Evidence that Src-type tyrosine kinase activity is necessary for initiation of calcium release at fertilization in sea urchin eggs

The initiation of Ca2+ release from internal stores in the egg is a hallmar k of egg activation. In sea urchins, PLC gamma activity is necessary for th e production of IP3, which leads to the initial rise in Ca2+. To examine th e possible function of a tyrosine kinase in activating PLC gamma at fertili zation, sea urchin eggs were treated with the specific Src kinase inhibitor PP1 or microinjected with recombinant Src-family SR2-domain proteins, whic h act as dominant interfering inhibitors of Src-family kinase function. Bot h modes of inhibiting Src-family kinases resulted in a specific and dose-de pendent delay in the onset of Ca2+ release from the endoplasmic reticulum a t fertilization. The rise in cytoplasmic pH at fertilization also was inhib ited by microinjection of Src-family SH2-domain proteins. Further, an antib ody directed against Src-type kinases recognized a protein of ca. M-r 57K t hat was enriched in the membrane fraction of eggs. The kinase activity of t his protein was stimulated rapidly and transiently at fertilization, as mea sured by autophosphorylation and by phosphorylation of an exogenous substra te. Together, these data indicate that a Src-type tyrosine kinase is necess ary for the initiation of Ca2+ release from the egg]ER at fertilization and identify a Src-type p57 protein as a candidate in the signaling pathway le ading to this Ca2+ release. (C) 2000 Academic Press.