PDK1 selectively phosphorylates Thr(308) on Akt and contributes to human platelet functional responses

Thrombosis and Haemostasis - Tập 112 Số 03 - Trang 508-517 - 2014
Carol Dangelmaier, Bhanu Kanth Manne, Elizabetta Liverani, Jianguo Jin, Paul F. Bray, Satya P. Kunapuli1
1Satya P. Kunapuli, PhD, Department of Physiology, Temple University, Rm. 217 MRB, 3420 N. Broad Street, Philadelphia, Pennsylvania 19140, USA, Tel.: +1 215 707 4615, Fax: +1 215 707 4003, E-mail: [email protected].

Tóm tắt

Summary3-phosphoinositide-dependent protein kinase 1 (PDK1), a member of the protein A,G and C (AGC) family of proteins, is a Ser/Thr protein kinase that can phosphorylate and activate other protein kinases from the AGC family, including Akt at Thr308, all of which play important roles in mediating cellular responses. The functional role of PDK1 or the importance of phosphorylation of Akt on Thr308 for its activity has not been investigated in human platelets. In this study, we tested two pharmacological inhibitors of PDK1, BX795 and BX912, to assess the role of Thr308 phosphorylation on Akt. PAR4-induced phosphorylation of Akt on Thr308 was inhibited by BX795 without affecting phosphorylation of Akt on Ser473. The lack of Thr308 phosphorylation on Akt also led to the inhibition of PAR4-induced phosphorylation of two downstream substrates of Akt, viz. GSK3β and PRAS40. In vitro kinase activity of Akt was completely abolished if Thr308 on Akt was not phosphorylated. BX795 caused inhibition of 2-MeSADP-induced or collagen-induced aggregation, ATP secretion and thromboxane generation. Primary aggregation induced by 2-MeSADP was also inhibited in the presence of BX795. PDK1 inhibition also resulted in reduced clot retraction indicating its role in outside-in signalling. These results demonstrate that PDK1 selectively phosphorylates Thr308 on Akt thereby regulating its activity and plays a positive regulatory role in platelet physiological responses.

Từ khóa


Tài liệu tham khảo

LF Brass, 2003, Thrombin and platelet activation, Chest, 124, 18S, 10.1378/chest.124.3_suppl.18S

S Kulkarni, 2000, A revised model of platelet aggregation, J Clin Invest, 105, 783, 10.1172/JCI7569

MH Kroll, 1996, Platelets and shear stress, Blood, 88, 1525, 10.1182/blood.V88.5.1525.1525

B Savage, 1996, Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor, Cell, 84, 289, 10.1016/S0092-8674(00)80983-6

B Nieswandt, 2003, Platelet-collagen interaction: is GPVI the central receptor?, Blood, 102, 449, 10.1182/blood-2002-12-3882

SP Watson, 1999, Collagen receptor signalling in platelets and megakaryocytes, Thromb Haemost, 82, 365, 10.1055/s-0037-1615855

F Rendu, 2001, The platelet release reaction: granules’ constituents, secretion and functions, Platelets, 12, 261, 10.1080/09537100120068170

PJ Sims, 1991, Effect of platelet activation on the conformation of the plasma membrane glycoprotein IIb-IIIa complex, J Biol Chem, 266, 7345, 10.1016/S0021-9258(20)89452-6

S Kim, 2004, Akt activation in platelets depends on Gi signalling pathways, J Biol Chem, 279, 4186, 10.1074/jbc.M306162200

B Vanhaesebroeck, 2000, The PI3K-PDK1 connection: more than just a road to PKB, Biochem J, 346, 561, 10.1042/bj3460561

V Calleja, 2007, Intramolecular and intermolecular interactions of protein kinase B define its activation in vivo, PLoS Biol, 5, e95, 10.1371/journal.pbio.0050095

DR Alessi, 1997, Characterisation of a 3-phosphoinosi-tide-dependent protein kinase which phosphorylates and activates protein ki-nase Balpha, Curr Biol, 7, 261, 10.1016/S0960-9822(06)00122-9

C Kroner, 2000, Dual regulation of platelet protein kinase B, J Biol Chem, 275, 27790, 10.1074/jbc.M000540200

J Chen, 2004, Impaired platelet responses to thrombin and collagen in AKT-1-deficient mice, Blood, 104, 1703, 10.1182/blood-2003-10-3428

D Woulfe, 2004, Defects in secretion, aggregation, and thrombus formation in platelets from mice lacking Akt2, J Clin Invest, 113, 441, 10.1172/JCI200420267

KA O’Brien, 2011, An important role for Akt3 in platelet activation and thrombosis, Blood, 118, 4215, 10.1182/blood-2010-12-323204

KA O’Brien, 2012, ADP-stimulated activation of Akt during integrin outside-in signalling promotes platelet spreading by inhibiting gly-cogen synthase kinase-3beta, Arterioscler Thromb Vasc Biol, 32, 2232, 10.1161/ATVBAHA.112.254680

MA Lawlor, 2002, Essential role of PDK1 in regulating cell size and development in mice, Embo J, 21, 3728, 10.1093/emboj/cdf387

N Chalhoub, 2009, Cell type specificity of PI3K signalling in Pdk1- and Pten-deficient brains, Genes Dev, 23, 1619, 10.1101/gad.1799609

A Najafov, 2011, Characterisation of GSK2334470, a novel and highly specific inhibitor of PDK1, Biochem J, 433, 357, 10.1042/BJ20101732

X Chen, 2013, PDK1 regulates platelet activation and arterial thrombosis, Blood, 121, 3718, 10.1182/blood-2012-10-461897

RE Hubbard, 2008, Fragment approaches in structure-based drug discovery, J Synchrotron Radiation, 15, 227, 10.1107/S090904950705666X

VP Krymskaya, 2007, Targeting the phosphatidylinositol 3-kinase pathway in airway smooth muscle: rationale and promise, BioDrugs, 21, 85, 10.2165/00063030-200721020-00003

T Tamguney, 2008, Analysis of 3-phosphoinositide-depend-ent kinase-1 signalling and function in ES cells, Exp Cell Res, 314, 2299, 10.1016/j.yexcr.2008.04.006

RI Feldman, 2005, Novel small molecule inhibitors of 3-phosphoinositide-dependent kinase-1, J Biol Chem, 280, 19867, 10.1074/jbc.M501367200

S Kim, 2009, Role of phosphoinositide 3-kinase beta in glycoprotein VI-mediated Akt activation in platelets, J Biol Chem, 284, 33763, 10.1074/jbc.M109.048553

BK Manne, 2013, Fucoidan Is a Novel Platelet Agonist for the C-type Lectin-like Receptor 2 (CLEC-2), J Biol Chem, 288, 7717, 10.1074/jbc.M112.424473

A Garcia, 2010, Role of phosphoinositide 3-kinase beta in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways, Biochem J, 429, 369, 10.1042/BJ20100166

L Buitrago, 2011, Tyrosine phosphorylated c-Cbl regulates platelet functional responses mediated by outside-in signalling, Blood, 118, 5631, 10.1182/blood-2011-01-328807

A Toker, 2000, Cellular signalling: pivoting around PDK-1, Cell, 103, 185

SF Moore, 2011, mTORC2 protein complex-mediated Akt (Protein Kinase B) Serine 473 Phosphorylation is not required for Akt1 activity in human platelets [corrected], J Biol Chem, 286, 24553, 10.1074/jbc.M110.202341

SM Schoenwaelder, 2010, Phosphoinositide 3-kinase p110 beta regulates integrin alpha IIb beta 3 avidity and the cellular transmission of contractile forces, J Biol Chem, 285, 2886, 10.1074/jbc.M109.029132

C Raimondi, 2011, Targeting PDK1 in cancer, Curr Med Chem, 18, 2763, 10.2174/092986711796011238

D Stokoe, 1997, Dual role of phosphatidylinosi-tol-3,4,5-trisphosphate in the activation of protein kinase B, Science, 277, 567, 10.1126/science.277.5325.567

A Mora, 2004, PDK1, the master regulator of AGC kinase signal transduction, Semin Cell Dev Biol, 15, 161, 10.1016/j.semcdb.2003.12.022

C Belham, 1999, Intracellular signalling: PDK1-- a kinase at the hub of things, Curr Biol, 9, R93, 10.1016/S0960-9822(99)80058-X

DA Cross, 1995, Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B, Nature, 378, 785, 10.1038/378785a0

EJ McManus, 2005, Role that phosphorylation of GSK3 plays in insulin and Wnt signalling defined by knockin analysis, Embo J, 24, 1571, 10.1038/sj.emboj.7600633

Q Wang, 2006, Glycogen synthase kinase-3 is a negative regulator of extracellular signal-regulated kinase, Oncogene, 25, 43, 10.1038/sj.onc.1209004

L Rossig, 2002, Glycogen synthase kinase-3 couples AKT-dependent signalling to the regulation of p21Cip1 degradation, J Biol Chem, 277, 9684, 10.1074/jbc.M106157200

M Pap, 2002, Role of translation initiation factor 2B in control of cell survival by the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3beta signalling pathway, Mol Cell Biol, 22, 578, 10.1128/MCB.22.2.578-586.2002

D Li, 2008, GSK3beta is a negative regulator of platelet function and thrombosis, Blood, 111, 3522, 10.1182/blood-2007-09-111518

SF Moore, 2013, Dual regulation of glycogen synthase kinase 3 (GSK3)alpha/beta by protein kinase C (PKC)alpha and Akt promotes thrombin-mediated integrin alphaIIbbeta3 activation and granule secretion in platelets, J Biol Chem, 288, 3918, 10.1074/jbc.M112.429936

RM Biondi, 2004, Phosphoinositide-dependent protein kinase 1, a sensor of protein conformation, Trends Biochem Sci, 29, 136, 10.1016/j.tibs.2004.01.005

A Balendran, 2000, Further evidence that 3-phosphoinosi-tide-dependent protein kinase-1 (PDK1) is required for the stability and phos-phorylation of protein kinase C (PKC) isoforms, FEBS Lett, 484, 217, 10.1016/S0014-5793(00)02162-1

D Komander, 2004, Interactions of LY333531 and other bisindolyl maleimide inhibitors with PDK1, Structure, 12, 215, 10.1016/j.str.2004.01.005

K Clark, 2009, Use of the pharmacological inhibitor BX795 to study the regulation and physiological roles of TBK1 and IkappaB kinase epsilon: a distinct upstream kinase mediates Ser-172 phosphorylation and activation, J Biol Chem, 284, 14136, 10.1074/jbc.M109.000414

KA Fitzgerald, 2003, IKKepsilon and TBK1 are essential components of the IRF3 signalling pathway, Nat Immunol, 4, 491, 10.1038/ni921

H Hacker, 2006, Regulation and function of IKK and IKK-related kinases, Sci STKE, 2006, re13, 10.1126/stke.3572006re13

T Kawai, 2007, Signalling to NF-kappaB by Toll-like receptors, Trends Mol Med., 13, 460, 10.1016/j.molmed.2007.09.002

YH Ou, 2011, TBK1 directly engages Akt/PKB survival signalling to support oncogenic transformation, Mol Cell, 41, 458, 10.1016/j.molcel.2011.01.019

X Xie, 2011, IkappaB kinase epsilon and TANK-binding ki-nase 1 activate AKT by direct phosphorylation, Proc Natl Acad Sci USA, 108, 6474, 10.1073/pnas.1016132108

A Iwanami, 2009, Striking the balance between PTEN and PDK1: it all depends on the cell context, Genes Dev, 23, 1699, 10.1101/gad.1832909

J Polgar, 1997, Platelet activation and signal transduc-tion by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor, J Biol Chem, 272, 13576, 10.1074/jbc.272.21.13576

Thông tin
Thông tin xuất bản

Thrombosis and Haemostasis

Tập 112 Số 03

508-517

Thông tin tác giả