|Budget Amount *help
¥7,000,000 (Direct Cost : ¥7,000,000)
Fiscal Year 1992 : ¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1991 : ¥4,600,000 (Direct Cost : ¥4,600,000)
We have reported a porcine gene syk encoding a non-receptor type 72-kDa PTK (p72^<syk>) which has a distinct structural character, that is the presence of the second src homology region 2 (SH2) instead of SH3, from other members of this group of PTK. The expression of p72^<syk> has been detected in porcine splenocytes, platelets, peripheral blood lymphocytes, polymorphonuclear leukocytes and tonsilocytes on immunoblot. In the cases of splenocytes and platelets, the activation of p72^<syk> was seen by the stimulation of wheat germ agglutinin. When washed porcine platelets was stimulated with thrombin and the activation of p72^<syk> was assessed in immunoprecipitation kinase assay, employing anti-CPTK40 antibodies, the activity of p72^<syk> increased within 5s, reached maximum at 10s and decreased to basal level within 60 s after 0.5 U/ml thrombin stimulation. This activation was enhanced dose dependently and was completely cancelled by the pretreatment of platelet suspension with hirudin, a specific antagonist of thrombin. In the Ca^<2+>-depleted condition both extra- and intracellularly, the activation of p72^<syk> was not affected at all but the deactivation was completely abrogated even at 120s after thrombin stimulation. In addition, the replenishment of Ca^<2+> resulted in a similar deactivation pattern as seen in Ca^<2+>-rich condition. Furthermore, this deactivation was also cancelled by the pretreatment of platelets with W7, a calmodulin antagonist, as well as ML9, a myosin-light-chain kinase inhibitor. These results have suggested that p72^<syk> is rapidly activated following thrombin stimulation with a peak at 10s and is negatively regulated mainly through a calmodulin-dependent protein kinase, myosin- light-chain kinase, in thrombin-stimulated platelets.