<i>src</i>
            gene product from different strains of avain sarcoma virus: Kinetics and possible mechanism of heat inactivation of protein kinase activity from cells infected by transformation-defective, temperature-sensitive mutant and wild-type virus

Rübsamen, Helga; Friis, Robert R.; Bauer, Heinz

doi:10.1073/pnas.76.2.967

Cited by 85 publications

(33 citation statements)

References 19 publications

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“…This preference for MnZt is in contrast to pphO' -"' " (Riibsamen et al, 1979) and to v-abl produced in bacteria (Foulkes et al, 1985), both of which showed a marked preference for Mg2+. A preference for Mn"+, however.…”

Section: Discussionmentioning

confidence: 88%

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A strong protein‐tyrosine kinase activity is associated with a baculovirus‐expressed chicken tkl gene

Gärtner

Raab

et al. 1992

European Journal of Biochemistry

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We have previously described a gene named tkl (tyrosine kinase related to lck). It belongs to the src family of protein‐tyrosine kinases and among these it has significant homology to the lck gene (lymphoide cell kinase). The tkl gene product may represent the avian homolog of Lck, which is believed to participate in a lymphocyte‐specific signal transduction pathway by association with a membrane receptor. To study the biochemical properties of the protein, a nearly complete tkl gene (isolated from a cDNA library from chicken spleen cells) was expressed in a baculovirus system. Approximately 10% of the extracted protein consisted of the soluble 51‐kDa Tkl protein (p51tkl) at 40 h post‐infection. This protein was found to be phosphorylated on tyrosine and serine residues at a ratio of 5:1. As expected, glycosylation or myristoylation could not be detected. Immunocomplex kinase assays indicated strong autophosphorylation of p51tkl at tyrosine residues and phosphorylation of exogenous substrates such as D‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), histones H2b and H4, and casein. This protein‐tyrosine kinase activity also exhibited a marked preference for Mn2+ compared to Mg2+. The high level expression of enzymatically active Tkl should provide an excellent tool to further study the biological functions of this class of enzymes.

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Section: Discussionmentioning

confidence: 88%

“…6). No reaction was observed when CaZf was used, as has been shown for pp60'-"'" (Riibsamen et al, 1979). In further analogy to pp60"-src, GTP was accepted as phosphate donor but was less efficient than ATP (data not shown).…”

Section: Characterization Of the P51fk' Protein-tyrosine Kinase Activitymentioning

confidence: 78%

A strong protein‐tyrosine kinase activity is associated with a baculovirus‐expressed chicken tkl gene

Gärtner

Raab

et al. 1992

European Journal of Biochemistry

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“…The pp60Src_associated kinase activity was assayed essentially as previously reported (21 Cells were lysed in RIPA buffer, clarified, and preabsorbed with antiserum against viral structural proteins, and pp6(Wc was precipitated with TBR serum. The labeled proteins were resolved on 6 to 18% acrylamide gradient gels and detected by fluorography.…”

Section: Materiais and Methodsmentioning

confidence: 99%

Half-life of the Rous sarcoma virus transforming protein pp60src and its associated kinase activity.

Ziemiecki¹,

Friis²,

Bauer³

1982

Mol. Cell. Biol.

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The half-life of metabolically labeled pp6(Yrc of the Prague A strain of Rous sarcoma virus and of several transformation-defective, temperature-sensitive mutants was investigated by pulse-labeling infected cells with [35S]methionine, chasing for different times, and immunoprecipitating pp6(Yrc with tumor-bearing rabbit serum. These experiments showed that pp6(Yrc has a short half-life of approximately 60 min under normal physiological conditions and that the mutant pp6(Src proteins have similar half-lives to the wild type, irrespective of whether the cells are kept at the nonpermissive (42°C) or permissive (35°C) temperature. The half-life of the ppWsr-associated kinase activity was determined by monitoring its decay by the immunoglobulin G heavy chain assay after the cells had been treated with several inhibitors of protein synthesis. In these experiments the kinase half-life was much longer than expected from the half-life of pp6O!rc. The apparent contradiction between the half-lives of the kinase activity and the 135S]methionine-labeled pp6fysc protein could be resolved by the observation that treatment of cells with inhibitors of protein synthesis stabilized pp6(Yrc, resulting in a greatly extended half-life. Inhibitors of protein synthesis also extended the half-life of the gag precursor polypeptide, Pr76, suggesting that a host factor(s) may be required for the efficient intracellular processing of this polypeptide to the gag proteins.The continual expression of the src gene of Rous sarcoma virus (RSV) is required for maintenance of the transformed phenotype in infected cells (19). A 60,000-dalton phosphoprotein, pp61)Src, has been shown to be a product of the src gene (4,5,18), and a protein kinase activity capable of phosphorylating in vitro several substrates at tyrosine has been demonstrated to be intimately associated with pp6&Wrc (6,7,10,13). Despite extensive biochemical investigation of pp60srC, little is known about the in vivo h'alf-life of the molecule itself and its associated kinase activity (25). In this paper we describe experiments aimed at determining the half-lives of metabolically labeled pp6(sYc and the associated protein kinase activity. We show that pp60Yrc metabolically labeled with [35S]methionine has a short half-life of approximately 60 min. In experiments in which the decay of the pp6&frc-associated kinase activity was followed in cells treated with cycloheximide and other inhibitors of protein synthesis, a long half-life of many hours was observed. These results could be reconciled by our observation that the protein synthesis inhibitors used to demonstrate the decay of pp64src-associated kinase activity greatly prolonged the half-life of metabolically labeled pp60sc.

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“…To prove that the kinase reaction observed actually utilized the same antibodies in TBR serum as those which took part in the reaction with pp60v-src an assay was performed in which a limiting amount of serum was reacted with heat-inactivated (50°C for 40 min) sponge extracts (ca. 0.2 mg of protein) before the addition of RSVinfected chicken cell lysates, followed by an in vitro kinase reaction (10). Preincubation with the heat-inactivated sponge extracts greatly reduced the pp6O-src kinase reaction (Fig.…”

mentioning

confidence: 99%

Cellular src gene product detected in the freshwater sponge Spongilla lacustris.

Barnekow¹,

Schartl²

1984

Mol. Cell. Biol.

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Serum from Rous sarcoma virus tumor-bearing rabbits immunoprecipitated from extracts of the freshwater sponge Spongilla lacustris a tyrosine-specific protein kinase with characteristics similar to the chicken pp60c-src kinase activity. An immune competition assay confirmed the relationship between the protein from sponges and viral pp6O-sr'.The transforming gene of Rous sarcoma virus (RSV), vsrc, codes for a 60,000-dalton phosphoprotein, pp6O-src (1), which functions as a tyrosine-specific protein kinase (2, 6). The corresponding normal cellular counterpart, c-src, also codes for a protein, pp60c-rs, which shares structural and functional homology with viral pp60-src. Sequences homologous to v-src are present in the genomes of all vertebrates tested so far (7, 9, 13-15) and have been detected in the genus Drosophila (12). In this communication, we demonstrate the presence of a protein immunologically and functionally related to pp6Ov-src in the freshwater sponge Spongilla lacustris. Sponge cell extracts were tested for a tyrosine-specific kinase activity similar to the avian pp60sr-associated kinase activity. It was shown that the immunoglobulin G heavy chain of tumor-bearing rabbit (TBR) serum could be phosphorylated upon reaction (11) with a sponge cell extract, whereas the reaction with normal rabbit serum was negative (Fig. la). That the 32P-labeled 53,000 (53K) band was indeed the heavy chain of immunoglobulin G was established by running parallel portions of each sample with and without mercaptoethanol, so that under nonreducing conditions, a high-molecular-weight band of 150K could be observed (Fig. la). To prove that the kinase reaction observed actually utilized the same antibodies in TBR serum as those which took part in the reaction with pp60v-src an assay was performed in which a limiting amount of serum was reacted with heat-inactivated (50°C for 40 min) sponge extracts (ca. 0.2 mg of protein) before the addition of RSVinfected chicken cell lysates, followed by an in vitro kinase reaction (10). Preincubation with the heat-inactivated sponge extracts greatly reduced the pp6O-src kinase reaction (Fig. lb). Finally, the 53K product of the in vitro protein kinase reaction seen in Fig. la was cut out of the gel and eluted, and phosphoamino acids were analyzed by the method of Hunter and Sefton (6). The phosphorylated immunoglobulin G heavy chain is exclusively labeled in phosphotyrosine (Fig. lc).Immunoprecipitations with increasing amounts of TBR serum and a constant amount of sponge extract resulted in a maximum kinase activity with 5 ,lI of serum, whereas after the time dependence of the phosphorylation of heavy chain a * Corresponding author. maximum incorporation was revealed between 4 and 5 min ( Fig. ld and e).With respect to all reaction characteristics, the protein kinase activity as detected in sponge extracts could not be distinguished from the activity associated with the chicken cell pp60csrc (A. Barnekow and H. Bauer, Biochim. Biophys. Acta, in press). Thus, it was reasonable to expect ho...

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src gene product from different strains of avain sarcoma virus: Kinetics and possible mechanism of heat inactivation of protein kinase activity from cells infected by transformation-defective, temperature-sensitive mutant and wild-type virus

Cited by 85 publications

References 19 publications

A strong protein‐tyrosine kinase activity is associated with a baculovirus‐expressed chicken tkl gene

A strong protein‐tyrosine kinase activity is associated with a baculovirus‐expressed chicken tkl gene

Half-life of the Rous sarcoma virus transforming protein pp60src and its associated kinase activity.

Cellular src gene product detected in the freshwater sponge Spongilla lacustris.

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