A cyclin D1/cyclin-dependent kinase 4 binding site within the C domain of the retinoblastoma protein

Phosphorylation of the retinoblastoma protein (Rb) by the cyclin D1/cyclin- dependent kinase (cdk) 4 complex (cdk4/D1) is a key regulatory step for mai ntaining the orderly progression of the cell cycle. The B domain of Rb cont ains a site that recognizes and binds the LXCXE motif found in D-type cycli ns. This interaction is important for phosphorylation of Rb by cdk4/D1, alt hough irt vitro the Rb C domain alone is efficiently phosphorylated by cdk4 /D1. A mutation in the C domain of Rb, L901Q, has been identified that comp letely abolishes cdk4/D1 phosphorylation of the isolated C domain. By contr ast, the L901Q mutation has no effect on phosphorylation by either cyclin E /cdk2 or cyclin B/cdk1, suggesting that the interaction between L901Q and c dk4/D1 is specific. Introduction of the L901Q mutation into Rb containing t he A, B, and C domains results in phosphorylation becoming predominantly de pendent on the LXCXE binding region. However, when the LXCXE binding region of Rb is mutated, phosphorylation becomes dependent on the L901 site withi n the C domain. The L901 binding site can supplant the LXCXE binding site f or the cdk4/D1-dependent phosphorylation of S780 and S795 but not S807/S811 . Despite the limited homology between C domains of Rb, p107, and p130, the L901 site is conserved and introduction of the L925Q mutation into the iso lated C domain of p107 also inhibits phosphorylation by cdk4/D1. These data support a model for cdk4/D1 recognizing two independent binding sites in R b and suggests a conservation of this C domain binding motif for cyclin D1/ cdk4 kinase among the Rb family of proteins.