No abstract

Simian virus 40 largetumor antigen (T Ag) can be separated by sucrose gradient sedimentation into a rapidly sedimenting, maximally phosphorylated fraction and a slowly sedimenting, less phosphorylated fraction. The Mr 48,000 host tumor antigen (48,000 HTA, also called nonviral T Ag) is preferentially complexed with the maximally phosphorylated T Ag. Pulse-labeled T Ag sediments as a 5-6S monomer, whereas T Ag radiolabeled for progressively longer periods slowly increases in sedimentation coefficient to give a, broad distribution between 5 S and greater than 28 S. Mutation in the viral A locus causes a decrease in T Ag phosphorylation and a marked decrease in 48,000 HTA binding, shifting the sedimentation coefficient of T Ag to the monomer value. The more highly phosphorylated T Ag also has the highest affinity for chromatin.Simian virus 40 (SV40) large tumor antigen (T Ag) is encoded by the viral A locus (1, 2), which governs initiation of DNA synthesis and the establishment of viral transformation (3, 4). Found in the nuclei of cells infected (5) and transformed (6) by SV40, T Ag is a DNA-binding protein (7-11) that binds DNA with heterogeneous affinities (7,12). Mutation in the viral A locus has been shown to decrease the DNA-binding affinity of T Ag (13,14). T Ag also sediments heterogeneously with sedimentation coefficients from 5S to greater than 22S (7,(15)(16)(17)(18)(19)(20). Mutation of the A locus causes T Ag to sediment as a 5-6S species (17, 18), corresponding to the 94,000 dalton monomer (13). Furthermore, T Ag, a phosphoprotein (21), is heterogeneously phosphorylated (22), generating multiple isoelectric focusing species with differing contents of phosphate. Differential phosphorylation may play a physiological role in generating the heterogeneous aggregating and DNA-binding forms of T Ag. Similarly, the addition and removal of the phosphate groups of acidic chromatin-associated proteins of eukaryotic cells has been suggested as a control mechanism for the regulation of their DNA-binding properties (23). In addition, differential phosphorylation states can affect the binding affinities of subunits for each other, as in glycogen phosphorylase (24).Two recently discovered host gene products, the Mr 48,000 and 55,000 host tumor antigens (HTA), also termed nonviral T Ags, have been shown to complex with T Ag in vitro (19,20,25,26 McCormick and Harlow (20) have proposed that the "53K NVT"-associated form oflarge T Ag becomes more highly phosphorylated than the free form. The 48,000 and 55,000 HTAs have been shown to be unrelated immunologically and structurally (19). The broad physiological significance of these nuclear phosphoproteins has been established by the fact that they are induced in mouse, rat, hamster, and human cells transformed by SV40 (19). They are expressed, in the absence of SV40, in cells transformed by a variety ofcarcinogenic agentsincluding the RNA tumor virus, murine sarcoma virus, and the chemical carcinogen methyicholanthrene (27)-and in spontaneously transformed fibro...

We report here the isolation and identification of the RNA specifically immunoprecipitated and covalently linked to the tumor suppressor gene product p53. After treatment with proteinase K, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) band of p53 yields a single, discrete 157-nucleotide RNA, which was cloned, sequenced, and identified as 5.8S rRNA. 5.8S rRNA was obtained only after proteolysis of the p53 SDS-PAGE band. Free 5.8S rRNA did not comigrate with p53 in SDS-PAGE. This RNA was only immunoprecipitated from cells containing p53. Protein-free RNA obtained by proteolysis of the p53 band hybridized to the single-stranded DNA vector containing the antisense sequence of 5.8S rRNA. The covalence of the p53-5.8S rRNA linkage was demonstrated by the following findings: (i) p53 and the linked 5.8S rRNA comigrated in SDS-PAGE; (ii) only after treatment of the p53-RNA complex with proteinase K did the 5.8S rRNA migrate differently from p53-linked 5.8S rRNA; and (iii) this isolated RNA was found linked to phosphoserine, presumably at the 5' end. Covalent linkage to the single, specific RNA suggests that p53 may be involved in regulating the expression or function of 5.8S rRNA.Inactivation of the tumor suppressor p53 is implicated mechanistically in both human tumorigenesis and the replication of the small DNA viruses. Both of these processes involve the abrogation of normal cellular growth control. Although the molecular mechanism of cell cycle inhibition by p53 has not been worked out, several of its biochemical properties have been demonstrated and can be correlated with specific domains of p53. The amino-terminal third of the molecule is rich in hydrophobic and acidic residues. p53-GAL4 fusion proteins containing the amino terminus of p53 have been shown previously to have transcriptional transactivating activity in vitro (7,25). p53 has also been shown previously to down-regulate various promoters (11). Fur polypeptide. Murine p53 is phosphorylated both at multiple sites at the amino terminus (17, 28) and at Ser-312 and Ser-389 (17,18,28) at the carboxyl terminus.We have previously postulated that p53 is covalently linked to RNA on the basis of our initial observations that Ser-389 is not dephosphorylated by alkaline phosphatase and that the carboxyl-terminal tryptic peptide containing Ser-389 coeluted from reverse-phase high-pressure liquid chromatography with ribonucleotides (28). By using a proteinchemical approach to characterization of the complex, we subsequently showed covalent linkage of p53 to some undefined RNA at the levels of both the tryptic carboxyl-terminal peptide and the intact protein (29). Stable and transient protein-nucleic acid covalent bonds have been described.The genome-linked proteins 4X protein A (27) and adenovirus terminal protein (3) are found covalently linked to DNA, and the poliovirus genome-linked protein, VpG, is covalently linked to RNA (9,15

The T antigen of simian virus 40, which may play a role in the control of viral DNA replication, is recovered from nuclei of cells transformed by simian virus 40 in several forms sedimenting at different rates. The large molecular weight forms are converted to the smallest (5 S) form by high salt, suggesting that they differ in the degree of aggregation. All the forms of the antigen bind efficiently to double-stranded DNA-cellulose columns at pH 6.2 and low salt, and elute in two fractions: one at pH 8.0 and low salt, the other at pH 8.0 and high salt. The antigen has little affinity for single-stranded DNA.Simian virus 40 (SV40) T antigen is detected by complement fixation or immunofluorescence in the nuclei of cells transformed (1) or lytically infected (2) by SV40. In lytic infection it is detectable before the onset of viral DNA synthesis, and its appearance is not inhibited if DNA synthesis is stopped by FdU and arabinosylcytosine (3,4). Hence, its appearance is controlled by the "early" region of the viral genome. The appearance of a normal T antigen requires the activity of the viral A gene of both SV40 and of the related polyoma virus. In fact, Osborn and Weber have reported an alteration in the sedimentation pattern of the SV40 T antigen in cells infected with a tsA mutant at the nonpermissive temperature (abstract in Cold Spring Harbor virus meeting 1973), and in cells infected or transformed by the tsA mutant of polyoma virus, the T antigen is not detectable by immunofluorescence at the nonpermissive temperature (5). These findings show that the T antigen may be the product of the A gene, or related to it. This relationship makes the antigen especially interesting, because the A-gene function has been shown to be required for viral DNA replication in both SV40 (6) and polyoma virus (7) infection and, with polyoma virus, for initiation of transformation (8).Not much is known about the properties of the T antigen. SV40 T antigen has been reported to sediment as multiple species with molecular weights of about 300,000, 120,000, and 70,000 by Potter et al. (9) (10).In this report we show that the larger molecular weight forms can be dissociated to yield the smallest form, and that all forms bind strongly to double-stranded DNA. MATERIALS AND METHODSCell Cultures. Cells of the SV40-transformed mouse line SV3T3 (11) were grown in rotating 2.5-liter bottles in 200 ml of Dulbecco's modified Eagle's medium with 10% calf serum at 37°. Before they were sealed, the bottles were gassed for 15 seconds with 20% C02-80% air. The cells were grown to confluency and then harvested by detaching them with trypsin-EDTA.Cells of the SV40-transformed hamster cell line SV28 (12), derived from BHK cells, and of the SV40-transformed BSC-1 line were grown and harvested by the above procedure.Preparation of T Antigen. Nuclei were prepared by homogenization of fresh SV3T3 cells (about 12 g wet cells) with a 40-ml Dounce homogenizer in 30 ml of 0.25 M sucrose, 3 mM CaCl2. The nuclei were pelleted by centrifugation for 5 min...

The oncogene product p53, isolated from SV3T3 cells where it forms a complex with simian virus 40 large tumor antigen (T antigen) in the nucleus, has been found to be phosphorylated at at least four distinct sites on the 390 amino acid protein. Separation of tryptic phosphopeptides has permitted identification of two sites as Ser-312 and Ser-389, and permitted analysis of the types of phosphate bonds. The peptide containing Ser-312 separates electrophoretically into three charged forms; two are resistant to dephosphorylation by both alkaline phosphatase and alkaline hydrolysis, suggesting a phosphodiester. The carboxyl-terminal phosphopeptide containing Ser-389 was alkaline phosphatase-resistant and liberated four ribonucleoside monophosphates upon base or RNase hydrolysis, suggesting that Ser-389 may be covalently linked to RNA. Phosphorylation of Ser-389 decreased markedly at the nonpermissive temperature in simian virus 40 tsA58-transformed cells, indicating a dependence on native T antigen function and a possible role in transformation by T antigen. Two additional phosphorylation sites, one involving serine and one involving threonine, probably reside in the amino-terminal segment of p53 and appear to be peptide-phosphate monoesters.