Dual phosphorylation of protein phosphatase PPM1H promotes dephosphorylation of Smad1 in cellulo

Osawa, Jin; Akizuki, Kazutoshi; Kashimura, Akari; Ueta, Saki; Nakatani, Misato; Inui, Yuiko; Shigeri, Yasushi; Ishida, Atsuhiko; Kameshita, Isamu; Sueyoshi, Noriyuki

doi:10.1016/j.bbrc.2020.05.082

Cited by 9 publications

(4 citation statements)

References 27 publications

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“…PPM1H has also been reported to be phosphorylated (13); non-phosporylatable S124A and S211A PPM1H proteins were not altered in their localizations (Supplemental Figure 2).…”

Section: Resultsmentioning

confidence: 98%

Localization of PPM1H phosphatase tunes Parkinson’s disease-linked LRRK2 kinase-mediated Rab GTPase phosphorylation and ciliogenesis

Yeshaw

Adhikari

Chiang

et al. 2023

Preprint

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PPM1H phosphatase reverses Parkinson's disease-associated, LRRK2-mediated Rab GTPase phosphorylation. We show here that PPM1H relies on an N-terminal amphipathic helix for Golgi localization. The amphipathic helix enables PPM1H to bind to liposomes in vitro, and small, highly curved liposomes stimulate PPM1H activity. We artificially anchored PPM1H to the Golgi, mitochondria, or mother centriole. Our data show that regulation of Rab10 GTPase phosphorylation requires PPM1H access to Rab10 at or near the mother centriole. Moreover, poor co-localization of Rab12 explains in part why it is a poor substrate for PPM1H in cells but not in vitro. These data support a model in which localization drives PPM1H substrate selection and centriolar PPM1H is critical for regulation of Rab GTPase-regulated ciliogenesis. Moreover, Golgi localized PPM1H maintains active Rab GTPases on the Golgi to carry out their non-ciliogenesis-related functions in membrane trafficking.

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“…PPM1H has also been reported to be phosphorylated (13); non-phosporylatable S124A and S211A PPM1H proteins were not altered in their localizations (Supplemental Figure 2).…”

Section: Resultsmentioning

confidence: 98%

Localization of PPM1H phosphatase tunes Parkinson’s disease-linked LRRK2 kinase-mediated Rab GTPase phosphorylation and ciliogenesis

Yeshaw

Adhikari

Chiang

et al. 2023

Preprint

View full text Add to dashboard Cite

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“…This loop is distant from the active site, but in a docked model of a heterotetrameric complex, this PPM1H loop is situated in close proximity to pRab8a at the active site of the dimeric partner (Figs 2B and 3A). At least one of the residues in this loop (Ser210) is phosphorylated (Osawa et al , 2020), and in future studies, it would be important to investigate the role that dimerization plays in controlling PPM1H function in cells. However, conversion of the enzyme into a monomer by mutagenesis through abolition of flap/α2 contacts does not markedly affect catalysis in vitro .…”

Section: Discussionmentioning

confidence: 99%

Structural basis for the specificity of PPM1H phosphatase for Rab GTPases

et al. 2021

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LRRK2 serine/threonine kinase is associated with inherited Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases within their switch 2 motif to control their interactions with effectors. Recent work has shown that the metal‐dependent protein phosphatase PPM1H counteracts LRRK2 by dephosphorylating Rabs. PPM1H is highly selective for LRRK2 phosphorylated Rabs, and closely related PPM1J exhibits no activity towards substrates such as Rab8a phosphorylated at Thr72 (pThr72). Here, we have identified the molecular determinant of PPM1H specificity for Rabs. The crystal structure of PPM1H reveals a structurally conserved phosphatase fold that strikingly has evolved a 110‐residue flap domain adjacent to the active site. The flap domain distantly resembles tudor domains that interact with histones in the context of epigenetics. Cellular assays, crosslinking and 3‐D modelling suggest that the flap domain encodes the docking motif for phosphorylated Rabs. Consistent with this hypothesis, a PPM1J chimaera with the PPM1H flap domain dephosphorylates pThr72 of Rab8a both in vitro and in cellular assays. Therefore, PPM1H has acquired a Rab‐specific interaction domain within a conserved phosphatase fold.

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“…However, modulation of kinase activity downstream of the cAMP binding protein, EPAC is also to be expected in most cells (Robichaux and Cheng, 2018). Moreover, a series of different protein phosphatases can reverse these phosphorylations some of which can be modulated directly by cAMP-dependent phosphorylation (Leslie and Nairn, 2019;Osawa et al, 2020). Finally, there are many different substrates for PKA, some of which are other kinases.…”

Section: General Background For Camp and Camp-dependent Phosphorylation As Regulators Of Cellularmentioning

confidence: 99%

Phosphoproteomic Analysis as an Approach for Understanding Molecular Mechanisms of cAMP-Dependent Actions

et al. 2021

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In recent years, highly sensitive mass spectrometry-based phosphoproteomic analysis is beginning to be applied to identification of protein kinase substrates altered downstream of increased cyclic 3',5'adenosine monophosphate (cAMP). Such studies identify a very large number of phosphorylation sites regulated in response to increased cAMP. Therefore, we now are tasked with the challenge of determining how many of these altered phosphorylation sites are relevant to regulation of function in the cell. This mini-review describes the use of phosphoproteomic analysis to monitor the effects of cyclic nucleotide phosphodiesterase (PDE) inhibitors on cAMP-dependent phosphorylation events. More specifically, it describes two examples of this approach carried out in the authors' laboratories using the selective PDE inhibitor approach. After a short discussion of several likely conclusions suggested by these analyses of cAMP function in steroid hormone producing cells and also in T-cells, it expands into a discussion about some newer and more speculative interpretations of the data. These include the idea that multiple phosphorylation sites and not a single rate limiting step likely regulate these and by analogy many other cAMP-dependent pathways. In addition, the idea that meaningful regulation requires a high stoichiometry of phosphorylation in order to be important is discussed and suggested to be untrue in many instances. These new interpretations have important implications for drug design, especially for targeting pathway agonists.

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Dual phosphorylation of protein phosphatase PPM1H promotes dephosphorylation of Smad1 in cellulo

Cited by 9 publications

References 27 publications

Localization of PPM1H phosphatase tunes Parkinson’s disease-linked LRRK2 kinase-mediated Rab GTPase phosphorylation and ciliogenesis

Localization of PPM1H phosphatase tunes Parkinson’s disease-linked LRRK2 kinase-mediated Rab GTPase phosphorylation and ciliogenesis

Structural basis for the specificity of PPM1H phosphatase for Rab GTPases

Phosphoproteomic Analysis as an Approach for Understanding Molecular Mechanisms of cAMP-Dependent Actions

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