Distinct roles for the α, β and γ1 isoforms of protein phosphatase 1 in the outside-in α_IIbβ₃ integrin signalling-dependent functions

 

 Buy Article Permissions and Reprints

Summary

Although protein kinases and phosphatases participate in integrin α_IIbβ₃ signalling, whether integrin functions are regulated by the catalytic subunit of protein phosphatase 1 (PP1c) isoforms are unclear. We show that siRNA mediated knockdown of all PP1c isoforms (α, β and γ1) in 293 α_IIbβ₃ cells decreased adhesion to immobilised fibrinogen and fibrin clot retraction. Selective knockdown of only PP1cγ1 did not alter adhesion or clot retraction, while depletion of PP1cβ decreased both functions. Unexpectedly, knockdown of PP1cα enhanced α_IIbβ₃ adhesion to fibrinogen and clot retraction. Protein interaction studies revealed that all PP1c isoforms can interact with the integrin α_IIb subunit. Phospho-profiling studies revealed an enhanced activation of mitogen- activated protein kinase (MAPK) p38 in the PP1cα depleted cells. Enhanced adhesive phenotype displayed by the PP1cα-depleted 293 α_IIbβ₃ cells was blocked by pharmacological inhibition of p38. Conversely, the decreased adhesion of PP1cα overexpressing cells was rescued by the expression of constitutively active p38α or p38γ. Thus, PP1c isoforms have distinct contribution to the outside-in α_IIbβ3 signalling- dependent functions in 293 α_IIbβ₃ cells. Moreover, PP1cα negatively regulates integrin function by suppressing the p38 pathway.

^* These authors contributed equally to this manuscript and are designated as co-first authors.

• References

• 1 Shattil SJ, Newman PJ. Integrins: dynamic scaffolds for adhesion and signalling in platelets. Blood 2004; 104: 1606-1615. DOI:10.1182/blood-2004-04-1257.
  CrossrefPubMedGoogle Scholar
• 2 Coller BS, Shattil SJ. The GPIIb/IIIa (integrin a IIb b 3) odyssey: a technology-driven saga of a receptor with twists, turns, and even a bend. Blood 2008; 112: 3011-3025. DOI:10.1182/blood-2008-06-077891.
  CrossrefPubMedGoogle Scholar
• 3 Shattil SJ. The b 3 integrin cytoplasmic tail: protein scaffold and control freak. J.Thromb. Haemost 2009; 07: 210-213. DOI:10.1111/j.1538-7836.2009.03397.x.
  PubMedGoogle Scholar
• 4 Vijayan KV, Liu Y, Li TT. et al. Protein phosphatase 1 associates with the integrin a IIb subunit and regulates signalling. J Biol Chem 2004; 279: 33039-33042. DOI:10.1074/jbc.C400239200.
  CrossrefPubMedGoogle Scholar
• 5 Raab M, Daxecker H, Edwards RJ. et al. Protein interactions with the platelet integrin a IIb regulatory motif. Proteomics 2010; 10: 2790-2800. DOI:10.1002/pmic.200900621.
  CrossrefPubMedGoogle Scholar
• 6 Cohen PT. Protein phosphatase 1--targeted in many directions. J. Cell Sci 2002; 115: 241-256.
  PubMedGoogle Scholar
• 7 Ceulemans H, Bollen M. Functional diversity of protein phosphatase-1, a cellular economizer and reset button. Physiol Rev 2004; 84: 1-39. DOI:10.1152/physrev.00013.2003.
  CrossrefPubMedGoogle Scholar
• 8 Lerea KM, Cordero KP, Sakariassen KS. et al. Phosphorylation sites in the integrin b 3 cytoplasmic domain in intact platelets. J Biol Chem 1999; 274: 1914-1919. DOI:10.1074/jbc.274.4.1914.
  CrossrefPubMedGoogle Scholar
• 9 Nishikawa M, Toyoda H, Saito M. et al. Calyculin A and okadiac acid inhibit human platelet aggregation by blocking protein phosphatases types 1 and 2A. Cell Signal 1994; 06: 59-71. DOI:10.1016/0898-6568(94)90061-2.
  CrossrefPubMedGoogle Scholar
• 10 Mauco G, Artcanuthurry V, Pidard D. et al. Total inhibition of phospholipase C and phosphatidylinositol 3-kinase by okadaic acid in thrombin-stimulated platelets. Cell Signal 1997; 09: 117-124. DOI:10.1016/S0898-6568(96)00119-2.
  CrossrefPubMedGoogle Scholar
• 11 Hoyt CH, Lerea KM. Aggregation-dependent signalling in human platelets is sensitive to protein serine/threonine phosphatase inhibitors. Biochemistry 1995; 34: 9565-9570. DOI:10.1021/bi00029a033.
  CrossrefPubMedGoogle Scholar
• 12 Gushiken FC, Hyojeong H, Pradhan S. et al. The catalytic subunit of protein phosphatase 1 gamma regulates thrombin-induced murine platelet a IIb b 3 function. PLoS One 2009; 04: e8304. DOI:10.1371/journal.pone.0008304.
  CrossrefPubMedGoogle Scholar
• 13 Vijayan KV, Goldschmidt-Clermont PJ, Roos C. et al. The Pl A2 polymorphism of integrin b 3 enhances outside-in signalling and adhesive functions. J Clin Invest 2000; 105: 793-802. DOI:10.1172/JCI6982.
  CrossrefPubMedGoogle Scholar
• 14 Eto M, Kirkbride JA, Brautigan DL. Assembly of MYPT1 with protein phosphatase-1 in fibroblasts redirects localization and reorganizes the actin cytoskeleton. Cell Motil Cytoskeleton 2005; 62: 100-109. DOI:10.1002/cm.20088.
  CrossrefPubMedGoogle Scholar
• 15 Qi X, Pohl NM, Loesch M. et al. p38 a antagonizes p38 g activity through c-Jun-dependent ubiquitin-proteasome pathways in regulating Ras transformation and stress response. J Biol Chem 2007; 282: 31398-31408. DOI:10.1074/jbc.M703857200.
  CrossrefPubMedGoogle Scholar
• 16 Gushiken FC, Patel V, Liu Y. et al. Protein phosphatase 2A negatively regulates integrin a IIb b 3 signalling. J Biol Chem 2008; 283: 12862-12869. DOI:10.1074/jbc.M708804200.
  CrossrefPubMedGoogle Scholar
• 17 O’Toole TE, Loftus JC, Du XP. et al. Affinity modulation of the a IIb b 3 integrin (platelet GPIIb- IIIa) is an intrinsic property of the receptor. Cell Regul 1990; 01: 883-893.
  CrossrefPubMedGoogle Scholar
• 18 Han J, Lim CJ, Watanabe N. et al. Reconstructing and deconstructing agonist-induced activation of integrin a IIb b 3. Curr Biol 2006; 16: 1796-1806. DOI:10.1016/j.cub.2006.08.035.
  CrossrefPubMedGoogle Scholar
• 19 Pradhan S, Alrehani N, Patel V. et al. Cross-talk between serine/threonine protein phosphatase 2A and protein tyrosine phosphatase 1B regulates Src activation and adhesion of integrin a IIb b 3 to fibrinogen. J Biol Chem 2010; 285: 29059-29068. DOI:10.1074/jbc.M109.085167.
  CrossrefPubMedGoogle Scholar
• 20 Robinson MJ, Stippec SA, Goldsmith E. et al. A constitutively active and nuclear form of the MAP kinase ERK2 is sufficient for neurite outgrowth and cell transformation. Curr Biol 1998; 08: 1141-1150. DOI:10.1016/S0960-9822(07)00485-X.
  CrossrefPubMedGoogle Scholar
• 21 Zheng C, Xiang J, Hunter T. et al. The JNKK2-JNK1 fusion protein acts as a constitutively active c-Jun kinase that stimulates c-Jun transcription activity. J Biol Chem 1999; 274: 28966-28971. DOI:10.1074/jbc.274.41.28966.
  CrossrefPubMedGoogle Scholar
• 22 McCluskey A, Sim AT, Sakoff JA. Serine-threonine protein phosphatase inhibitors: development of potential therapeutic strategies. J Med Chem 2002; 45: 1151-1175. DOI:10.1021/jm010066k.
  CrossrefPubMedGoogle Scholar
• 23 Mitsuhashi S, Matsuura N, Ubukata M. et al. Tautomycetin is a novel and specific inhibitor of serine/threonine protein phosphatase type 1, PP1. Biochem Biophys Res Commun 2001; 287: 328-331. DOI:10.1006/bbrc.2001.5596.
  CrossrefPubMedGoogle Scholar
• 24 Swingle M, Ni L, Honkanen RE. Small-molecule inhibitors of ser/thr protein phosphatases: specificity, use and common forms of abuse. Methods Mol Biol 2007; 365: 23-38.
  PubMedGoogle Scholar
• 25 Okada T, Fujii T, Tanuma N. et al. Analysis of isoform specific function of PP1 catalytic subunits in mammalian cells using siRNA. Int J Oncol 2004; 25: 1383-1388.
  PubMedGoogle Scholar
• 26 Aoyama H, Ikeda Y, Miyazaki Y. et al. Isoform-specific roles of protein phosphatase 1 catalytic subunits in sarcoplasmic reticulum-mediated Ca2+ cycling. Cardiovasc Res 2011; 89: 79-88. DOI:10.1093/cvr/cvq252.
  CrossrefPubMedGoogle Scholar
• 27 Eto M, Kirkbride J, Elliott E. et al. Association of the tensin N-terminal protein-tyrosine phosphatase domain with the a isoform of protein phosphatase-1 in focal adhesions. J Biol Chem 2007; 282: 17806-17815. DOI:10.1074/jbc.M700944200.
  CrossrefPubMedGoogle Scholar
• 28 Bianchi M, De Lucchini S, Vietri M. et al. Reciprocally interacting domains of protein phosphatase 1 and focal adhesion kinase. Mol Cell Biochem 2005; 272: 85-90. DOI:10.1007/s11010-005-7639-z.
  CrossrefPubMedGoogle Scholar
• 29 Moorhead G, Johnson D, Morrice N. et al. The major myosin phosphatase in skeletal muscle is a complex between the b isoform of protein phosphatase 1 and the MYPT2 gene product. FEBS Lett 1998; 438: 141-144. DOI:10.1016/S0014-5793(98)01276-9.
  CrossrefPubMedGoogle Scholar
• 30 Allen PB, Ouimet CC, Greengard P. Spinophilin, a novel protein phosphatase 1 binding protein localized to dendritic spines. Proc Natl Acad Sci USA 1997; 94: 9956-9961. DOI:10.1073/pnas.94.18.9956.
  CrossrefPubMedGoogle Scholar
• 31 Harper MT, Poole AW. Diverse functions of protein kinase C isoforms in platelet activation and thrombus formation. J Thromb Haemost 2010; 08: 454-462. DOI:10.1111/j.1538-7836.2009.03722.x.
  CrossrefPubMedGoogle Scholar
• 32 Flevaris P, Li Z, Zhang G. et al. Two distinct roles of mitogen-activated protein kinases in platelets and a novel Rac1-MAPK-dependent integrin outside-in retractile signalling pathway. Blood 2009; 113: 893-901. DOI:10.1182/blood-2008-05-155978.
  CrossrefPubMedGoogle Scholar
• 33 Mazharian A, Roger S, Berrou E. et al. Protease-activating receptor-4 induces full platelet spreading on a fibrinogen matrix: involvement of ERK2 and p38 and Ca2+ mobilization. J Biol Chem 2007; 282: 5478-5487. DOI:10.1074/jbc.M609881200.
  CrossrefPubMedGoogle Scholar
• 34 Hall EH, Balsbaugh JL, Rose K. et al. Comprehensive Analysis of Phosphorylation Sites in Tensin1 Reveals Regulation by p38MAPK. Mol Cell Proteomics 2010; 12: 2853-2863.
  PubMedGoogle Scholar

• Supplementary Material