Rb inhibits the intrinsic kinase activity of TATA-binding protein-associated factor TAFII250.
The retinoblastoma tumor suppressor protein, Rb, interacts directly with the largest TATA-binding protein-associated factor, TAFII250, 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 TAFII250. Here, we report that Rb is able to inhibit the kinase activity of immunopurified and gel-purified recombinant TAFII250. Rb inhibits the autophosphorylation of TAFII250 as well as its phosphorylation of the RAP74 subunit of TFIIF in a dose-responsive manner. Inhibition of TAFII250 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 TAFII250 through a direct protein-protein interaction. We further demonstrate that two different tumor-derived Rb pocket mutants, C706F and Deltaex22, are functionally defective for kinase inhibition, even though they are able to bind the amino terminus of TAFII250. Our results suggest a novel mechanism of transcriptional regulation by Rb, involving direct interaction with TAFII250 and inhibition of its ability to phosphorylate itself, RAP74, and possibly other targets.
Siegert
J L
JL
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Robbins
P D
PD
D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D051548:Histone Acetyltransferases; D009154:Mutation; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D051548:Histone Acetyltransferases; D011494:Protein Kinases; C069001:TATA-binding protein associated factor 250 kDa