Phosphorylation of tyrosine-416 is not required for the transforming properties and kinase activity of pp60v-src

Snyder, Mark A.; Bishop, J. Michael; Colby, Wendy W.; Levinson, Arthur D.

doi:10.1016/0092-8674(83)90074-0

Cited by 142 publications

(58 citation statements)

References 36 publications

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“…This is the major phosphorylated tyrosine residue found in pp60v sr, in vivo and is located in the carboxy-terminal region of the polypeptide. This residue is also the major site labeled in vitro when autophosphorylation is carried out at low ATP concentrations (28 (29). It is interesting that tyrosine-specific phosphorylation of the mutant pp6Ov-sr polypeptide was still detected, indicating that pp6Ov-src may be phosphorylated at tyrosine sites other than residue 416.…”

Section: Discussionmentioning

confidence: 90%

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Increase in the phosphotransferase specific activity of purified Rous sarcoma virus pp60v-src protein after incubation with ATP plus Mg2+.

Purchio¹,

Wells²,

Collett³

1983

Mol. Cell. Biol.

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pp6O-vsc, the product of the Rous sarcoma virus src gene, was partially purified by immunoaffinity chromatography from extracts of Rous sarcoma virus-transformed field vole cells. Incubation of this preparation with ATP plus Mg2+ and subsequent repurification by chromatography on hexylamine-agarose resulted in a net increase in the specific activity of the src protein kinase. This increase in phosphotransferase activity was detected by using a variety of substrates including casein, tubulin, and a 34,000-dalton protein presumed to be an in vivo target substrate of pp6O-src. In all cases, the phosphorylation was at tyrosine residues, and the kinase activity was inhibited by preincubation of the enzyme with immunoglobulin G prepared from tumor-bearing rabbit sera. The implications of these results for the regulation and control of pp6vOsr,'-associated kinase activity are discussed.The product of the Rous sarcoma virus (RSV) src gene is a 60,000-dalton (60K) phosphoprotein termed pp60vsrc (2, 22). Early experimental results suggested that a tentative function of this transforming protein was that of a protein kinase. This assignment was based on the ability of immune complexes containing pp6Ovsrc to phosphorylate the heavy chain of immunoglobulin G (IgG) molecules directed against the pp60V-sr( protein (5, 16). Subsequent purification of pp60v-sr from cytoplasmic extracts of RSVtransformed cells indicated that pp6Ov-sr either was itself a protein kinase or was tightly associated with a protein kinase (9, 10). More recently, the src gene has been molecularly cloned to expression in Escherichia coli (11,12,18). The polypeptide produced in bacteria appears to possess protein phosphotransferase activity (11, 18), clearly indicating that the RSV-transforming protein pp6Ov-sr is indeed a protein kinase. In all cases studied to date, the pp60v-src protein kinase activity appears to be specific for only tyrosine residues (6, 14, 17).Early experiments with pp6OvsrL indicated that this enzyme was capable of apparent autophosphorylation (9); this was based on the observation that the addition of [-y-32P]ATP to partially purified preparations of pp60v-sr, resulted in the phosphorylation of pp6Ov-src itself, although other workers have been unable to detect this activity (17). Experiments with highly purified preparations of pp6Ov-srC suggest that pp60v-src either does phosphorylate itself or is very tightly associated with a second kinase which is responsible for phosphorylating pp60v-src (21). Analyses of pp6Ov-.r( phosphorylation in vitro indicate that the same major tyrosine residue is phosphorylated on pp6Ov-src in vivo (28).Understanding the regulation of the pp6Ov-sr( kinase activity is an important step in understanding the mechanism of transformation by RSV. In this study, partially purified pp60v5sr( was incubated with ATP plus Mg2+ (ATP/Mg2+) and, after separation from all unlabeled ATP, was analyzed for its kinase activity. We observed a significant increase in the tyrosinespecific protein kinase activity of the enz...

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Section: Discussionmentioning

confidence: 90%

“…Snyder et al (29) recently constructed mutants of RSV in which the tyrosine residue at position 416 in pp,60vsrc was replaced by phenylalanine. This is the major phosphorylated tyrosine residue found in pp60v sr, in vivo and is located in the carboxy-terminal region of the polypeptide.…”

Section: Discussionmentioning

confidence: 99%

Increase in the phosphotransferase specific activity of purified Rous sarcoma virus pp60v-src protein after incubation with ATP plus Mg2+.

Purchio¹,

Wells²,

Collett³

1983

Mol. Cell. Biol.

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“…Therefore, although middle T is required for transformation, phosphorylation of middle T is not. It has also been demonstrated that phosphorylation of the transforming protein of RSV is not required for transformation [25]. The present studies ( fig.2 [29], which exhibits tyrosine-specific protein kinase activity [30].…”

Section: Febsletters January 1984mentioning

confidence: 96%

Decreased adenylate cyclase responsiveness of transformed cells correlates with the presence of a viral transforming protein

Beckner

1984

FEBS Letters

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The adenylate cyclase responsiveness of transformed fibroblastic and epithelial cell lines to forskolin, fluoride, guanine nucleotides and cholera toxin was reduced compared to their parental counterparts. This phenomenon was observed in lines transformed by either RNA or DNA tumor viruses, and in the case of polyoma virus, coincided with the expression of middle T antigen. The data suggest that decreased responsiveness of adenylate cyclase to non-hormone activators is a general consequence of viral transformation and may be related to viral regulation of protein kinase activity.

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“…Several points should be considered in understanding this observation. 1) The autophosphorylation site was chosen as a convenient low-affinity peptide; the fact that autophosphorylation is not required for transformation by wt pp6ov-src (Snyder et al, 1983) implies it is not the relevant P-Tyr in vivo. Binding by the true in vivo target protein may differ in subtle but important ways.…”

Section: Discussionmentioning

confidence: 99%

pp60v-src transformation of rat cells but not chicken cells strongly correlates with low-affinity phosphopeptide binding by the SH2 domain.

Verderame

1997

MBoC

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Substrates critical for transformation by pp6Ov-src remain unknown, as does the precise role of the src homology 2 (SH2) domain in this process. To continue exploring the role of the SH2 domain in pp60vsrc-mediated transformation, site-directed mutagenesis was used to create mutant v-src alleles predicted to encode proteins with overall structural integrity intact but with reduced ability to bind phosphotyrosine-containing peptides. Arginine-175, which makes critical contacts in the phosphotyrosine-binding pocket, was mutated to lysine or alanine. Unexpectedly, both mutations created v-src alleles that transform chicken cells with wild-type (wt) efficiency and are reduced for transformation of rat cells; these alleles are host dependent for transformation. Additionally, these alleles resulted in a round morphological transformation of chicken cells, unlike 12 of the 13 known host-dependent src SH2 mutations that result in a fusiform morphology. Analysis of phosphopeptide binding by the mutant SH2 domains reveal that the in vitro ability to bind phosphopeptides known to have a high affinity for wt src SH2 correlates with wt (round) morphological transformation in chicken cells and the in vitro ability to bind phosphopeptides known to have a low affinity for wt src SH2 correlates with rat cell transformation. These results suggest that the search for critical substrates in rat cells should be among proteins that interact with pp6ov-src with low affinity.

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Phosphorylation of tyrosine-416 is not required for the transforming properties and kinase activity of pp60v-src

Cited by 142 publications

References 36 publications

Increase in the phosphotransferase specific activity of purified Rous sarcoma virus pp60v-src protein after incubation with ATP plus Mg2+.

Increase in the phosphotransferase specific activity of purified Rous sarcoma virus pp60v-src protein after incubation with ATP plus Mg2+.

Decreased adenylate cyclase responsiveness of transformed cells correlates with the presence of a viral transforming protein

pp60v-src transformation of rat cells but not chicken cells strongly correlates with low-affinity phosphopeptide binding by the SH2 domain.

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