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

G protein-coupled receptors (GPCRs) That Signal via Protein Kinase A (PKA) Cross-talk at Insulin Receptor Substrate 1 (IRS1) to Activate the phosphatidylinositol 3-kinase (PI3K)/AKT Pathway

“…Phosphoproteomic analysis of L6 skeletal muscle cells previously revealed the interaction of MYPT1 and IRS1 (62) as well as the potential role of MYPT1 in insulin responses (63); however, the role of MYPT1 was unknown. We show that CA MYPT1 (in the absence of FSH) stimulates IRS1 Ser dephosphorylation, IRS1 YXXM phosphorylation, and PI3K/AKT activation.…”

Myosin phosphatase Fine-tunes Zebrafish Motoneuron Position during Axonogenesis

“…Besides regulating myosin II phosphorylation, the MYPT1-containing holoenzyme has also been shown to interact with a large host of putative substrates including moesin, tau, MAP2, Polo like kinase and the transcriptional repressor HDAC7, suggesting that mypt1 likely has broader functions than myosin regulation [18, 47, 48]. Furthermore, mypt1 has recently been shown to interact with the insulin receptor substrate-1, and to regulate other pathways including mTOR signaling [49, 50]. Thus, it will be interesting to determine whether mypt1 restricts axonal branching through a ‘canonical’ pathway such as myosin II or moesin phosphorylation, or through one of these recently identified substrates and pathways.…”

Optimization of Search Engines and Postprocessing Approaches to Maximize Peptide and Protein Identification for High-Resolution Mass Data

“…The data sets for Velos HCD–OT yeast and QE HCD–OT human were described in a previous publication by Michalski et al, 24 and Q-TOF E. coli was described in Wenger et al; 8 these three data sets are available at http://www.chem.wisc.edu/~coon/Downloads/Morpheus/. The data set for Fusion HCD–IT yeast is available at https://chorusproject.org/anonymous/download/experiment/449795368199176159, as described in the report by Hebert et al 25 The sample preparation and MS analysis of data set Elite CID–IT human (MCF7 cells) were performed as previously described 26 and also analyzed in triplicate. The remained data sets were described in detail as below.…”