Getting in and out of mitosis (Video Lecture)
Cell Cycle Control Laboratory, Cancer Research UK, South Mimms, UK
Cells enter mitosis (more generally, M-phase, and much of our work has been in frog oocytes and eggs and extracts thereof) when CDK1/cyclin complexes are activated. Phosphorylation by these, and other mitotic protein kinases, is responsible for reorganizing the cell and initiating progression to metaphase.
We would like to know how many proteins needs to be phosphorylated how much to bring about this state of affairs, and have been trying to enumerate the mitotic targets for various cyclin-CDK combinations for some time. I’ll talk about our approaches, difficulties and findings.
Exit from mitosis, starting at the metaphase to anaphase transition, occurs when the anaphasepromoting factor (APC/ C) is activated and tags a small number of target proteins, including cyclins and securin, with polyubiquitin chains that signal their proteolysis by the proteasome. Chromatids part and move to opposite poles of the cell where they decondense and re-form a functional nucleus.
Cytokinesis separates the two daughter cells. Mitotic phosphoproteins revert to their interphase un- or hypo-phosphorylated state.
We recently made the accidental discovery that the activity responsible for this postmitotic dephosphorylation is almost completely inactive in M-phase cell extracts, and is reactivated when cells exit mitosis. This explains how proteins can become almost completely converted to hyperphosphorylated states: not only are kinases activated, but the counteracting phosphatase(s) are concomitantly shut down. I will present the evidence that has led us to this conclusion. It stems from studies of frog egg extracts released from cytostatic factor (CSF) arrest by added CaCl2, and the discovery that calcineurin (protein phosphatase 2B) plays a role in escaping the clutches of CSF. But the real work of restoring proteins to their interphase state of hypophosphorylation is performed by an activity we call ‘Phosphatase X’, whose identity and regulation I shall discuss.
Mitosis, oocytes, cytokynesis, phosphatase