Rb Inhibits the Intrinsic Kinase Activity of TATA-Binding Protein-Associated Factor TAF_II250

Abstract

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...

Cyclin D1 Associates with the TBP-associated factor TAFII250 to regulate Sp1-mediated transcription

“…We recently have shown that Rb is able to inhibit the TAF II 250 amino terminal kinase activity in vitro through association with the amino terminus of TAF II 250 (Siegert and Robbins, 1998). Indeed, Rb is able both TAF II 250 autophosphorylation as well as phosphorylation of RAP74.…”

Cyclin D1 suppresses retinoblastoma protein-mediated inhibition of TAFII250 kinase activity

“…As shown in Figure 3, the addition of Rb alone to the TAF II 250 membrane slice (lane 3) resulted in a signi®cant reduction in the kinase activity of TAF II 250, as has been previously demonstrated (Siegert and Robbins, 1999). However, when TAF II 250 was incubated with both Rb and cyclin D1 (Figure 3, lane 4), Rb was no longer able to inhibit the phosphorylation of RAP74 by TAF II 250.…”

“…Very recently, it has been shown that the histone acetyltransferase and kinase activities of TAF II 250 modulate expression of distinct subsets of mammalian genes, with the kinase activity apparently necessary for expression of approximately 6% of genes in ts13 cells and the acetyltransferase activity needed for expression of a di erent set of genes, constituting approximately 1% of the ts13 genes (O'Brien and Tjian, 2000). Interestingly, we have demonstrated that the Rb protein can directly inhibit the kinase activity of TAF II 250, through its interaction with the amino terminus of TAF II 250 (Siegert and Robbins, 1999).…”

“…As a negative control, the corresponding region of membrane was also cut from a lane loaded only with Laemmli bu er, and this was subsequently subjected to the same conditions as the TAF II 250-containing membrane slice. Note that this control has been previously tested and was found to give results identical to those achieved by cutting a corresponding region of membrane from a lane loaded with extract from mock-infected Sf9 cells (Siegert and Robbins, 1999). The TAF II 250-containing slices were then incubated either alone or in the presence of a variety of combinations of puri®ed GST-Rb (large pocket), GST-cyclin D1, and GST.…”

“…A modi®ed version of the denaturationrenaturation assay described by Dikstein et al (1996) was used, as described previously (Siegert and Robbins, 1999), in order to ensure the use of a highly puri®ed form of TAF II 250. Baculovirus-expressed HAhTAF II 250 was immunopuri®ed from Sf9 cell extract, divided into equal aliquots, subjected to SDS ± PAGE, and transferred to nitrocellulose.…”