Analysis with specific polyclonal antiserum indicates that the E1A-associated 300-kDa product is a stable nuclear phosphoprotein that undergoes cell cycle phase-specific modification

Binding of a 300-kDa host cell protein (p300) is tightly correlated with the ability of the adenovirus E1A products to induce quiescent baby rat kidney cells to proliferate. We have generated rabbit polyclonal antibodies against p300 to characterize this protein further. We have found p300 to be a nuclear phosphoprotein that is actively synthesized in both quiescent and proliferating baby rat kidney cells. In partially purified mitotic cell populations, we observe a form of p300 with decreased electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels that shares a nearly identical partial proteolytic digest pattern with p300. The slower-migrating form of p300 is greatly reduced by treating immune complexes with potato acid phosphatase. The relative stability and presence of p300 even in resting cells suggests that p300 has a basal cell function, but the appearance of differentially modified forms during the cell cycle suggests the possibility that p300 function is modulated specifically in growing cells.</jats:p

Analysis with specific polyclonal antiserum indicates that the E1A-associated 300-kDa product is a stable nuclear phosphoprotein that undergoes cell cycle phase-specific modification.

Binding of a 300-kDa host cell protein (p300) is tightly correlated with the ability of the adenovirus E1A products to induce quiescent baby rat kidney cells to proliferate. We have generated rabbit polyclonal antibodies against p300 to characterize this protein further. We have found p300 to be a nuclear phosphoprotein that is actively synthesized in both quiescent and proliferating baby rat kidney cells. In partially purified mitotic cell populations, we observe a form of p300 with decreased electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels that shares a nearly identical partial proteolytic digest pattern with p300. The slower-migrating form of p300 is greatly reduced by treating immune complexes with potato acid phosphatase. The relative stability and presence of p300 even in resting cells suggests that p300 has a basal cell function, but the appearance of differentially modified forms during the cell cycle suggests the possibility that p300 function is modulated specifically in growing cells

Features of the pp60v-src carboxyl terminus that are required for transformation.

Analysis of the biological and biochemical activities of pp60recombinant-src proteins encoded by 12 carboxyl-terminal mutants showed that a wide family of alternate src carboxyl termini permit complete transforming and kinase activities. src proteins having carboxyl termini which are up to 10 amino acids longer than that of pp60c-src (17 amino acids longer than that of pp60v-src) still permit transformation. Transformation-positive mutations preserve leucine-516, a residue which is highly conserved in protein-tyrosine kinase sequences; removal causes in vivo protein instability. Successive deletion mutants show that this residue is at the boundary of a region required for kinase activity. pp60src which is truncated just outside this point still transforms cells and binds both pp50 and pp90 cellular proteins

DNA cleavage by restriction endonuclease PflMI is inhibited in recognition sites modified by dcm methylation.

Plasmids pBSoc£-l + (1) and pALTEla (described below*) both contain two canonical P/IMI recognition sites [CCANNNNNTGG (2)]. These plasmids, when purified from Escherichia coli dam/dcm~ strain RB404 (3). release a 362- or 337-bp fragment, respectively, after digestion with P/IMI (Fig. 1A, lanes 1 and 6). However, when purified from dam*/dcm* E. coli strains, these plasmids cleaved only once after digestion with P/IMI (Fig. 1A, lanes 2 and 7). Digestion of unmethylated (lane 3) and methylated (lane 4) pBSocM* DNA with P/IMI and EcoRI Identified the oct-1 P/IMI site at nucleotide position 1680 (1) (see Fig. IB) as the recognition site that is resistant to cleavage. Digestion of unmethylated (lane 8) and methylated (lane 9) pALTEla DNA with P/IMI and PuuII identified the adenovirus P/IMI site at nucleotide position 1461 (4) (see Fig 1C) as the resistant site. Both cleavage-resistant P/IMI sites contain the dcm methylation sequence, CmCWGG (5). These results suggest that a subset of P/IMI recognition sequences that contain a dcm methylation site are resistant to P/IMI cleavage when methylated

Characterization of monoclonal antibodies raised against p300: both p300 and CBP are present in intracellular TBP complexes

The amino terminus of the adenovirus E1A protein is involved in E1A transforming functions, repression of tissue-specific gene expression, and E1A-mediated enhancer repression. These N-terminal functions are associated with the ability of this region of E1A to bind to p300 and CBP, two closely related cellular proteins thought to function as transcriptional adaptor molecules. Here we describe the characterization of a panel of 11 monoclonal antibodies raised against E1A-affinity-purified 300-kDa proteins. The panel can be divided into two groups based on immunoprecipitation patterns. The first group consists of five p300/CBP-cross-reactive and two p300-specific monoclonal antibodies, all of which immunoprecipitate p300 and/or CBP without associated cellular proteins. In contrast, the second group immunoprecipitates p300 or both p300 and CBP in association with a complex of at least seven other cellular proteins. Taking advantage of the specificities of these monoclonal antibodies, we have identified both p300 and CBP in in vivo complexes with TBP, a finding consistent with a role for both p300 and CBP in promoting interactions between upstream promoter elements and the basal transcription apparatus.</jats:p

Mutation of amino acids in pp60c-src that are phosphorylated by protein kinases C and A

The product of the c-src proto-oncogene, pp60c-src, is phosphorylated at Ser-17 by cyclic AMP-dependent protein kinase A and at Ser-12 by calcium-phospholipid-dependent protein kinase C (when stimulated by 12-O-tetradecanoyl phorbol acetate). We tested the effects of Ser----Ala and Ser----Glu mutations at these sites in pp60c-src and in pp60c-src(F527) (a mutant whose transforming activities are enhanced by Tyr-527----Phe mutation) by transfecting single-, double-, and triple-mutant src expression plasmids into NIH 3T3 cells. Tryptic phosphopeptide analyses of the mutant proteins confirmed prior biochemical identifications of the phosphorylation sites and showed that neither separate nor coordinate mutations at Ser-12 and Ser-17 affected Tyr-416, Tyr-527, or Ser-48 phosphorylation or prevented mitosis-specific phosphorylations of either pp60c-src or pp60c-src(F527). Ser-12 mutation did not affect phosphorylation of the Ser-17-containing peptide, but mutation of Ser-17 significantly increased phosphorylation at Ser-12. Specific kinase activities (both with and without in vivo 12-O-tetradecanoyl phorbol acetate treatment) and the abilities of pp60c-src and pp60c-src(F527) to induce foci, transformed morphologies, and anchorage-independent growth were unaffected by any of the serine mutations. Thus, pp60c-src transforming activity in NIH 3T3 cells is relatively insensitive to phosphorylation at these sites, but there is a suggestion that Ser-17 phosphorylation may have a subtle regulatory effect.</jats:p