p13SUC1 and the WW domain of PIN1 bind to the same phosphothreonine-proline epitope

Abstract

The WW domain of the human PIN1 and p13SUC1, a subunit of the cyclin-dependent kinase complex, were previously shown to be involved in the regulation of the cyclin-dependent kinase complex activity at the entry into mitosis, by an unresolved molecular mechanism. We report here experimental evidence for the direct interaction of p13SUC1 with a model CDC25 peptide, dependent on the phosphorylatlon state of its threonine. Chemical shift perturbation of backbone 1HN, 15N, and 13Cα resonances during NMR titration experiments allows accurate identification of the binding site, primarily localized around the anion-binding site, occupied in the crystal structure of the homologous p9CKSHs2 by a sulfate molecule. The epitope recognized by p13SUC1 includes the proline at position +1 of the phosphothreonine, as was shown by the decrease in affinity for a mutated CDC25 phosphopeptide, containing an alanine/praline substitution. No direct interaction between the PIN1 WW domain or its catalytic praline cis/trans-isomerase domain and p13SUC1 was detected, but our study showed that in vitro the WW domain of the human PIN1 antagonizes the binding of the p13SUC1 to the CDC25 phosphopeptide, by binding to the same phosphoepitope. We thus propose that the full cyclin-dependent kinase complex stimulates the phosphorylation of CDC25 through binding of its p13SUC1 module to the phosphoepitope of the substrate and that the reported WW antagonism of pl3SUC1-stimulated CDC25 phosphorylation is caused by competitive binding of both protein modules to the same phosphoepitope.