Investigation of Factors That Influence Phosphorylation of pp60<sup>c-<i>src</i></sup> on Tyrosine 527

Schuh, S M; Brugge, Joan S.

doi:10.1128/mcb.8.6.2465-2471.1988

Cited by 8 publications

(7 citation statements)

References 34 publications

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“…We transiently overexpressed either a kinase inactive c-Src (K298M) as a control or a constitutively active c-Src (Y527F) kinase in 293T cells. Phosphorylation of the C-terminal tyrosine by C-terminal Src Kinase (CSK) allows inactivation of c-Src. – Hence, mutation of this tyrosine residue to phenylalanine allows c-Src kinase to be constitutively active by preventing its folding and by allowing the kinase domain access to its substrates. , Inhibition of c-Src activity is often achieved by coexpression of the c-Src-inactivating C-terminal Src Kinase (CSK), the kinase-inactive Src mutant Src K298 M, or by treatment of the cells with c-Src inhibitors.…”

Section: Resultsmentioning

confidence: 99%

“…[20][21][22] Hence, mutation of this tyrosine residue to phenylalanine allows c-Src kinase to be constitutively active by preventing its folding and by allowing the kinase domain access to its substrates. 23,24 Inhibition of c-Src activity is often achieved by coexpression of the c-Src-inactivating C-terminal Src Kinase (CSK), the kinase-inactive Src mutant Src K298 M, 25 or by treatment of the cells with c-Src inhibitors.…”

Section: Resultsmentioning

confidence: 99%

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Identification of c-Src Tyrosine Kinase Substrates Using Mass Spectrometry and Peptide Microarrays

et al. 2008

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c-Src tyrosine kinase plays a critical role in signal transduction downstream of growth factor receptors, integrins and G protein-coupled receptors. We used stable isotope labeling with amino acids in cell culture (SILAC) approach to identify additional substrates of c-Src tyrosine kinase in human embryonic kidney 293T cells. We have identified 10 known substrates and interactors of c-Src and Src family kinases along with 26 novel substrates. We have experimentally validated 4 of the novel proteins (NICE-4, RNA binding motif 10, FUSE-binding protein 1 and TRK-fused gene) as direct substrates of c-Src using in vitro kinase assays and cotransfection experiments. Significantly, using a c-Src specific inhibitor, we were also able to implicate 3 novel substrates (RNA binding motif 10, EWS1 and Bcl-2 associated transcription factor) in PDGF signaling. Finally, to identify the exact tyrosine residues that are phosphorylated by c-Src on the novel c-Src substrates, we designed custom peptide microarrays containing all possible tyrosine-containing peptides (312 unique peptides) and their mutant counterparts containing a Tyr → Phe substitution from 14 of the identified substrates. Using this platform, we identified 34 peptides that are phosphorylated by c-Src. We have demonstrated that SILAC-based quantitative proteomics approach is suitable for identification of substrates of nonreceptor tyrosine kinases and can be coupled with peptide microarrays for high-throughput identification of substrate phosphopeptides.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Identification of c-Src Tyrosine Kinase Substrates Using Mass Spectrometry and Peptide Microarrays

et al. 2008

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“…One potentially significant biochemical difference between pp6E c-src and the EGF receptor is the mechanism by which C-terminal tyrosine becomes phosphorylated. In pp6Jcsrc, Tyr-527 is phosphorylated via an intermolecular reaction whose kinase is not another pp6fc-src molecule (21,34). The phosphorylation of Tyr-1173 in the EGF receptor occurs as an EGF-dependent autophosphorylation.…”

Section: Discussionmentioning

confidence: 99%

Functional Heterogeneity of Proto-Oncogene Tyrosine Kinases: the C Terminus of the Human Epidermal Growth Factor Receptor Facilitates Cell Proliferation

Velu

Vass

Lowy

et al. 1989

Molecular and Cellular Biology

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Previous reports have indicated that the C termini of the membrane-associated tyrosine kinases encoded by c-src and c-fms proto-oncogenes have a negative effect on their biological activity and that this effect is mediated by their C-terminal tyrosine residue. To determine whether this was true for the human epidermal growth factor (EGF) receptor, which is also a membrane-associated tyrosine kinase proto-oncogene, we have constructed two premature termination mutants, dc19 and dc63, that delete the C-terminal 19 and 63 amino acids, respectively, from the human full-length receptor (hEGFR). The smaller deletion removes the C-terminal tyrosine residue, while the larger deletion removes the two most C-terminal tyrosines; similar deletions are found in v-erbB. As previously shown for the gene encoding the full-length EGF receptor, the two C-terminal mutants induced EGF-dependent focal transformation and anchorage-independent growth of NIH 3T3 cells. However, both dc19 and dc63 were quantitatively less efficient than the gene encoding the full-length receptor, with dc63 being less active than dc19. Although the C-terminal mutants displayed lower biological activity than the gene encoding the full-length receptor, the mutant receptors were found to be similar in several respects to the full-length receptor. These parameters included receptor localization, stability in the absence of EGF, receptor half-life in the presence of EGF, EGF binding, extent of EGF-dependent autophosphorylation in vitro, and EGF-dependent phosphorylation of an exogenous substrate in vitro. Therefore, the C-terminal 63 amino acids of the human receptor have no detectable influence on EGF-dependent early events. We conclude that in contrast

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“…To determine the relative phosphorylation on Y-416 (autophosphorylation) and Y-527, phosphorylated Src proteins were cleaved with cyanogen bromide and analyzed on a high-percentage polyacrylamide gel. This analysis separates a 4-kDa peptide containing Y-527 from a 10-kDa peptide containing Y-416 (43). In the absence of CSK, Y-416 was the major site of phosphorylation in wild-type and mutant c-Src proteins (Fig.…”

Section: Methodsmentioning

confidence: 93%

Suppression of c-Src Activity by C-Terminal Src Kinase Involves the c-Src SH2 and SH3 Domains: Analysis with Saccharomyces cerevisiae

Murphy

Bergman

Morgan

1993

Molecular and Cellular Biology

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The kinase activity of c-Src is normally repressed in vertebrate cells by extensive phosphorylation of Y-527. C-terminal Src kinase (CSK) is a candidate for the enzyme that catalyzes this phosphorylation. We have used budding yeast to study the regulation of c-Src activity by CSK in intact cells. Expression of c-Src in Saccharomyces cerevisiae, which lacks endogenous c-Src and Y-527 kinases, induces a kinase-dependent growth inhibition. Coexpression of CSK in these cells results in phosphorylation of c-Src on Y-527 and suppression of the c-Src phenotype. CSK does not fully suppress the activity of c-Src mutants lacking portions of the SH2 or SH3 domains, even though these mutant proteins are phosphorylated on Y-527 by CSK both in vivo and in vitro. These results suggest that both the SH2 and SH3 domains of c-Src are required for the suppression of c-Src activity by Y-527 phosphorylation.

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Investigation of Factors That Influence Phosphorylation of pp60^c-src on Tyrosine 527

Cited by 8 publications

References 34 publications

Identification of c-Src Tyrosine Kinase Substrates Using Mass Spectrometry and Peptide Microarrays

Identification of c-Src Tyrosine Kinase Substrates Using Mass Spectrometry and Peptide Microarrays

Functional Heterogeneity of Proto-Oncogene Tyrosine Kinases: the C Terminus of the Human Epidermal Growth Factor Receptor Facilitates Cell Proliferation

Suppression of c-Src Activity by C-Terminal Src Kinase Involves the c-Src SH2 and SH3 Domains: Analysis with Saccharomyces cerevisiae

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Investigation of Factors That Influence Phosphorylation of pp60c-src on Tyrosine 527

Cited by 8 publications

References 34 publications

Identification of c-Src Tyrosine Kinase Substrates Using Mass Spectrometry and Peptide Microarrays

Identification of c-Src Tyrosine Kinase Substrates Using Mass Spectrometry and Peptide Microarrays

Functional Heterogeneity of Proto-Oncogene Tyrosine Kinases: the C Terminus of the Human Epidermal Growth Factor Receptor Facilitates Cell Proliferation

Suppression of c-Src Activity by C-Terminal Src Kinase Involves the c-Src SH2 and SH3 Domains: Analysis with Saccharomyces cerevisiae

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Investigation of Factors That Influence Phosphorylation of pp60^c-src on Tyrosine 527