p13(SUC1) and the WW domain of PIN1 bind to the same phosphothreonine-proline epitope

The WW domain of the human PIN1 and p13SUC1, a subunit of the cyclin-depend ent 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 p13(SUC1) With a model CDC25 peptide, depende nt on the phosphorylation state of its threonine, Chemical shift perturbati on of backbone H-1(N), N-15, and C-13 alpha, resonances during MMR titratio n experiments allows accurate identification of the binding site, primarily localized around the anion-binding site, occupied in the crystal structure of the homologous p9(CKSHs2) by a sulfate molecule. The epitope recognized by p13(SUC1) 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/ proline substitution. No direct interaction betwee n the PIN1 WW domain or its catalytic proline cis/transisomerase domain and p13(SUC1) was detected, but our study showed that in vitro the WW domain o f the human PIN1 antagonizes the binding of the p13(SUC1) to the CDC25 phos phopeptide, by binding to the same phosphoepitope. We thus propose that the full cyclin-dependent kinase complex stimulates the phosphorylation of CDC 25 through binding of its p13(SUC1) module to the phosphoepitope of the sub strate and that the reported WW antagonism of p13(SUC1)-stimulated CDC25 ph osphorylation is caused by competitive binding of both protein modules to t he same phosphoepitope.