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 controlled positively and negatively by phosphorylation, and by association with B-type mitotic cyclins. In addition, activation and inactivation of p34(cdc2) are induced by Ca(2+) and prevented by Ca(2+) chelators in permeabilized cells and cell-free systems. This suggests that intracellular Ca(2+) transients may play an important physiological role in the control of p34(cdc2) kinase activity. We have found that activators of protein kinase C can be used to block cell cycle-related alterations in intracellular Ca(2+) concentration ([Ca(2+)](i)) in early sea urchin embryos without altering the normal resting level of Ca(2+). We have used this finding to investigate whether [Ca(2+)](i) transients control p34(cdc2) kinase activity in living cells via a mechanism that involves cyclin B or the phosphorylation state of p34(cdc2). In the present study we show that the elimination of [Ca(2+)](i) transients during interphase blocks p34(cdc2) activation and entry into mitosis, while the elimination of mitotic [Ca(2+)](i) transients prevents p34(cdc2) inactivation and exit from mitosis. Moreover, we find that [Ca(2+)](i) transients are not required for the synthesis of cyclin B, its binding to p34(cdc2) or its destruction during anaphase. However, in the absence of interphase [Ca(2+)](i) transients p34(cdc2) does not undergo the tyrosine dephosphorylation that is required for activation, and in the absence of mitotic [Ca(2+)](i) transients p34(cdc2) does not undergo threonine dephosphorylation that is normally associated with inactivation. These results provide evidence that intracellular [Ca(2+)](i) transients trigger the dephosphorylation of p34(cdc2) at key regulatory sites, thereby controlling the timing of mitosis entry and exit.
Suprynowicz
F A
FA
Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, Bethesda, MD 20892, USA.
Groigno
L
L
Whitaker
M
M
Miller
F J
FJ
Sluder
G
G
Sturrock
J
J
Whalley
T
T
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism* / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D002273:Carcinogens / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D019926:Cyclin B / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000201:enzymology; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D008938:Mitosis / Q000187:drug effects / Q000502:physiology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D012617:Sea Urchins / Q000196:embryology / Q000201:enzymology* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors
D002273:Carcinogens; D019926:Cyclin B; D020536:Enzyme Activators; D011493:Protein Kinase C; D016203:CDC2 Protein Kinase; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium