Phosphorylation-dependent and -independent functions of p130 cooperate to evoke a sustained G(1) block

The retinoblastoma (pRb)-related p130 pocket protein is a regulator of cell growth and differentiation, and a candidate tumour suppressor. Both pRb an d p130 operate through interactions with cellular proteins, including the E 2F transcription factors. While such interactions are controlled by phospho rylation of multiple sites of pRb, regulation of p130 remains poorly unders tood. We now identify 22 in vivo phosphorylation sites of p130, targeted by diverse kinases, and present evidence for three cyclin-dependent kinase 4( 6) [Cdk4(6)] specific phosphorylations, which appear critical for controlli ng the growth-restraining activity of p130, When expressed in U20S cells, t he phosphorylation-deficient mutant p130(Delta Cdk4), in which the Cdk4 spe cific sites were mutated to alanine residues, imposed a more sustained G(1) arrest than a constitutively active pRb(Delta Cdk), known to repress all c ellular E2F activity. Experiments using p130(Delta Cdk4).,d another phospho rylation-deficient mutant, p130(PM19A) with 19 phosphorylation sites mutate d, revealed that the p130-imposed G(1) block reflects cooperative growth-su ppressive effects of phosphorylation-regulated E2F binding and phosphorylat ion-independent sequestration of cyclin E(A)-Cdk2 through the N-terminal cy clin binding motif of p130.