PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins

Mutations that activate LRRK2 protein kinase cause Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif controlling interaction with effectors. An siRNA screen of all human protein phosphatases revealed that a poorly studied protein phosphatase, PPM1H, counteracts LRRK2 signaling by specifically dephosphorylating Rab proteins. PPM1H knockout increased endogenous Rab phosphorylation and inhibited Rab dephosphorylation in human A549 cells. Overexpression of PPM1H suppressed LRRK2-mediated Rab phosphorylation. PPM1H also efficiently and directly dephosphorylated Rab8A in biochemical studies. A “substrate-trapping” PPM1H mutant (Asp288Ala) binds with high affinity to endogenous, LRRK2-phosphorylated Rab proteins, thereby blocking dephosphorylation seen upon addition of LRRK2 inhibitors. PPM1H is localized to the Golgi and its knockdown suppresses primary cilia formation, similar to pathogenic LRRK2. Thus, PPM1H acts as a key modulator of LRRK2 signaling by controlling dephosphorylation of Rab proteins. PPM1H activity enhancers could offer a new therapeutic approach to prevent or treat Parkinson’s disease.

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PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins

10.1101/711176 ◽

2019 ◽

Author(s):

Kerryn Berndsen ◽

Pawel Lis ◽

Wondwossen Yeshaw ◽

Paulina S. Wawro ◽

Raja S. Nirujogi ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Protein Phosphatase ◽

Primary Cilia ◽

Rab Proteins ◽

Rab Gtpases ◽

Knock Out ◽

High Affinity ◽

Biochemical Studies ◽

Cilia Formation

AbstractMutations that activate LRRK2 protein kinase cause Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif controlling interaction with effectors. An siRNA screen of all protein phosphatases revealed that a poorly studied protein phosphatase, PPM1H, counteracts LRRK2 signaling by specifically dephosphorylating Rab proteins. PPM1H knock out increased endogenous Rab phosphorylation and inhibited Rab dephosphorylation. Overexpression of PPM1H suppressed LRRK2-mediated Rab phosphorylation. PPM1H also efficiently and directly dephosphorylated Rab8A in biochemical studies. A “substrate-trapping” PPM1H mutant (Asp288Ala) binds with high affinity to endogenous, LRRK2-phosphorylated Rab proteins, thereby blocking dephosphorylation seen upon addition of LRRK2 inhibitors. PPM1H is localized to the Golgi and its knockdown suppresses primary cilia formation, similar to pathogenic LRRK2. Thus, PPM1H acts as a key modulator of LRRK2 signaling by controlling dephosphorylation of Rab proteins. PPM1H activity enhancers could offer a new therapeutic approach to prevent or treat Parkinson’s disease.

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Back to the future: new target-validated Rab antibodies for evaluating LRRK2 signalling in cell biology and Parkinson's disease

Biochemical Journal ◽

10.1042/bcj20170870 ◽

2018 ◽

Vol 475 (1) ◽

pp. 185-189 ◽

Cited By ~ 3

Author(s):

Patrick A. Eyers

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Cell Biology ◽

Human Neutrophils ◽

Rab Proteins ◽

Rab Gtpases ◽

Biochemical Journal ◽

The Future ◽

Brain Extracts ◽

Sporadic Cases

The addition of phosphate groups to substrates allows protein kinases to regulate a myriad of biological processes, and contextual analysis of protein-bound phosphate is important for understanding how kinases contribute to physiology and disease. Leucine-rich repeat kinase 2 (LRRK2) is a Ser/Thr kinase linked to familial and sporadic cases of Parkinson's disease (PD). Recent work established that multiple Rab GTPases are physiological substrates of LRRK2, with Rab10 in particular emerging as a human substrate whose site-specific phosphorylation mirrors hyperactive LRRK2 lesions associated with PD. However, current assays to quantify Rab10 phosphorylation are expensive, time-consuming and technically challenging. In back-to-back studies reported in the Biochemical Journal, Alessi and colleagues teamed up with clinical colleagues and collaborators at the Michael J. Fox Foundation (MJFF) for Parkinson's research to develop, and validate, a panel of exquisitely sensitive phospho-specific Rab antibodies. Of particular interest, the monoclonal antibody-designated MJFF-pRAB10 detects phosphorylated Rab 10 on Thr73 in a variety of cells, brain extracts, PD-derived samples and human neutrophils, the latter representing a previously unrecognised biological resource for LRRK2 signalling analysis. In the future, these antibodies could become universal resources in the fight to understand and quantify connections between LRRK2 and Rab proteins, including those associated with clinical PD.

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A pathway for Parkinson’s Disease LRRK2 kinase to block primary cilia and Sonic hedgehog signaling in the brain

eLife ◽

10.7554/elife.40202 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 58

Author(s):

Herschel S Dhekne ◽

Izumi Yanatori ◽

Rachel C Gomez ◽

Francesca Tonelli ◽

Federico Diez ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Sonic Hedgehog ◽

Transcriptional Activation ◽

Hedgehog Signaling ◽

Primary Cilia ◽

Cultured Cells ◽

Editorial Note ◽

Rab Gtpases ◽

Lrrk2 Kinase

Parkinson’s disease-associated LRRK2 kinase phosphorylates multiple Rab GTPases, including Rab8A and Rab10. We show here that LRRK2 kinase interferes with primary cilia formation in cultured cells, human LRRK2 G2019S iPS cells and in the cortex of LRRK2 R1441C mice. Rab10 phosphorylation strengthens its intrinsic ability to block ciliogenesis by enhancing binding to RILPL1. Importantly, the ability of LRRK2 to interfere with ciliogenesis requires both Rab10 and RILPL1 proteins. Pathogenic LRRK2 influences the ability of cells to respond to cilia-dependent, Hedgehog signaling as monitored by Gli1 transcriptional activation. Moreover, cholinergic neurons in the striatum of LRRK2 R1441C mice show decreased ciliation, which will decrease their ability to sense Sonic hedgehog in a neuro-protective circuit that supports dopaminergic neurons. These data reveal a molecular pathway for regulating cilia function that likely contributes to Parkinson’s disease-specific pathology.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

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Pathogenic LRRK2 regulates ciliation probability upstream of Tau Tubulin kinase 2

10.1101/2020.04.07.029983 ◽

2020 ◽

Cited By ~ 4

Author(s):

Yuriko Sobu ◽

Paulina S. Wawro ◽

Herschel S. Dhekne ◽

Suzanne R. Pfeffer

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Protein Kinase ◽

Kinase Activity ◽

Cell Types ◽

Cell Physiology ◽

Rab Gtpases ◽

Cilia Formation ◽

Mother Centriole ◽

Lrrk2 Protein

ABSTRACTMutations that activate LRRK2 protein kinase cause Parkinson’s disease. We have shown previously that Rab10 phosphorylation by LRRK2 enhances its binding to RILPL1 and together, these proteins block cilia formation in a variety of cell types including patient derived iPS cells. We have used live cell fluorescence microscopy to identify, more precisely, the effect of LRRK2 kinase activity on both the formation of cilia triggered by serum starvation and loss of cilia seen upon serum re-addition. LRRK2 activity decreases the overall probability of ciliation without changing the rates of cilia formation in R1441C LRRK2 MEF cells. Cilia loss in these cells is accompanied by ciliary decapitation. Kinase activity does not change the timing or frequency of decapitation or the rate of cilia loss, but increases the percent of cilia that are lost upon serum addition. LRRK2 activity, or overexpression of RILPL1 protein, blocks release of CP110 from the mother centriole, a step normally required for early ciliogenesis. In both cases, failure of CP110 uncapping was due to failure to recruit TTBK2, a kinase needed for CP110 release. In contrast, recruitment of EHD1, another step important for ciliogenesis, appears unaltered. These experiments provide critical detail to our understanding of the cellular consequences of pathogenic LRRK2 mutation, and indicate that LRRK2 blocks ciliogenesis upstream of TTBK2 and enhances the deciliation process in response to serum addition.SIGNIFICANCE STATEMENTMutations that activate LRRK2 protein kinase cause Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases, in particular Rab8 and Rab10. Phosphorylated Rabs bind preferentially to a distinct set of effectors and block in primary ciliation in multiple cell types. We show here that the cilia blockade is upstream of the recruitment of TTBK2 kinase to the mother centriole, a step required for the release of CP110 and subsequent cilia formation. This study provides fundamental information related to how pathogenic LRRK2 interferes with normal cell physiology.

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Multiple Pathways of LRRK2-G2019S/Rab10 Interaction in Dopaminergic Neurons

Journal of Parkinson s Disease ◽

10.3233/jpd-202421 ◽

2021 ◽

pp. 1-16

Author(s):

Alison Fellgett ◽

C. Adam Middleton ◽

Jack Munns ◽

Chris Ugbode ◽

David Jaciuch ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Dopaminergic Neurons ◽

Genetic Interaction ◽

In Vitro Assays ◽

Rab Proteins ◽

Circadian Activity ◽

Loss Of Function ◽

Lrrk2 G2019s

Background: Inherited mutations in the LRRK2 protein are the common causes of Parkinson’s disease, but the mechanisms by which increased kinase activity of mutant LRRK2 leads to pathological events remain to be determined. In vitro assays (heterologous cell culture, phospho-protein mass spectrometry) suggest that several Rab proteins might be directly phosphorylated by LRRK2-G2019S. An in vivo screen of Rab expression in dopaminergic neurons in young adult Drosophila demonstrated a strong genetic interaction between LRRK2-G2019S and Rab10. Objective: To determine if Rab10 is necessary for LRRK2-induced pathophysiological responses in the neurons that control movement, vision, circadian activity, and memory. These four systems were chosen because they are modulated by dopaminergic neurons in both humans and flies. Methods: LRRK2-G2019S was expressed in Drosophila dopaminergic neurons and the effects of Rab10 depletion on Proboscis Extension, retinal neurophysiology, circadian activity pattern (‘sleep’), and courtship memory determined in aged flies. Results: Rab10 loss-of-function rescued LRRK2-G2019S induced bradykinesia and retinal signaling deficits. Rab10 knock-down, however, did not rescue the marked sleep phenotype which results from dopaminergic LRRK2-G2019S. Courtship memory is not affected by LRRK2, but is markedly improved by Rab10 depletion. Anatomically, both LRRK2-G2019S and Rab10 are seen in the cytoplasm and at the synaptic endings of dopaminergic neurons. Conclusion: We conclude that, in Drosophila dopaminergic neurons, Rab10 is involved in some, but not all, LRRK2-induced behavioral deficits. Therefore, variations in Rab expression may contribute to susceptibility of different dopaminergic nuclei to neurodegeneration seen in people with Parkinson’s disease.

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Development of phospho-specific Rab protein antibodies to monitor in vivo activity of the LRRK2 Parkinson's disease kinase

Biochemical Journal ◽

10.1042/bcj20170802 ◽

2018 ◽

Vol 475 (1) ◽

pp. 1-22 ◽

Cited By ~ 53

Author(s):

Pawel Lis ◽

Sophie Burel ◽

Martin Steger ◽

Matthias Mann ◽

Fiona Brown ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Protein Kinase ◽

Therapeutic Benefit ◽

Rab Proteins ◽

Specific Antibodies ◽

Rab Protein ◽

The Impact ◽

Inhibitor Treatment

Mutations that activate the LRRK2 (leucine-rich repeat protein kinase 2) protein kinase predispose to Parkinson's disease, suggesting that LRRK2 inhibitors might have therapeutic benefit. Recent work has revealed that LRRK2 phosphorylates a subgroup of 14 Rab proteins, including Rab10, at a specific residue located at the centre of its effector-binding switch-II motif. In the present study, we analyse the selectivity and sensitivity of polyclonal and monoclonal phospho-specific antibodies raised against nine different LRRK2-phosphorylated Rab proteins (Rab3A/3B/3C/3D, Rab5A/5B/5C, Rab8A/8B, Rab10, Rab12, Rab29[T71], Rab29[S72], Rab35 and Rab43). We identify rabbit monoclonal phospho-specific antibodies (MJFF-pRAB10) that are exquisitely selective for LRRK2-phosphorylated Rab10, detecting endogenous phosphorylated Rab10 in all analysed cell lines and tissues, including human brain cingulate cortex. We demonstrate that the MJFF-pRAB10 antibodies can be deployed to assess enhanced Rab10 phosphorylation resulting from pathogenic (R1441C/G or G2019S) LRRK2 knock-in mutations as well as the impact of LRRK2 inhibitor treatment. We also identify rabbit monoclonal antibodies displaying broad specificity (MJFF-pRAB8) that can be utilised to assess LRRK2-controlled phosphorylation of a range of endogenous Rab proteins, including Rab8A, Rab10 and Rab35. The antibodies described in the present study will help with the assessment of LRRK2 activity and examination of which Rab proteins are phosphorylated in vivo. These antibodies could also be used to assess the impact of LRRK2 inhibitors in future clinical trials.

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Decision letter: Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases

10.7554/elife.12813.022 ◽

2015 ◽

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Rab Gtpases

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Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A): Could It Be a Promising Biomarker and Therapeutic Target in Parkinson’s Disease?

Molecular Neurobiology ◽

10.1007/s12035-021-02670-w ◽

2022 ◽

Author(s):

Sijia Yin ◽

Chao Han ◽

Yun Xia ◽

Fang Wan ◽

Junjie Hu ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Protein Phosphatase ◽

Essential Role ◽

Protein Phosphatase 2A ◽

Area Under The Curve ◽

Alpha Synuclein ◽

Phosphatase 2A ◽

Induced Aggregation ◽

Age And Sex

AbstractParkinson’s disease (PD) is an incurable neurodegenerative disease characterized by aggregation of pathological alpha-synuclein (α-syn) and loss of dopaminergic neuron in the substantia nigra. Inhibition of phosphorylation of the α-syn has been shown to mediate alleviation of PD-related pathology. Protein phosphatase 2A (PP2A), an important serine/threonine phosphatase, plays an essential role in catalyzing dephosphorylation of the α-syn. Here, we identified and validated cancerous inhibitor of PP2A (CIP2A), as a potential diagnostic biomarker for PD. Our data showed that plasma CIP2A concentrations in PD patients were significantly lower compared to age- and sex-matched controls, 1.721 (1.435–2.428) ng/ml vs 3.051(2.36–5.475) ng/ml, p < 0.0001. The area under the curve of the plasma CIP2A in distinguishing PD from the age- and sex-matched controls was 0.776. In addition, we evaluated the role of CIP2A in PD-related pathogenesis in PD cellular and MPTP-induced mouse model. The results demonstrated that CIP2A is upregulated in PD cellular and MPTP-induced mouse models. Besides, suppression of the CIP2A expression alleviates rotenone induced aggregation of the α-syn as well as phosphorylation of the α-syn in SH-SY5Y cells, which is associated with increased PP2A activity. Taken together, our data demonstrated that CIP2A plays an essential role in the mechanisms related to PD development and might be a novel PD biomarker.

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