CYCLIN-MEDIATED INHIBITION OF MUSCLE GENE-EXPRESSION VIA A MECHANISM THAT IS INDEPENDENT OF PRB HYPERPHOSPHORYLATION

It was recently demonstrated that ectopic expression of cyclin D1 inhi bits skeletal muscle differentiation and, conversely, that expression of cyclin-dependent kinase (cdk) inhibitors facilitates activation of this differentiation program (S. S. Rao, C. Chu, and D. S. Kohtz, Mol. Cell. Biol. 14:5259-5267, 1994; S. S. Rao and D. S. Kohtz, J. Biol. C hem. 270:4093-4100, 1995; S. X. Skapek, J. Rhee, D. B. Spicer, and A. B. Lassar, Science 267:1022-1024, 1995). Here we demonstrate that cycl in D1 inhibits muscle gene expression without affecting MyoD DNA bindi ng activity, Ectopic expression of cyclin D1 inhibits muscle gene acti vation by both MyoD and myogenin, including a mutated form of myogenin in which two potential inhibitory cdk phosphorylation sites are absen t. Because the retinoblastoma gene product, pRB, is a known target for cyclin D1-cdk phosphorylation, we determined whether cyclin D1-mediat ed inhibition of myogenesis was due to hyperphosphorylation of pRB In pRB-deficient fibroblasts, the ability of MyoD to activate the express ion of muscle-specific genes requires coexpression of ectopic pRB (B. G. Novitch, G. J. Mulligan, T. Jacks, and A. B. Lassar, J. Cell Biol., 135:441-456, 1996). In these cells, the expression of cyclins A and E can lead to pRB hyperphosphorylation and can inhibit muscle gene expr ession. The negative effects of cyclins A or E on muscle gene expressi on are, however, reversed by the presence of a mutated form of pRB whi ch cannot be hyperphosphorylated. In contrast, cyclin D1 can inhibit m uscle gene expression in the presence of the nonhyperphosphorylatable form of pRB. On the basis of these results we propose that G(1) cyclin -cdk activity blocks the initiation of skeletal muscle differentiation by two distinct mechanisms: one that is dependent on pRB hyperphospho rylation and one that is independent of pRB hyperphosphorylation.