IDENTIFICATION OF MITOSIS-SPECIFIC P65 DIMER AS A COMPONENT OF HUMAN-PHASE-PROMOTING FACTOR

Antisera raised against two mitosis-specific protein kinases from human cells recognized a single 65-kDa polypeptide (p65) that is present in similar amounts in interphase and mitotic cell extracts. Immunoblot analysis of reduced and unreduced extracts revealed that p65 exists as a 65-kDa monomer during interphase but forms a 130-kDa disulfide-linked homodimer during mitosis. Several different antibodies recognizing the p34(cdc2) protein kinase and cyclin B components of M phase-promoting factor (MPF) coprecipitated p65 from mitotic but not from interphase extracts. In addition, an anti-p65 immunoaffinity column substantially depleted mitotic extracts of histone H1 kinase activity assayed under conditions diagnostic for MPF. These results suggest that active human MPF may be a complex of p34(cdc2), cyclin B, and dimeric p65. A sulfhydryl cycle, proposed in the earlier literature on the biochemistry of mitosis, might underlie the dimerization of p65 and formation of active MPF.