mutation cohort and protein levels of the receptor were correlated to levels of LRRK2. These results provide new data on LRRK2 protein expression in brain tissue from LRRK2 mutation carriers and support a relationship between LRRK2 and retromer dysfunction in LRRK2-associated Parkinson's disease brain.
View details for DOI 10.1093/brain/awx344
View details for PubMedID 29253086
View details for PubMedCentralID PMC5837795
Advances in neurobiology
2017; 14: 51-70
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene were discovered in 2004 and have been found to be the most frequently mutated gene in Parkinson's disease. LRRK2 is a large multi-domain protein with a functional GTPase and kinase domain. The signal transduction pathways in which LRRK2 is dysfunctional in the disease state are only now being resolved, but we do know that LRRK2 is, itself, a substrate of multiple kinases and phosphatases and exists in variable phosphorylated states. Autophosphorylation of LRRK2 can impact GTPase and pathological outcomes. LRRK2 serines (910/935/955/973) are differentially phosphorylated in pathogenic PD mutations and after LRRK2 kinase inhibition. The phosphorylation status of LRRK2 can therefore provide key insight into the mechanisms of kinase dysfunction during disease. This chapter will describe the identification of LRRK2 phosphorylation sites and how phosphoregulation of LRRK2 reveals its own kinase activity and regulates its ubiquitination and localization in vitro, in cells, and in tissues.
View details for DOI 10.1007/978-3-319-49969-7_3
View details for PubMedID 28353278
LRRK2 levels and phosphorylation in Parkinson's disease brain and cases with restricted Lewy bodies.
Movement disorders : official journal of the Movement Disorder Society
2017; 32 (3): 423-432
Leucine rich repeat kinase 2 (LRRK2) is a promising target for the treatment of Parkinson's disease; however, little is known about the expression of LRRK2 in human brain and if/how LRRK2 protein levels are altered in Parkinson's disease.We measured the protein levels of LRRK2 as well as its phosphorylation on serines 910, 935, and 973 in the postmortem brain tissue of Parkinson's disease patients and aged controls with and without Lewy bodies.LRRK2 and its phosphorylation were measured by immunoblot in brain regions differentially affected in Parkinson's disease (n = 30) as well as subjects with Lewy bodies restricted to the periphery and lower brain stem (n = 25) and matched controls without pathology (n = 25).LRRK2 levels were increased in cases with restricted Lewy bodies, with a 30% increase measured in the substantia nigra. In clinical Parkinson's disease, levels of LRRK2 negatively correlated to disease duration and were comparable with controls. LRRK2 phosphorylation, however, particularly at serine 935, was reduced with clinical Parkinson's disease with a 36% reduction measured in the substantia nigra.Our data show that LRRK2 phosphorylation is reduced with clinical PD, whereas LRRK2 expression is increased in early potential prodromal stages. These results contribute to a better understanding of the role of LRRK2 in idiopathic Parkinson's disease and may aid efforts aimed at therapeutically targeting the LRRK2 protein. © 2016 International Parkinson and Movement Disorder Society.
View details for DOI 10.1002/mds.26892
View details for PubMedID 27911006
Multisystem Lewy body disease and the other parkinsonian disorders
2015; 47 (12): 1378–84
Here we prioritize as multisystem Lewy body disease (MLBD) those genetic forms of Parkinson's disease that point the way toward a mechanistic understanding of the majority of sporadic disease. Pathological diagnosis of genetic subtypes offers the prospect of distinguishing different mechanistic trajectories with a common mutational etiology, differing outcomes from varying allelic bases, and those disease-associated variants that can be used in gene-environment analysis. Clearly delineating parkinsonian disorders into subclasses on the basis of molecular mechanisms with well-characterized outcome expectations is the basis for refining these forms of neurodegeneration as research substrate through the use of cell models derived from affected individuals while ensuring that clinically collected data can be used for therapeutic decisions and research without increasing the noise and confusion engendered by the collection of data against a range of historically defined criteria.
View details for DOI 10.1038/ng.3454
View details for Web of Science ID 000365813200006
View details for PubMedID 26620112
LRRK2 dephosphorylation increases its ubiquitination.
The Biochemical journal
2015; 469 (1): 107-20
Activating mutations in the leucine rich repeat protein kinase 2 (LRRK2) gene are the most common cause of inherited Parkinson's disease (PD). LRRK2 is phosphorylated on a cluster of phosphosites including Ser(910), Ser(935), Ser(955) and Ser(973), which are dephosphorylated in several PD-related LRRK2 mutants (N1437H, R1441C/G, Y1699C and I2020T) linking the regulation of these sites to PD. These serine residues are also dephosphorylated after kinase inhibition and lose 14-3-3 binding, which serves as a pharmacodynamic marker for inhibited LRRK2. Loss of 14-3-3 binding is well established, but the consequences of dephosphorylation are only now being uncovered. In the present study, we found that potent and selective inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(935) then ubiquitination and degradation of a significant fraction of LRRK2. GNE1023 treatment decreased the phosphorylation and stability of LRRK2 in expression systems and endogenous LRRK2 in A549 cells and in mouse dosing studies. We next established that LRRK2 is ubiquitinated through at least Lys(48) and Lys(63) ubiquitin linkages in response to inhibition. To investigate the link between dephosphorylation induced by inhibitor treatment and LRRK2 ubiquitination, we studied LRRK2 in conditions where it is dephosphorylated such as expression of PD mutants [R1441G, Y1699C and I2020T] or by blocking 14-3-3 binding to LRRK2 via difopein expression, and found LRRK2 is hyper-ubiquitinated. Calyculin A treatment prevents inhibitor and PD mutant induced dephosphorylation and reverts LRRK2 to a lesser ubiquitinated species, thus directly implicating phosphatase activity in LRRK2 ubiquitination. This dynamic dephosphorylation-ubiquitination cycle could explain detrimental loss-of-function phenotypes found in peripheral tissues of LRRK2 kinase inactive mutants, LRRK2 KO (knockout) animals and following LRRK2 inhibitor administration.
View details for DOI 10.1042/BJ20141305
View details for PubMedID 25939886
View details for PubMedCentralID PMC4613513
LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status.
Frontiers in molecular neuroscience
2014; 7: 54
Missense mutations in the Leucine-Rich Repeat protein Kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson's disease (PD) (Farrer et al., 2005; Skipper et al., 2005; Di Fonzo et al., 2006; Healy et al., 2008; Paisan-Ruiz et al., 2008; Lesage et al., 2010). LRRK2 is a large multi-domain phosphoprotein with a GTPase domain and a serine/threonine protein kinase domain whose activity is implicated in neuronal toxicity; however the precise mechanism is unknown. LRRK2 autophosphorylates on several serine/threonine residues across the enzyme and is found constitutively phosphorylated on Ser910, Ser935, Ser955, and Ser973, which are proposed to be regulated by upstream kinases. Here we investigate the phosphoregulation at these sites by analyzing the effects of disease-associated mutations Arg1441Cys, Arg1441Gly, Ala1442Pro, Tyr1699Cys, Ile2012Thr, Gly2019Ser, and Ile2020Thr. We also studied alanine substitutions of phosphosite serines 910, 935, 955, and 973 and specific LRRK2 inhibition on autophosphorylation of LRRK2 Ser1292, Thr1491, Thr2483 and phosphorylation at the cellular sites. We found that mutants in the Roc-COR domains, including Arg1441Cys, Arg1441His, Ala1442Pro, and Tyr1699Cys, can positively enhance LRRK2 kinase activity, while concomitantly inducing the dephosphorylation of the cellular sites. Mutation of the cellular sites individually did not affect LRRK2 intrinsic kinase activity; however, Ser910/935/955/973Ala mutations trended toward increased kinase activity of LRRK2. Increased cAMP levels did not lead to increased LRRK2 cellular site phosphorylation, 14-3-3 binding or kinase activity. In cells, inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser1292 by Calyculin A and Okadaic acid sensitive phosphatases, while the cellular sites are dephosphorylated by Calyculin A sensitive phosphatases. These findings indicate that comparative analysis of both Ser1292 and Ser910/935/955/973 phosphorylation sites will provide important and distinct measures of LRRK2 kinase and biological activity in vitro and in vivo.
View details for DOI 10.3389/fnmol.2014.00054
View details for PubMedID 25009464
View details for PubMedCentralID PMC4068021
Parkinson disease-associated mutation R1441H in LRRK2 prolongs the "active state" of its GTPase domain.
Proceedings of the National Academy of Sciences of the United States of America
2014; 111 (11): 4055-60
Mutation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD). A disease-causing point mutation R1441H/G/C in the GTPase domain of LRRK2 leads to overactivation of its kinase domain. However, the mechanism by which this mutation alters the normal function of its GTPase domain [Ras of complex proteins (Roc)] remains unclear. Here, we report the effects of R1441H mutation (RocR1441H) on the structure and activity of Roc. We show that Roc forms a stable monomeric conformation in solution that is catalytically active, thus demonstrating that LRRK2 is a bona fide self-contained GTPase. We further show that the R1441H mutation causes a twofold reduction in GTPase activity without affecting the structure, thermal stability, and GDP-binding affinity of Roc. However, the mutation causes a twofold increase in GTP-binding affinity of Roc, thus suggesting that the PD-causing mutation R1441H traps Roc in a more persistently activated state by increasing its affinity for GTP and, at the same time, compromising its GTP hydrolysis.
View details for DOI 10.1073/pnas.1323285111
View details for PubMedID 24591621
View details for PubMedCentralID PMC3964117
Identification of protein phosphatase 1 as a regulator of the LRRK2 phosphorylation cycle.
The Biochemical journal
2013; 456 (1): 119-28
A cluster of phosphorylation sites in LRRK2 (leucine-rich repeat kinase 2), including Ser910, Ser935, Ser955 and Ser973, is important for PD (Parkinson's disease) pathogenesis as several PD-linked LRRK2 mutants are dephosphorylated at these sites. LRRK2 is also dephosphorylated in cells after pharmacological inhibition of its kinase activity, which is currently proposed as a strategy for disease-modifying PD therapy. Despite this importance of LRRK2 dephosphorylation in mutant LRRK2 pathological mechanism(s) and in LRRK2's response to inhibition, the mechanism by which this occurs is unknown. Therefore we aimed to identify the phosphatase for LRRK2. Using a panel of recombinant phosphatases, we found that PP1 (protein phosphatase 1) efficiently dephosphorylates LRRK2 in vitro. PP1 activity on LRRK2 dephosphorylation was confirmed in cells using PP1 inhibition to reverse LRRK2 dephosphorylation induced by the potent LRRK2 kinase inhibitor LRRK2-IN1 as well as in R1441G mutant LRRK2. We also found that PP1 and LRRK2 can form a complex in cells. Furthermore, we observed that PP1 inhibition modulates LRRK2's cellular phenotype by reducing skein-like LRRK2-positive structures associated with dephosphorylation. In conclusion, the present study reveals PP1 as the physiological LRRK2 phosphatase, responsible for LRRK2 dephosphorylation observed in PD mutant LRRK2 and after LRRK2 kinase inhibition.
View details for DOI 10.1042/BJ20121772
View details for PubMedID 23937259
View details for PubMedCentralID PMC5141581
Pharmacological inhibition of LRRK2 cellular phosphorylation sites provides insight into LRRK2 biology.
Biochemical Society transactions
2012; 40 (5): 1158-62
Mutations in LRRK2 (leucine-rich repeat kinase 2) have been linked to inherited forms of PD (Parkinson's disease). Substantial pre-clinical research and drug discovery efforts have focused on LRRK2 with the hope that small-molecule inhibitors of the enzyme may be valuable for the treatment or prevention of the onset of PD. The pathway to develop therapeutic or neuroprotective agents based on LRRK2 function (i.e. kinase activity) has been facilitated by the development of both biochemical and cell-based assays for LRRK2. LRRK2 is phosphorylated on Ser910, Ser935, Ser955 and Ser973 in the N-terminal domain of the enzyme, and these sites of phosphorylation are likely to be regulated by upstream enzymes in an LRRK2 kinase-activity-dependent manner. Knowledge of these phosphorylation sites and their regulation can be adapted to high-throughput-screening-amenable platforms. The present review describes the utilization of LRRK2 phosphorylation as indicators of enzyme inhibition, as well as how such assays can be used to deconvolute the pathways in which LRRK2 plays a role.
View details for DOI 10.1042/BST20120137
View details for PubMedID 22988882
Screening for novel LRRK2 inhibitors using a high-throughput TR-FRET cellular assay for LRRK2 Ser935 phosphorylation.
2012; 7 (8): e43580
Mutations in the leucine-rich repeat kinase-2 (LRRK2) have been linked to Parkinson's disease. Recent studies show that inhibition of LRRK2 kinase activity decreased the level of phosphorylation at its own Ser910 and Ser935, indicating that these sites are prime targets for cellular readouts of LRRK2 inhibition.Using Time-Resolved Förster Resonance Energy Transfer (TR-FRET) technology, we developed a high-throughput cellular assay for monitoring LRRK2 phosphorylation at Ser935. LRRK2-Green Fluorescence Protein (GFP) fusions were expressed in cells via BacMam. Phosphorylation at Ser935 in these cells is detected using a terbium labeled anti-phospho-Ser935 antibody that generates a TR-FRET signal between terbium and GFP. LRRK2 wild-type and G2019S are constitutively phosphorylated at Ser935 in cells as measured by TR-FRET. The phosphorylation level is reduced for the R1441C mutant and little could be detected for the kinase-dead mutant D1994A. The TR-FRET cellular assay was further validated using reported LRRK2 inhibitors including LRRK2-IN-1 and our results confirmed that inhibition of LRRK2 can reduce the phosphorylation level at Ser935. To demonstrate the utility of this assay for screening, we profiled a small library of 1120 compounds. Three known LRRK2 inhibitors were identified and 16 hits were followed up in the TR-FRET and a cytotoxicity assay. Interestingly, out of the top 16 hits, five are known inhibitors of IκB phosphorylation, two CHK1 and two CDC25 inhibitors. Thirteen hits were further tested in a biochemical LRRK2 kinase activity assay and Western blot analysis for their effects on the phosphorylation of Ser910, Ser935, Ser955 and Ser973.We developed a TR-FRET cellular assay for LRRK2 Ser935 phosphorylation that can be applied to the screening for LRRK2 inhibitors. We report for the first time that several compounds such as IKK16, CHK1 inhibitors and GW441756 can inhibit LRRK2 Ser935 phosphorylation in cells and LRRK2 kinase activity in vitro.
View details for DOI 10.1371/journal.pone.0043580
View details for PubMedID 22952710
View details for PubMedCentralID PMC3429506
Phosphorylation of LRRK2 serines 955 and 973 is disrupted by Parkinson's disease mutations and LRRK2 pharmacological inhibition.
Journal of neurochemistry
2012; 120 (1): 37-45
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. An amino terminal cluster of constitutively phosphorylated residues, serines 860, 910, 935, 955, and 973, appears to be biologically relevant. Phosphorylation of serines 910 and 935 is regulated in response to LRRK2 kinase activity and is responsible for interaction with 14-3-3 and maintaining LRRK2 in a non-aggregated state. We examined the phosphorylation status of two other constitutive phosphorylation sites, serines 955 and 973. Treatment of LRRK2 expressing cells with the selective LRRK2 inhibitor LRRK2-IN1 revealed that, like Ser910/Ser935, phosphorylation of Ser955 and Ser973 is disrupted by acute inhibition of LRRK2 kinase activity. Additionally, phosphorylation of Ser955 and 973 is disrupted in the context of several Parkinson's disease associated mutations [R1441G/C, Y1699C, and I2020T]. We observed that modification of Ser973 is dependent on the modification of Ser910/Ser935. Ser955Ala and Ser973Ala mutations do not induce relocalization of LRRK2; however, all phosphomutants exhibited similar localization patterns when exposed to LRRK2-IN1. We conclude that the mechanisms of regulation of Ser910/935/955/973 phosphorylation are similar and physiologically relevant. These sites can be utilized as biomarkers for LRRK2 activity as well as starting points for the elucidation of upstream and downstream enzymes that regulate LRRK2.
View details for DOI 10.1111/j.1471-4159.2011.07537.x
View details for PubMedID 22004453
14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization.
The Biochemical journal
2010; 430 (3): 393-404
LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinson's disease patients, but still little is understood about how it is regulated or functions. In the present study we have demonstrated that 14-3-3 protein isoforms interact with LRRK2. Consistent with this, endogenous LRRK2 isolated from Swiss 3T3 cells or various mouse tissues is associated with endogenous 14-3-3 isoforms. We have established that 14-3-3 binding is mediated by phosphorylation of LRRK2 at two conserved residues (Ser910 and Ser935) located before the leucine-rich repeat domain. Our results suggests that mutation of Ser910 and/or Ser935 to disrupt 14-3-3 binding does not affect intrinsic protein kinase activity, but induces LRRK2 to accumulate within discrete cytoplasmic pools, perhaps resembling inclusion bodies. To investigate links between 14-3-3 binding and Parkinson's disease, we studied how 41 reported mutations of LRRK2 affected 14-3-3 binding and cellular localization. Strikingly, we found that five of the six most common pathogenic mutations (R1441C, R1441G, R1441H, Y1699C and I2020T) display markedly reduced phosphorylation of Ser910/Ser935 thereby disrupting interaction with 14-3-3. We have also demonstrated that Ser910/Ser935 phosphorylation and 14-3-3 binding to endogenous LRRK2 is significantly reduced in tissues of homozygous LRRK2(R1441C) knock-in mice. Consistent with 14-3-3 regulating localization, all of the common pathogenic mutations displaying reduced 14-3-3-binding accumulated within inclusion bodies. We also found that three of the 41 LRRK2 mutations analysed displayed elevated protein kinase activity (R1728H, ~2-fold; G2019S, ~3-fold; and T2031S, ~4-fold). These results provide the first evidence suggesting that 14-3-3 regulates LRRK2 and that disruption of the interaction of LRRK2 with 14-3-3 may be linked to Parkinson's disease.
View details for DOI 10.1042/BJ20100483
View details for PubMedID 20642453
View details for PubMedCentralID PMC2932554
Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization.
The Biochemical journal
2010; 430 (3): 405-13
LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinson's disease patients. Since a common mutation that replaces Gly2019 with a serine residue enhances kinase catalytic activity, small-molecule LRRK2 inhibitors might have utility in treating Parkinson's disease. However, the effectiveness of inhibitors is difficult to assess, as no physiological substrates or downstream effectors have been identified that could be exploited to develop a robust cell-based assay. We recently established that LRRK2 bound 14-3-3 protein isoforms via its phosphorylation of Ser910 and Ser935. In the present study we show that treatment of Swiss 3T3 cells or lymphoblastoid cells derived from control or a Parkinson's disease patient harbouring a homozygous LRRK2(G2019S) mutation with two structurally unrelated inhibitors of LRRK2 (H-1152 or sunitinib) induced dephosphorylation of endogenous LRRK2 at Ser910 and Ser935, thereby disrupting 14-3-3 interaction. Our results suggest that H-1152 and sunitinib induce dephosphorylation of Ser910 and Ser935 by inhibiting LRRK2 kinase activity, as these compounds failed to induce significant dephosphorylation of a drug-resistant LRRK2(A2016T) mutant. Moreover, consistent with the finding that non-14-3-3-binding mutants of LRRK2 accumulated within discrete cytoplasmic pools resembling inclusion bodies, we observed that H-1152 causes LRRK2 to accumulate within inclusion bodies. These findings indicate that dephosphorylation of Ser910/Ser935, disruption of 14-3-3 binding and/or monitoring LRRK2 cytoplasmic localization can be used as an assay to assess the relative activity of LRRK2 inhibitors in vivo. These results will aid the elaboration and evaluation of LRRK2 inhibitors. They will also stimulate further research to understand how phosphorylation of Ser910 and Ser935 is controlled by LRRK2, and establish any relationship to development of Parkinson's disease.
View details for DOI 10.1042/BJ20100784
View details for PubMedID 20659021
View details for PubMedCentralID PMC3631100
Mice deficient in the serine/threonine protein kinase VRK1 are infertile due to a progressive loss of spermatogonia.
Biology of reproduction
2010; 82 (1): 182-93
The VRK1 protein kinase has been implicated as a pro-proliferative factor. Genetic analyses of mutant alleles of the Drosophila and Caenorhabditis elegans VRK1 homologs have revealed phenotypes ranging from embryonic lethality to mitotic and meiotic defects with resultant sterility. Herein, we describe the first genetic analysis of murine VRK1. Two lines of mice containing distinct gene-trap integrations into the Vrk1 locus were established. Insertion into intron 12 (GT12) spared VRK1 function, enabling the examination of VRK1 expression in situ. Insertion into intron 3 (GT3) disrupted VRK1 function, but incomplete splicing to the gene trap rendered this allele hypomorphic (approximately 15% of wild-type levels of VRK1 remain). GT3/GT3 mice are viable, but both males and females are infertile. In testes, VRK1 is expressed in Sertoli cells and spermatogonia. The infertility of GT3/GT3 male mice results from a progressive defect in spermatogonial proliferation or differentiation, culminating in the absence of mitotic and meiotic cells in adult testis. These data demonstrate an important role for VRK1 in cell proliferation and confirm that the need for VRK1 during gametogenesis is evolutionarily conserved.
View details for DOI 10.1095/biolreprod.109.079095
View details for PubMedID 19696012
View details for PubMedCentralID PMC2802121
Substrate specificity and inhibitors of LRRK2, a protein kinase mutated in Parkinson's disease.
The Biochemical journal
2009; 424 (1): 47-60
The LRRK2 (leucine-rich repeat protein kinase-2) is mutated in a significant number of Parkinson's disease patients, but little is known about its regulation and function. A common mutation changing Gly2019 to serine enhances catalytic activity, suggesting that small-molecule inhibitors might have utility in treating Parkinson's disease. We employed various approaches to explore the substrate-specificity requirements of LRRK2 and elaborated a peptide substrate termed Nictide, that had 20-fold lower Km and nearly 2-fold higher Vmax than the widely deployed LRRKtide substrate. We demonstrate that LRRK2 has marked preference for phosphorylating threonine over serine. We also observed that several ROCK (Rho kinase) inhibitors such as Y-27632 and H-1152, suppressed LRRK2 with similar potency to which they inhibited ROCK2. In contrast, GSK429286A, a selective ROCK inhibitor, did not significantly inhibit LRRK2. We also identified a mutant LRRK2[A2016T] that was normally active, but resistant to H-1152 and Y-27632, as well as sunitinib, a structurally unrelated multikinase inhibitor that, in contrast with other compounds, suppresses LRRK2, but not ROCK. We have also developed the first sensitive antibody that enables measurement of endogenous LRRK2 protein levels and kinase activity as well as shRNA (short hairpin RNA) methods to reduce LRRK2 expression. Finally, we describe a pharmacological approach to validate whether substrates are phosphorylated by LRRK2 and use this to provide evidence that LRRK2 may not be rate-limiting for the phosphorylation of the proposed substrate moesin. The findings of the present study will aid with the investigation of LRRK2.
View details for DOI 10.1042/BJ20091035
View details for PubMedID 19740074
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Functional characterization of the vaccinia virus I5 protein.
2008; 5: 148
The I5L gene is one of approximately 90 genes that are conserved throughout the chordopoxvirus family, and hence are presumed to play vital roles in the poxvirus life cycle. Previous work had indicated that the VP13 protein, a component of the virion membrane, was encoded by the I5L gene, but no additional studies had been reported. Using a recombinant virus that encodes an I5 protein fused to a V5 epitope tag at the endogenous locus (vI5V5), we show here that the I5 protein is expressed as a post-replicative gene and that the approximately 9 kDa protein does not appear to be phosphorylated in vivo. I5 does not appear to traffic to any cellular organelle, but ultrastructural and biochemical analyses indicate that I5 is associated with the membranous components of assembling and mature virions. Intact virions can be labeled with anti-V5 antibody as assessed by immunoelectron microscopy, indicating that the C' terminus of the protein is exposed on the virion surface. Using a recombinant virus which encodes only a TET-regulated copy of the I5V5 gene (vDeltaindI5V5), or one in which the I5 locus has been deleted (vDeltaI5), we also show that I5 is dispensable for replication in tissue culture. Neither plaque size nor the viral yield produced in BSC40 cells or primary human fibroblasts are affected by the absence of I5 expression.
View details for DOI 10.1186/1743-422X-5-148
View details for PubMedID 19077320
View details for PubMedCentralID PMC2621143
The vaccinia virus gene I2L encodes a membrane protein with an essential role in virion entry.
Journal of virology
2008; 82 (20): 10247-61
The previously unstudied vaccinia virus gene I2L is conserved in all orthopoxviruses. We show here that the 8-kDa I2 protein is expressed at late times of infection, is tightly associated with membranes, and is encapsidated in mature virions. We have generated a recombinant virus in which I2 expression is dependent upon the inclusion of tetracycline in the culture medium. In the absence of I2, the biochemical events of the viral life cycle progress normally, and virion morphogenesis culminates in the production of mature virions. However, these virions show an approximately 400-fold reduction in specific infectivity due to an inability to enter target cells. Several proteins that have been previously identified as components of an essential entry/fusion complex are present at reduced levels in I2-deficient virions, although other membrane proteins, core proteins, and DNA are encapsidated at normal levels. A preliminary structure/function analysis of I2 has been performed using a transient complementation assay: the C-terminal hydrophobic domain is essential for protein stability, and several regions within the N-terminal hydrophilic domain are essential for biological competency. I2 is thus yet another component of the poxvirus virion that is essential for the complex process of entry into target cells.
View details for DOI 10.1128/JVI.01035-08
View details for PubMedID 18701587
View details for PubMedCentralID PMC2566298
LRRK2 phosphorylates moesin at threonine-558: characterization of how Parkinson's disease mutants affect kinase activity.
The Biochemical journal
2007; 405 (2): 307-17
Mutations in the LRRK2 (leucine-rich repeat kinase-2) gene cause late-onset PD (Parkinson's disease). LRRK2 contains leucine-rich repeats, a GTPase domain, a COR [C-terminal of Roc (Ras of complex)] domain, a kinase and a WD40 (Trp-Asp 40) motif. Little is known about how LRRK2 is regulated, what its physiological substrates are or how mutations affect LRRK2 function. Thus far LRRK2 activity has only been assessed by autophosphorylation and phosphorylation of MBP (myelin basic protein), which is catalysed rather slowly. We undertook a KESTREL (kinase substrate tracking and elucidation) screen in rat brain extracts to identify proteins that were phosphorylated by an activated PD mutant of LRRK2 (G2019S). This led to the discovery that moesin, a protein which anchors the actin cytoskeleton to the plasma membrane, is efficiently phosphorylated by LRRK2, at Thr558, a previously identified in-vivo-phosphorylation site that regulates the ability of moesin to bind actin. LRRK2 also phosphorylated ezrin and radixin, which are related to moesin, at the residue equivalent to Thr558, as well as a peptide (LRRKtide: RLGRDKYKTLRQIRQ) encompassing Thr558. We exploited these findings to determine how nine previously reported PD mutations of LRRK2 affected kinase activity. Only one of the mutations analysed, namely G2019S, stimulated kinase activity. Four mutations inhibited LRRK2 kinase activity (R1941H, I2012T, I2020T and G2385R), whereas the remainder (R1441C, R1441G, Y1699C and T2356I) did not influence activity. Therefore the manner in which LRRK2 mutations induce PD is more complex than previously imagined and is not only caused by an increase in LRRK2 kinase activity. Finally, we show that the minimum catalytically active fragment of LRRK2 requires an intact GTPase, COR and kinase domain, as well as a WD40 motif and a C-terminal tail. The results of the present study suggest that moesin, ezrin and radixin may be LRRK2 substrates, findings that have been exploited to develop the first robust quantitative assay to measure LRRK2 kinase activity.
View details for DOI 10.1042/BJ20070209
View details for PubMedID 17447891
View details for PubMedCentralID PMC1904520
The vaccinia-related kinases phosphorylate the N' terminus of BAF, regulating its interaction with DNA and its retention in the nucleus.
Molecular biology of the cell
2006; 17 (5): 2451-64
The vaccinia-related kinases (VRKs) comprise a branch of the casein kinase family whose members are characterized by homology to the vaccinia virus B1 kinase. The VRK orthologues encoded by Caenorhabditis elegans and Drosophila melanogaster play an essential role in cell division; however, substrates that mediate this role have yet to be elucidated. VRK1 can complement the temperature sensitivity of a vaccinia B1 mutant, implying that VRK1 and B1 have overlapping substrate specificity. Herein, we demonstrate that B1, VRK1, and VRK2 efficiently phosphorylate the extreme N' terminus of the BAF protein (Barrier to Autointegration Factor). BAF binds to both DNA and LEM domain-containing proteins of the inner nuclear membrane; in lower eukaryotes, BAF has been shown to play an important role during the reassembly of the nuclear envelope at the end of mitosis. We demonstrate that phosphorylation of ser4 and/or thr2/thr3 abrogates the interaction of BAF with DNA and reduces its interaction with the LEM domain. Coexpression of VRK1 and GFP-BAF greatly diminishes the association of BAF with the nuclear chromatin/matrix and leads to its dispersal throughout the cell. Cumulatively, our data suggest that the VRKs may modulate the association of BAF with nuclear components and hence play a role in maintaining appropriate nuclear architecture.
View details for DOI 10.1091/mbc.e05-12-1179
View details for PubMedID 16495336
View details for PubMedCentralID PMC1446082
Characterization of three paralogous members of the Mammalian vaccinia related kinase family.
The Journal of biological chemistry
2004; 279 (9): 7934-46
Members of the novel vaccinia related kinase (VRK) protein family are characterized by notable sequence homology to the vaccinia virus-encoded B1 kinase (vvB1). vvB1 plays an essential role in viral DNA replication, and Boyle and Traktman have demonstrated that VRK1 enzymes complement the replication defect of a temperature-sensitive viral mutant defective in vvB1 (Boyle, K., and Traktman, P. (2004) J. Virol. 78, 1992-2005). This mammalian kinase family comprises three members, VRK1, VRK2, and VRK3. We have annotated the gene structure for the members of this family and have characterized the enzyme activity and subcellular localization for the human and mouse proteins. VRK1 enzymes show robust autophosphorylation activity and will phosphorylate casein; VRK2 enzymes show modest autophosphorylation activity and will also phosphorylate casein. The VRK3 proteins have key amino acid substitutions that disrupt invariant motifs required for catalytic activity, rendering them enzymatically inert. The VRK1 and VRK2 proteins contain COOH-terminal extracatalytic sequences that mediate intracellular localization. VRK1 proteins possess a basic nuclear localization signal and are indeed nuclear; the extreme C termini of the VRK2 proteins are highly hydrophobic, and the proteins are membrane-associated and colocalize with markers of the endoplasmic reticulum. The NH(2)-terminal region of the VRK3s contains a bipartite nuclear localization signal, which directs these proteins to the nucleus. Our findings provide the basis for further studies of the structure and function of this newly discovered family of protein kinases.
View details for DOI 10.1074/jbc.M310813200
View details for PubMedID 14645249