We have identified the single phosphorylated tyrosine in p60, the transforming protein of Rous sarcoma virus, as part of the sequence NH2-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr(P)-Thr-Ala-Arg-COOH.Therefore,. this is a sequence that is recognized efficiently by a tyrosine protein kinase in vivo. Phosphorylation of tyrosine in cellular proteins appears to play a role in malignant transformation by four classes of genetically distinct RNA tumor viruses. Phosphorylated tyrosines in several other proteins resemble the tyrosine in p60 in that they are located 7 residues to the COOHterminal side of a basic amino acid and either 4 residues to the COOH-terminal side of, or in close proximity to, a glutamic acid residue. Therefore, it is possible that these features play a role in the selection of sites of phosphorylation by some tyrosine protein kinases. However, several clear exceptions to this rule exist. Rous sarcoma virus (RSV) transforms cells to a malignant state through the expression of a single gene, src (reviewed in ref. 1). This transforming gene encodes a single 60,000-dalton phosphoprotein, p6Orc (2). p6Osrc is a protein kinase with the unusual specificity ofphosphorylating tyrosine residues in its target proteins (3-6). This unscheduled phosphorylation of tyrosine almost certainly plays a role in cellular transformation by this virus (7) and probably also by three other groups of RNA tumor viruses genetically distinct from RSV (8-10). Tyrosine protein kinases may also play an important role in normal metabolism because they are implicated in the cellular response to epidermal growth factor (11).An important question is how tyrosine protein kinases select and recognize their substrates. In the case of the well-characterized protein kinases that phosphorylate serine and threonine, the amino acid sequence of the substrate plays a crucial role in selection of the phosphorylation site. The cAMP-dependent protein kinase has a strong preference for serines that are located 2-5 residues to the COOH-terminal side of one or two basic amino acids, most commonly arginine (12, 13). In contrast, the casein protein kinases as a class show a strong preference for sites in the vicinity ofacidic residues (14, 15). In casein itself, all but one of the sites ofphosphorylation are located 2 residues to the NH2-terminal side of an acidic amino acid (14).Because of these precedents, we have characterized the sequence of amino acids at sites of tyrosine phosphorylation. We deduced the complete amino acid sequence surrounding the single phosphorylated tyrosine in p6(5rc and analyzed indirectly the sequence at sites of tyrosine phosphorylation in a number of other proteins. (4,16,(19)(20)(21). MATERIALS AND METHODSGel Electrophoresis, Elution of Proteins, and Tryptic Digestion. Immunoprecipitated proteins were purified on preparative NaDodSO4polyacrylamide gels (22) and localized by autoradiography. The position of 3H-labeled proteins was identified by 32p_ or 3S-labeled markers that were run in parallel. All other procedures we...

Eukaryotic cells contain genes termed proto-oncogenes (c-onc) which have the potential to transform cells in culture and induce tumours in vivo. Most of these genes have been identified by their occasional incorporation into retroviral genomes which can act as natural transducing vectors for these and perhaps other cellular genes. Cell-derived oncogenes of retroviruses (v-onc) are associated mostly with the induction of mesenchymal tumours whereas carcinoma induction is rare. One of these rare carcinoma-inducing viruses is the acutely transforming avian retrovirus MH2 (refs 3-5). Recently we and others have shown that this virus carries a novel putative oncogene, v- mil , in addition to the known oncogene v-myc. While the transforming ability of v- mil has not been directly established, we have recently discovered by hybridization analysis that v- mil is homologous to v-raf (ref. 9), the transforming gene of the murine retrovirus 3611 MSV (ref. 10). Both viruses express the mil /raf oncogene product as a gag-fusion polyprotein, while the myc oncogene of MH2 is expressed via a subgenomic mRNA. Here we report the complete nucleotide sequence of v- mil and compare it with that of v-raf. The 80% homology between the nucleotide sequences and the 94% homology between the predicted amino acid sequences of the two viral genes clearly indicate that these are the avian and murine forms of the same gene. Comparison of the two sequences with that of the human cellular homologue (T. I. Bonner et al., manuscript in preparation) indicates that v-raf has more 3' untranslated sequences while v- mil has additional sequences from two 5' exons of the cellular homologue. Although the mil /raf amino acid sequences reveal partial homology to that of the v-src product, no tyrosine-specific protein kinase activity is detected for the gag- mil and the gag-raf hybrid proteins.

The Gag-derived protein p6 of human immunodeficiency virus type 1 (HIV-1) plays a crucial role in the release of virions from the membranes of infected cells. It is presumed that p6 and functionally related proteins from other viruses act as adapters, recruiting cellular factors to the budding site. This interaction is mediated by so-called late domains within the viral proteins. Previous studies had suggested that virus release from the plasma membrane shares elements with the cellular endocytosis machinery. Since protein phosphorylation is known to be a regulatory mechanism in these processes, we have investigated the phosphorylation of HIV-1 structural proteins. Here we show that p6 is the major phosphoprotein of HIV-1 particles. After metabolic labeling of infected cells with [ortho-32 P]phosphate, we found that phosphorylated p6 from infected cells and from virus particles consisted of several forms, suggesting differential phosphorylation at multiple sites. Apparently, phosphorylation occurred shortly before or after the release of p6 from Gag and involved only a minor fraction of the total virion-associated p6 molecules. Phosphoamino acid analysis indicated phosphorylation at Ser and Thr, as well as a trace of Tyr phosphorylation, supporting the conclusion that multiple phosphorylation events do occur. In vitro experiments using purified virus revealed that endogenous or exogenously added p6 was efficiently phosphorylated by virion-associated cellular kinase(s). Inhibition experiments suggested that a cyclin-dependent kinase or a related kinase, most likely ERK2, was involved in p6 phosphorylation by virion-associated enzymes.

The LSTRA murine thymoma cell line contains an elevated level of tyrosine protein kinase activity. When a microsomal preparation from these cells is incubated in vitro with ATP, the principal tyrosine protein kinase substrate is a 56,000-dalton protein, p56. We have found that an activity phosphorylating p56 on tyrosine can also be detected at low levels in microsomes from most, but not all, T lymphoma cell lines and from normal thymic tissue. Only 1 of 30 other lymphoma cell lines was found to contain an elevated level of such a tyrosine protein kinase. An activity that phosphorylated p56 in vitro was not detectable in the cells of other hematopoietic lineages. Anti-peptide antibodies reactive with the site of in vitro tyrosine phosphorylation of p56 allowed us to determine that the apparent abundance of the p56 polypeptide parallels closely the level of the tyrosine protein kinase activity in the cell lines examined. This suggests that p56 is the protein kinase responsible for the elevated tyrosine protein kinase activity in LSTRA cells and that the phosphorylation of p56 observed in vitro results from autophosphorylation. Two-dimensional tryptic peptide mapping revealed that p56 is distinct from the proteins encoded by the cellular genes which are the progenitors of retroviral tyrosine protein kinases, src, yes, fgr, abl, fes, and ros. Additionally, none of these proto-oncogenes was found to be transcribed at elevated levels in LSTRA or Thy19 cells. Like the catalytic subunit of the cyclic AMP-dependent protein kinase, the cellular and viral forms of p60src, and the protein phosphatase calcineurin B, p56 contains covalently bound fatty acid.