Antibodies against synthetic peptides as a tool for functional analysis of the transforming protein pp60src

Tamura, Teruko; Bauer, Heinz; Birr, Christian; Pipkorn, Rüdiger

doi:10.1016/0092-8674(83)90391-4

Cited by 107 publications

(48 citation statements)

References 38 publications

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“…Since the amino acid sequences of pp60c-'""c and pp60""" are almost identical except for the carboxy terminus [24], and since previous studies using antibodies to individual ~~60'" determinants prepared with synthetic peptides suggested that the binding site for pp50 and pp90 should be at the carboxy terminus [23,25,26], it is reasonable that pp6@-"" should fail to show the complexing typical of pp60"". The binding of pp60""' to DEAE was strongly influenced by whether or not the 50-and 90-kDa phosphoproteins were complexed to the pp60""'.…”

Section: Resultsmentioning

confidence: 99%

pp60^c‐src is a substrate for phosphorylation when cells are stimulated to enter cycle

1984

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The endogenous cellular oncogene products, pp60 c‐src , exhibits a protein kinase activity, but is itself a phosphoprotein. Based on the assumption that pp60 c‐src might play a role in the control of cell proliferation, we have studied its behaviour as a substrate for phosphorylation known to occur when quiescent, serum‐deprived cells are stimulated to enter cell cycle following addition of either serum, platelet‐derived growth factor or the phorbol ester derivative, 12‐0‐tetradecanoyl‐phorbol‐13‐acetate. For this purpose a partial purification of pp60 c‐src on DEAE ion‐exchange chromatography was combined with immune precipitation. A 2–4‐fold increase in serine phosphorylation of pp60 c‐src was consistently observed after stimulation of quiescent cells to growth.

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Section: Resultsmentioning

confidence: 99%

pp60^c‐src is a substrate for phosphorylation when cells are stimulated to enter cycle

1984

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confidence: 99%

“…Mutants generated by in vitro mutagenesis of the cloned src gene are being used to define important regions of the molecule with respect to kinase activity and transforming ability (7,20,58). Such studies, taken together, suggest that pp605r( has perhaps more than one domain (of which the kinase site is one) that are implicated in full expression of the transformed phenotype (26,34,38,44,59).We have previously isolated and mapped a number of temperature-sensitive src mutations of the Prague strain of RSV, finding that the lesions were distributed along the length of the gene (25,62 Immunoprecipitation and gel electrophoresis. Cells were washed three times in phosphate-buffered saline and lysed in buffer A (0.1 M NaCl, 1 mM EDTA; 10 mM Tris-hydrochloride [pH 7], 1% Nonidet P-40, 1 mg of bovine serum albumin per ml, 40 mM NaF, 50 mM ZnCl,) or buffer B (0.15 M NaCl, 20 mM Tris-EDTA [pH 7], 1% Nonidet P-40, 1 mg of bovine serum albumin per ml).…”

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Functional domains of the pp60v-src protein as revealed by analysis of temperature-sensitive Rous sarcoma virus mutants.

Stoker¹,

Enrietto²,

Wyke³

1984

Mol. Cell. Biol.

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Four temperature-sensitive (ts) Rous sarcoma virus src gene mutants with lesions in different parts of the gene represent three classes of alteration in pp6Osrc. These classes are composed of mutants with (i) heat-labile protein kinase activities both in vitro and in vivo (tsLA27 and tsLA29), (ii) heat-labile kinases in vivo but not in vitro (tsLA33), and (iii) neither in vivo nor in vitro heat-labile kinases (tsLA32). The latter class indicates the existence of structural or functional pp6Osrc domains that are required for transformation but do not grossly affect tyrosine kinase activity.The transforming src gene of Rous sarcoma virus (RSV) encodes a phosphoprotein with a molecular weight of 60,000, pp6Osr , which is required for the transformation of chicken embryo fibroblasts (CEF) in vitro (30,47). pp605'' is a cyclic-AMP-independent tyrosine protein kinase (6,10,12,24,31,36,37), the presence of which leads to an increase in the to, al cellular phosphotyrosine levels in transformed cells (13,14). The transformation mechanism of this protein is still unknown, but the bulk of pp6O0s appears to be localized at the inner surface of the plasma membrane (27,29,45,50), suggesting that it exerts its effects in this cellular compartment. Moreover, several proteins showing increased phosphotyrosine levels have been identified in RSV-transformed cells (4,15,39,49). These include vinculin (53), a 50-kilodalton (Kd) protein (28), a 36-Kd protein (23, 48), and three non-rate-determining glycolytic enzymes (17). Although the presence of several of these phosphoproteins may not be a prerequisite for morphological transformation or other transformation parameters, individually they could be involved in the appearance of specific features of transformation, as certain parameters may be divorced from each other and thus can be controlled independently (1-3, 16, 40, 51, 56, 61).The mechanism by which pp6os5' transforms cells has been studied with mutants of src. Work with temperaturesensitive (ts) mutants helped to establish the importance of the src gene and its kinase activity in maintenance of the transformed state (27,33,55). Mutants generated by in vitro mutagenesis of the cloned src gene are being used to define important regions of the molecule with respect to kinase activity and transforming ability (7,20,58). Such studies, taken together, suggest that pp605r( has perhaps more than one domain (of which the kinase site is one) that are implicated in full expression of the transformed phenotype (26,34,38,44,59).We have previously isolated and mapped a number of temperature-sensitive src mutations of the Prague strain of RSV, finding that the lesions were distributed along the length of the gene (25,62 Immunoprecipitation and gel electrophoresis. Cells were washed three times in phosphate-buffered saline and lysed in buffer A (0.1 M NaCl, 1 mM EDTA; 10 mM Tris-hydrochloride [pH 7], 1% Nonidet P-40, 1 mg of bovine serum albumin per ml, 40 mM NaF, 50 mM ZnCl,) or buffer B (0.15 M NaCl, 20 mM Tris-EDTA [pH 7], 1% Nonidet P-...

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“…lb), or 1 mCi Of 32p; per ml ( Fig. lc) in the presence or absence of vanadate and analyzed cleared cellular lysates by immunoprecipitation as described previously (15).…”

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gp140v-fms molecules expressed at the surface of cells transformed by the McDonough strain of feline sarcoma virus are phosphorylated in tyrosine and serine.

Tamura¹,

Simon²,

Niemann³

et al. 1986

Mol. Cell. Biol.

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Cells transformed by the McDonough strain of feline sarcoma virus express at their surface a v-fms-specific transmembrane glycoprotein designated gpl40v-fns. By labeling with 32p;, gpl40v-fFs was shown to be phosphorylated 30-fold more in serine residues than were the cytosolic v-fms polypeptides gpl8]w-finS and gp120v-f1". By using the phosphotyrosine phosphatase-specffic inhibitor sodium orthovanadate, an additional tyrosine phosphorylation was observed in vivo, again involving predominantly gpl40v-fFs. In vitro studies showed that the v-fms proteins were phosphorylated by protein kinase C in a calcium-and phosphatidylserinedependent manner.The McDonough strain of feline sarcoma virus (SM-FeSV) encodes a gag-fms fusion protein (gpl80(a9-fms) which exhibits the typical features of a transmembrane glycoprotein: it is cotranslationally N-glycosylated and processed by proteolytic cleavage to generate p555a5 and gpl2Ov-fms. gpl20v-fms is specified entirely by the viral oncogene v-fms and remains membrane associated. About 10 to 15% of the molecules are transported to the plasma membrane and concomitantly processed by additional glycosylation of the amino-terminal domain to yield gpl40v-fms (1,8,10,12). It has been reported previously that the extracellular domain of gpl40-fms binds the colony-stimulating factor 1 of macrophages (13). In addition, this domain plays a crucial role in regulating the growth offms-transformed cells (3). Although the cytoplasmic domain of the fms gene product exhibits partial sequence homology with other retroviral transforming proteins that exhibit tyrosine-specific kinase activity (4), tyrosine kinase activity has previously only been demonstrated in vitro (2).Effects of sodium orthovanadate on the biosynthesis and the phosphorylation of v-fms-molecules in vivo. Sodium orthovanadate is a potent inhibitor of phosphotyrosinespecific phosphatases (7,14). To study the effects of vanadate on the biosynthesis and phosphorylation of the v-fmsspecific glycoproteins in vivo, we labeled sister cultures of SM-FeSV-transformed NRK cells (G2-NRK-CJS) with 100 pCi of [3H]leucine per ml (Fig. la), 100 ,uCi of [6-3H]glucosamine per ml (Fig. lb), or 1 mCi Of 32p; per ml ( Fig. lc) in the presence or absence of vanadate and analyzed cleared cellular lysates by immunoprecipitation as described previously (15).Figure la shows that gpl2ov-fms represented the most abundantfms species. About 10% of thefms molecules were present as the mature form gpl4Ov-fms which is associated with the plasma membrane (1, 8). The presence of sodium orthovanadate in the labeling medium (Fig. la, lane 3) did not alter the rate of synthesis or the proportions of the individual fms polypeptides. Metabolic labeling under simi-* Corresponding author. lar conditions with glucosamine indicated that glycosylation and processing of the carbohydrate side chains were also not affected by the presence of the phosphatase inhibitor (Fig. lb). This finding suggests that the intracellular transport is not affected by vanadate.Two surprisi...

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Antibodies against synthetic peptides as a tool for functional analysis of the transforming protein pp60src

Cited by 107 publications

References 38 publications

pp60^c‐src is a substrate for phosphorylation when cells are stimulated to enter cycle

pp60^c‐src is a substrate for phosphorylation when cells are stimulated to enter cycle

Functional domains of the pp60v-src protein as revealed by analysis of temperature-sensitive Rous sarcoma virus mutants.

gp140v-fms molecules expressed at the surface of cells transformed by the McDonough strain of feline sarcoma virus are phosphorylated in tyrosine and serine.

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Antibodies against synthetic peptides as a tool for functional analysis of the transforming protein pp60src

Cited by 107 publications

References 38 publications

pp60c‐src is a substrate for phosphorylation when cells are stimulated to enter cycle

pp60c‐src is a substrate for phosphorylation when cells are stimulated to enter cycle

Functional domains of the pp60v-src protein as revealed by analysis of temperature-sensitive Rous sarcoma virus mutants.

gp140v-fms molecules expressed at the surface of cells transformed by the McDonough strain of feline sarcoma virus are phosphorylated in tyrosine and serine.

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pp60^c‐src is a substrate for phosphorylation when cells are stimulated to enter cycle

pp60^c‐src is a substrate for phosphorylation when cells are stimulated to enter cycle