Phosphorylation of LRRK2: from kinase to substrate

The PD (Parkinson's disease) protein LRRK2 (leucine-rich repeat kinase 2) occurs in cells as a highly phosphorylated protein, with the majority of phosphosites clustering in the region between the ankyrin repeat and leucine-rich repeat domains. The observation that several pathogenic variants of LRRK2 display strongly reduced cellular phosphorylation suggests that phosphorylation of LRRK2 is involved in the PD pathological process. Furthermore, treatment of cells with inhibitors of LRRK2 kinase activity, which are currently considered as potential disease-modifying therapeutics for PD, leads to a rapid decrease in the phosphorylation levels of LRRK2. For these reasons, understanding the cellular role and regulation of LRRK2 as a kinase and as a substrate has become the focus of intense investigation. In the present review, we discuss what is currently known about the cellular phosphorylation of LRRK2 and how this relates to its function and dysfunction.

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P62/SQSTM1 is a novel leucine-rich repeat kinase 2 (LRRK2) substrate that enhances neuronal toxicity

Biochemical Journal ◽

10.1042/bcj20170699 ◽

2018 ◽

Vol 475 (7) ◽

pp. 1271-1293 ◽

Cited By ~ 26

Author(s):

Alexia F. Kalogeropulou ◽

Jing Zhao ◽

Marc F. Bolliger ◽

Anna Memou ◽

Shreya Narasimha ◽

...

Keyword(s):

Kinase Activity ◽

Rab Proteins ◽

Missense Mutations ◽

Leucine Rich Repeat ◽

Lrrk2 Gene ◽

Ubiquitin Binding Domain ◽

Lrrk2 Kinase ◽

Lrrk2 Kinase Activity ◽

In Cells

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.

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Pharmacological inhibition of LRRK2 cellular phosphorylation sites provides insight into LRRK2 biology

Biochemical Society Transactions ◽

10.1042/bst20120137 ◽

2012 ◽

Vol 40 (5) ◽

pp. 1158-1162 ◽

Cited By ~ 13

Author(s):

Jing Zhao ◽

Spencer B. Hermanson ◽

Coby B. Carlson ◽

Steven M. Riddle ◽

Kurt W. Vogel ◽

...

Keyword(s):

High Throughput Screening ◽

Kinase Activity ◽

Phosphorylation Sites ◽

Dependent Manner ◽

Neuroprotective Agents ◽

Leucine Rich Repeat ◽

Lrrk2 Kinase ◽

Activity Dependent ◽

Lrrk2 Kinase Activity ◽

Insight Into

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.

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Dependence of Leucine-rich Repeat Kinase 2 (LRRK2) Kinase Activity on Dimerization

Journal of Biological Chemistry ◽

10.1074/jbc.m109.025437 ◽

2009 ◽

Vol 284 (52) ◽

pp. 36346-36356 ◽

Cited By ~ 124

Author(s):

Saurabh Sen ◽

Philip J. Webber ◽

Andrew B. West

Keyword(s):

Kinase Activity ◽

Leucine Rich Repeat ◽

Lrrk2 Kinase ◽

Lrrk2 Kinase Activity

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Targeting leucine-rich repeat kinase 2 (LRRK2) for the treatment of Parkinson's disease

Future Medicinal Chemistry ◽

10.4155/fmc-2018-0484 ◽

2019 ◽

Vol 11 (15) ◽

pp. 1953-1977 ◽

Cited By ~ 4

Author(s):

Sofia Domingos ◽

Teresa Duarte ◽

Lucília Saraiva ◽

Rita C Guedes ◽

Rui Moreira

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Kinase Activity ◽

Leucine Rich Repeat ◽

Serine Threonine Kinase ◽

Drug Candidates ◽

Cellular Processes ◽

Pathogenic Variants ◽

Lrrk2 Kinase ◽

Relationship Of

Leucine-rich repeat kinase 2 (LRRK2) is a serine-threonine kinase involved in multiple cellular processes and signaling pathways. LRRK2 mutations are associated with autosomal-inherited Parkinson's disease (PD), and evidence suggests that LRRK2 pathogenic variants generally increase kinase activity. Therefore, inhibition of LRRK2 kinase function is a promising therapeutic strategy for PD treatment. The search for drug-like molecules capable of reducing LRRK2 kinase activity in PD led to the design of selective LRRK2 inhibitors predicted to be within the CNS drug-like space. This review highlights the journey that translates chemical tools for interrogating the role of LRRK2 in PD into promising drug candidates, addressing the challenges in discovering selective and brain-penetrant LRRK2 modulators and exploring the structure–activity relationship of distinct LRRK2 inhibitors.

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Allosteric inhibition of LRRK2, where are we now

Biochemical Society Transactions ◽

10.1042/bst20200424 ◽

2020 ◽

Vol 48 (5) ◽

pp. 2185-2194

Author(s):

Ahmed Soliman ◽

Fatma Nihan Cankara ◽

Arjan Kortholt

Keyword(s):

Recent Progress ◽

Kinase Activity ◽

Kinase Inhibitors ◽

High Selectivity ◽

Gtpase Activity ◽

Leucine Rich Repeat ◽

Allosteric Inhibitors ◽

Lrrk2 Kinase ◽

Lrrk2 Kinase Activity

Parkinson's disease (PD) is the second most common neurodegenerative disease. In recent years, it has been shown that leucine-rich repeat kinase 2 (LRRK2) has a crucial function in both familial and sporadic forms of PD. LRRK2 pathogenic mutations are thought to result in an increase in LRRK2 kinase activity. Thus, inhibiting LRRK2 kinase activity has become a main therapeutic target. Many compounds capable of inhibiting LRRK2 kinase activity with high selectivity and brain availability have been described. However, the safety of long-term use of these ATP-competitive LRRK2 kinase inhibitors has been challenged by several studies. Therefore, alternative ways of targeting LRRK2 activity will have a great benefit. In this review, we discuss the recent progress in the development of allosteric inhibitors of LRRK2, mainly via interfering with GTPase activity, and propose potential new intra and interprotein interactions targets that can lead to open doors toward new therapeutics.

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