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

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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.

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