Interaction between the cell-cycle-control proteins p34cdc2 and p9CKShs2. Evidence for two cooperative binding domains in p9CKShs2.
A universal intracellular factor, the 'M-phase-promoting factor' (MPF), displaying histone H1 kinase activity and constituted of at least two subunits, p34cdc2 and cyclin Bcdc13, triggers the G2----M transition of the cell cycle in all organisms. The yeast p13suc1 and p18CKS1 subunits and their functionally interchangeable human homologues, p9CKShs1 and p9CKShs2, directly interact with p34cdc2 and may actually be part of the MPF complex. We have chemically synthesized p9CKShs2 and several of its peptide domains in order to investigate the binding of p9CKShs2 and p34cdc2. Several arguments support the hypothesis that the N-terminal half (peptide B) and the C-terminal half (peptide E) each contain a p34cdc2-binding site and that these two binding domains cooperate in establishing a stable p9CKShs2-p34cdc2 complex: (a) only the combination of peptides B + E, and not B or E alone, is able to elute the cdc2 kinase from p9CKShs1-Sepharose beads; (b) only immobilized peptides B + E, and not immobilized B or E, bind the cdc2 kinase; (c) only the peptides B + E combination, and not B or E alone, can compete with p9CKShs1 for cdc2 kinase binding; (d) only when supplemented with E or B free peptide does the cdc2 kinase bind to B- or E-Sepharose beads, respectively. No binding occurs in the absence of free peptide. This additivity cannot be attributed to the formation of a B-E complex mimicking the full-length p9CKShs2. The cyclin B subunit is not required for the formation of the p9CKShs2-p34cdc2 complex through these two binding domains. The implications of the existence of two cooperative p34cdc2-binding domains in p9CKShs2 on the structure of the active M-phase-specific kinase is discussed.
Azzi
L
L
CNRS, Station Biologique, Roscoff, France.
Meijer
L
L
Reed
S I
SI
Pidikiti
R
R
Tung
H Y
HY
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002352:Carrier Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle; D016213:Cyclins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D006656:Protamine Kinase / Q000378:metabolism; D012617:Sea Urchins / Q000378:metabolism; D013215:Starfish / Q000378:metabolism
D002352:Carrier Proteins; D016213:Cyclins; D047908:Intracellular Signaling Peptides and Proteins; C072739:p9CKShs2 protein, human; D006656:Protamine Kinase; D016203:CDC2 Protein Kinase