Activation of p34cdc2 protein kinase by microinjection of human cdc25C into mammalian cells. Requirement for prior phosphorylation of cdc25C by p34cdc2 on sites phosphorylated at mitosis.

International audienceHuman cdc25C protein, a specific tyrosine phosphatase that activates the p34cdc2 protein kinase at mitosis, is itself a phosphoprotein that shows increased phosphorylation during the G2-M transition. In vitro, cdc25C protein is substantially phosphorylated by purified p34cdc2-cyclin B protein kinase. Of seven putative phosphorylation sites for p34cdc2 protein kinase present in human cdc25C, five are phosphorylated by p34cdc2 protein kinase in vitro, as assessed by tryptic phosphopeptide mapping and peptide sequencing. These same sites are also phosphorylated in vivo during the G2-M transition in normal mammalian fibroblasts and have been precisely mapped. The cdc25C phosphorylated in vitro by p34cdc2 protein kinase exhibits a 2-3-fold higher activity than the nonphosphorylated cdc25C, as assayed by activation of inactive cdc2 prokinase. Microinjection of purified cdc25C proteins into living fibroblasts reveals that only the phosphorylated form of cdc25 is highly effective in activating G2 cells into premature prophase in a manner similar to microinjection of purified active p34cdc2 protein kinase. Together these data show that multisite phosphorylation of cdc25C by p34cdc2-cyclin B protein kinase occurs at the G2-M transition and is sufficient to induce the autoamplification of cdc2/M-phase promoting factor necessary to drive somatic mammalian cells into mitosis

The C-terminal domain but not the tyrosine 723 of human DNA topoisomerase I active site contributes to kinase activity

International audienceHuman DNA topoisomerase I not only has DNA relaxing activity, but also splicing factors phosphorylating activity. Topo I shows strong preference for ATP as the phosphate donor. We used photoaffinity labeling with the ATP analogue [α-32 P] 8-azidoadenosine-5′-triphos-phate combined with limited proteolysis to characterize Topo I domains involved in ATP binding. The majority of incorporated analogue was associated with two fragments derived from N-terminal and C-terminal regions of Topo I, respectively. However, mutational analysis showed that deletion of the first 138 N-terminal residues, known to be dispensable for topoisomerase activity, did not change the binding of ATP or the kinase activity. In contrast, deletion of 162 residues from the C-terminal domain was deleterious for ATP binding, kinase and topoisomerase activities. Furthermore, a C-terminal tyrosine 723 mutant lacking topoisomerase activity is still able to bind ATP and to phosphorylate SF2/ASF, suggesting that the two functions of Topo I can be separated. These findings argue in favor of the fact that Topo I is a complex enzyme with a number of potential intra-cellular functions

Maitotoxin triggers the cortical reaction and phosphatidylinositol-4,5-bisphosphate breakdown in amphibian oocytes

International audienceMaitotoxin, a potent marine toxin extracted from peredinians, was found to mimic fertilization in Xenopus oocytes and to trigger the breakdown of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2, the precursor of inositol 1,4,5-trisphosphate], an increase of intracellular pCa and the cortical reaction, including the exocytosis of cortical granules and a wave-like propagation of contraction in the animal hemisphere. All these effects of maitotoxin required the presence of external calcium. Moreover, the toxin considerably increased Ca2+ influx in amphibian oocytes arrested at first meiotic prophase, due to the permanent activation of voltage-dependent Ca2+ channels. Nevertheless it is doubtful that maitotoxin acts primarily as a Ca2+ ionophore or at the level of Ca2+ channels. Indeed no stimulation of Ca2+ uptake was observed in metaphase-II-arrested oocytes, although maitotoxin readily triggered the breakdown of PtdIns(4,5)P2 as well as the cortical reaction in such cells. On the other hand, PtdIns(4,5)P2 breakdown was not reduced in oocytes microinjected with EGTA, although the calcium chelator prevented the oocytes from undergoing the cortical reaction. Taken together, these findings support the view that the toxin might act primarily by increasing PtdIns(4,5)P2 phosphodiesterase activity