Identification of mitosis-specific p65 dimer as a component of human M 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 p34cdc2 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 histon H1 kinase activity assayed under conditions diagnostic for MPF. These results suggest that active human MPF may be a complex of p34cdc2, 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.
Meikrantz
W
W
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Suprynowicz
F A
FA
Halleck
M S
MS
Schlegel
R A
RA
D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D004220:Disulfides / Q000378:metabolism; D006801:Humans; D007106:Immune Sera; D007074:Immunoglobulin G; D007399:Interphase; D046911:Macromolecular Substances; D008938:Mitosis; D008970:Molecular Weight; D006656:Protamine Kinase / Q000378:metabolism; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*
D004220:Disulfides; D007106:Immune Sera; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D011494:Protein Kinases; D006656:Protamine Kinase