Activation of protein kinase C alters p34(cdc2) phosphorylation state and kinase activity in early sea urchin embryos by abolishing intracellular Ca2+ transients

The p34(cdc2) protein kinase, a universal regulator of mitosis, is controll ed positively and negatively by phosphorylation, and by association with B- type mitotic cyclins. In addition, activation and inactivation of p34(cdc2) are induced by Ca2+ and prevented by Ca2+ chelators in permeabilized cells and cell-free systems. This suggests that intracellular Ca2+ transients ma y play an important physiological role in the control of p34(cdc2) kinase a ctivity. We have found that activators of protein kinase C can be used to b lock cell cycle-related alterations in intracellular Ca2+ concentration ([C a2+](i)) in early sea urchin embryos without altering the normal resting le vel of Ca2+. We have used this finding to investigate whether [Ca2+](i) tra nsients control p34(cdc2) kinase activity in living cells via a mechanism t hat involves cyclin B or the phosphorylation state of p34(cdc2). In the pre sent study we show that the elimination of [Ca2+](i), transients during int erphase blocks p34(cdc2) activation and entry into mitosis, while the elimi nation of mitotic [Ca2+](i) transients prevents p34(cdc2) inactivation and exit from mitosis. Moreover, we find that [Ca2+](i) transients are not requ ired for the synthesis of cyclin B, its binding to p34(cdc2) Or its destruc tion during anaphase. However, in the absence of interphase [Ca2+](i) trans ients p34(cdc2) does not undergo the tyrosine dephosphorylation that is req uired for activation, and in the absence of mitotic [Ca2+](i) transients p3 4(cdc2) does not undergo threonine dephosphorylation that is normally assoc iated with inactivation. These results provide evidence that intracellular [Ca2+](i) transients trigger the dephosphorylation of p34(cdc2) at key regu latory sites, thereby controlling the timing of mitosis entry and exit.