Coupling of mitosis to the completion of S phase in Xenopus occurs via modulation of the tyrosine kinase that phosphorylates p34cdc2.
In cell-free extracts derived from Xenopus eggs which oscillate between S phase and mitosis, incompletely replicated DNA blocks the activation of p34cdc2-cyclin by maintaining p34cdc2 in a tyrosine-phosphorylated form. We used a recombinant cyclin fusion protein to generate a substrate to measure the ability of the tyrosine kinase(s) to phosphorylate and inactivate p34cdc2 in the absence of tyrosine phosphatase activity. p34cdc2 tyrosine phosphorylation is highly regulated during the cell cycle, being elevated in S phase and attenuated in mitosis. The elevation in p34cdc2 tyrosine phosphorylation rate occurs in response to the presence of incompletely replicated DNA. Moreover, okadaic acid and caffeine, which uncouple the dependence of mitosis on the completion of S phase, increase unphosphorylated p34cdc2 by attenuating tyrosine kinase function. These data indicate that the control system, which monitors the state of DNA replication, modulates the function of the tyrosine kinase by a phosphorylation/dephosphorylation mechanism, ensuring that mitosis occurs only when S phase is complete.
Smythe
C
C
Department of Biology, University of California, San Diego, La Jolla 92093.
Newport
J W
JW
D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002110:Caffeine / Q000494:pharmacology; D002474:Cell-Free System; D016213:Cyclins / Q000378:metabolism; D004261:DNA Replication; D004988:Ethers, Cyclic / Q000494:pharmacology; D005246:Feedback; D005982:Glutathione Transferase / Q000378:metabolism; D008938:Mitosis; D008958:Models, Molecular; D019319:Okadaic Acid; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016196:S Phase; D014981:Xenopus
D016213:Cyclins; D004988:Ethers, Cyclic; D011993:Recombinant Fusion Proteins; D019319:Okadaic Acid; D016590:Aphidicolin; D002110:Caffeine; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D016203:CDC2 Protein Kinase