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

Chao, Alex; Zhang, Xiangmin; Ma, Dongzhu; Langlais, Paul; Luo, Moulun; Mandarino, Lawrence J.; Zingsheim, Morgan; Pham, Kimberly; Dillon, J. L.; Yi, Zhengping

doi:10.1016/j.jprot.2012.03.043

Cited by 14 publications

(15 citation statements)

References 37 publications

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“…Inhibitor experiments indicated that the interaction of PPP1R12A and PP1cδ with IRS1 is diminished upon inhibition of Akt and mTOR/Raptor [20]. Moreover, we have demonstrated that insulin stimulates or suppresses multiple phosphorylation sites of PPP1R12A [21]. These results provided the first evidence that PPP1R12A may regulate skeletal muscle insulin action through tuning PP1cδ activity and specificity.…”

Section: Introductionmentioning

confidence: 84%

“…However, a mechanism for serine/threonine phosphatase action in insulin signal transduction is largely unknown. Recent evidences from our group suggest that PPP1R12A, a regulatory subunit of protein phosphatase 1, is involved in insulin signaling [19–21]. Moreover, the majority of research on the abnormalities of phosphorylation in skeletal muscle insulin action focuses a few known phosphorylation targets.…”

Section: Discussionmentioning

confidence: 99%

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Quantitative phosphoproteomics reveals novel phosphorylation events in insulin signaling regulated by protein phosphatase 1 regulatory subunit 12A

Zhang¹,

Ma²,

Caruso³

et al. 2014

Journal of Proteomics

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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

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Section: Introductionmentioning

confidence: 84%

Section: Discussionmentioning

confidence: 99%

Quantitative phosphoproteomics reveals novel phosphorylation events in insulin signaling regulated by protein phosphatase 1 regulatory subunit 12A

Zhang¹,

Ma²,

Caruso³

et al. 2014

Journal of Proteomics

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“…PP1R12A is a member of the major group of phosphatases in eukaryotes. Therefore, its phosphorylation seems to play a key role in the insulin signal transduction, because this hormone stimulates PP1R12A phosphorylation at several sites, including Ser 507 (80). To determine over-represented sequence motifs from the whole as well as regulated phosphoproteome dataset, we performed the motif-X algorithm (41) using the Homo sapiens database as background.…”

Section: Phosphorylation and N-linked Sialylation As Important Ptms Fmentioning

confidence: 99%

Comprehensive Quantitative Comparison of the Membrane Proteome, Phosphoproteome, and Sialiome of Human Embryonic and Neural Stem Cells

Melo‐Braga

Schulz

Liu

et al. 2014

Molecular & Cellular Proteomics

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Human embryonic stem cells (hESCs) can differentiate into neural stem cells (NSCs), which can further be differentiated into neurons and glia cells. Therefore, these cells have huge potential as source for treatment of neurological diseases. Membrane-associated proteins are very important in cellular signaling and recognition, and their function and activity are frequently regulated by posttranslational modifications such as phosphorylation and glycosylation. To obtain information about membraneassociated proteins and their modified amino acids potentially involved in changes of hESCs and NSCs as well as to investigate potential new markers for these two cell stages, we performed large-scale quantitative membrane-proteomic of hESCs and NSCs. This approach employed membrane purification followed by peptide dimethyl labeling and peptide enrichment to study the membrane subproteome as well as changes in phosphorylation and sialylation between hESCs and NSCs. Combining proteomics and modification specific proteomics we identified a total of 5105 proteins whereof 57% contained transmembrane domains or signal peptides. The enrichment strategy yielded a total of 10,087 phosphorylated peptides in which 78% of phosphopeptides were identified with >99% confidence in site assignment and 1810 unique formerly sialylated N-linked glycopeptides. Several proteins were identified as significantly regulated in hESCs and NSC, including proteins involved in the early embryonic and neural development. In the latter group of proteins, we could identify potential NSC markers as Pluripotent embryonic stem cell (ESC)1 -derived neural stem cells (NSCs) can differentiate into neurons and glia cells of the central nervous system (1), including specialized neuron types like dopaminergic, representing a potential source for treatment of neurological diseases, such as ParkinsonЈs disease. Therefore, a better understanding of the cellular processes behind the changes of hESCs into NSCs, including solid markers for each cell type, is fundamental to move forward with a successful regenerative cell therapy and to investigate the early human neurogenesis processes.Many markers have been reported for the two types of stem cells (2, 3), however several of these markers are also identified in other stem or progenitor cells such as CD133 (Prominin-1) (4). Discovery of cell surface specific markers for differentiated stem cells is highly relevant for future clinical applications. In particular being able to distinguish the developmental stages of the differentiation from parental stem cells to fully mature cells would allow a correct manipulation and isolation of the cell type of interest. Moreover, such study 1 The abbreviations used are: CID, collision-induced dissociation; FDR, false discovery rate; FLR, false localization rate; HCD, highenergy collision induced dissociation; hESC, human embryonic stem cell; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MSA, multistage activation; NSC, neural stem cell; PTM, post-translational modificat...

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“…This is one of several sites in MYPT1 reported by Cell Signaling Technology using LC-MS/MS platform to be phosphorylated in a number of human cancers [48]. Insulin has also been shown to result in MYPT1 phosphorylation at Ser668 [49], although neither the function of this phosphorylation nor the responsible kinase have been identified. Protein kinase G (PKG1α) known to selectively phosphorylate MYPT1 isoforms that include a C-terminal leucine zipper has also been shown to phosphorylate MYPT1 on Ser668 in vitro [50].…”

Section: Discussionmentioning

confidence: 96%

The p90 Ribosomal S6 Kinase (RSK) Is a Mediator of Smooth Muscle Contractility

et al. 2013

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In the canonical model of smooth muscle (SM) contraction, the contractile force is generated by phosphorylation of the myosin regulatory light chain (RLC20) by the myosin light chain kinase (MLCK). Moreover, phosphorylation of the myosin targeting subunit (MYPT1) of the RLC20 phosphatase (MLCP) by the RhoA-dependent ROCK kinase, inhibits the phosphatase activity and consequently inhibits dephosphorylation of RLC20 with concomitant increase in contractile force, at constant intracellular [Ca2+]. This pathway is referred to as Ca2+-sensitization. There is, however, emerging evidence suggesting that additional Ser/Thr kinases may contribute to the regulatory pathways in SM. Here, we report data implicating the p90 ribosomal S6 kinase (RSK) in SM contractility. During both Ca2+- and agonist (U46619) induced SM contraction, RSK inhibition by the highly selective compound BI-D1870 (which has no effect on MLCK or ROCK) resulted in significant suppression of contractile force. Furthermore, phosphorylation levels of RLC20 and MYPT1 were both significantly decreased. Experiments involving the irreversible MLCP inhibitor microcystin-LR, in the absence of Ca2+, revealed that the decrease in phosphorylation levels of RLC20 upon RSK inhibition are not due solely to the increase in the phosphatase activity, but reflect direct or indirect phosphorylation of RLC20 by RSK. Finally, we show that agonist (U46619) stimulation of SM leads to activation of extracellular signal-regulated kinases ERK1/2 and PDK1, consistent with a canonical activation cascade for RSK. Thus, we demonstrate a novel and important physiological function of the p90 ribosomal S6 kinase, which to date has been typically associated with the regulation of gene expression.

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

Cited by 14 publications

References 37 publications

Quantitative phosphoproteomics reveals novel phosphorylation events in insulin signaling regulated by protein phosphatase 1 regulatory subunit 12A

Quantitative phosphoproteomics reveals novel phosphorylation events in insulin signaling regulated by protein phosphatase 1 regulatory subunit 12A

Comprehensive Quantitative Comparison of the Membrane Proteome, Phosphoproteome, and Sialiome of Human Embryonic and Neural Stem Cells

The p90 Ribosomal S6 Kinase (RSK) Is a Mediator of Smooth Muscle Contractility

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