Identification of the domains in cyclin A required for binding to, and activation of, p34cdc2 and p32cdk2 protein kinase subunits.
The binding of cyclin A to p34cdc2 and p32cdk2 and the protein kinase activity of the complexes has been measured by cell-free translation of the corresponding mRNA in extracts of frog eggs, followed by immunoprecipitation. A variety of mutant cyclin A molecules have been constructed and tested in this assay. Small deletions and point mutations of highly conserved residues in the 100-residue "cyclin box" abolish binding and activation of both p34cdc2 and p32cdk2. By contrast, large deletions at the N-terminus have no effect on kinase binding and activation, until they remove residues beyond 161, where the first conserved amino acids are found in all known examples of cyclin A. At the C-terminus, removal of 14 or more amino acids abolishes activity. We also demonstrate that deletion of, or point mutations, in the cyclin A homologue of the 10-residue "destruction box," previously described in cyclin B (Glotzer et al., 1991) abolish cyclin proteolysis at the transition from M-phase to interphase.
Kobayashi
H
H
ICRF Clare Hall Laboratories, South Mimms, Herts, England.
Stewart
E
E
Poon
R
R
Adamczewski
J P
JP
Gannon
J
J
Hunt
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D042846:CDC2-CDC28 Kinases; D002478:Cells, Cultured; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D016213:Cyclins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000737:chemistry; D017354:Point Mutation; D011233:Precipitin Tests; D006656:Protamine Kinase / Q000378:metabolism; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger / Q000737:chemistry; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000378:metabolism
D016213:Cyclins; D012333:RNA, Messenger; D029867:Xenopus Proteins; D011494:Protein Kinases; D006656:Protamine Kinase; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; C495891:Cdk2 protein, Xenopus; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases