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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 MET activation.(A) SH2 domains correlated with MET phosphorylation (p<0.01, q<0.1). Bar plot of SH2 signal for high and low MET phosphorylation. Mean and standard errors are shown. For this analysis “Low” includes both intermediate and low/negative categories (Fig. 5). (B) Far-Western domain-specific bands correlated with MET phosphorylation. Colored boxes indicate statistical significance in Mann-Whitney test for differences (q<0.1). (C) H1648 cells were exposed to control (DMSO), 1000 nM erlotinib (E), 1000 nM PHA665752 (P), or combination (E+P) for 3 h and analyzed by immunoblotting. Lysates from untreated H820 cells served as control for p-MET. Anti-β-actin was used to confirm equal loading. (D) Cell viability for H1648 cells exposed to 60 nM erlotinib (E), 300 nM PHA665752 (P), or combination (E+P).
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pone-0013470-g006: SH2 domains correlated with MET activation.(A) SH2 domains correlated with MET phosphorylation (p<0.01, q<0.1). Bar plot of SH2 signal for high and low MET phosphorylation. Mean and standard errors are shown. For this analysis “Low” includes both intermediate and low/negative categories (Fig. 5). (B) Far-Western domain-specific bands correlated with MET phosphorylation. Colored boxes indicate statistical significance in Mann-Whitney test for differences (q<0.1). (C) H1648 cells were exposed to control (DMSO), 1000 nM erlotinib (E), 1000 nM PHA665752 (P), or combination (E+P) for 3 h and analyzed by immunoblotting. Lysates from untreated H820 cells served as control for p-MET. Anti-β-actin was used to confirm equal loading. (D) Cell viability for H1648 cells exposed to 60 nM erlotinib (E), 300 nM PHA665752 (P), or combination (E+P).

Mentions: Next we tested whether the binding of any individual SH2 domain probes was associated with MET activation. By rosette analysis we found that the binding of 46 SH2 domains was significantly associated with MET phosphorylation (Fig. 6A). Similarly, far-Western analysis showed a large number of bands associated with MET phosphorylation status (Fig. 6B). The number of SH2 domains and markers that correlate with MET activation is larger than those associated with EGFR mutation, suggesting MET activity has a more profound effect on overall pTyr patterns than EGFR mutation.


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 MET activation.(A) SH2 domains correlated with MET phosphorylation (p<0.01, q<0.1). Bar plot of SH2 signal for high and low MET phosphorylation. Mean and standard errors are shown. For this analysis “Low” includes both intermediate and low/negative categories (Fig. 5). (B) Far-Western domain-specific bands correlated with MET phosphorylation. Colored boxes indicate statistical significance in Mann-Whitney test for differences (q<0.1). (C) H1648 cells were exposed to control (DMSO), 1000 nM erlotinib (E), 1000 nM PHA665752 (P), or combination (E+P) for 3 h and analyzed by immunoblotting. Lysates from untreated H820 cells served as control for p-MET. Anti-β-actin was used to confirm equal loading. (D) Cell viability for H1648 cells exposed to 60 nM erlotinib (E), 300 nM PHA665752 (P), or combination (E+P).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2957407&req=5

pone-0013470-g006: SH2 domains correlated with MET activation.(A) SH2 domains correlated with MET phosphorylation (p<0.01, q<0.1). Bar plot of SH2 signal for high and low MET phosphorylation. Mean and standard errors are shown. For this analysis “Low” includes both intermediate and low/negative categories (Fig. 5). (B) Far-Western domain-specific bands correlated with MET phosphorylation. Colored boxes indicate statistical significance in Mann-Whitney test for differences (q<0.1). (C) H1648 cells were exposed to control (DMSO), 1000 nM erlotinib (E), 1000 nM PHA665752 (P), or combination (E+P) for 3 h and analyzed by immunoblotting. Lysates from untreated H820 cells served as control for p-MET. Anti-β-actin was used to confirm equal loading. (D) Cell viability for H1648 cells exposed to 60 nM erlotinib (E), 300 nM PHA665752 (P), or combination (E+P).
Mentions: Next we tested whether the binding of any individual SH2 domain probes was associated with MET activation. By rosette analysis we found that the binding of 46 SH2 domains was significantly associated with MET phosphorylation (Fig. 6A). Similarly, far-Western analysis showed a large number of bands associated with MET phosphorylation status (Fig. 6B). The number of SH2 domains and markers that correlate with MET activation is larger than those associated with EGFR mutation, suggesting MET activity has a more profound effect on overall pTyr patterns than EGFR mutation.

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