Direct binding of the signal-transducing adaptor Grb2 facilitates down-regulation of the cyclin-dependent kinase inhibitor p27(Kip1)

Ectopic expression of Jab1/CSN5 induces specific down-regulation of the cyc lin-dependent kinase (Cdk) inhibitor p27 (p27(Kip1)) in a manner dependent upon transportation from the nucleus to the cytoplasm. Here we show that Gr b2 and Grb3-3, the molecules functioning as an adaptor in the signal transd uction pathway, specifically and directly bind to p27 in the cytoplasm and participate in the regulation of p27. The interaction requires the C-termin al SH3-domain of Grb2/3-3 and the proline-rich sequence contained in p27 im mediately downstream of the Cdk binding domain. In living cells, enforcemen t of the cytoplasmic localization of p27, either by artificial manipulation of the nuclear/cytoplasmic transport signal sequence or by coexpression of ectopic Jab1/CSN5, markedly enhances the stable interaction between p27 an d Grb2. Overexpression of Grb2 accelerates Jab1/CSN5-mediated degradation o f p27, while Grb3-3 expression suppresses if. A p27 mutant unable to bind t o Grb2 is transported into the cytoplasm in cells ectopically expressing Ja b1/CSN5 but is refractory to the subsequent degradation. These findings ind icate that Grb2 participates in a negative regulation of p27 and may direct ly link the signal transduction pathway with the cell cycle regulatory mach inery.