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

Carol Dangelmaier; Bhanu Kanth Manne; Elizabetta Liverani; Jianguo Jin; Paul Bray; Satya P. Kunapuli

doi:10.1160/TH13-06-0484

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2014; 111(03): 508-517
DOI: 10.1160/TH13-06-0484

Platelets and Blood Cells

Schattauer GmbH

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

Carol Dangelmaier

¹Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Temple University Hospital, Philadelphia, Pennsylvania, USA

,

Bhanu Kanth Manne

¹Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Temple University Hospital, Philadelphia, Pennsylvania, USA

,

Elizabetta Liverani

¹Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Temple University Hospital, Philadelphia, Pennsylvania, USA

,

Jianguo Jin

¹Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Temple University Hospital, Philadelphia, Pennsylvania, USA

,

Paul Bray

²Cardeza Foundation for Hematologic Research, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA

,

Satya P. Kunapuli

¹Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Temple University Hospital, Philadelphia, Pennsylvania, USA

› Author Affiliations

Abstract

Summary

3-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.

Keywords

Kinases - platelet pharmacology - signal transduction

Full Text

References

References
1 Brass LF. Thrombin and platelet activation. Chest 2003; 124: 18S-25S.
2 Kulkarni S, Dopheide SM, Yap CL. et al. A revised model of platelet aggregation. J Clin Invest 2000; 105: 783-791.
3 Kroll MH, Hellums JD, McIntire LV. et al. Platelets and shear stress. Blood 1996; 88: 1525-1541.
4 Savage B, Saldivar E, Ruggeri ZM. Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor. Cell 1996; 84: 289-97.
5 Nieswandt B, Watson SP. Platelet-collagen interaction: is GPVI the central receptor?. Blood 2003; 102: 449-461.
6 Watson SP. Collagen receptor signalling in platelets and megakaryocytes. Thromb Haemost 1999; 82: 365-376.
7 Rendu F, Brohard-Bohn B. The platelet release reaction: granules’ constituents, secretion and functions. Platelets 2001; 12: 261-273.
8 Sims PJ, Ginsberg MH, Plow EF. et al. Effect of platelet activation on the conformation of the plasma membrane glycoprotein IIb-IIIa complex. J Biol Chem 1991; 266: 7345-7352.
9 Kim S, Jin J, Kunapuli SP. Akt activation in platelets depends on Gi signalling pathways. J Biol Chem 2004; 279: 4186-4195.
10 Vanhaesebroeck B, Alessi DR. The PI3K-PDK1 connection: more than just a road to PKB. Biochem J 2000; 346: 561-576.
11 Calleja V, Alcor D, Laguerre M. et al. Intramolecular and intermolecular interactions of protein kinase B define its activation in vivo. PLoS Biol 2007; 5: e95
12 Alessi DR, James SR, Downes CP. et al. Characterisation of a 3-phosphoinosi-tide-dependent protein kinase which phosphorylates and activates protein ki-nase Balpha. Curr Biol 1997; 7: 261-269.
13 Kroner C, Eybrechts K, Akkerman JW. Dual regulation of platelet protein kinase B. J Biol Chem 2000; 275: 27790-27798.
14 Chen J, De S, Damron DS. et al. Impaired platelet responses to thrombin and collagen in AKT-1-deficient mice. Blood 2004; 104: 1703-1710.
15 Woulfe D, Jiang H, Morgans A. et al. Defects in secretion, aggregation, and thrombus formation in platelets from mice lacking Akt2. J Clin Invest 2004; 113: 441-450.
16 O’Brien KA, Stojanovic-Terpo A, Hay N. et al. An important role for Akt3 in platelet activation and thrombosis. Blood 2011; 118: 4215-4223.
17 O’Brien KA, Gartner TK, Hay N. et al. ADP-stimulated activation of Akt during integrin outside-in signalling promotes platelet spreading by inhibiting gly-cogen synthase kinase-3beta. Arterioscler Thromb Vasc Biol 2012; 32: 2232-2240.
18 Lawlor MA, Mora A, Ashby PR. et al. Essential role of PDK1 in regulating cell size and development in mice. Embo J 2002; 21: 3728-3738.
19 Chalhoub N, Zhu G, Zhu X. et al. Cell type specificity of PI3K signalling in Pdk1- and Pten-deficient brains. Genes Dev 2009; 23: 1619-1624.
20 Najafov A, Sommer EM, Axten JM. et al. Characterisation of GSK2334470, a novel and highly specific inhibitor of PDK1. Biochem J 2011; 433: 357-369.
21 Chen X, Zhang Y, Wang Y. et al. PDK1 regulates platelet activation and arterial thrombosis. Blood 2013; 121: 3718-3726.
22 Hubbard RE. Fragment approaches in structure-based drug discovery. J Synchrotron Radiation 2008; 15: 227-230.
23 Krymskaya VP. Targeting the phosphatidylinositol 3-kinase pathway in airway smooth muscle: rationale and promise. BioDrugs 2007; 21: 85-95.
24 Tamguney T, Zhang C, Fiedler D. et al. Analysis of 3-phosphoinositide-depend-ent kinase-1 signalling and function in ES cells. Exp Cell Res 2008; 314: 2299-2312.
25 Feldman RI, Wu JM, Polokoff MA. et al. Novel small molecule inhibitors of 3-phosphoinositide-dependent kinase-1. J Biol Chem 2005; 280: 19867-19874.
26 Kim S, Mangin P, Dangelmaier C. et al. Role of phosphoinositide 3-kinase beta in glycoprotein VI-mediated Akt activation in platelets. J Biol Chem 2009; 284: 33763-33772.
27 Manne BK, Getz TM, Hughes CE. et al. Fucoidan Is a Novel Platelet Agonist for the C-type Lectin-like Receptor 2 (CLEC-2). J Biol Chem 2013; 288: 7717-7726.
28 Garcia A, Kim S, Bhavaraju K. et al. Role of phosphoinositide 3-kinase beta in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways. Biochem J 2010; 429: 369-377.
29 Buitrago L, Langdon WY, Sanjay A, Kunapuli SP. Tyrosine phosphorylated c-Cbl regulates platelet functional responses mediated by outside-in signalling. Blood 2011; 118: 5631-5640.
30 Toker A, Newton AC. Cellular signalling: pivoting around PDK-1. Cell 2000; 103: 185-188.
31 Moore SF, Hunter RW, Hers I. mTORC2 protein complex-mediated Akt (Protein Kinase B) Serine 473 Phosphorylation is not required for Akt1 activity in human platelets [corrected]. J Biol Chem 2011; 286: 24553-24560.
32 Schoenwaelder SM, Ono A, Nesbitt WS. et al. Phosphoinositide 3-kinase p110 beta regulates integrin alpha IIb beta 3 avidity and the cellular transmission of contractile forces. J Biol Chem 2010; 285: 2886-2896.
33 Raimondi C, Falasca M. Targeting PDK1 in cancer. Curr Med Chem 2011; 18: 2763-2769.
34 Stokoe D, Stephens LR, Copeland T. et al. Dual role of phosphatidylinosi-tol-3,4,5-trisphosphate in the activation of protein kinase B. Science 1997; 277: 567-570.
35 Mora A, Komander D, van Aalten DM. et al. PDK1, the master regulator of AGC kinase signal transduction. Semin Cell Dev Biol 2004; 15: 161-170.
36 Belham C, Wu S, Avruch J. Intracellular signalling: PDK1-- a kinase at the hub of things. Curr Biol 1999; 9: R93-96.
37 Cross DA, Alessi DR, Cohen P. et al. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 1995; 378: 785-789.
38 McManus EJ, Sakamoto K, Armit LJ. et al. Role that phosphorylation of GSK3 plays in insulin and Wnt signalling defined by knockin analysis. Embo J 2005; 24: 1571-1583.
39 Wang Q, Zhou Y, Wang X. et al. Glycogen synthase kinase-3 is a negative regulator of extracellular signal-regulated kinase. Oncogene 2006; 25: 43-50.
40 Rossig L, Badorff C, Holzmann Y. et al. Glycogen synthase kinase-3 couples AKT-dependent signalling to the regulation of p21Cip1 degradation. J Biol Chem 2002; 277: 9684-9689.
41 Pap M, Cooper GM. 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 2002; 22: 578-586.
42 Li D, August S, Woulfe DS. GSK3beta is a negative regulator of platelet function and thrombosis. Blood 2008; 111: 3522-3530.
43 Moore SF, van den Bosch MT, Hunter RW. et al. 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 2013; 288: 3918-3928.
44 Biondi RM. Phosphoinositide-dependent protein kinase 1, a sensor of protein conformation. Trends Biochem Sci 2004; 29: 136-142.
45 Balendran A, Hare GR, Kieloch A. et al. 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 2000; 484: 217-223.
46 Komander D, Kular GS, Schuttelkopf AW. et al. Interactions of LY333531 and other bisindolyl maleimide inhibitors with PDK1. Structure 2004; 12: 215-226.
47 Clark K, Plater L, Peggie M. et al. 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 2009; 284: 14136-14146.
48 Fitzgerald KA, McWhirter SM, Faia KL. et al. IKKepsilon and TBK1 are essential components of the IRF3 signalling pathway. Nat Immunol 2003; 4: 491-496.
49 Hacker H, Karin M. Regulation and function of IKK and IKK-related kinases. Sci STKE 2006; 2006: re13
50 Kawai T, Akira S. Signalling to NF-kappaB by Toll-like receptors. Trends Mol Med. 2007; 13: 460-469.
51 Ou YH, Torres M, Ram R. et al. TBK1 directly engages Akt/PKB survival signalling to support oncogenic transformation. Mol Cell 2011; 41: 458-470.
52 Xie X, Zhang D, Zhao B. et al. IkappaB kinase epsilon and TANK-binding ki-nase 1 activate AKT by direct phosphorylation. Proc Natl Acad Sci USA 2011; 108: 6474-6479.
53 Iwanami A, Cloughesy TF, Mischel PS. Striking the balance between PTEN and PDK1: it all depends on the cell context. Genes Dev 2009; 23: 1699-1704.
54 Polgar J, Clemetson JM, Kehrel BE. et al. 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 1997; 272: 13576-13583.