In vitro binding of free cdc2 and raf kinase to membrane vesicles: a possible new regulatory mechanism for cdc2 kinase activation in Xenopus oocyte.
The G2-M transition of the cell cycle is under the control of the M-phase promoting factor (MPF) formed of cdc2 kinase and cyclin B. The Xenopus prophase-blocked oocyte contains a stockpile of cyclin B2-cdc2 complexes that are maintained inactive by a double inhibitory phosphorylation on Thr-14 and Tyr-15 of cdc2. Free cdc2 molecules that are not associated with cyclin, are present in excess as compared to cyclin B2-associated cdc2. This pool of free cdc2 is permanently recruited to associate with neosynthetized cyclin B2 in the resting prophase oocyte, to feed up the pre-MPF stockpile. During re-entry into meiosis, free cdc2 could generate with newly synthesized cyclin B a small level of active MPF, that could serve as starter to initiate the conversion of pre-MPF into MPF. It was, therefore, of high interest to investigate whether free cdc2 interacts with other proteins and what could be its intracellular localization. To address these questions, we developed an in vitro system of membrane vesicles. We demonstrate here that free cdc2 is recovered in association with the external layer of membrane vesicles, whereas cyclin B2-associated cdc2 is not. Cyclin is able to associate in vitro with cdc2-containing membrane vesicles. This association does not induce the inhibitory cdc2 phosphorylations. However, it does not lead to active complexes, suggesting that membrane vesicles prevent cdc2 activation. C-Raf1, another kinase activated during reentry into meiosis, is also totally recovered in association with the membrane vesicles.
De Smedt
V
V
Laboratoire de Physiologie de la Reproduction, ESA-CNRS 7080, Université Pierre et Marie Curie, Paris, France.
Crozet
N
N
Jessus
C
C
D000818:Animals; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D019926:Cyclin B / Q000378:metabolism; D004789:Enzyme Activation; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D008677:Metaphase; D009865:Oocytes / Q000378:metabolism*; D011418:Prophase; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D013696:Temperature; D014982:Xenopus laevis; D018631:ras Proteins / Q000378:metabolism
D019926:Cyclin B; D018841:HSP90 Heat-Shock Proteins; D019908:Proto-Oncogene Proteins c-raf; D016203:CDC2 Protein Kinase; D018631:ras Proteins