Cyclin A Is a Functional Target of Retinoblastoma Tumor Suppressor Protein-mediated Cell Cycle Arrest

Abstract

Although RB inhibits the G 1 -S transition, the mechanism through which RB prevents cell cycle advancement remains unidentified. To delineate the mechanism(s) utilized by RB to exert its anti-proliferative activity, constitutively active RB proteins (which cannot be inactivated by phosphorylation) or p16ink4a (which prevents RB inactivation) were utilized. Both proteins inhibited the G 1 -S transition, whereas wildtype RB did not. We show that active RB acts to attenuate cyclin A promoter activity, and that overexpression of cyclin E reverses RB-mediated repression of the cyclin A promoter. Although cyclin A is an E2F-regulated gene, and it has been long hypothesized that RB mediates cell cycle advancement through binding to E2F and attenuating its transactivation potential, cyclin E does not reverse dominant negative E2F-mediated repression of the cyclin A promoter. Although active RB repressed both cyclin A and two other paradigm E2F-regulated promoters, only cyclin A transcription was restored upon co-expression of cyclin E. Additionally, we show that RB but not dominant negative E2F regulates the cyclin A promoter through the CCRE element. These data identify cyclin A as a downstream target of RBmediated arrest. Consistent with this idea, ectopic expression of cyclin A reversed RB-mediated G 1 arrest. The findings presented suggest a pathway wherein cyclin A is a downstream target of RB, and cyclin E functions to antagonize this aspect of RB-mediated G 1 -S inhibition.The retinoblastoma tumor suppressor protein (RB), 1 is functionally inactivated in over 60% of human tumors (1-3). The role of RB as a tumor suppressor has been well established, and it is known that RB can inhibit cellular proliferation by halting cell cycle progression (4 -6). RB carries out this growth inhibition through its ability to assemble and modulate a host of multiprotein complexes (5,7,8). At least four distinct proteinbinding domains of RB have been identified and extensively characterized, including: the A/B pocket, the large A/B pocket, the C-pocket, and the N-terminal domain (5, 7, 9). The large A/B pocket is the minimal growth suppressing region of RB and is required to bind the E2F family of transcription factors (10, 11).Binding of RB to proteins such as E2F is regulated by cyclindependent kinase (Cdk)-mediated phosphorylation (4, 5, 12). The full-length RB protein contains 16 consensus Cdk-phosphorylation sites, and phosphorylation at specific sites inhibits the binding of RB to cellular proteins, thereby disrupting the anti-proliferative activity of RB (13-19). Not surprisingly, therefore, overexpression of proteins which cause excessive or deregulated phosphorylation of RB is a common event in human tumors (2,3,20). For example, amplification of Cdk4 and/or its regulatory partner, cyclin D1, are frequently observed in human tumors. In either case, excessive Cdk4/cyclin D kinase activity results in deregulated phosphorylation and inactivation of RB. Similarly, loss of the tumor suppressor p16ink4a, which acts to attenuate C...

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