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Identification of mitosis-specific p65 dimer as a component of human M phase-promoting factor
W Meikrantz1, F A Suprynowicz, M S Halleck
1Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Abstract:
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.
Insights
Human cells utilize a protein kinase, p65, which dimerizes during mitosis. This dimerization is crucial for forming active M phase-promoting factor (MPF), a key regulator of cell division.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Mitosis is regulated by complex protein interactions.
- M phase-promoting factor (MPF) is a critical regulator of mitosis.
- The precise composition of active MPF in human cells requires further elucidation.
Purpose of the Study:
- To characterize a novel protein, p65, identified in human mitotic extracts.
- To investigate the role of p65 in the formation and activity of M phase-promoting factor (MPF).
- To explore the structural changes of p65 during the cell cycle.
Main Methods:
- Immunoblot analysis using antisera against mitosis-specific protein kinases.
- Immunoprecipitation assays with antibodies against p34cdc2 and cyclin B.
- Immunoaffinity chromatography using anti-p65 antibodies.
- Histone H1 kinase activity assays.
Main Results:
- A 65-kDa polypeptide (p65) was identified in both interphase and mitotic human cell extracts.
- p65 exists as a monomer in interphase and forms a disulfide-linked 130-kDa homodimer during mitosis.
- p65 was coprecipitated with p34cdc2 and cyclin B from mitotic extracts.
- Anti-p65 immunoaffinity columns depleted histone H1 kinase activity characteristic of MPF.
Conclusions:
- Active human MPF likely comprises a complex of p34cdc2, cyclin B, and dimeric p65.
- The dimerization of p65, potentially via a sulfhydryl cycle, is implicated in the formation of active MPF.
- p65 represents a novel component of the human MPF regulatory machinery.