Three clones for the human tumor antigen p53 were isolated from a cDNA library prepared from A431 cells. One of these clones, pR4-2, contains the entire coding region for human p53. This clone directs the synthesis of a polypeptide with the correct molecular weight and immunological epitopes of an authentic p53 molecule in an in vitro transcription-translation reaction. Although the pR4-2 clone contains the coding region for p53, it is not a full-length copy of the human p53 mRNA. Northern analysis showed that the p53 mRNA is approximately 2,500 nucleotides long, whereas the pR4-2 insert is only 1,760 base pairs in length. Analysis of the DNA sequence of this clone suggests that the human p53 polypeptide has 393 amino acids. We compared the predicted amino acid sequence of the pR4-2 clone with similar clones for the mouse p53 and found long regions of amino acid homology between these two molecules.A number of studies have shown that primary rat cells can be transformed in vitro by cotransfecting an activated ras gene with any of a series of cellular or viral genes (28, 50). Transformed foci have been observed after the cotransfection of an activated ras gene with the gene for the cellular myc protein, polyoma large-T antigen, adenovirus ElA proteins, or MC29 gag-myc fusion protein. Recent studies by three groups demonstrated that cotransfection of the gene that encodes the mouse cellular tumor antigen p53 with an activated human ras gene also yields transformed foci (12,21,42). Results of earlier work had suggested that p53 might play a role in some types of transformation. These suggestions were based not only on comparison of the biochemistry of p53 in normal and transformed cells but also on studies of the immune response of animals to some types of tumors. This work has shown that (i) sera from laboratory animals with tumors or from human patients with some types of neoplasia often contain circulating antibodies specific for p53 (9,11,29,32,49); (ii) transformed cells often have higher levels of p53 than their normal cell counterparts (3,10,11,20,48,53); (iii) in cells transformed by simian virus 40 (SV40) or adenovirus, p53 is found in a stable, highmolecular-weight complex with either the SV40-coded large-T antigen or the adenovirus-coded E1B 57-kilodalton protein (29,32,35,52); (iv) p53 appears to play an important role in the movement of quiescent cells into the S phase after serum stimulation (37, 38); and (v) the synthesis of p53 is temporally regulated after stimulation of cells with mitogens (40,46). These studies suggest that p53 may play an important role in the regulation of cell division in some cell types, but how p53 may be involved in these processes is not known. We present here the isolation, characterization, and nucleic acid sequence of a p53 cDNA clone from human A431 cells that has the coding potential for a full-length p53. * Corresponding author. MATERIALS AND METHODSCells and antibodies. All cells were grown in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum. ...

We have observed a modification of the cellular protein kinase pp601, elicited in muriwe 3T3 fibroblasts by platelet-derived growth factor (PDGF). The modification occurred rapidly after addition of PDGF to the culture medium and was first detected as a reduction in the electrophoretic mobility of a portion of the pp60c1 molecules. A similarly modified form of the viral bomologue pp6OvM occurs in vivo in the absence of stimulation by PDGF. The'occurrence of modified forms of both pp6O' and pp6Ov' was associated with a novel phosphorylation at tyrosine in the amino-terminal domains of the proteins. The time-course and dose-response for this modification of pp6O0 paralleled PDGF-induced increases in phosphorylation of pp36, a major cellular substrate for several tyrosine-specific protein kinases. In parallel experiments, treatment of cells with PDGF increased the kinase activity of pp6OC-r in an immunocomplex assay. These results suggest pp60c may play a role in the mitogenic response to PDGF.Continuous growth of cells in culture requires several protein factors present in serum. One of the major polypeptide mitogens in serum for fibroblasts is platelet-derived growth factor (PDGF) (1, 2). PDGF is released from the alpha granules of platelets during clot formation (2) and is believed to stimulate fibroblast growth in wound healing. The cellular response to PDGF displays a number of parallels with the effects of expression of the v-src oncogene (the transforming gene of Rous sarcoma virus). These similarities include phosphorylation of cellular proteins at tyrosine residues (3-5), phosphorylation of the ribosomal protein S6 (6, 7), increased turnover of phosphatidylinositol (8, 9), reorganization of actin cables (2, 10), mitogenesis (2,11,12), and even neoplastic transformation (by v-sis, which encodes one subunit of PDGF) (13,14).Expression of v-src induces mitosis in fibroblasts that have been growth-arrested by serum starvation (11,12). The v-src oncogene has a closely related cellular homologue, c-src (15). This gene is expressed in normal fibroblasts (16) and encodes a 60-kDa protein, pp60csrc, that has been shown to possess tyrosine-specific protein kinase activity in vitro (17). Because of the similarities in the responses to PDGF stimulation and v-src expression, we investigated the possible role ofpp60-src in the mitogenic response to PDGF. We found that the -amino-terminal domain ofpp60c-src rapidly becomes modified when quiescent Swiss 3T3 cells are stimulated with PDGF. The modification is associated with phosphorylation of tyrosine residues in the amino-terminal portion of the protein and is accompanied by both increased kinase activity of pp60c-src in vitro and increased phosphorylation ofthe cellular substrate pp36 (18) were incubated an additional 4 hr. Growth factors were added directly to this medium and incubation was continued for various times. The PDGF used in these experiments, purified to apparent homogeneity (19), was a gift of T. Deuel.General Procedures. Immunoprecipitation pp...

Structural and functional homologies have been found among proteins encoded by several retroviral oncogenes, demonstrating the existence of families of these genes. Because the retroviral oncogenes have cellular homologues, the existence of similar families among these 'cellular oncogenes' is also implied (for a review, see ref. 2). Cellular genes belonging to these families have been found in such evolutionarily distant species as humans, fruit flies, nematodes and brewer's yeast (E. Scolnick and S. Reed, personal communications), consistent with the hypothesis that these genes have evolved from a small number of ancestral sequences. We extend these observations by showing here that the proteins encoded by the oncogenes myc, myb and adenovirus E1a are structurally related. Our findings suggest that oncogenes of RNA and DNA tumour viruses may in at least some instances share evolutionary origins and function according to common principles.

A 36,000-dalton cellular protein (p36) has been identified previously as an abundant substrate for phosphorylation by tyrosine-specific protein kinases. Since several of the responsible kinases are associated with the plasma membrane, we explored the subcellular distribution of p36. Biochemical fractionations located p36 on the plasma membrane of both normal and retrovirus-transformed cells. Approximately half of the p36 was bound to the membrane with the affinity of a peripheral membrane protein; the remainder was even more tightly bound. The distribution of p36 among subcellular fractions and its affinity for the plasma membrane were not affected by tyrosine phosphorylation. We determined that p36 is synthesized in the soluble compartment of the cell and then moves rapidly to the membranous compartment. Immunofluorescence microscopy with antibodies directed against p36 revealed two distinct distributions of the antigen: (i) a sharply demarcated crenelated pattern within or immediately beneath the plasma membrane, which we presume to be a correlary of the distribution of p36 in biochemical fractionations; and (ii) diffuse staining in a cytoplasmic location that could not be attributed to a specific feature of cytoarchitecture and could not be easily reconciled with the results of biochemical fractionations. Efforts to detect the secretion of p36 were unsuccessful. No evidence was obtained for exposure of p36 on the cell surface, and no changes in localization were observed as a consequence of neoplastic transformation. During the course of this study, we had the opportunity to pursue a previous report that p36 is a component of the enzyme malate dehydrogenase (Rubsamen et al., Proc. Natl. Acad. Sci. U.S. A. 79:228-232, 1982). We were unable to substantiate this claim. We conclude that at least a substantial fraction of p36 is located on the cytoplasmic aspect of the plasma membrane, where it could be well situated to serve as a substrate for several identified tyrosine-specific kinases. But the function of p36 and its role, if any, in neoplastic transformation of cells by retroviruses possessing tyrosine-specific kinases remain enigmatic.