Inhibition of retinoblastoma protein phosphorylation by myogenesis-induced changes in the subunit composition of the cyclin-dependent kinase 4 complex.

The retinoblastoma protein (Rb) is essential for the maintenance of the postmitotic state in terminally differentiated myocytes. Upon C2C12 myogenesis, the level of the cyclin-dependent kinase 4 (CDK4) protein does not change, but its Rb kinase activity is down-regulated markedly. Here, we show that the reduction in CDK4 activity results from (a) the irreversible induction and association of the p21 CDK inhibitor with the CDK4 complex and (b) a decline in overall D-type cyclin expression. Immunoprecipitation-coupled immunoblot analyses demonstrated that myocyte differentiation produces alterations in the subunit interactions within the CDK4 complex, including a diminished interaction with cyclin D1 and enhanced interactions with cyclin D3 and p21. The significance of the p21 interaction with CDK4 was indicated by the ability of anti-p21 antibodies to specifically immunodeplete a Rb kinase inhibitory activity that was bound to the CDK4 complex in myotubes. Furthermore, the restimulation of myotubes with serum did not lead to the re-activation of CDK4 or disrupt the CDK4-p21 interaction. Despite the increase in cyclin D3 expression during myogenesis, quantitative immunoblot analyses revealed that the combined levels of cyclin D1 and D3 decline during this process and that CDK4 is expressed at much higher levels than either of these cyclin subunits in postmitotic myotubes. These results suggest that the myogenesis-induced up-regulation of p21 and down-regulation of the total D-type cyclin expression contribute to the inhibition of the CDK4 Rb kinase activity, leading to conditions that favor the accumulation of the hypophosphorylated Rb and growth arrest upon terminal differentiation.