Phosphorylation of a 36,000 Mr cellular protein in cells infected with partial transformation mutants of rous sarcoma virus.

Nakamura, Kenji; Weber, Michael J.

doi:10.1128/mcb.2.2.147

Cited by 35 publications

(27 citation statements)

References 24 publications

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“…Cells that exhibited low levels of phosphotyrosine on vinculin grew poorly in soft agar (i.e., CU2, tsCUll grown at 42°C, tsNY68 grown at 42°C), and those with more elevated levels of phosphotyrosine on vinculin grew much better (tsCUll grown at 35°C, CU12, RD10) regardless of pp6Osrc localization, stress fiber integrity, or expression of cell surface fibronectin. Similar results regarding the relationship of anchorage-independent growth to tyrosine-specific kinase activity have been obtained from other studies of the level of phosphotyrosine in the 36K-dalton substrate protein in the CU mutant-infected cells (31). Together, these results support the notion that the tyrosine-specific kinase activity of pp6osrc is needed for transformation and that the specific phosphorylation of vinculin and the 36K-dalton protein is a necessary part of that mechanism.…”

Section: Resultssupporting

confidence: 79%

Transformation parameters and pp60src localization in cells infected with partial transformation mutants of Rous sarcoma virus.

Rohrschneider¹,

Rosok²

1983

Mol. Cell. Biol.

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Rous sarcoma virus (RSV)-induced transformation is mediated by the action of the viral src gene product pp6OSrc. This transforming protein is found at several cytoplasmic locations, including the adhesion plaques of RSV-transformed cells. In these studies, we have focused on the adhesion plaque location of pp6Osrc and determined whether any of the induced transformation parameters correlate with the presence of pp6&src in the adhesion plaques. A series of partial transformation mutants of RSV that induce distinct transformation phenotypes were used, and infected chicken embryo cells were examined for (i) intracellular pp605rc location, (ii) vinculin localization, (iii) abundance of phosphotyrosine on vinculin, (iv) integrity of stress fibers, and (v) expression of cell surface fibronectin. The results indicate that, among the limited number of mutants studied here, the presence of pp60src in adhesion plaques is independent of growth in soft agar and the increased phosphorylation of vinculin on tyrosine, but it does correlate with the loss of cell surface fibronectin. An elevated abundance of phosphotyrosine on vinculin is insufficient to cause stress fiber dissolution and is independent of the loss of fibronectin from the extracellular matrix. However, the increased relative amount of phosphotyrosine on vinculin is related to the ability of the cells to grow in soft agar. The adhesion plaque binding and tyrosine-specific kinase activities seem to represent two independent functions of pp6ors.Neoplastic transformation results in numerous cellular alterations whose cumulative effect defines the total transformed phenotype. These cellular alterations often represent a rather di-

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Section: Resultssupporting

confidence: 79%

Transformation parameters and pp60src localization in cells infected with partial transformation mutants of Rous sarcoma virus.

Rohrschneider¹,

Rosok²

1983

Mol. Cell. Biol.

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“…Again, we have shown, by looking at a putative target of pp60''', that the kinase of LA32 is not obviously heat labile. We have also divorced 36K phosphorylation from the parameter of morphological transformation, in agreement with Nakamura and Weber (40). We plan to carry out similar studies with other putative substrates of pp6OArc.…”

Section: Resultsmentioning

confidence: 57%

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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“…The isolation of partial transformation mutants of RSV has provided support for the second model (3,5,35). More recently (22) we have shown that a prominent cellular protein which becomes phosphorylated on tyrosine during RSV-mediated oncogenic transformation (the 36 x 103-molecular-weight ["36K"] protein) is preferentially underphosphorylated in cells infected with a partial transformation mutant of RSV (CU2), providing direct biochemical evidence that the kinase substrate specificity of pp60Orc must be directed towards one or more additional proteins besides the 36K protein.…”

mentioning

confidence: 99%

“…However, as we will describe, these methods have not revealed the full complexity of transformation-specific phosphorylations on tyrosine. The identification of phosphotyrosine-containing proteins is a difficult task due to the low levels of cellular phosphotyrosine (1 to 2% of the total phosphorylhydroxyamino acid levels in RSV-transformed cells [22]). In this paper we describe a methodology to identify by molecular weight proteins which are phosphorylated on tyrosine residues.…”

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confidence: 99%

Identification of Phosphotyrosine-Containing Proteins in Untransformed and Rous Sarcoma Virus-Transformed Chicken Embryo Fibroblasts

1982

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Phosphorylation on tyrosine residues mediated by pp60src appears to be a primary biochemical event leading to the establishment of the transformed phenotype in Rous sarcoma virus (RSV)-infected cells. To identify the cellular proteins that undergo tyrosine phosphorylation during transformation, a 32p_ labeled RSV-transformed chicken embryo cell extract was analyzed by electrophoresis on a polyacrylamide gel. After slicing the gel into approximately 60 slices, phosphoamino acid analyses were carried out on the protein recovered from each gel slice. Phosphotyrosine was found in every gel slice, with two major peaks of this phosphoamino acid around Mr's of 59 and 36 kilodaltons. When the same analysis was performed with cells infected with a transformation-defective src deletion mutant of RSV (tdNY101), significant and reproducible peaks of phosphotyrosine were found in only 2 of 60 gel slices. These gel slices corresponded to Mr's of 42 and 40 kilodaltons. Identical results were obtained with normal uninfected chicken embryo fibroblasts. We conclude from these observations that pp60`Yc or the combined action of pp60`c and pp60&rc-activated cellular protein kinases cause the tyrosine-specific phosphorylation of a very large number of cellular polypeptides in RSV-transformed cells. In addition, untransformed cells appear to possess one or more active tyrosine-specific protein kinases which are responsible for the phosphorylation of a limited number of proteins. These proteins are different from the major phosphotyrosine-containing proteins of the transformed cells.Infection of chicken embryo fibroblasts (CEF) by Rous sarcoma virus (RSV) leads to the rapid appearance in these cells of a new spectrum of phenotypic properties collectively referred to as the transformed phenotype (14). The finding that the transforming protein of RSV (pp6(Y'9 possesses an intrinsic protein kinase activity with tyrosine substrate specificity (8,9,11,13,16,20,21) and the rapid rise in cellular phosphotyrosine levels seen during the first hour of the transformation process (28, 29) strongly suggest that pp60src_mediated phosphorylation of specific cellular targets represents a primary event in the establishment of the transformed phenotype.We have previously discussed (3, 35) two models with regard to the mechanism of action of pp6(Yrc: a single target hypothesis in which the action of pp6jsrc on a single protein could trigger a biochemical cascade leading to the transformed phenotype; and a multitarget hypothesis invoking the action of pp6(Yrc on two or more cellular proteins. The isolation of partial transformation mutants of RSV has provided support for the second model (3,5, 35). More recently (22) we have shown that a prominent cellular protein which becomes phosphorylated on tyrosine during RSV-mediated oncogenic transformation (the 36 x 103-molecular-weight ["36K"] protein) is preferentially underphosphorylated in cells infected with a partial transformation mutant of RSV (CU2), providing direct biochemical evidence that the kinase...

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Phosphorylation of a 36,000 Mr cellular protein in cells infected with partial transformation mutants of rous sarcoma virus.

Cited by 35 publications

References 24 publications

Transformation parameters and pp60src localization in cells infected with partial transformation mutants of Rous sarcoma virus.

Transformation parameters and pp60src localization in cells infected with partial transformation mutants of Rous sarcoma virus.

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

Identification of Phosphotyrosine-Containing Proteins in Untransformed and Rous Sarcoma Virus-Transformed Chicken Embryo Fibroblasts

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