EXPRESSION AND ACTIVITY OF THE RETINOBLASTOMA PROTEIN (PRB)-FAMILY PROTEINS, P107 AND P130, DURING L(6) MYOBLAST DIFFERENTIATION

The activity of the E2 F-family of transcription factors is tightly li nked to control of the cell cycle. p107 and p130, two closely related members of the retinoblastoma protein-family of negative cell cycle re gulators, modulate the activity of the E2F-family proteins by direct i nteraction with these factors. To understand the role of p107 and p130 in progression through or exit from the cell cycle, we have character ized the expression, phosphorylation state, cyclin-binding, and E2F-bi nding activity of p107 and p130 during terminal differentiation of rat myoblast cells into immature skeletal muscle (myotubes). In exponenti ally growing L(6) myoblasts, p107 is phosphorylated in a cell cycle-de pendent manner, and E2F-site binding complexes containing p107 can be observed throughout the cell cycle. During differentiation of L(6) cel ls, p107 levels are reduced, while p130 protein levels are increased 8 -fold. Despite both p107 and p130 becoming hypophosphorylated during m yogenesis, the E2F-site DNA-binding complexes containing p107 observed in exponentially growing myoblasts are quantitatively replaced in myo tubes with complexes containing only p130. In myotubes, p107 is not as sociated with E2F-family proteins that are capable of binding DNA. The failure to observe p107-containing complexes in myotubes appears to b e due to the differentiation-specific induction of both p130 and cycli n D3. p107 is found in complexes with cyclin D3 in myotubes, and the a ddition of exogenous cyclin D3 or p130 to lysates from undifferentiate d L, cells was able to disrupt p107-containing E2F-site binding comple xes. In myotubes, p130 also forms complexes with cyclin D3 as well as cyclin E, cdk2, and cdk4. We are able to copurify cyclin D3 with cycli n E from myotubes, indicating the presence of a macromolecular complex containing both cyclin E and cyclin D3 simultaneously bound to p130. Thus, in myoblasts, p107 is normally involved in regulation of E2F-fam ily proteins during cell cycle progression, while p130 is a differenti ation-specific regulator of E2F activity. Our results also provide evi dence that the apparent positive regulator of cell cycle progression, cyclin D3, has a function in terminally differentiated muscle cells.