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Identification of phosphatases that dephosphorylate Tom6 using synthetic trap-peptides

ABSTRACT: The identification of phosphatases that dephosphorylate specific sites in proteins remains a major challenge, particularly for the major class of serine/threonine-specific phosphatases, which function as holoenzymes. Here, we report the development of synthetic trap-peptides to identify phosphatases that bind to Tom6, a subunit of the mitochondrial translocase of the outer membrane (TOM complex). The TOM complex is regulated by reversible phosphorylation, and while responsible kinases have been identified, the corresponding phosphatases so far remain unknown. Here, the trap-peptides enriched PP2A and PP4 as full holoenzymes from yeast cytosolic-fractions. We observed that their interaction with Tom6 was mediated through their regulatory subunits Psy2reg and Cdc55reg, and that PP2A was able to dephosphorylate Ser16 of Tom6 in vitro. In summary, synthetic trap-peptides facilitate the identification of complete holoenzymes that bind to the target sequence and reveal PP2A as the first TOM phosphatase.

INSTRUMENT(S):

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Christina Ludwig

LAB HEAD: Christina Ludwig

PROVIDER: PXD058384 | Pride | 2025-06-16

REPOSITORIES: Pride

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	BBM_559_P177_04_FRH_001.raw	Raw
	BBM_559_P177_04_FRH_002.raw	Raw
	BBM_559_P177_04_FRH_003.raw	Raw
	BBM_559_P177_04_FRH_004.raw	Raw
	BBM_559_P177_04_FRH_005.raw	Raw

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Phosphatases modified by LH signaling in ovarian follicles: testing their role in regulating the NPR2 guanylyl cyclase

Project description:In response to luteinizing hormone (LH), multiple proteins in rat and mouse granulosa cells are rapidly dephosphorylated, but the responsible phosphatases remain to be identified. Because the phosphorylation state of phosphatases can regulate their interaction with substrates, we searched for phosphatases that might function in LH signaling by using quantitative mass spectrometry. We identified all proteins in rat ovarian follicles whose phosphorylation state changed detectably in response to a 30-min exposure to LH, and within this list, identified protein phosphatases or phosphatase regulatory subunits that showed changes in phosphorylation. Phosphatases in the phosphoprotein phosphatase (PPP) family were of particular interest because of their requirement for dephosphorylating the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase in the granulosa cells, which triggers oocyte meiotic resumption. Among the PPP family regulatory subunits, PPP1R12A and PPP2R5D showed the largest increases in phosphorylation, with 4–10 fold increases in signal intensity on several sites. Although follicles from mice in which these phosphorylations were prevented by serine-to-alanine mutations in either Ppp1r12a or Ppp2r5d showed normal LH-induced NPR2 dephosphorylation, these regulatory subunits and others could act redundantly to dephosphorylate NPR2. Our identification of phosphatases and other proteins whose phosphorylation state is rapidly modified by LH provides clues about multiple signaling pathways in ovarian follicles.

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Project description:Phosphorylation of the aquaporin-2 (AQP2) water channel at four COOH-terminal serines plays a central role in the regulation of water permeability of the renal collecting duct. The level of phosphorylation at these sites is determined by a balance between phosphorylation by protein kinases and dephosphorylation by phosphatases. The phosphatases that dephosphorylate AQP2 have not been identified. Here, we use large-scale data integration techniques to identify serine-threonine phosphatases likely to interact with AQP2 in renal collecting duct principal cells. As a first step, we have created a comprehensive list of 38 S/T phosphatase catalytic subunits present in the mammalian genome. Then we used Bayes’ theorem to integrate available information from large-scale data sets from proteomic and transcriptomic studies in order to rank the known S/T phosphatases with regard to the likelihood that they interact with AQP2 in renal collecting duct cells. To broaden the analysis, we have generated new proteomic data (LC-MS/MS) identifying 4538 distinct proteins including 22 S/T phosphatases in cytoplasmic fractions from native inner medullary collecting duct cells from rats. The official gene symbols corresponding to the top-ranked phosphatases (common names in parentheses) were: Ppp1cb (PP1-beta), Ppm1g (PP2C), Ppp1ca (PP1-alpha), Ppp3ca (PP2-B or calcineurin), Ppp2ca (PP2A-alpha), Ppp1cc (PP1-gamma), Ppp2cb (PP2A-beta), Ppp6c (PP6C) and Ppp5c (PP5). This ranking correlates well with results of prior reductionist studies of ion and water channels in renal collecting duct cells.

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