Analysis of β₃-endonexin mutants for their ability to interact with cyclin A

Abstract.

We have recently identified β₃-endonexin as a molecule that interacts with cyclin A-associated kinase. In this study, β₃-endonexin mutants were constructed by PCR-based site-directed mutagenesis, and characterized. β₃-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 β₃-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-β₃-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-β₃-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 β₃-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 β₃-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 β₃-endonexin is phosphorylated by cyclinA/Cdk2 in vitro and that cyclin A-associated kinase activity is inhibited by the binding of β₃-endonexin to the kinase complex.