Differential Regulation of Retinoblastoma Tumor Suppressor Protein by G 1 Cyclin-Dependent Kinase Complexes In Vivo

Abstract

ABSTRACT The retinoblastoma tumor suppressor protein (pRB) negatively regulates early-G 1 cell cycle progression, in part, by sequestering E2F transcription factors and repressing E2F-responsive genes. Although pRB is phosphorylated on up to 16 cyclin-dependent kinase (Cdk) sites by multiple G 1 cyclin-Cdk complexes, the active form(s) of pRB in vivo remains unknown. pRB is present as an unphosphorylated protein in G 0 quiescent cells and becomes hypophosphorylated (∼2 mol of PO 4 to 1 mol of pRB) in early G 1 and hyperphosphorylated (∼10 mol of PO 4 to 1 mol of pRB) in late G 1 phase. Here, we report that hypophosphorylated pRB, present in early G 1 , represents the biologically active form of pRB in vivo that is assembled with E2Fs and E1A but that both unphosphorylated pRB in G 0 and hyperphosphorylated pRB in late G 1 fail to become assembled with E2Fs and E1A. Furthermore, using transducible dominant-negative TAT fusion proteins that differentially target cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes, namely, TAT-p16 and TAT–dominant-negative Cdk2, respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G 1 and that cyclin E-Cdk2 complexes inactivate pRB by hyperphosphorylation in late G 1 . Moreover, we found that cycling human tumor cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion of the p16 INK4a gene, contained hypophosphorylated pRB that was bound to E2Fs in early G 1 and that E2F-responsive genes, including those for dihydrofolate reductase and cyclin E, were transcriptionally repressed. Thus, we conclude that, physiologically, pRB is differentially regulated by G 1 cyclin-Cdk complexes.