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Characterizing tyrosine phosphorylation signaling in lung cancer using SH2 profiling.

Machida K, Eschrich S, Li J, Bai Y, Koomen J, Mayer BJ, Haura EB - PLoS ONE (2010)

Bottom Line: Binding of specific SH2 domains, most prominently RAS pathway activators Grb2 and ShcA, correlated with EGFR mutation and sensitivity to the EGFR inhibitor erlotinib.SH2 binding patterns also reflected MET activation and could identify cells driven by multiple kinases.The pTyr responses of cells treated with kinase inhibitors provided evidence of distinct mechanisms of inhibition.

View Article: PubMed Central - PubMed

Affiliation: Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut, United States of America.

ABSTRACT

Background: Tyrosine kinases drive the proliferation and survival of many human cancers. Thus profiling the global state of tyrosine phosphorylation of a tumor is likely to provide a wealth of information that can be used to classify tumors for prognosis and prediction. However, the comprehensive analysis of tyrosine phosphorylation of large numbers of human cancer specimens is technically challenging using current methods.

Methodology/principal findings: We used a phosphoproteomic method termed SH2 profiling to characterize the global state of phosphotyrosine (pTyr) signaling in human lung cancer cell lines. This method quantifies the phosphorylated binding sites for SH2 domains, which are used by cells to respond to changes in pTyr during signaling. Cells could be grouped based on SH2 binding patterns, with some clusters correlated with EGF receptor (EGFR) or K-RAS mutation status. Binding of specific SH2 domains, most prominently RAS pathway activators Grb2 and ShcA, correlated with EGFR mutation and sensitivity to the EGFR inhibitor erlotinib. SH2 binding patterns also reflected MET activation and could identify cells driven by multiple kinases. The pTyr responses of cells treated with kinase inhibitors provided evidence of distinct mechanisms of inhibition.

Conclusions/significance: This study illustrates the potential of modular protein domains and their proteomic binding profiles as powerful molecular diagnostic tools for tumor classification and biomarker identification.

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Related in: MedlinePlus

SH2 domains correlated with EGFR mutation.(A) SH2 domains whose binding is significantly correlated with EGFR mutation (q<0.1). Bar plot of SH2 signal for mutant and wild-type EGFR cell lines is shown as mean with standard error bars. "SH2 domain" is used in figures for all probes, including PTB domains and anti-pTyr antibody. (B) Protein-protein interaction map for EGFR (gray circle) and proteins with SH2 domains whose binding is significantly correlated with EGFR mutation (white circles). Lines indicate reported direct binding interactions. (C) Far-Western domain-specific bands significantly correlated with EGFR mutation. Colored boxes indicate the results of statistical significance in a Mann-Whitney test for differences (q<0.1) among the 22 cell lines shown in Figure 2B. Arrow indicates bin corresponding to EGFR family members. The numbers adjacent to bins indicate apparent MW, e.g. "291.0"  =  MW between 256–291 kDa.
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pone-0013470-g003: SH2 domains correlated with EGFR mutation.(A) SH2 domains whose binding is significantly correlated with EGFR mutation (q<0.1). Bar plot of SH2 signal for mutant and wild-type EGFR cell lines is shown as mean with standard error bars. "SH2 domain" is used in figures for all probes, including PTB domains and anti-pTyr antibody. (B) Protein-protein interaction map for EGFR (gray circle) and proteins with SH2 domains whose binding is significantly correlated with EGFR mutation (white circles). Lines indicate reported direct binding interactions. (C) Far-Western domain-specific bands significantly correlated with EGFR mutation. Colored boxes indicate the results of statistical significance in a Mann-Whitney test for differences (q<0.1) among the 22 cell lines shown in Figure 2B. Arrow indicates bin corresponding to EGFR family members. The numbers adjacent to bins indicate apparent MW, e.g. "291.0"  =  MW between 256–291 kDa.

Mentions: We next asked whether the binding of any individual SH2 domain probes was highly associated with activating EGFR mutations. From rosette binding experiments we identified 7 probes whose binding was correlated in a statistically significant fashion with EGFR mutation status: Grb2, ShcA(ptb), Grap2, Brk, Txk, CblB and CblA (Fig. 3A) (refer to Suppl. Table S3 for SH2 domain names and corresponding proteins). We input these domains into PPI Spider, a tool for interpreting proteomics data in the context of known protein-protein interaction networks. This analysis showed that five of these proteins (ShcA, Grb2, CblA, CblB, and Brk) have been reported to bind directly to EGFR, while Grap2 is potentially linked to EGFR through ShcA (Fig. 3B). The fact that binding sites for Grb2 and ShcA (and the close Grb2 relative Grap2) are closely associated with EGFR mutation is particularly intriguing, as increased binding of these SH2 domains would be strongly predicted to lead to activation of the RAS signaling pathway via recruitment of the RAS activator Sos [32], [33].


Characterizing tyrosine phosphorylation signaling in lung cancer using SH2 profiling.

Machida K, Eschrich S, Li J, Bai Y, Koomen J, Mayer BJ, Haura EB - PLoS ONE (2010)

SH2 domains correlated with EGFR mutation.(A) SH2 domains whose binding is significantly correlated with EGFR mutation (q<0.1). Bar plot of SH2 signal for mutant and wild-type EGFR cell lines is shown as mean with standard error bars. "SH2 domain" is used in figures for all probes, including PTB domains and anti-pTyr antibody. (B) Protein-protein interaction map for EGFR (gray circle) and proteins with SH2 domains whose binding is significantly correlated with EGFR mutation (white circles). Lines indicate reported direct binding interactions. (C) Far-Western domain-specific bands significantly correlated with EGFR mutation. Colored boxes indicate the results of statistical significance in a Mann-Whitney test for differences (q<0.1) among the 22 cell lines shown in Figure 2B. Arrow indicates bin corresponding to EGFR family members. The numbers adjacent to bins indicate apparent MW, e.g. "291.0"  =  MW between 256–291 kDa.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2957407&req=5

pone-0013470-g003: SH2 domains correlated with EGFR mutation.(A) SH2 domains whose binding is significantly correlated with EGFR mutation (q<0.1). Bar plot of SH2 signal for mutant and wild-type EGFR cell lines is shown as mean with standard error bars. "SH2 domain" is used in figures for all probes, including PTB domains and anti-pTyr antibody. (B) Protein-protein interaction map for EGFR (gray circle) and proteins with SH2 domains whose binding is significantly correlated with EGFR mutation (white circles). Lines indicate reported direct binding interactions. (C) Far-Western domain-specific bands significantly correlated with EGFR mutation. Colored boxes indicate the results of statistical significance in a Mann-Whitney test for differences (q<0.1) among the 22 cell lines shown in Figure 2B. Arrow indicates bin corresponding to EGFR family members. The numbers adjacent to bins indicate apparent MW, e.g. "291.0"  =  MW between 256–291 kDa.
Mentions: We next asked whether the binding of any individual SH2 domain probes was highly associated with activating EGFR mutations. From rosette binding experiments we identified 7 probes whose binding was correlated in a statistically significant fashion with EGFR mutation status: Grb2, ShcA(ptb), Grap2, Brk, Txk, CblB and CblA (Fig. 3A) (refer to Suppl. Table S3 for SH2 domain names and corresponding proteins). We input these domains into PPI Spider, a tool for interpreting proteomics data in the context of known protein-protein interaction networks. This analysis showed that five of these proteins (ShcA, Grb2, CblA, CblB, and Brk) have been reported to bind directly to EGFR, while Grap2 is potentially linked to EGFR through ShcA (Fig. 3B). The fact that binding sites for Grb2 and ShcA (and the close Grb2 relative Grap2) are closely associated with EGFR mutation is particularly intriguing, as increased binding of these SH2 domains would be strongly predicted to lead to activation of the RAS signaling pathway via recruitment of the RAS activator Sos [32], [33].

Bottom Line: Binding of specific SH2 domains, most prominently RAS pathway activators Grb2 and ShcA, correlated with EGFR mutation and sensitivity to the EGFR inhibitor erlotinib.SH2 binding patterns also reflected MET activation and could identify cells driven by multiple kinases.The pTyr responses of cells treated with kinase inhibitors provided evidence of distinct mechanisms of inhibition.

View Article: PubMed Central - PubMed

Affiliation: Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut, United States of America.

ABSTRACT

Background: Tyrosine kinases drive the proliferation and survival of many human cancers. Thus profiling the global state of tyrosine phosphorylation of a tumor is likely to provide a wealth of information that can be used to classify tumors for prognosis and prediction. However, the comprehensive analysis of tyrosine phosphorylation of large numbers of human cancer specimens is technically challenging using current methods.

Methodology/principal findings: We used a phosphoproteomic method termed SH2 profiling to characterize the global state of phosphotyrosine (pTyr) signaling in human lung cancer cell lines. This method quantifies the phosphorylated binding sites for SH2 domains, which are used by cells to respond to changes in pTyr during signaling. Cells could be grouped based on SH2 binding patterns, with some clusters correlated with EGF receptor (EGFR) or K-RAS mutation status. Binding of specific SH2 domains, most prominently RAS pathway activators Grb2 and ShcA, correlated with EGFR mutation and sensitivity to the EGFR inhibitor erlotinib. SH2 binding patterns also reflected MET activation and could identify cells driven by multiple kinases. The pTyr responses of cells treated with kinase inhibitors provided evidence of distinct mechanisms of inhibition.

Conclusions/significance: This study illustrates the potential of modular protein domains and their proteomic binding profiles as powerful molecular diagnostic tools for tumor classification and biomarker identification.

Show MeSH
Related in: MedlinePlus