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Dissection of the insulin signaling pathway via quantitative phosphoproteomics.

ABSTRACT: The insulin signaling pathway is of pivotal importance in metabolic diseases, such as diabetes, and in cellular processes, such as aging. Insulin activates a tyrosine phosphorylation cascade that branches to create a complex network affecting multiple biological processes. To understand the full spectrum of the tyrosine phosphorylation cascade, we have defined the tyrosine-phosphoproteome of the insulin signaling pathway, using high resolution mass spectrometry in combination with phosphotyrosine immunoprecipitation and stable isotope labeling by amino acids in cell culture (SILAC) in differentiated brown adipocytes. Of 40 identified insulin-induced effectors, 7 have not previously been described in insulin signaling, including SDR, PKCdelta binding protein, LRP-6, and PISP/PDZK11, a potential calcium ATPase binding protein. A proteomic interaction screen with PISP/PDZK11 identified the calcium transporting ATPase SERCA2, supporting a connection to calcium signaling. The combination of quantitative phosphoproteomics with cell culture models provides a powerful strategy to dissect the insulin signaling pathways in intact cells.

SUBMITTER: Kruger M

PROVIDER: S-EPMC2268157 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Dissection of the insulin signaling pathway via quantitative phosphoproteomics.

Krüger Marcus M Kratchmarova Irina I Blagoev Blagoy B Tseng Yu-Hua YH Kahn C Ronald CR Mann Matthias M

Proceedings of the National Academy of Sciences of the United States of America 20080211 7

The insulin signaling pathway is of pivotal importance in metabolic diseases, such as diabetes, and in cellular processes, such as aging. Insulin activates a tyrosine phosphorylation cascade that branches to create a complex network affecting multiple biological processes. To understand the full spectrum of the tyrosine phosphorylation cascade, we have defined the tyrosine-phosphoproteome of the insulin signaling pathway, using high resolution mass spectrometry in combination with phosphotyrosin ...[more]

PMID: 18268350

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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.

Dataset Information

Dissection of the insulin signaling pathway via quantitative phosphoproteomics.

Publications

Dissection of the insulin signaling pathway via quantitative phosphoproteomics.

Similar Datasets

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