Biochemical Journal

Research article

Activation of protein kinase C alters p34cdc2 phosphorylation state and kinase activity in early sea urchin embryos by abolishing intracellular Ca2+ transients

Frank A. SUPRYNOWICZ, Laurence GROIGNO, Michael WHITAKER, Frederick J. MILLER, Greenfield SLUDER, John STURROCK, Tim WHALLEY

Abstract

The p34cdc2 protein kinase, a universal regulator of mitosis, is controlled positively and negatively by phosphorylation, and by association with B-type mitotic cyclins. In addition, activation and inactivation of p34cdc2 are induced by Ca2+ and prevented by Ca2+ chelators in permeabilized cells and cell-free systems. This suggests that intracellular Ca2+ transients may play an important physiological role in the control of p34cdc2 kinase activity. We have found that activators of protein kinase C can be used to block cell cycle-related alterations in intracellular Ca2+ concentration ([Ca2+]i) in early sea urchin embryos without altering the normal resting level of Ca2+. We have used this finding to investigate whether [Ca2+]i transients control p34cdc2 kinase activity in living cells via a mechanism that involves cyclin B or the phosphorylation state of p34cdc2. In the present study we show that the elimination of [Ca2+]i transients during interphase blocks p34cdc2 activation and entry into mitosis, while the elimination of mitotic [Ca2+]i transients prevents p34cdc2 inactivation and exit from mitosis. Moreover, we find that [Ca2+]i transients are not required for the synthesis of cyclin B, its binding to p34cdc2 or its destruction during anaphase. However, in the absence of interphase [Ca2+]i transients p34cdc2 does not undergo the tyrosine dephosphorylation that is required for activation, and in the absence of mitotic [Ca2+]i transients p34cdc2 does not undergo threonine dephosphorylation that is normally associated with inactivation. These results provide evidence that intracellular [Ca2+]i transients trigger the dephosphorylation of p34cdc2 at key regulatory sites, thereby controlling the timing of mitosis entry and exit.

  • cyclin B
  • embryonic cell cycle
  • Ins(1,4,5)P3
  • mitosis
  • phorbol esters