A role for Gab1/SHP2 in thrombin activation of PAK1: gene transfer of kinase-dead PAK1 inhibits injury-induced restenosis.
To understand the role of epidermal growth factor receptor (EGFR) transactivation in G protein-coupled receptor (GPCR) agonist-induced signaling events, we have studied the capacity of thrombin in the activation of Gab1-SHP2 in vascular smooth muscle cells (VSMCs). Thrombin activated both Gab1 and SHP2 in EGFR-dependent manner. Similarly, thrombin induced Rac1 and Cdc42 activation, and these responses were suppressed when either Gab1 or SHP2 stimulation is blocked. Thrombin also induced PAK1 activation in a time- and EGFR-Gab1-SHP2-Rac1/Cdc42-dependent manner. Inhibition of activation of EGFR, Gab1, SHP2, Rac1, Cdc42, or PAK1 by pharmacological or genetic approaches attenuated thrombin-induced VSMC stress fiber formation and motility. Thrombin activated RhoA in a time-dependent manner in VSMCs. LARG, a RhoA-specific GEF (guanine nucleotide exchange factor), was found to be associated with Gab1 and siRNA-mediated depletion of its levels suppressed RhoA, Rac1 and PAK1 activation. Dominant negative mutant-mediated interference of RhoA activation inhibited thrombin-induced Rac1 and PAK1 stimulation in VSMCs and their stress fiber formation and migration. Balloon injury induced PAK1 activity and interference with its activation led to attenuation of SMC migration from media to intima, resulting in reduced neointima formation and increased lumen size. Inhibition of thrombin signaling by recombinant hirudin also blocked balloon injury-induced EGFR tyrosine phosphorylation and PAK1 activity. These results show that thrombin-mediated PAK1 activation plays a crucial role in vascular wall remodeling and it could be a potential target for drug development against these vascular lesions.
Department of Physiology, University of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163, USA.
Dong
D
Wang
Biman C
BC
Paria
Qiuhua
Q
Zhang
Manjula
M
Karpurapu
Quanyi
Q
Li
William T
WT
Gerthoffer
Yoshikazu
Y
Nakaoka
Gadiparthi N
GN
Rao
D000800:Angioplasty, Balloon / Q000009:adverse effects; D000818:Animals; D002340:Carotid Artery Diseases / Q000201:enzymology / Q000209:etiology / Q000235:genetics / Q000628:therapy*; D016893:Carotid Stenosis / Q000201:enzymology / Q000209:etiology / Q000235:genetics / Q000517:prevention & control*; D002465:Cell Movement; D002478:Cells, Cultured; D004195:Disease Models, Animal; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000494:pharmacology; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000235:genetics / Q000378:metabolism*; D011799:Quinazolines; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D064067:Rho Guanine Nucleotide Exchange Factors; D022502:Stress Fibers / Q000201:enzymology; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D020032:Tyrphostins / Q000494:pharmacology; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D054462:p21-Activated Kinases / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
C577224:Arhgef12 protein, rat; D005343:Fibrinolytic Agents; C490896:Gab1 protein, rat; D020662:Guanine Nucleotide Exchange Factors; D006629:Hirudins; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D034741:RNA, Small Interfering; D064067:Rho Guanine Nucleotide Exchange Factors; D020032:Tyrphostins; C101044:RTKI cpd; C483803:Egfr protein, rat; D066246:ErbB Receptors; C515906:Pak1 protein, rat; D054462:p21-Activated Kinases; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; C516727:Ptpn11 protein, rat; D013917:Thrombin; C499134:Rac1 protein, rat; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein