Insulin-stimulated phosphorylation of protein phosphatase 1 regulatory subunit 12B revealed by HPLC-ESI-MS/MS

Proteome Science

Zhengping Yi²  Morgan Zingsheim¹  Alex Chao¹  Xiangmin Zhang²  Paul Langlais¹  Kimberly Pham¹ 

[1] Center for Metabolic and Vascular Biology, Arizona State University, Tempe, AZ, USA;Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy/Health Sciences, Wayne State University, 259 Mack Ave., Detroit, MI, USA

关键词: Quantification;    Label-free;    Insulin signaling;    HPLC-ESI-MS/MS;    Phosphorylation;    PPP1R12B;   

Others  :  817198
DOI  :  10.1186/1477-5956-10-52

 received in 2012-02-13, accepted in 2012-07-31,  发布年份 2012

【 摘 要 】

Background

Protein phosphatase 1 (PP1) is one of the major phosphatases responsible for protein dephosphorylation in eukaryotes. Protein phosphatase 1 regulatory subunit 12B (PPP1R12B), one of the regulatory subunits of PP1, can bind to PP1cδ, one of the catalytic subunits of PP1, and modulate the specificity and activity of PP1cδ against its substrates. Phosphorylation of PPP1R12B on threonine 646 by Rho kinase inhibits the activity of the PP1c-PPP1R12B complex. However, it is not currently known whether PPP1R12B phosphorylation at threonine 646 and other sites is regulated by insulin. We set out to identify phosphorylation sites in PPP1R12B and to quantify the effect of insulin on PPP1R12B phosphorylation by using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry.

Results

14 PPP1R12B phosphorylation sites were identified, 7 of which were previously unreported. Potential kinases were predicted for these sites. Furthermore, relative quantification of PPP1R12B phosphorylation sites for basal and insulin-treated samples was obtained by using peak area-based label-free mass spectrometry of fragment ions. The results indicate that insulin stimulates the phosphorylation of PPP1R12B significantly at serine 29 (3.02 ± 0.94 fold), serine 504 (11.67 ± 3.33 fold), and serine 645/threonine 646 (2.34 ± 0.58 fold).

Conclusion

PPP1R12B was identified as a phosphatase subunit that undergoes insulin-stimulated phosphorylation, suggesting that PPP1R12B might play a role in insulin signaling. This study also identified novel targets for future investigation of the regulation of PPP1R12B not only in insulin signaling in cell models, animal models, and in humans, but also in other signaling pathways.

【 授权许可】

   
2012 Pham et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1 .

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【 参考文献 】

• [1]Bollen M, Peti W, Ragusa MJ, Beullens M: The extended PP1 toolkit: designed to create specificity. Trends Biochem Sci 2010, 35:450-458.
• [2]Cohen PT: Protein phosphatase 1-targeted in many directions. J Cell Sci 2002, 115:241-256.
• [3]Virshup DM, Shenolikar S: From promiscuity to precision: protein phosphatases get a makeover. Mol Cell 2009, 33:537-545.
• [4]Grassie ME, Moffat LD, Walsh MP, Macdonald JA: The myosin phosphatase targeting protein (MYPT) family: a regulated mechanism for achieving substrate specificity of the catalytic subunit of protein phosphatase type 1δ. Arch Biochem Biophys 2011, 510:147-159.
• [5]Okamoto R, Kato T, Mizoguchi A, Takahashi N, Nakakuki T, Mizutani H, Isaka N, Imanaka-Yoshida K, Kaibuchi K, Lu Z, et al.: Characterization and function of MYPT2, a target subunit of myosin phosphatase in heart. Cell Signal 2006, 18:1408-1416.
• [6]Ito M, Nakano T, Erdodi F, Hartshorne DJ: Myosin phosphatase: structure, regulation and function. Mol Cell Biochem 2004, 259:197-209.
• [7]Shichi D, Arimura T, Ishikawa T, Kimura A: Heart-specific small subunit of myosin light chain phosphatase activates rho-associated kinase and regulates phosphorylation of myosin phosphatase target subunit 1. J Biol Chem 2010, 285:33680-33690.
• [8]Geetha T, Langlais P, Luo M, Mapes R, Lefort N, Chen SC, Mandarino LJ, Yi Z: Label-free proteomic identification of endogenous, insulin-stimulated interaction partners of insulin receptor substrate-1. J Am Soc Mass Spectrom 2011, 22:457-466.
• [9]Matsumura F, Hartshorne DJ: Myosin phosphatase target subunit: Many roles in cell function. Biochem Biophys Res Commun 2008, 369:149-156.
• [10]Chao A, Xiangmin Z, Danjun M, Langlais P, Moulun-Luo LJM, Zingsheim M, Pham K, Dillon J, Zhengping Y: Site-Specific Phosphorylation of Protein Phosphatase 1 Regulatory Subunit 12A Stimulated or Suppressed by Insulin. Journal of Proteomics 2012, 75:3342-3350.
• [11]Langlais P, Mandarino LJ, Yi Z: Label-free relative quantification of co-eluting isobaric phosphopeptides of insulin receptor substrate-1 by HPLC-ESI-MS/MS. J Am Soc Mass Spectrom 2010, 21:1490-1499.
• [12]Moritz A, Li Y, Guo A, Villen J, Wang Y, MacNeill J, Kornhauser J, Sprott K, Zhou J, Possemato A, et al.: Akt-RSK-S6 kinase signaling networks activated by oncogenic receptor tyrosine kinases. Sci Signal 2010, 3:ra64.
• [13]Pan C, Gnad F, Olsen JV, Mann M: Quantitative phosphoproteome analysis of a mouse liver cell line reveals specificity of phosphatase inhibitors. Proteomics 2008, 8:4534-4546.
• [14]Huttlin EL, Jedrychowski MP, Elias JE, Goswami T, Rad R, Beausoleil SA, Villen J, Haas W, Sowa ME, Gygi SP: A tissue-specific atlas of mouse protein phosphorylation and expression. Cell 2010, 143:1174-1189.
• [15]Hoffert JD, Pisitkun T, Wang G, Shen RF, Knepper MA: Quantitative phosphoproteomics of vasopressin-sensitive renal cells: regulation of aquaporin-2 phosphorylation at two sites. Proc Natl Acad Sci U S A 2006, 103:7159-7164.
• [16]Zanivan S, Gnad F, Wickstrom SA, Geiger T, Macek B, Cox J, Fassler R, Mann M: Solid tumor proteome and phosphoproteome analysis by high resolution mass spectrometry. J Proteome Res 2008, 7:5314-5326.
• [17]Imami K, Sugiyama N, Kyono Y, Tomita M, Ishihama Y: Automated phosphoproteome analysis for cultured cancer cells by two-dimensional nanoLC-MS using a calcined titania/C18 biphasic column. Anal Sci 2008, 24:161-166.
• [18]Linding R, Jensen LJ, Ostheimer GJ, van Vugt MA, Jorgensen C, Miron IM, Diella F, Colwill K, Taylor L, Elder K, et al.: Systematic discovery of in vivo phosphorylation networks. Cell 2007, 129:1415-1426.
• [19]Linding R, Jensen LJ, Pasculescu A, Olhovsky M, Colwill K, Bork P, Yaffe MB, Pawson T: NetworKIN: a resource for exploring cellular phosphorylation networks. Nucleic Acids Res 2008, 36:D695-699.
• [20]Sun J, Khalid S, Rozakis-Adcock M, Fantus IG, Jin T: P-21-activated protein kinase-1 functions as a linker between insulin and Wnt signaling pathways in the intestine. Oncogene 2009, 28:3132-3144.
• [21]Tanasijevic MJ, Myers MG, Thoma RS, Crimmins DL, White MF, Sacks DB: Phosphorylation of the insulin receptor substrate IRS-1 by casein kinase II. J Biol Chem 1993, 268:18157-18166.
• [22]Llagostera E, Catalucci D, Marti L, Liesa M, Camps M, Ciaraldi TP, Kondo R, Reddy S, Dillmann WH, Palacin M, et al.: Role of myotonic dystrophy protein kinase (DMPK) in glucose homeostasis and muscle insulin action. PLoS One 2007, 2:e1134.
• [23]Furukawa N, Ongusaha P, Jahng WJ, Araki K, Choi CS, Kim HJ, Lee YH, Kaibuchi K, Kahn BB, Masuzaki H, et al.: Role of Rho-kinase in regulation of insulin action and glucose homeostasis. Cell metabolism 2005, 2:119-129.
• [24]Terrak M, Kerff F, Langsetmo K, Tao T, Dominguez R: Structural basis of protein phosphatase 1 regulation. Nature 2004, 429:780-784.
• [25]Toth A, Kiss E, Herberg FW, Gergely P, Hartshorne DJ, Erdodi F: Study of the subunit interactions in myosin phosphatase by surface plasmon resonance. Eur J Biochem 2000, 267:1687-1697.
• [26]Yi Z, Langlais P, De Filippis EA, Luo M, Flynn CR, Schroeder S, Weintraub ST, Mapes R, Mandarino LJ: Global assessment of regulation of phosphorylation of insulin receptor substrate-1 by insulin in vivo in human muscle. Diabetes 2007, 56:1508-1516.
• [27]Langlais P, Yi Z, Mandarino LJ: The Identification of Raptor as a Substrate for p44/42 MAPK. Endocrinology 2011, 152:1264-1273.
• [28]Højlund K, Yi Z, Lefort N, Langlais P, Bowen B, Levin K, Beck-Nielsen H, Mandarino L: Human ATP synthase beta is phosphorylated at multiple sites and shows abnormal phosphorylation at specific sites in insulin-resistant muscle. Diabetologia 2010, 53:541-551.
• [29]Siddle K: Signalling by insulin and IGF receptors: supporting acts and new players. J Mol Endocrinol 2011, 47:R1-10.
• [30]Yamashiro S, Yamakita Y, Totsukawa G, Goto H, Kaibuchi K, Ito M, Hartshorne DJ, Matsumura F: Myosin phosphatase-targeting subunit 1 regulates mitosis by antagonizing polo-like kinase 1. Developmental cell 2008, 14:787-797.
• [31]Ando A, Momomura K, Tobe K, Yamamoto-Honda R, Sakura H, Tamori Y, Kaburagi Y, Koshio O, Akanuma Y, Yazaki Y, et al.: Enhanced insulin-induced mitogenesis and mitogen-activated protein kinase activities in mutant insulin receptors with substitution of two COOH-terminal tyrosine autophosphorylation sites by phenylalanine. J Biol Chem 1992, 267:12788-12796.
• [32]Yi Z, Luo M, Mandarino LJ, Reyna SM, Carroll CA, Weintraub ST: Quantification of phosphorylation of insulin receptor substrate-1 by HPLC-ESI-MS/MS. J Am Soc Mass Spectrom 2006, 17:562-567.
• [33]Beausoleil SA, Villen J, Gerber SA, Rush J, Gygi SP: A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat Biotechnol 2006, 24:1285-1292.
• [34]Zhai B, Villen J, Beausoleil SA, Mintseris J, Gygi SP: Phosphoproteome analysis of Drosophila melanogaster embryos. J Proteome Res 2008, 7:1675-1682.