Amino acid substitutions sufficient to convert the nontransforming p60c-src protein to a transforming protein

1986 ◽  
Vol 6 (12) ◽  
pp. 4155-4160
Author(s):  
J Y Kato ◽  
T Takeya ◽  
C Grandori ◽  
H Iba ◽  
J B Levy ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Tumor Formation ◽  
Protein Kinase Activity ◽  
Focus Formation ◽  
Rous Sarcoma ◽  
Src Gene ◽  
Cellular Homolog ◽  
Sarcoma Virus ◽  
Hybrid Genes

We have previously shown that Rous sarcoma virus variants that carry the cellular homolog (c-src) of the viral src gene (v-src) do not transform chicken embryo fibroblasts. We also have shown that replacement of sequences upstream or downstream from the BglI site of the cellular src gene with the corresponding regions of v-src restored transforming activity to the hybrid genes. Since there are only six amino acid changes between p60c-src and p60v-src within the sequences upstream from BglI, we constructed chimeric molecules involving v-src and c-src to determine the effect of each amino acid substitution on the biological activities of the gene product. We found that the change from Thr to Ile at position 338 or the replacement of a fragment of c-src containing Gly-63, Arg-95, and Thr-96 with a corresponding fragment of v-src containing Asp-63, Trp-95, and Ile-96 converted p60c-src into a transforming protein by the criteria of focus formation, anchorage-independent growth, and tumor formation in newborn chickens. These mutations also resulted in elevation of the protein kinase activity of p60c-src.

scholarly journals Amino acid substitutions sufficient to convert the nontransforming p60c-src protein to a transforming protein.

1986 ◽  
Vol 6 (12) ◽  
pp. 4155-4160 ◽  
Author(s):  
J Y Kato ◽  
T Takeya ◽  
C Grandori ◽  
H Iba ◽  
J B Levy ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Tumor Formation ◽  
Protein Kinase Activity ◽  
Focus Formation ◽  
Rous Sarcoma ◽  
Src Gene ◽  
Cellular Homolog ◽  
Sarcoma Virus ◽  
Hybrid Genes

We have previously shown that Rous sarcoma virus variants that carry the cellular homolog (c-src) of the viral src gene (v-src) do not transform chicken embryo fibroblasts. We also have shown that replacement of sequences upstream or downstream from the BglI site of the cellular src gene with the corresponding regions of v-src restored transforming activity to the hybrid genes. Since there are only six amino acid changes between p60c-src and p60v-src within the sequences upstream from BglI, we constructed chimeric molecules involving v-src and c-src to determine the effect of each amino acid substitution on the biological activities of the gene product. We found that the change from Thr to Ile at position 338 or the replacement of a fragment of c-src containing Gly-63, Arg-95, and Thr-96 with a corresponding fragment of v-src containing Asp-63, Trp-95, and Ile-96 converted p60c-src into a transforming protein by the criteria of focus formation, anchorage-independent growth, and tumor formation in newborn chickens. These mutations also resulted in elevation of the protein kinase activity of p60c-src.

Lack of induction of neuroretinal cell proliferation by Rous sarcoma virus variants that carry the c-src gene

1985 ◽  
Vol 5 (10) ◽  
pp. 2856-2859
Author(s):  
H Iba ◽  
R Jove ◽  
H Hanafusa
Keyword(s):  
Cell Proliferation ◽  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Cell Populations ◽  
Protein Kinase Activity ◽  
Morphological Transformation ◽  
Rous Sarcoma ◽  
Src Gene ◽  
Cellular Homolog ◽  
Sarcoma Virus

Expression of p60v-src of Rous sarcoma virus in cultured chicken embryo neuroretinal cells was previously shown to result in the transformation and sustained proliferation of normally quiescent cell populations. We show here that Rous sarcoma virus variants that encode p60c-src, the cellular homolog of p60v-src, lack the ability to induce morphological transformation and cell proliferation of cultured neuroretinal cells. Neuroretinal cells infected with c-src-containing viruses, however, possess no less p60 protein kinase activity assayed in the immune complex than those infected with the transformation-defective Rous sarcoma virus mutants PA101 or PA104, which do stimulate the growth of these cells.

Amino acid alterations within a highly conserved region of the Rous sarcoma virus src gene product pp60src inactivate tyrosine protein kinase activity

1984 ◽  
Vol 4 (5) ◽  
pp. 862-866
Author(s):  
D L Bryant ◽  
J T Parsons
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Mutant Virus ◽  
Protein Kinase Activity ◽  
Rous Sarcoma ◽  
Src Gene ◽  
Tyrosine Protein Kinase ◽  
Sarcoma Virus

Bisulfite mutagenesis techniques have been used to introduce single-point mutations within a region of the Rous sarcoma virus src gene defined by a BglI restriction endonuclease cleavage site. The mutants of Rous sarcoma virus that are produced by these techniques encode src proteins which contain single amino acid changes within a highly conserved amino acid sequence encompassing residues 430 to 433. DNA from the mutants CHpm26 ( Ala430 to Val), CHpm9 ( Pro431 to Ser), CHpm6 ( Glu432 to Lys), and CHpm65 ( Ala433 to Thr) each failed to transform chicken cells upon transfection, whereas DNA from CHpm59 (a third base alteration in the codon for Glu432 ) readily transformed chicken cells. Analysis of immune complexes containing the altered src proteins indicates that these proteins have decreased tyrosine protein kinase activity in vitro. In vivo labeling of cells infected with the mutant virus revealed diminished levels of the tyrosine-phosphorylated 34,000-molecular-weight protein. These data indicate that mutations within the sequence Ala430 - Pro431 - Glu432 - Ala433 lead to alterations in pp60src-specific tyrosine protein kinase activity and a concomitant loss of transforming potential of the mutant virus.

scholarly journals Lack of induction of neuroretinal cell proliferation by Rous sarcoma virus variants that carry the c-src gene.

1985 ◽  
Vol 5 (10) ◽  
pp. 2856-2859 ◽  
Author(s):  
H Iba ◽  
R Jove ◽  
H Hanafusa
Keyword(s):  
Cell Proliferation ◽  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Cell Populations ◽  
Protein Kinase Activity ◽  
Morphological Transformation ◽  
Rous Sarcoma ◽  
Src Gene ◽  
Cellular Homolog ◽  
Sarcoma Virus

Expression of p60v-src of Rous sarcoma virus in cultured chicken embryo neuroretinal cells was previously shown to result in the transformation and sustained proliferation of normally quiescent cell populations. We show here that Rous sarcoma virus variants that encode p60c-src, the cellular homolog of p60v-src, lack the ability to induce morphological transformation and cell proliferation of cultured neuroretinal cells. Neuroretinal cells infected with c-src-containing viruses, however, possess no less p60 protein kinase activity assayed in the immune complex than those infected with the transformation-defective Rous sarcoma virus mutants PA101 or PA104, which do stimulate the growth of these cells.

scholarly journals Amino acid alterations within a highly conserved region of the Rous sarcoma virus src gene product pp60src inactivate tyrosine protein kinase activity.

1984 ◽  
Vol 4 (5) ◽  
pp. 862-866 ◽  
Author(s):  
D L Bryant ◽  
J T Parsons
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Mutant Virus ◽  
Protein Kinase Activity ◽  
Rous Sarcoma ◽  
Src Gene ◽  
Tyrosine Protein Kinase ◽  
Sarcoma Virus

Bisulfite mutagenesis techniques have been used to introduce single-point mutations within a region of the Rous sarcoma virus src gene defined by a BglI restriction endonuclease cleavage site. The mutants of Rous sarcoma virus that are produced by these techniques encode src proteins which contain single amino acid changes within a highly conserved amino acid sequence encompassing residues 430 to 433. DNA from the mutants CHpm26 ( Ala430 to Val), CHpm9 ( Pro431 to Ser), CHpm6 ( Glu432 to Lys), and CHpm65 ( Ala433 to Thr) each failed to transform chicken cells upon transfection, whereas DNA from CHpm59 (a third base alteration in the codon for Glu432 ) readily transformed chicken cells. Analysis of immune complexes containing the altered src proteins indicates that these proteins have decreased tyrosine protein kinase activity in vitro. In vivo labeling of cells infected with the mutant virus revealed diminished levels of the tyrosine-phosphorylated 34,000-molecular-weight protein. These data indicate that mutations within the sequence Ala430 - Pro431 - Glu432 - Ala433 lead to alterations in pp60src-specific tyrosine protein kinase activity and a concomitant loss of transforming potential of the mutant virus.

scholarly journals Expression of the chicken c-src gene in COS cells

1984 ◽  
Vol 4 (5) ◽  
pp. 846-851
Author(s):  
T M Gilmer
Keyword(s):  
Rous Sarcoma Virus ◽  
Recombinant Plasmid ◽  
Expression System ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cos Cells ◽  
Rous Sarcoma ◽  
Src Gene ◽  
Cellular Homolog ◽  
Sarcoma Virus

The cellular homolog of the Rous sarcoma virus transforming gene (v-src) was cloned into a plasmid containing the simian virus 40 origin of replication and transcriptional signals. This recombinant plasmid, designated pSVOHCS11 , directs the synthesis of relatively high levels of c-src mRNA and c-src protein ( pp60c -src), when the plasmid is studied 48 to 72 h after calcium phosphate-mediated DNA transfection of COS (monkey) cells. The level of c-src mRNA synthesis is 50-fold higher than the amount of c-src RNA produced in uninfected chicken embryo fibroblasts. Furthermore, the level of pp60c -src expressed in pSVOHCS11 -transfected COS cells is approximately the same as that of pp60v -src in Rous sarcoma virus-transformed cells. Using this recombinant plasmid, we demonstrated that c-src mRNA contains sequences which map 3' to the previously identified c-src-v-src regions of homology. In view of the small amount of c-src mRNA and protein that can be isolated from uninfected cells, this transient expression system offers a convenient source of material for further analyses of the c-src gene product.

scholarly journals Human cellular src gene: nucleotide sequence and derived amino acid sequence of the region coding for the carboxy-terminal two-thirds of pp60c-src.

1985 ◽  
Vol 5 (5) ◽  
pp. 1122-1129 ◽  
Author(s):  
S K Anderson ◽  
C P Gibbs ◽  
A Tanaka ◽  
H J Kung ◽  
D J Fujita
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Rous Sarcoma Virus ◽  
Cellular Growth ◽  
Tyrosine Kinase Domain ◽  
Rous Sarcoma ◽  
Src Gene ◽  
Sarcoma Virus ◽  
Carboxy Terminal

The nucleotide sequence of the 3' two-thirds of a highly conserved, molecularly cloned human cellular src gene (c-src) has been determined. This region of the c-src gene encodes the tyrosine kinase domain of the cellular src protein (pp60c-src) and corresponds to exons 6 through 12 of the chicken c-src gene, as well as nucleotides 545 to 1542 of the Rous sarcoma virus src gene (v-src). The human c-src sequence is very strongly conserved with respect to both the chicken c-src and the Rous sarcoma virus v-src genes, with nearly 90% nucleotide homology observed in this region. Amino acid sequence conservation in this region is even greater; 98% of the amino acids are conserved between human and chicken c-src. Furthermore, the exon sizes and the locations of the exon-intron boundaries are identical in the human and chicken c-src genes. However, sequences within the introns have not been conserved, and the introns within the human c-src gene are significantly larger than the corresponding introns within the chicken c-src gene. The strong amino acid conservation between the carboxy-terminal two-thirds of pp60c-src of species as divergent as humans and chickens suggests that this portion of the pp60c-src protein specifies one or more functional domains that are of great importance to some aspect of normal cellular growth or differentiation.

scholarly journals Transformation of NIH 3T3 cells by cotransfection with c-src and nuclear oncogenes.

1987 ◽  
Vol 7 (10) ◽  
pp. 3582-3590 ◽  
Author(s):  
D Shalloway ◽  
P J Johnson ◽  
E O Freed ◽  
D Coulter ◽  
W A Flood
Keyword(s):  
3T3 Cells ◽  
Focus Formation ◽  
Adenovirus E1a ◽  
Rous Sarcoma ◽  
Cellular Homolog ◽  
Sarcoma Virus ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

pp60c-src, the cellular homolog of the Rous sarcoma virus transforming protein, does not completely transform cells even when present at high levels, but has been shown to be involved in polyomavirus-induced transformation when activated by polyomavirus middle T (pmt)-antigen binding. Here we show that cotransfection, but not solo transfection, of expression plasmids for c-src and either adenovirus E1A, v-myc, c-myc, or the 5' half of polyomavirus large T (pltN) antigen into NIH 3T3 cells induces anchorage-independent growth, enhanced focus formation, and, for pltN cotransfection, tumorigenicity in adult NFS mice. Enhancement of transformation was not observed with polyomavirus small t (pst) antigen. Cotransfection of c-src with pltN induced modification of pp60c-src that altered its electrophoretic mobility and in vivo phosphorylation state and stimulated its in vitro kinase activity. Similar alterations were not seen after c-src-E1A cotransfection, suggesting that at least two different mechanisms of enhancement are involved.

scholarly journals N-terminal deletions in Rous sarcoma virus p60src: effects on tyrosine kinase and biological activities and on recombination in tissue culture with the cellular src gene.

1985 ◽  
Vol 5 (10) ◽  
pp. 2789-2795 ◽  
Author(s):  
F R Cross ◽  
E A Garber ◽  
H Hanafusa
Keyword(s):  
Amino Acids ◽  
Tyrosine Kinase ◽  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Biological Activities ◽  
Tyrosine Kinase Activity ◽  
Rous Sarcoma ◽  
Src Gene ◽  
Sarcoma Virus ◽  
Transforming Activity

We have constructed deletions within the region of cloned Rous sarcoma virus DNA coding for the N-terminal 30 kilodaltons of p60src. Infectious virus was recovered after transfection. Deletions of amino acids 15 to 149, 15 to 169, or 149 to 169 attenuated but did not abolish transforming activity, as assayed by focus formation and anchorage-independent growth. These deletions also had only slight effects on the tyrosine kinase activity of the mutant src protein. Deletion of amino acids 169 to 264 or 15 to 264 completely abolished transforming activity, and src kinase activity was reduced at least 10-fold. However, these mutant viruses generated low levels of transforming virus by recombination with the cellular src gene. The results suggest that as well as previously identified functional domains for p60src myristylation and membrane binding (amino acids 1 to 14) and tyrosine kinase activity (amino acids 250 to 526), additional N-terminal sequences (particularly amino acids 82 to 169) can influence the transforming activity of the src protein.

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