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Site-specific phosphorylation of protein phosphatase 1 regulatory subunit 12A stimulated or suppressed by insulin.

ABSTRACT: Protein phosphatase 1 (PP1) is one of the major phosphatases responsible for protein dephosphorylation in eukaryotes. So far, only few specific phosphorylation sites of PP1 regulatory subunit 12A (PPP1R12A) have been shown to regulate the PP1 activity. The effect of insulin on PPP1R12A phosphorylation is largely unknown. Utilizing a mass spectrometry based phosphorylation identification and quantification approach, we identified 21 PPP1R12A phosphorylation sites (7 novel sites, including Ser20, Thr22, Thr453, Ser478, Thr671, Ser678, and Ser680) and quantified 16 of them under basal and insulin stimulated conditions in hamster ovary cells overexpressing the insulin receptor (CHO/IR), an insulin sensitive cell model. Insulin stimulated the phosphorylation of PPP1R12A significantly at Ser477, Ser478, Ser507, Ser668, and Ser695, while simultaneously suppressing the phosphorylation of PPP1R12A at Ser509 (more than 2-fold increase or decrease compared to basal). Our data demonstrate that PPP1R12A undergoes insulin stimulated/suppressed phosphorylation, suggesting that PPP1R12A phosphorylation may play a role in insulin signal transduction. The novel PPP1R12A phosphorylation sites as well as the new insulin-responsive phosphorylation sites of PPP1R12A in CHO/IR cells provide targets for investigation of the regulation of PPP1R12A and the PPP1R12A-PP1c? complex in insulin action and other signaling pathways in other cell models, animal models, and humans.

SUBMITTER: Chao A

PROVIDER: S-EPMC3367048 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Site-specific phosphorylation of protein phosphatase 1 regulatory subunit 12A stimulated or suppressed by insulin.

Chao Alex A Zhang Xiangmin X Ma Danjun D Langlais Paul P Luo Moulun M Mandarino Lawrence J LJ Zingsheim Morgan M Pham Kimberly K Dillon James J Yi Zhengping Z

Journal of proteomics 20120406 11

Protein phosphatase 1 (PP1) is one of the major phosphatases responsible for protein dephosphorylation in eukaryotes. So far, only few specific phosphorylation sites of PP1 regulatory subunit 12A (PPP1R12A) have been shown to regulate the PP1 activity. The effect of insulin on PPP1R12A phosphorylation is largely unknown. Utilizing a mass spectrometry based phosphorylation identification and quantification approach, we identified 21 PPP1R12A phosphorylation sites (7 novel sites, including Ser20, ...[more]

PMID: 22516431

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Project description:Serine/threonine protein phosphatase 1 regulatory subunit 12A (PPP1R12A) modulates the activity and specificity of the catalytic subunit of protein phosphatase 1, regulating various cellular processes via dephosphorylation. Nonetheless, little is known about phosphorylation events controlled by PPP1R12A in skeletal muscle insulin signaling. Here, we used quantitative phosphoproteomics to generate a global picture of phosphorylation events regulated by PPP1R12A in a L6 skeletal muscle cell line, which were engineered for inducible PPP1R12A knockdown. Phosphoproteomics revealed 3876 phosphorylation sites (620 were novel) in these cells. Furthermore, PPP1R12A knockdown resulted in increased overall phosphorylation in L6 cells at the basal condition, and changed phosphorylation levels for 698 sites (assigned to 295 phosphoproteins) at the basal and/or insulin-stimulated conditions. Pathway analysis on the 295 phosphoproteins revealed multiple significantly enriched pathways related to insulin signaling, such as mTOR signaling and RhoA signaling. Moreover, phosphorylation levels for numerous regulatory sites in these pathways were significantly changed due to PPP1R12A knockdown. These results indicate that PPP1R12A indeed plays a role in skeletal muscle insulin signaling, providing novel insights into the biology of insulin action. This new information may facilitate the design of experiments to better understand mechanisms underlying skeletal muscle insulin resistance and type 2 diabetes.Biological significanceThese results identify a large number of potential new substrates of serine/threonine protein phosphatase 1 and suggest that serine/threonine protein phosphatase 1 regulatory subunit 12A indeed plays a regulatory role in multiple pathways related to insulin action, providing novel insights into the biology of skeletal muscle insulin signaling. This information may facilitate the design of experiments to better understand the molecular mechanism responsible for skeletal muscle insulin resistance and associated diseases, such as type 2 diabetes and cardiovascular diseases.

Project description:BackgroundType 2A protein phosphatase (PP2A) enzymes are serine/threonine phosphatases which comprise a scaffold A subunit, a regulatory B subunit and a catalytic C subunit, and have been implicated in the dephosphorylation of multiple cardiac phosphoproteins. B subunits determine subcellular targeting, substrate specificity and catalytic activity, and can themselves be regulated by post-translational modifications. We explored potential β-adrenergic regulation of PP2A in cardiomyocytes through phosphorylation of the regulatory B subunit isoform B56δ.Methods and resultsPhosphate affinity SDS-PAGE and immunoblot analysis revealed increased phosphorylation of B56δ in adult rat ventricular myocytes (ARVM) exposed to the β-adrenergic receptor (βAR) agonist isoprenaline (ISO). Phosphorylation of B56δ occurred at S573, primarily through stimulation of the β1AR subtype, and was dependent on PKA activity. The functional role of the phosphorylation was explored in ARVM transduced with adenoviruses expressing wild type (WT) or non-phosphorylatable (S573A) B56δ, fused to GFP at the N-terminus. C subunit expression was increased in ARVM expressing GFP-B56δ-WT or GFP-B56δ-S573A, both of which co-immunoprecipitated with endogenous C and A subunits. PP2A activity in cell lysates was increased in response to ISO in ARVM expressing GFP-B56δ-WT but not GFP-B56δ-S573A. Immunoblot analysis of the phosphoproteome in ARVM expressing GFP-B56δ-WT or GFP-B56δ-S573A with antibodies detecting (i) phospho-serine/threonine residues in distinct kinase substrate motifs or (ii) specific phosphorylated residues of functional importance in selected proteins revealed a comparable phosphorylation profile in the absence or presence of ISO stimulation.ConclusionsIn cardiomyocytes, βAR stimulation induces PKA-mediated phosphorylation of the PP2A regulatory subunit isoform B56δ at S573, which increases associated PP2A catalytic activity. This is likely to regulate the phosphorylation status of specific B56δ-PP2A substrates, which remain to be identified.

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Site-specific phosphorylation of protein phosphatase 1 regulatory subunit 12A stimulated or suppressed by insulin.

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Site-specific phosphorylation of protein phosphatase 1 regulatory subunit 12A stimulated or suppressed by insulin.

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