Glutamic acid mutagenesis of retinoblastoma protein phosphorylation sites has diverse effects on function

The retinoblastoma tumor suppressor gene (Rb) has many functions within the cell including regulation of transcription, differentiation, apoptosis, an d the cell cycle. Regulation of these functions is mediated by phosphorylat ion at as many as 16 cyclin-dependent kinase (CDK) phosphorylation sites in vivo. The contribution of these sites to the regulation of the various Rb functions is not well understood. To characterize the effect of phosphoryla tion at these sites, we systematically mutagenized the serines or threonine s to glutamic acid. Thirty-five mutants with different combinations of modi fied phosphorylation sites were assayed for their ability to arrest the cel l cycle and for their potential to induce differentiation. Only the most hi ghly substituted mutants failed to arrest cell cycle progression, However, mutants with as few as four modified phosphorylation sites were unable to p romote differentiation. Other mutants had increased activity in this assay. We conclude that modification of Rb phosphorylation sites can increase or decrease protein activity, that different Rb functions can be regulated ind ependently by distinct combinations of sites, and that the effects of modif ication at any one site are context dependent.