[23] M phase-specific cdc2 kinase: Preparation from Starfish oocytes and properties

The M phase-specific cdc2 kinase (also called M phase-specific H1 histone kinase) is a serine/threonine kinase. It is Ca2+, diacylglycerol, and cyclic nucleotide independent, and was first discovered in maturing oocytes of starfish and amphibian oocytes. This kinase demonstrates dramatic changes in its activity during the course of the eukaryotic cell cycle. It is identical to MPF (the M phase-promoting factor) and is thus sufficient to push G2-arrested cells into M phase or to induce a variety of mitotic events in cell-free systems. This chapter describes a rapid and efficient procedure to purify the M phase-specific cdc2 kinase from starfish oocytes at first meiotic metaphase. One milligram of the final preparation catalyzes the transfer of about 5 μmol of phosphate from adenosine triphosphate (ATP) to H1 histone in one min under standard conditions. The kinase is a complex formed by the stoichiometric association of one molecule of cdc2 (a 34-kDa catalytic subunit) with one molecule of cyclin B. The main step in this procedure is affinity chromatography on the yeast p13sucl protein, which binds to the yeast cdc2/CDC28 proteins and their homologs in higher eukaryotes. Microinjection of the homogeneous M phase-specific cdc2 kinase into oocytes arrested at first meiotic prophase induces nuclear envelope breakdown, chromosome condensation, and spindle formation even in the absence of protein synthesis and is active across phylogenetic boundaries, working so far in all tested species, both vertebrate and invertebrate.