Activation of mitogen activated protein kinase in human platelets by genistein.
Genistein, a putative tyrosine kinase inhibitor, stimulated human platelet mitogen activated protein kinase (MAPK) activity in a dose- and time-dependent manner. When MAPK was maximally stimulated by phorbol 12-myristate 13-acetate (PMA), genistein still elicited the increase in MAPK activity. Staurosporine (50 nm), significantly decreased the PMA-induced MAPK activity, but had little inhibitory effect on the genistein-induced MAPK activity. Both these observations indicated a protein kinase C (PKC) independent pathway for the genistein-stimulated MAPK activity. When other tyrosine kinase inhibitors (methyl-2,5-dihydroxycinnamate, and tyrphostin) were employed, similar increases in the MAPK activity were observed. Addition of genistein to cytosolic fraction of platelets had no effect on the MAPK activity and indicated that this effect is not due to direct physical interaction between genistein and MAPK and that intact platelets are required for it. MAPK activity of platelets from rabbit and pig was also stimulated by genistein. This effect of genistein was not observed in other cell types tested (BNLCL2, HEL and U937 cells). Forskolin, which increases cyclic AMP had little effect on the basal platelet MAPK activity or the genistein activated MAPK, while it decreased by half the PMA-induced MAPK activity. The inactive analog of genistein, daidzein, which does not inhibit tyrosine kinase had little effect on MAPK. Genistein caused a decrease in basal tyrosine phosphorylation of pp60(c-src) protein as detected with anti-phosphotyrosine (anti-PTyr) Ab. Thus, inhibition of basal tyrosine kinase results in an increase in MAPK activity. This study demonstrates for the first time a novel mechanism for regulation of MAPK in platelets in which inhibition of tyrosine kinase results in activation of MAPK, independent of PKC and cAMP pathways.
Department of Pharmacology, School of Medicine, University of Missouri-Columbia, M517B Medical Sciences Building, Columbia, MO, 65212, USA.
S V
SV
Kansra
M A
MA
Reddy
Y I
YI
Weng
S D
SD
Shukla
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology; D015153:Blotting, Western; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D019950:Mitogen-Activated Protein Kinase 1 / Q000096:biosynthesis*; D011493:Protein Kinase C / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011817:Rabbits; D019311:Staurosporine / Q000494:pharmacology; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004791:Enzyme Inhibitors; D019833:Genistein; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate