Identification of a viral kinase that phosphorylates specific E2Fs and pocket proteins.
The transcription factor E2F and its regulation by pRB and related pocket proteins are central to cell cycle control in higher eukaryotes. Much of our knowledge of this regulation has come from studies using immediate-early proteins of DNA tumor viruses. Previously, we reported that the 72-kDa immediate-early region 1 gene product of the human cytomegalovirus, IE72, transactivates the dihydrofolate reductase promoter through the E2F site and that it physically interacts with E2F1 (M. J. Margolis, S. Pajovic, E. L. Wong, M. Wade, R. Jupp, J. A. Nelson, and J. C. Azizkhan, J. Virol. 69:7759-7767, 1995). In this study, we further characterized the mechanism by which IE72 modulates E2F-dependent transcription. In vitro phosphorylation reactions using gel-purified bacterially expressed proteins revealed that IE72 is a kinase that autophosphorylates and phosphorylates E2F1, -2, and -3 (but not E2F4 or -5) and the RB-related pocket proteins p130 and p107 (but not pRB). The region of IE72 spanning amino acids 173 to 197 shows a high level of homology to the ATP binding sites in over 500 kinases. The kinase-negative protein IE72deltaATP, from which this region has been deleted, cannot activate E2F-dependent transcription. The kinase activity of IE72 is also required for its ability to reduce the association of E2F4 with p107 and p130. Taken together, these data suggest that the kinase activity of IE72 is required for E2F-dependent transcriptional activation and that this is likely to result from phosphorylation of specific members of the E2F and pocket protein families by IE72.
Pajovic
S
S
Department of Experimental Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Wong
E L
EL
Black
A R
AR
Azizkhan
J C
JC
D000595:Amino Acid Sequence; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D003587:Cytomegalovirus / Q000201:enzymology*; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D017874:Immediate-Early Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins; D056465:Retinoblastoma-Binding Protein 1; D050717:Retinoblastoma-Like Protein p130; D013379:Substrate Specificity; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C088645:IE1 protein, cytomegalovirus; D017874:Immediate-Early Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011506:Proteins; D056465:Retinoblastoma-Binding Protein 1; D050717:Retinoblastoma-Like Protein p130; D050680:Transcription Factor DP1; D014157:Transcription Factors; D014764:Viral Proteins; D011494:Protein Kinases