The retinoblastoma tumor suppressor gene product (Rb) binds directly to the largest TFIID subunit, TATAbinding protein associated factor TAF II 250, ®rst identi®ed as the cell cycle regulatory protein CCG1. Here we map the domains in Rb and TAF II 250 important for their interaction in vitro and in vivo. Both the amino terminus and the large pocket of Rb are able to associate independently with TAF II 250. The binding domain(s) within the large pocket are distinct from the viral oncoprotein and E2F binding region since certain pocket mutations, which abolish E1A binding, do not abolish TAF II 250 binding. Consistent with the large pocket of Rb binding to TAF II 250, the large pocket domains of both p107 and p130 are able to bind to TAF II 250 in vivo. We also demonstrate that at least two regions of TAF II 250 are able to bind to the large pocket of Rb independently whereas the amino terminus of Rb binds to a distinct domain in TAF II 250. We further demonstrate that Rb can bind to TFIID in vitro, presumably in part through an interaction with TAF II 250. Our results suggest a complex interaction between Rb and TAF II 250 and imply that TAF II 250, TFIID, and potentially other basal transcription factors are targets for regulation by Rb and Rb-related proteins.

The retinoblastoma tumor suppressor protein, Rb, interacts directly with the largest TATA-binding proteinassociated factor, TAF II 250, through multiple regions in each protein. To define the potential role(s) of this interaction, we examined whether Rb could regulate the intrinsic, bipartite kinase activity of TAF II 250. Here, we report that Rb is able to inhibit the kinase activity of immunopurified and gel-purified recombinant TAF II 250. Rb inhibits the autophosphorylation of TAF II 250 as well as its phosphorylation of the RAP74 subunit of TFIIF in a dose-responsive manner. Inhibition of TAF II 250 kinase activity involves the Rb pocket (amino acids 379 to 928) but not its amino terminus. In addition, Rb appears to specifically inhibit the amino-terminal kinase domain of TAF II 250 through a direct protein-protein interaction. We further demonstrate that two different tumor-derived Rb pocket mutants, C706F and ⌬ex22, are functionally defective for kinase inhibition, even though they are able to bind the amino terminus of TAF II 250. Our results suggest a novel mechanism of transcriptional regulation by Rb, involving direct interaction with TAF II 250 and inhibition of its ability to phosphorylate itself, RAP74, and possibly other targets.The retinoblastoma protein, Rb, is a tumor suppressor whose mutational inactivation has been implicated in a variety of sporadic and familial human cancers (17). Rb is a key regulator of cell growth and differentiation (47), and although the exact mechanism by which it acts as a tumor suppressor is unclear, the ability of Rb to regulate transcription in a cell cycle-dependent manner is likely to be central to its function. Rb has been shown to either repress or stimulate the activity of specific promoters (23, 30), apparently through physical or functional interaction with transcription factors (15,19,22,36,44). Indeed, Rb can repress E2F-mediated transcription by binding directly to the E2F transcription factor (12), supposedly inhibiting transcription by blocking the transactivation domain of E2F (15). However, recent evidence from our laboratory and others has suggested that Rb can function as a general repressor of activated transcription, independent of an interaction with a specific transcription factor, when targeted to a promoter through fusion to a heterologous DNA-binding domain (1,4,35,48). Consistent with this finding is the recent observation that Rb can repress transcription by recruiting the histone deacetylase protein HDAC1 (3, 26). Thus, it is likely that Rb, once recruited to a promoter, does not simply repress the function of a transcription factor but may regulate transcription via some alternative mechanism, perhaps through either deacetylase recruitment or interactions with the transcription initiation complex. Given that promoter-targeted Rb appears to repress activated rather than basal transcription, we examined the ability of Rb to interact with the TFIID coactivator proteins, TATA-binding protein (TBP)-associated factors (TAFs), as they are belie...

The retinoblastoma tumor suppressor protein has been shown to bind directly and inhibit a transcriptionallyimportant amino-terminal kinase domain of TATAbinding protein-associated factor TAF II 250. Cyclin D1 also is able to associate with the amino terminus of TAF II 250 in a region very similar to or overlapping the Rb-binding site. In this study, we have examined whether cyclin D1 a ects the functional interaction between Rb and TAF II 250. We observed that when cyclin D1 is coincubated with Rb and TAF II 250, the ability of Rb to inhibit TAF II 250 kinase activity is e ectively blocked. However, cyclin D1 by itself has no apparent e ect on TAF II 250 kinase activity. We further found that the Rbrelated protein p107 can inhibit TAF II 250 kinase activity, and this inhibition is likewise alleviated by cyclin D1. Cyclin D1 prevents the kinase-inhibitory e ect of an Rb mutant unable to bind to D-type cyclins, indicating that it is acting through its association with TAF II 250 and not with Rb. However, we found no evidence of TAF II 250-binding competition between Rb and cyclin D1 in vitro. The adenovirus E1A protein, which also binds to both Rb and TAF II 250, exhibited a suppressive e ect on Rb-mediated kinase inhibition similar to that seen with cyclin D1. Our results suggest a novel means by which cyclin D1 may be able to independently regulate the activity of Rb. Oncogene (2000) 19, 5703 ± 5711.