M-phase-specific protein kinase from mitotic sea urchin eggs: Cyclic activation depends on protein synthesis and phosphorylation but does not require DNA or RNA synthesis

Abstract

Histone H 1 kinase (H 1K) undergoes a transient activation at each early M phase of both meiotic and mitotic cell cycles. The mechanisms underlying the transient activation of this protein kinase were investigated in mitotic sea urchin eggs. Translocation of active H 1K from particulate to soluble fraction does not seem to be responsible for this activation. H 1K activation cannot be accounted for by the transient disappearance of a putative H 1K inhibitor present in soluble fractions of homogenates. Aphidicolin, an inhibitor of DNA synthesis, and actinomycin D, an inhibitor of RNA synthesis, do not impede the transient appearance of H 1K activity. H 1K activation therefore does not require DNA or RNA synthesis. Fertilization triggers a rise in intracellular pH responsible for the increase of protein synthesis. H 1K activation is highly dependent on the intracellular pH. Ammonia triggers an increase of intracellular pH and stimulates protein synthesis and H 1K activation. Acetate lowers the intracellular pH, decreases protein synthesis, and blocks H 1K activation. Protein synthesis is an absolute requirement for H 1K activation as demonstrated by their identical sensitivities to emetine concentration and to time of emetine addition. About 60 min after fertilization, H 1K activation and cleavage become independent of protein synthesis. The concentration of p34, a homolog of the yeast cdc2 gene product which has been recently shown to be a subunit of H 1K, does not vary during the cell cycle and remains constant in emetine-treated cells. H 1K activation thus requires the synthesis of either a p34 postranslational modifying enzyme or another subunit. Finally, phosphatase inhibitors and ATP slow down in the in vitro inactivation rate of H 1K. These results suggest that a subunit or an activator of H 1K is stored as an mRNA in the egg before mitosis and that full activation of H 1K requires a phosphorylation.