Transduction of a cellular oncogene: the genesis of Rous sarcoma virus.

Analysis of the biological and biochemical activities of pp6orecombinant-src proteins encoded by 12 carboxylterminal 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 pp60 c-src (17 amino acids longer than that of pp6Ov-sc) 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. pp6OSrC which is truncated just outside this point still transforms cells and binds both pp5O and pp9O cellular proteins.The v-src retroviral oncogene arose by transduction and modification of the c-src cellular gene (for a review, see reference 3). Unlike the viral product pp60v-", the cellular product pp6oc-rc does not induce transformation even when overexpressed in fibroblasts (23,40,50) although it cafi induce focus formation at high expression levels (24). This may be due to the reduced protein-tyrosine kinase activity of pp6Oc-s relative to pp6oV-"' (7,10,22). The catalytic domain for this activity resides in the carboxyl half of the molecule which is strongly homologous to domains in the other sequenced protein-tyrosine kinases (for a review, see reference 21). Two cellular phosphoproteins, pp90 (a major cytoplasmic heat shock protein [38]) and pp50, specifically interact with pp60v-" (4) much more extensively than with pp6Oc-s" (22).Structurally, the viral and cellular proteins differ by the substitution of 12 different carboxyl-terminal amino acids in pp6Ov-"" for the 19 carboxyl-terminal amino acids of pp6Oc-'( and, depending on the strain of v-src, 8 to 15 isolated substitutions (11,46,59) scattered throughout the remainder of the proteins. In pp60c'r', this carboxyl-terminal region contains the major site of in vivo tyrosine phosphorylation (6) which may play a role in negatively regulating pp60c-Xrc protein-tyrosine kinase activity (8). Most of the DNA sequence encoding the v-src carboxyl terminus is found in the chicken genome about 900 base pairs (bp) downstream from the c-src termination codon (57, 59).The functional significance of the existence of multiple mutations between v-src and c-src has not yet been resolved.Single point mutations in pp60c-%'r enable it to transform chicken embryo cells (J. B. Levy, H. Iba, and H. Hanafusa, Proc. Natl. Acad. Sci. USA, in press), and chimeric genes encoding the amino region of pp60c-A' and carboxyl region of pp60'r-"' (clv-src chimeras) or encoding the amino region of pp60v-s and the carboxyl region of pp60c-r (vlc-src chime- (49).We studied the carboxyl-terminal region using a series of deletion, substitution, and addition mutants of SR-D pp60"vA(. We report that a wide variety of SR-D pp6O-vS(' carboxyl-terminal mutants can transform fibroblasts efficiently and comple...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Yaciuk¹,

Shalloway²

1986

“…The best-studied such retrovirus is Rous sarcoma virus, whose transforming gene v-src was derived from a cellular tyrosine kinase gene, c-src (22,23). The protein-tyrosine kinase activity of the v-src gene product pp60vsrc has been activated by a C-terminal substitution and scattered point mutations (12).…”

mentioning

confidence: 99%

Platelet tyrosine-specific protein phosphorylation is regulated by thrombin.

Ferrell¹,

Martin²

1988

230

120

Intact human platelets, terminally differentiated cells with no growth potential, were found to possess unusually high levels of tyrosine-specific protein phosphorylation. The physiological platelet activator thrombin transiently elevated platelet phosphotyrosine content, apparently through stimulation of one or more tyrosine-specific protein kinases. Immunoblotting with antiphosphotyrosine antiserum showed that thrombin caused dramatic changes in the tyrosine phosphorylation of a number of individual protein bands and that these changes occurred in three distinct temporal waves. Most but not all of the protein bands phosphorylated at tyrosine in response to thrombin were also tyrosine phosphorylated in response to chilling or the combination of ionophore A23187 and tetradecanoylphorbol acetate. Thrombin stimulated the phosphorylation of the tyrosine kinase pp6OC-S', primarily at Ser-12 and Tyr-527, although the effects of these phosphorylations on platelet pp60csrC function were not apparent. Together, these results suggest that tyrosine-specific protein kinases of uncertain identity are involved in signal transduction in platelets.The phosphorylation of proteins at tyrosine residues is implicated in normal and abnormal cell growth: several growth factor receptors are tyrosine-specific protein kinases, as are the transforming proteins of one class of acutely oncogenic retroviruses (13). The best-studied such retrovirus is Rous sarcoma virus, whose transforming gene v-src was derived from a cellular tyrosine kinase gene, c-src (22, 23). The protein-tyrosine kinase activity of the v-src gene product pp60vsrc has been activated by a C-terminal substitution and scattered point mutations (12). The transforming capacity of pp60v-src results, at least in part, from its increased kinase activity.Through alternative splicing, the c-src gene gives rise to two distinct proteins, both of about 60 kilodaltons (kDa) (16,18). Little is known about the physiological function of either c-src protein. A number of other proto-oncogene products are involved in the transduction of normal mitogenic signals (e.g., c-erbB [epidermal growth factor receptor], c-fms [colony-stimulating factor 1 receptor], c-sis [platelet-derived growth factor]). However, the tissue distribution of pp6Oc-src expression suggests that this is not its function; those cells which express the highest levels of pp60csrc do not divide. For example, postmitotic differentiated or differentiating neurons express high levels of both c-src proteins (1,3,7,8,17,21). Mammalian platelets, terminally differentiated cells with no growth potential, express still higher levels of the smaller c-src protein (9). These observations suggest that tyrosine-specific protein kinases in general, and pp60c-sr in particular, may regulate cellular processes that are distinct from growth control.We have chosen human platelets as a system for investigating the role of protein-tyrosine phosphorylation in differentiated cells. Platelets are relatively simple cell fragments (they do not syn...

“…The gag AUG is used to initiate the translation of gag, gag-pol, and env mRNAs (14,15,26). However, src mRNA is initiated at an AUG that lies 90 bases downstream from the gag AUG in the spliced src mRNA (8,26,28,29 daltons. Cells infected with the mutant virus were found to express a 63,000-dalton src protein, indicating that in the mutant most, if not all, of the src protein is initiated at the gag AUG.…”

mentioning

confidence: 99%

Mutation of a termination codon affects src initiation.

Hughes¹,

Mellström²,

Kosik³

et al. 1984

The four Rous sarcoma virus messages gag, gag-pol, env, and src all derive from a fuli-length RNA precursor. All four messages contain the same 5' leader segment. Three of the messages, gag, gag-pol, and env, use an AUG present in this leader to initiate translation. The src AUG initiation codon lies 3' of the leader segment, 90 bases downstream of the gag initiation codon in the spliced src message. However, in the spliced src message a UGA termination codon lies between the gag AUG and the src AUG. All three codons are in the same reading frame. By using oligonucleotide-directed mutagenesis, the UGA termination codon has been converted to CGA. Cells infected with the mutant (called 1057 CGA) were spindle shaped, distinct from the rounded shape of cells infected with the parental Rous sarcoma virus. The mutant virus initiates src ranslation at the gag AUG, producing a 63,000-dalton src protein. We suggest that the wild-type src message produces two polypeptides, a very small (nine-amino acid) peptide that is initiated at the gag AUG and the 60,000-dalton src protein that is initiated at the src AUG.Rous sarcoma virus (RSV) is apparently transcribed into a single RNA species beginning at the left end of the R sequence in the left-hand long terminal repeat and terminating at the right end of the R sequence in the right-hand long terminal repeat. This transcript constitutes genomic RNA and serves as the precursor for RSV messenger RNAs. Four messenger RNAs are known: gag mRNA, which is apparently unspliced and is thought to be identical to genomic RNA; gag-pol mRNA, which may have a small region near the gag-pol junction removed by RNA splicing; env mRNA, which has most of gag and pol removed by splicing; and src mRNA, which has most of gag, pol, and env removed by splicing (30; Fig.