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

Abstract

The initiation of Ca2+ release from internal stores in the egg is a hallmark of egg activation. In sea urchins, PLCγ activity is necessary for the production of IP3, which leads to the initial rise in Ca2+. To examine the possible function of a tyrosine kinase in activating PLCγ at fertilization, sea urchin eggs were treated with the specific Src kinase inhibitor PP1 or microinjected with recombinant Src-family SH2-domain proteins, which act as dominant interfering inhibitors of Src-family kinase function. Both modes of inhibiting Src-family kinases resulted in a specific and dose-dependent delay in the onset of Ca2+ release from the endoplasmic reticulum at fertilization. The rise in cytoplasmic pH at fertilization also was inhibited by microinjection of Src-family SH2-domain proteins. Further, an antibody directed against Src-type kinases recognized a protein of ca. M(r) 57K that was enriched in the membrane fraction of eggs. The kinase activity of this protein was stimulated rapidly and transiently at fertilization, as measured by autophosphorylation and by phosphorylation of an exogenous substrate. Together, these data indicate that a Src-type tyrosine kinase is necessary 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 leading to this Ca2+ release. (C) 2000 Academic Press.