Defining the substrate specificity of cdk4 kinase-cyclin D1 complex

cdk4 kinase-cyclin D1 complex (cdk4/D1) does not phosphorylate all of the s ites within retinoblastoma protein (Rb) equally. Comparison of five phospho rylation sites within the 15 kDa C domain of Rb indicates that Ser795 is th e preferred site of phosphorylation by cdk4/D1, A series of experiments has been performed to determine the properties of this site that direct prefer ential phosphorylation, For cdk4/D1, the preferred amino acid at the third position C-terminal to the phosphorylated serine/threonine is arginine, Sub stitution of other amino acids, including a conservative change to lysine, has dramatic effects on the rates of phosphorylation, This information has been used to mutate less favorable sites in Rb, converting them to sites th at are now preferentially phosphorylated by cdk4/D1, A conserved site at Se r842 in the related pocket protein p107 is also preferentially phosphorylat ed by cdk4/D1, Although Rb and p107 differ significantly in sequence, the R b Ser795 site can replace the p107 Ser842 site without affecting the rate o f phosphorylation, These results suggest that although a determinant of spe cificity resides in the sequences surrounding the phosphorylated site, the structural context of the site is also a critical parameter of specificity.