Association of cyclin-bound p34cdc2 with subcellular structures in xenopus eggs.
Cell cycle progression is controlled by changes in kinase activity of homologs of the fission yeast protein p34cdc2. The p34cdc2 kinase is activated by its association with a cyclin subunit, followed by post-translational modifications. Here, we show that in Xenopus eggs stimulated to enter the early embryonic cell cycle by an electric shock, part of the p34cdc2 becomes associated with subcellular fractions as the eggs progress towards mitosis. This occurs as a result of cyclin accumulation because most of the B-type cyclins and some of the A-type cyclins are found in the particulate fraction. Moreover, as soon as cyclins are degraded, p34cdc2 is released in the soluble fraction. The p34cdc2-cyclin complex can be solubilised by 80 mM beta-glycerophosphate (in the standard MPF extraction buffer) or by high salt concentrations. The post-translational modifications leading to cdc2 kinase activation by cyclin occur in the insoluble form. Following fractionation of egg extracts by sucrose gradient centrifugation, the p34cdc2-cyclin B complex is found in several fractions, but especially in two discrete peaks. We present evidence that in the slow-sedimenting peak the p34cdc2-cyclin B complex is associated with the 60 S subunit of monoribosomes. It could be targeted in this fashion to substrates such as ribosomal proteins and maybe to cytoskeletal proteins, since ribosomes bind to microtubules and are present in the spindle. The p34cdc2-cyclin B complex is also found in a faster-migrating fraction containing various membranous structures, including Golgi stacks. Therefore, as observed by immunofluorescence in other systems, it seems that cyclin subunits target p34cdc2 to specific cellular sites and this is certainly important for its function. In addition, we present preliminary evidence suggesting that some component present in the ribosome-containing fraction is required for activation of the p34cdc2-cyclin B complex.
Leiss
D
D
Cell Biology Program, EMBL, Heidelberg, FRG.
Félix
M A
MA
Karsenti
E
E
D000595:Amino Acid Sequence; D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D016213:Cyclins / Q000378:metabolism*; D004492:Edetic Acid; D004789:Enzyme Activation; D007700:Kinetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism* / Q000648:ultrastructure; D015388:Organelles / Q000378:metabolism; D006656:Protamine Kinase / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012995:Solubility; D014981:Xenopus
D016213:Cyclins; D004492:Edetic Acid; D006656:Protamine Kinase; D016203:CDC2 Protein Kinase