scholarly journals Neither arginine nor histidine can carry out the function of lysine-295 in the ATP-binding site of p60src.

1986 ◽  
Vol 6 (3) ◽  
pp. 751-757 ◽  
Author(s):  
M P Kamps ◽  
B M Sefton
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Activity ◽  
Morphological Transformation ◽  
Atp Binding Site ◽  
Rous Sarcoma ◽  
Tyrosine Protein Kinase ◽  
Sarcoma Virus ◽  
Dependent Protein ◽  
Infected Cells

All 15 protein kinases whose amino acid sequence is known contain a lysine residue at a position homologous to that of lysine-295 in p60src, the transforming protein of Rous sarcoma virus. The ATP analog p-fluorosulfonyl 5'-benzoyl adenosine inactivates both p60src and the catalytic subunit of the cyclic AMP-dependent protein kinase by modification of this lysine. We used oligonucleotide-directed mutagenesis to examine the possible functions of this residue. Lysine-295 in p60src was replaced with a glutamic acid, an arginine, or a histidine residue, and mutant p60src proteins were characterized in chicken cells infected by mutant viruses. None of these three mutant p60src proteins had tyrosine protein kinase activity in vitro, and none induced morphological transformation of infected cells. Since neither a histidine nor an arginine residue can replace the function of lysine-295, we suggest that it carries out the specialized function of proton transfer in the phosphotransferase reaction. All three mutant viruses underwent reversion to wild type during passage in tissue culture. Because the rate with which this occurred differed significantly among the mutants, reversion appears to have resulted from errors in transcription, rather than from recombination with the cellular src gene.

Neither arginine nor histidine can carry out the function of lysine-295 in the ATP-binding site of p60src

1986 ◽  
Vol 6 (3) ◽  
pp. 751-757
Author(s):  
M P Kamps ◽  
B M Sefton
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Activity ◽  
Morphological Transformation ◽  
Atp Binding Site ◽  
Rous Sarcoma ◽  
Tyrosine Protein Kinase ◽  
Sarcoma Virus ◽  
Dependent Protein ◽  
Infected Cells

All 15 protein kinases whose amino acid sequence is known contain a lysine residue at a position homologous to that of lysine-295 in p60src, the transforming protein of Rous sarcoma virus. The ATP analog p-fluorosulfonyl 5'-benzoyl adenosine inactivates both p60src and the catalytic subunit of the cyclic AMP-dependent protein kinase by modification of this lysine. We used oligonucleotide-directed mutagenesis to examine the possible functions of this residue. Lysine-295 in p60src was replaced with a glutamic acid, an arginine, or a histidine residue, and mutant p60src proteins were characterized in chicken cells infected by mutant viruses. None of these three mutant p60src proteins had tyrosine protein kinase activity in vitro, and none induced morphological transformation of infected cells. Since neither a histidine nor an arginine residue can replace the function of lysine-295, we suggest that it carries out the specialized function of proton transfer in the phosphotransferase reaction. All three mutant viruses underwent reversion to wild type during passage in tissue culture. Because the rate with which this occurred differed significantly among the mutants, reversion appears to have resulted from errors in transcription, rather than from recombination with the cellular src gene.

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 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 Molecular events in cells transformed by Rous Sarcoma virus.

1980 ◽  
Vol 87 (2) ◽  
pp. 319-325 ◽  
Author(s):  
R L Erikson ◽  
A F Purchio ◽  
E Erikson ◽  
M S Collett ◽  
J S Brugge
Keyword(s):  
Protein Kinase ◽  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Dependent Protein ◽  
Transforming Gene

The Rous sarcoma virus (RSV) transforming gene product has been identified and characterized as a phosphoprotein with a molecular weight of 60,000, denoted pp60src. Partially purified pp60src displays a closely associated phosphotransferase activity with the unusual specificity of phosphorylating tyrosine residues in a variety of proteins. That the enzymatic activity observed is actually encoded by the RSV-transforming gene is indicated by the comparison of the pp60src-protein kinase isolated from cells tranformed by a wild-type RSV or by a RSV temperature-sensitive transformation mutant; these experiments revealed that the latter enzyme had a half-life of 3 min at 41 degrees C, whereas that of the wild-type enzyme was 20 min. Evidence is now beginning to accumulate showing that viral pp60src expresses its protein kinase activity in transformed cells as well as in vitro because at least one cellular protein has been identified as a substrate for this activity of pp60src. Although the protein kinase activity associated with pp60src is itself cyclic AMP (cAMP) independent, the molecule contains at least one serine residue that is directly phosphorylated by the cellular cAMP-dependent protein kinase, thus suggesting that the viral transforming gene product may be regulated indirectly by the level of cAMP. The significance of this latter observation must be regarded from the point of view that the RSV src gene is apparently derived from a normal cellular gene that seemingly expresses in normal uninfected cells a phosphoprotein structurally and functionally closely related to pp60src. This celluar protein, found in all vertebrate species tested, also is a substrate for a cAMP-dependent protein kinase of normal cells, and, therefore, may be evolved to function in a regulatory circuit involving cAMP.

scholarly journals Discrete primary locations of a tyrosine-protein kinase and of three proteins that contain phosphotyrosine in virally transformed chick fibroblasts.

1982 ◽  
Vol 94 (2) ◽  
pp. 287-296 ◽  
Author(s):  
J A Cooper ◽  
T Hunter
Keyword(s):  
Protein Kinase ◽  
High Speed ◽  
Divalent Cations ◽  
Rous Sarcoma ◽  
Nonionic Detergent ◽  
Primary Location ◽  
Hypotonic Buffer ◽  
Tyrosine Protein Kinase ◽  
Sarcoma Virus ◽  
High Speed Supernatant

We have studied the localization of three abundant cellular proteins which are substrates for tyrosine protein kinases in virally transformed chicken embryo fibroblasts. The primary location of each substrate is unaltered by transformation with Rous sarcoma virus (RSV). The tyrosine-phosphorylated species is localized with the nonphosphorylated species. Two of the proteins, of about 46,000 and 28,000 daltons, have a similar location. They are present in the high speed supernatant of cells homogenized in hypotonic buffer, and are soluble in nonionic detergent. The third protein, of about 39,000 daltons, is particulate when cells are homogenized in hypotonic buffer containing divalent cations, but approximately 30% is free in the high-speed supernatant when divalent cations are absent. This protein appears to be associated with the detergent-insoluble matrix when adherent cells are gently lysed in nonionic detergent in situ, but is soluble when the same cells are extracted with nonionic detergent in suspension. This suggests that one of the proteins are tightly associated with detergent-insoluble cytoskeletal structures, unlike the RSV transforming protein itself, which is the main tyrosine protein kinase known to be active in RSV-transformed cells.

scholarly journals Expression of the mammalian c-fes protein in hematopoietic cells and identification of a distinct fes-related protein.

1985 ◽  
Vol 5 (10) ◽  
pp. 2543-2551 ◽  
Author(s):  
I MacDonald ◽  
J Levy ◽  
T Pawson
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Human Peripheral Blood ◽  
Hematopoietic Cells ◽  
Specific Protein ◽  
Protein Kinase Activity ◽  
Sarcoma Virus ◽  
Human And Mouse ◽  
Specific Protein Kinase

The avian c-fps and mammalian c-fes proto-oncogenes are cognate cellular sequences. Antiserum raised against the P140gag-fps transforming protein of Fujinami avian sarcoma virus specifically recognized a 92,000-Mr protein in human and mouse hematopoietic cells which was closely related in structure to Snyder-Theilen feline sarcoma virus P87gag-fes. This polypeptide was apparently the product of the human c-fes gene and was therefore designated p92c-fes. Human p92c-fes was associated with a tyrosine-specific protein kinase activity in vitro and was capable of both autophosphorylation and phosphorylation of enolase as an exogenous protein substrate. The synthesis of human and mouse p92c-fes was largely, though not entirely, confined to myeloid cells. p92c-fes was expressed to relatively high levels in a multipotential murine myeloid cell line, in more mature human and mouse granulocyte-macrophage progenitors, and in differentiated macrophage like cells as well as in the mononuclear fraction of normal and leukemic human peripheral blood. p92c-fes was not found in erythroid cells, with the exception of a human erythroleukemia line which retains the capacity to differentiate into macrophage like cells. These results suggest a normal role for the p92c-fes tyrosine kinase in hematopoiesis, particularly in granulocyte-macrophage differentiation. In addition, a distinct 94,000-Mr polypeptide, antigenically related to p92c-fes, was identified in a number of hematopoietic and nonhematopoietic human and mouse cells and was also found to be associated with a tyrosine-specific protein kinase activity.

scholarly journals Tyrosine phosphorylation of a 50K cellular polypeptide associated with the Rous sarcoma virus transforming protein pp60src.

1982 ◽  
Vol 2 (2) ◽  
pp. 199-206 ◽  
Author(s):  
T D Gilmore ◽  
K Radke ◽  
G S Martin
Keyword(s):  
Protein Kinase ◽  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fold Increase ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Rous Sarcoma ◽  
Defective Mutant ◽  
Sarcoma Virus

We have examined the phosphorylation of a 50,000-dalton cellular polypeptide associated with the Rous sarcoma virus (FSV) transforming protein pp60-src. It has been shown that pp60src forms a complex with two cellular polypeptides, an 89,000-dalton heat-shock protein (89K) and a 50,000-dalton phosphoprotein (50K). The pp60src-associated protein kinase activity phosphorylates at tyrosine residues, and the 50K polypeptide present in the complex contains phosphotyrosine and phosphoserine. These observations suggest that the 50K polypeptide may be a substrate for the protein kinase activity of pp60src. To examine this possibility, we isolated the 50K polypeptide by two-dimensional polyacrylamide gel electrophoresis from lysates of uninfected or virally infected cells. Tryptic phosphopeptide analysis indicated that the 50K polypeptide isolated by this method was the same polypeptide as that complexed to pp60src. In uninfected cells or cells infected by a transformation-defective mutant, the 50K polypeptide contained phosphoserine but little or no phosphotyrosine. In cells infected by Schmidt-Ruppin or Prague RSV, there was a 40- to 50-fold increase in the quantity of phosphotyrosine in the 50K protein. Thus, the phosphorylation of the 50K polypeptide at tyrosine is dependent on the presence of pp60src. However, the 50K polypeptide isolated from cells infected by temperature-sensitive mutants of RSV was found to be phosphorylated at tyrosine at both permissive and nonpermissive temperatures; this behavior is different from that of other substrates or putative substrates of the pp60src kinase activity. It is possible that the 50K polypeptide is a high-affinity substrate of pp60src.

scholarly journals Phosphorylation of cellular proteins in Rous sarcoma virus-infected cells: analysis by use of anti-phosphotyrosine antibodies.

1988 ◽  
Vol 8 (8) ◽  
pp. 3035-3042 ◽  
Author(s):  
M Hamaguchi ◽  
C Grandori ◽  
H Hanafusa
Keyword(s):  
Gel Electrophoresis ◽  
Rous Sarcoma Virus ◽  
Cellular Proteins ◽  
Morphological Transformation ◽  
Protein Substrates ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Infected Cells ◽  
Avian Sarcoma ◽  
In Cells

The protein substrates for the tyrosine protein kinases in cells transformed by avian sarcoma viruses were analyzed by gel electrophoresis in combination with immunoblotting or immunoprecipitation by antibodies against phosphotyrosine. We found that greater than 90% of phosphotyrosine-containing cellular proteins can be immunoprecipitated by these antibodies. The level of phosphotyrosine-containing cellular proteins detectable by this method markedly increased upon transformation with Rous sarcoma virus, and more than 20 distinct bands of such proteins were found in lysates of Rous sarcoma virus-transformed cells. Most of these phosphotyrosine-containing proteins had not been identified by other methods, and their presence appeared to correlate with morphological transformation in cells infected with various Rous sarcoma virus mutants and Y73, PRCII, and Fujinami sarcoma viruses. However, considerably different patterns were obtained with cells infected with nontransforming Rous sarcoma virus mutants that encode nonmyristylated src kinases, indicating that most substrates that correlate with transformation can only be recognized by p60v-src associated with the plasma membrane.

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