Thrombin Rapidly Induces Protein Kinase D Phosphorylation, and Protein Kinase C δ Mediates the Activation

Mingqi Tan,Xuemin Xu,Motoi Ohba,Wataru Ogawa,Mei-Zhen Cui

doi:10.1074/jbc.m211523200

Abstract

Thrombin plays a critical role in hemostasis, thrombosis, and inflammation. However, the responsible intracellular signaling pathways triggered by thrombin are still not well defined. We report here that thrombin rapidly and transiently induces activation of protein kinase D (PKD) in aortic smooth muscle cells. Our data demonstrate that protein kinase C (PKC) inhibitors completely block thrombin-induced PKD activation, suggesting that thrombin induces PKD activation via a PKC-dependent pathway. Furthermore, our results show that thrombin rapidly induces PKC delta phosphorylation and that the PKC delta-specific inhibitor rottlerin blocks thrombin-induced PKD activation, suggesting that PKC delta mediates the thrombin-induced PKD activation. Using dominant negative approaches, we demonstrated that expression of a dominant negative PKC delta inhibits the phosphorylation and activation of PKD induced by thrombin, whereas neither PKC epsilon nor PKC zeta affects thrombin-induced PKD activation. In addition, our results of co-immunoprecipitation assays showed that PKD forms a complex with PKC delta in smooth muscle cells. Taken together, the findings of the present study demonstrate that thrombin induces activation of PKD and reveal a novel role of PKC delta in mediating thrombin-induced PKD activation in vascular smooth muscle cells.

Highlights

Thrombin belongs to the multifunctional serine protease family and plays an important role in the blood coagulation cascade through the cleavage of fibrinogen to fibrin (1, 2)
Serum-starved rat aortic smooth muscle cell (SMC) were exposed to 0.1 unit/ml thrombin for various periods of time, the cells were lysed, and Protein kinase D (PKD) was immunoprecipitated with a PKD-specific antibody
To determine whether protein kinase C (PKC) activation is involved in thrombin-induced PKD activation in SMC, we examined the effect of two PKC inhibitors, GF 109203X and Ro 31-8220, on PKD activation stimulated by thrombin

Summary

Introduction

Thrombin belongs to the multifunctional serine protease family and plays an important role in the blood coagulation cascade through the cleavage of fibrinogen to fibrin (1, 2). Thrombin exerts direct effects on cells to regulate platelet aggregation, endothelial cell activation, and smooth muscle cell (SMC) proliferation via interactions with members of the protease-activated receptor (PAR) family, such as PAR1, PAR2, PAR3, and PAR4, known as G-protein-coupled receptors (2, 3). PKD can be activated by a variety of stimuli including biologically active phorbol esters, growth factors, and T- and B-cell receptor agonists via PKC-dependent pathways (6, 7). PKD has been implicated in the regulation of a variety of cellular functions including NF␬B-mediated gene expression, Naϩ/Hϩ antiport activity, Golgi organization and function, and protein transport (7, 8). Our results demonstrate that thrombin rapidly and markedly induces PKD activation in SMC. Our finding that thrombin induces PKC␦-dependent PKD activation reveals a novel thrombin-induced signaling pathway in living cells

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