DEFINING THE MINIMAL PORTION OF THE RETINOBLASTOMA PROTEIN THAT SERVES AS AN EFFICIENT SUBSTRATE FOR CDK4 KINASE CYCLIN D1 COMPLEX/

We have determined the minimal portion of the retinoblastoma protein ( Rb) that can serve as an efficient substrate for in vitro phosphorylat ion by cdk4 kinase-D1 cyclin, Kinetic measurements indicate that in vi tro, a 15-kDa fragment that represents the C-terminus of Rb can serve equally well as a substrate when compared with the larger 56-kDa fragm ent of Rb, which contains the A, B and C domains, By comparison, pepti de substrates appear to be 1000-fold less efficient, Furthermore, muta tional analysis indicates that not all of the five phosphorylation sit es within this minimal C domain are phosphorylated equally by cdk4/D1, Ser(795) is the preferred phosphorylation site, whereas the four rema ining sites Ser(807), Ser(811), Thr(821) and Thr(826) are phosphorylat ed to a much lesser degree. Truncations of the C domain from the carbo xy terminus indicate that almost all of this domain is required for ef ficient phosphorylation, These data suggest that the structural contex t of the phosphorylation site within the substrate is critical for its phosphorylation by the cdk4/D1 kinase.