Tyrosine Phosphorylation Of P34cdc2 Research Articles

Cyclin promotes the tyrosine phosphorylation of p34cdc2 in a wee1+ dependent manner.

The regulation of p34cdc2 was investigated by overproducing p34cdc2, cyclin (A and B) and the wee1+ gene product (p107wee1) using a baculoviral expression system. p34cdc2 formed a functional complex with both cyclins as judged by co-precipitation, phosphorylation of cyclin in vitro, and activation of p34cdc2 histone H1 kinase activity. Co-production of p34cdc2 and p107wee1 in insect cells resulted in a minor population of p34cdc2 that was phosphorylated on tyrosine and displayed an altered electrophoretic mobility. When p34cdc2 and p107wee1 were co-produced with cyclin (A or B) in insect cells, there was a dramatic increase in the population of p34cdc2 that was phosphorylated on tyrosine and that displayed a shift in electrophoretic mobility. The phosphorylation of p34cdc2 on tyrosine was absolutely dependent upon the presence of kinase-active p107wee1. Tyrosine-specific as well as serine/threonine-specific protein kinase activities co-immunoprecipitated with p107wee1. These results suggest that cyclin functions to facilitate tyrosine phosphorylation of p34cdc2 and that p107wee1 functions to regulate p34cdc2, either directly or indirectly, by tyrosine phosphorylation.

Cell cycle tyrosine phosphorylation of p34cdc2 and a microtubule-associated protein kinase homolog in Xenopus oocytes and eggs.

We have examined the time course of protein tyrosine phosphorylation in the meiotic cell cycles of Xenopus laevis oocytes and the mitotic cell cycles of Xenopus eggs. We have identified two proteins that undergo marked changes in tyrosine phosphorylation during these processes: a 42-kDa protein related to mitogen-activated protein kinase or microtubule-associated protein-2 kinase (MAP kinase) and a 34-kDa protein identical or related to p34cdc2. p42 undergoes an abrupt increase in its tyrosine phosphorylation at the onset of meiosis 1 and remains tyrosine phosphorylated until 30 min after fertilization, at which point it is dephosphorylated. p42 also becomes tyrosine phosphorylated after microinjection of oocytes with partially purified M-phase-promoting factor, even in the presence of cycloheximide. These findings suggest that MAP kinase, previously implicated in the early responses of somatic cells to mitogens, is also activated at the onset of meiotic M phase and that MAP kinase can become tyrosine phosphorylated downstream from M-phase-promoting factor activation. We have also found that p34 goes through a cycle of tyrosine phosphorylation and dephosphorylation prior to meiosis 1 and mitosis 1 but is not detectable as a phosphotyrosyl protein during the 2nd through 12th mitotic cell cycles. It may be that the delay between assembly and activation of the cyclin-p34cdc2 complex that p34cdc2 tyrosine phosphorylation provides is not needed in cell cycles that lack G2 phases. Finally, an unidentified protein or group of proteins migrating at 100 to 116 kDa increase in tyrosine phosphorylation throughout maturation, are dephosphorylated or degraded within 10 min of fertilization, and appear to cycle between low-molecular-weight forms and high-molecular-weight forms during early embryogenesis.

Tyrosine phosphorylation of p34cdc2 and p42 during meiotic maturation of Xenopus oocyte: Antagonistic action of okadaic acid and 6-DMAP

The tyrosine phosphorylation/dephosphorylation of p34cdc2 was estimated by immunoblotting with antiphosphotyrosine antibody during meiotic maturation of Xenopus oocytes. At the time of germinal vesicle breakdown (GVBD), p34cdc2 is tyrosine dephosphorylated whereas a p42 protein, which might correspond to a MAP2 kinase, becomes tyrosine phosphorylated. No modification in the level of tyrosine phosphorylation of either proteins was noticed during the whole maturation process from GVBD until metaphase II. When added to prophase oocytes, 6-DMAP (6-dimethyl-aminopurine) blocks GVBC, M-phase-promoting factor (MPF) activation and H1-histone, kinase activation induced by either progesterone, MPF transfer or okadaic acid microinjection. In each case, the tyrosine dephosphorylation reaction of p34cdc2 is inhibited. In meiosis I oocytes (just after the initiation of GVBD), 6-DMAP provokes the rephosphorylation of p34cdc2 on tyrosine residue(s), inactivation of MPF and H1-histone kinase and re-entry of the cell into an interphase-like state. These processes are reversible by simply removing the agent. In contrast to the observations in prophase oocytes, okadaic acid is able to reverse the inhibitory effect of 6-DMAP in meiosis I oocytes on MPF and H1-histone kinase activities and to initiate dephosphorylation of p34cdc2 on tyrosyl residue(s) even in the presence of 6-DMAP. Altogether, our results show that 6-DMAP and okadaic acid antagonistically control in vivo the level of tyrosine phosphorylation of p34cdc2.