Analysis of beta3-endonexin mutants for their ability to interact with cyclin A.
We have recently identified beta(3)-endonexin as a molecule that interacts with cyclin A-associated kinase. In this study, beta(3)-endonexin mutants were constructed by PCR-based site-directed mutagenesis, and characterized. Beta(3)-endonexin has a cyclin binding motif, RxL, in its N-terminal region, and two SP sequences which resemble a known target site for cyclin-dependent kinases (Cdks). The R5A/L7A mutant of beta(3)-endonexin, in which the RxL motif has been changed to AxA, is unable to bind to cyclin A, as revealed by two-hybrid experiments and in vitro pull-down assays. A GST-beta(3)-endonexin fusion, but not the corresponding R5A/L7A mutant, inhibits phosphorylation of Rb protein by cyclin A/Cdk2 in vitro. A cyclin A/Cdk2 kinase complex produced in, and purified from, insect cells phosphorylated GST-beta(3)-endonexin in vitro. The S33A or S46A mutant is partially phosphorylated by cyclin A/Cdk2, whereas no phosphorylation of the S33A/S46A double mutant is detectable. This demonstrates that these two serine residues, each of which is followed by a proline residue, are target sites for phosphorylation by cyclin A-associated kinase. The R5A/L7A mutant form of beta(3)-endonexin, which is defective for binding to cyclin A, is also not phosphorylated by cyclin A/Cdk2, confirming that the phosphorylation requires binding to cyclin A in the kinase complex. The neutralizing effect of beta(3)-endonexin on the toxicity associated with the expression of full-length human cyclin A in budding yeast is correlated with its ability to bind to cyclin A. Taken together, these data suggest that beta(3)-endonexin is phosphorylated by cyclinA/Cdk2 in vitro and that cyclin A-associated kinase activity is inhibited by the binding of beta(3)-endonexin to the kinase complex.
Ohtoshi
A
A
Division of Biotecture, Hope-tecture Research Institute, Hyogo, Japan. aotoshi@mail.mdanderson.org
Otoshi
H
H
D042846:CDC2-CDC28 Kinases; D019925:Cyclin A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009154:Mutation; D009687:Nuclear Proteins; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D004718:Saccharomycetales / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D019925:Cyclin A; C403295:ITGB3BP protein, human; D009687:Nuclear Proteins; D011506:Proteins; D012694:Serine; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases