title: Quantitative phosphoproteomics reveals novel phosphorylation events in insulin signaling regulated by protein phosphatase 1 regulatory subunit 12A.
abstract: 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.
  
  
  CONCLUSIONS
  These 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.
authors: Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48202, USA.
authors: Xiangmin
authors: X
authors: Zhang
authors: Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48202, USA.
authors: Danjun
authors: D
authors: Ma
authors: Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48202, USA.
authors: Michael
authors: M
authors: Caruso
authors: Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48202, USA.
authors: Monique
authors: M
authors: Lewis
authors: Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48202, USA.
authors: Yue
authors: Y
authors: Qi
authors: Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48202, USA. Electronic address: zhengping.yi@wayne.edu.
authors: Zhengping
authors: Z
authors: Yi
mesh_terms: D000818:Animals; D002460:Cell Line; D003924:Diabetes Mellitus, Type 2 / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007333:Insulin Resistance; D009124:Muscle Proteins / Q000378:metabolism*; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D054645:Protein Phosphatase 1 / Q000378:metabolism*; D040901:Proteomics; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D007328:Insulin; D009124:Muscle Proteins; C066737:RHOA protein, human; C546842:MTOR protein, human; C546845:mTOR protein, rat; D058570:TOR Serine-Threonine Kinases; C499850:Ppp1r12a protein, rat; D054645:Protein Phosphatase 1; D043263:Myosin-Light-Chain Phosphatase; C490372:PPP1R12A protein, human; D020742:rhoA GTP-Binding Protein
keywords: Insulin signaling; Protein phosphatase 1 (PP1); Quantitative phosphoproteomics; SILAC; Serine/threonine protein phosphatase 1 regulatory subunit 12A (PPP1R12A); Skeletal muscle