ABSTRACT
The SNF2-related CBP activator protein, SrCap (pronounced "sir cap"), shares homology with the SNF2/SWI2 protein family. SrCap was cloned through its ability to bind CBP. SrCap can function as a CBP coactivator and can activate transcription in a reporter assay when expressed as a Gal-SrCap fusion protein. A monoclonal antibody raised against the carboxyl terminus of SrCap coimmunoprecipitates CBP/p300, supporting the model that SrCap is a CBP binding protein and that these proteins can be found together in a cellular protein complex. In addition, several cellular proteins are coimmunoprecipitated by the SrCap-specific antibody. Since adenovirus E1A proteins interact with CBP/p300 proteins, we examined what proteins could be copurified in a SrCap-specific coimmunoprecipitation assay from lysates of adenovirus-infected cells. While E1A proteins were not detected in this complex, to our surprise, we observed the presence of an infected-cell-specific band of 72 kDa, which we suspected might be the adenovirus DNA binding protein, DBP. The adenovirus DBP is a multifunctional protein involved in several aspects of the adenovirus life cycle, including an ability to modulate transcription. The identity of DBP was confirmed by DBP-specific Western blot analysis and by reimmunoprecipitating DBP from denatured SrCap-specific protein complexes. Using in vitro-translated DBP and SrCap proteins, we demonstrated that these proteins interact. To determine whether this interaction could affect SrCap-mediated transcription, we tested whether increasing amounts of DBP could modulate the Gal-SrCap transcription activity. We observed that DBP inhibited Gal-SrCap transcription activity in a dose-dependent manner. These data suggest a novel mechanism of adenovirus host cell control by which DBP binds to and inactivates SrCap, a member of the SNF2 chromatin-remodeling protein family.
Subject(s)
Adenosine Triphosphatases/antagonists & inhibitors , DNA-Binding Proteins/physiology , Transcription, Genetic , Viral Proteins/physiology , Animals , DNA/metabolism , E1A-Associated p300 Protein , HeLa Cells , Humans , Mice , Mice, Inbred BALB C , Nuclear Proteins/metabolism , Precipitin Tests , Trans-Activators/metabolismABSTRACT
pp60c-src kinase and transforming activities are negatively regulated by phosphorylation of Tyr 527, a residue 6 amino acids from its carboxyl terminus. Tyr 527 to Phe mutation has been shown to activate pp60c-src, yet pp60c-src(F527) is still less active than pp60v-src. To see if additional carboxyl terminal mutation can stimulate pp60c-src transforming activity to pp60v-src levels, we compared the properties of pp60c-src(Am517), a pp60c-src mutant in which the 17 carboxyl terminal amino acids were deleted, with those of pp60c-src(F527) and pp60c-src(F519), a protein in which the Tyr nearest to Tyr 527 was changed to Phe. Tyr 519 to Phe mutation did not affect pp60c-src activities while the Am517 mutation activated focus formation, growth in soft agarose, in vivo tumorigenicity and in vitro specific kinase activity to levels between those of pp60c-src and pp60c-src(F527). This contrasts with a previous study [Cartwright et al. (1987) Cell 49, 83-91] which reported that Am517 mutation enhances biological activities without enhanced kinase activity. These data support the hypotheses that (1) complete transformation by pp60c-src requires activation of its protein tyrosine kinase activity and (2) that downregulation by the pp60c-src carboxyl terminus is governed by phosphorylation of Tyr 527; additional changes beyond that needed to prevent this phosphorylation do not further enhance activity.
Subject(s)
Mutation , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/genetics , Animals , Cell Line , Mice , Phosphorylation , Proto-Oncogene Proteins pp60(c-src) , Substrate SpecificityABSTRACT
The complete DNA sequence of the human JC virus, which was found to consist of 5,130 nucleotide pairs, is presented. The amino acid sequence of six proteins could be deduced: the early, nonstructural proteins, large T and small t antigens; the late capsid proteins, VP1, VP2, and VP3; and the agnogene product encoded within the late leader sequence, called the agnoprotein in simian virus 40. The extent of homology between JC virus DNA and the genomes of simian virus 40 (69%) and BK virus (75%) confirmed the close evolutionary relationship of these three polyomaviruses. The sequences showing the greatest divergence in these viral DNAs occurred within the tandem repeats located to the late side of the replication origins.
