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Analysis of EGFR signaling pathway in nasopharyngeal carcinoma cells by quantitative phosphoproteomics.

Ruan L, Li XH, Wan XX, Yi H, Li C, Li MY, Zhang PF, Zeng GQ, Qu JQ, He QY, Li JH, Chen Y, Chen ZC, Xiao ZQ - Proteome Sci (2011)

Bottom Line: As a result, 33 nonredundant phosphoproteins including five known EGFR-regulated proteins and twenty-eight novel EGFR-regulated proteins in CNE2 were identified, three differential phosphoproteins were selectively validated, and two differential phosphoproteins (GSTP1 and GRB2) were showed interacted with phospho-EGFR.GSTP1, one of the EGFR-regulated proteins, associated with chemoresistance was analyzed.The results showed that GSTP1 could contribute to paclitaxel resistance in EGF-stimulated CNE2 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha 410008, China. zqxiao2001@hotmail.com.

ABSTRACT

Background: The epidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC) and is associated with pathogenesis of NPC. However, the downstream signaling proteins of EGFR in NPC have not yet been completely understood at the system level. The aim of this study was identify novel downstream proteins of EGFR signaling pathway in NPC cells.

Results: We analyzed EGFR-regulated phosphoproteome in NPC CNE2 cells using 2D-DIGE and mass spectrometry analysis after phosphoprotein enrichment. As a result, 33 nonredundant phosphoproteins including five known EGFR-regulated proteins and twenty-eight novel EGFR-regulated proteins in CNE2 were identified, three differential phosphoproteins were selectively validated, and two differential phosphoproteins (GSTP1 and GRB2) were showed interacted with phospho-EGFR. Bioinformatics analysis showed that 32 of 33 identified proteins contain phosphorylation modification sites, and 17 identified proteins are signaling proteins. GSTP1, one of the EGFR-regulated proteins, associated with chemoresistance was analyzed. The results showed that GSTP1 could contribute to paclitaxel resistance in EGF-stimulated CNE2 cells. Furthermore, an EGFR signaling network based on the identified EGFR-regulated phosphoproteins were constructed using Pathway Studio 5.0 software, which includes canonical and novel EGFR-regulated proteins and implicates the possible biological roles for those proteins.

Conclusion: The data not only can extend our knowledge of canonical EGFR signaling, but also will be useful to understand the molecular mechanisms of EGFR in NPC pathogenesis and search therapeutic targets for NPC.

No MeSH data available.


Related in: MedlinePlus

Enrichment of phosphoproteins in EGF-stimulated and unstimulated NPC cells. A, Detection of EGFR activation by Western blotting. CNE-2 cells were stimulated with 30 ng/mL EGF for different times, and Western blotting was performed to detect levels of phosphorylated EGFR in total cellular proteins. Total EGFR serves as a loading control. B, Enrichment of phosphoproteins. Phosphoprotein enrichment kit was used to enrich phosphoproteins from total cellular proteins of EGF-stimulated and unstimulated CNE2 cells, and Western blotting analysis of phosphoproteins using anti-phosphotyrosine antibody(4G10) in the total cellular protein, elution fraction and flow through fraction. β-actin serves as a loading control.
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Figure 1: Enrichment of phosphoproteins in EGF-stimulated and unstimulated NPC cells. A, Detection of EGFR activation by Western blotting. CNE-2 cells were stimulated with 30 ng/mL EGF for different times, and Western blotting was performed to detect levels of phosphorylated EGFR in total cellular proteins. Total EGFR serves as a loading control. B, Enrichment of phosphoproteins. Phosphoprotein enrichment kit was used to enrich phosphoproteins from total cellular proteins of EGF-stimulated and unstimulated CNE2 cells, and Western blotting analysis of phosphoproteins using anti-phosphotyrosine antibody(4G10) in the total cellular protein, elution fraction and flow through fraction. β-actin serves as a loading control.

Mentions: A commercial phosphoprotein enrichment kit based on phosphate metal affinity chromatography was used to enrich phosphoproteins from EGF-stimulated and unstimulated NPC CNE2 cells. Typically, the elution fraction contains highly-concentrated and purified phosphoproteins. As shown in Figure 1A, levels of phosphorylated EGFR in CNE2 reached the high peak 15 min after 30 ng/mL EGF-stimulated cells. Then the total proteins of cells treated by ng/mL EGF for 15 min were used to enrich phosphoproteins. As shown in Figure 1B, the elution fractions contained more phosphoproteins compared with the total cellular proteins and flow-through fractions, indicating that the elution fractions can be used to identify EGFR-regulated phosphoproteins.


Analysis of EGFR signaling pathway in nasopharyngeal carcinoma cells by quantitative phosphoproteomics.

Ruan L, Li XH, Wan XX, Yi H, Li C, Li MY, Zhang PF, Zeng GQ, Qu JQ, He QY, Li JH, Chen Y, Chen ZC, Xiao ZQ - Proteome Sci (2011)

Enrichment of phosphoproteins in EGF-stimulated and unstimulated NPC cells. A, Detection of EGFR activation by Western blotting. CNE-2 cells were stimulated with 30 ng/mL EGF for different times, and Western blotting was performed to detect levels of phosphorylated EGFR in total cellular proteins. Total EGFR serves as a loading control. B, Enrichment of phosphoproteins. Phosphoprotein enrichment kit was used to enrich phosphoproteins from total cellular proteins of EGF-stimulated and unstimulated CNE2 cells, and Western blotting analysis of phosphoproteins using anti-phosphotyrosine antibody(4G10) in the total cellular protein, elution fraction and flow through fraction. β-actin serves as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3141626&req=5

Figure 1: Enrichment of phosphoproteins in EGF-stimulated and unstimulated NPC cells. A, Detection of EGFR activation by Western blotting. CNE-2 cells were stimulated with 30 ng/mL EGF for different times, and Western blotting was performed to detect levels of phosphorylated EGFR in total cellular proteins. Total EGFR serves as a loading control. B, Enrichment of phosphoproteins. Phosphoprotein enrichment kit was used to enrich phosphoproteins from total cellular proteins of EGF-stimulated and unstimulated CNE2 cells, and Western blotting analysis of phosphoproteins using anti-phosphotyrosine antibody(4G10) in the total cellular protein, elution fraction and flow through fraction. β-actin serves as a loading control.
Mentions: A commercial phosphoprotein enrichment kit based on phosphate metal affinity chromatography was used to enrich phosphoproteins from EGF-stimulated and unstimulated NPC CNE2 cells. Typically, the elution fraction contains highly-concentrated and purified phosphoproteins. As shown in Figure 1A, levels of phosphorylated EGFR in CNE2 reached the high peak 15 min after 30 ng/mL EGF-stimulated cells. Then the total proteins of cells treated by ng/mL EGF for 15 min were used to enrich phosphoproteins. As shown in Figure 1B, the elution fractions contained more phosphoproteins compared with the total cellular proteins and flow-through fractions, indicating that the elution fractions can be used to identify EGFR-regulated phosphoproteins.

Bottom Line: As a result, 33 nonredundant phosphoproteins including five known EGFR-regulated proteins and twenty-eight novel EGFR-regulated proteins in CNE2 were identified, three differential phosphoproteins were selectively validated, and two differential phosphoproteins (GSTP1 and GRB2) were showed interacted with phospho-EGFR.GSTP1, one of the EGFR-regulated proteins, associated with chemoresistance was analyzed.The results showed that GSTP1 could contribute to paclitaxel resistance in EGF-stimulated CNE2 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha 410008, China. zqxiao2001@hotmail.com.

ABSTRACT

Background: The epidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC) and is associated with pathogenesis of NPC. However, the downstream signaling proteins of EGFR in NPC have not yet been completely understood at the system level. The aim of this study was identify novel downstream proteins of EGFR signaling pathway in NPC cells.

Results: We analyzed EGFR-regulated phosphoproteome in NPC CNE2 cells using 2D-DIGE and mass spectrometry analysis after phosphoprotein enrichment. As a result, 33 nonredundant phosphoproteins including five known EGFR-regulated proteins and twenty-eight novel EGFR-regulated proteins in CNE2 were identified, three differential phosphoproteins were selectively validated, and two differential phosphoproteins (GSTP1 and GRB2) were showed interacted with phospho-EGFR. Bioinformatics analysis showed that 32 of 33 identified proteins contain phosphorylation modification sites, and 17 identified proteins are signaling proteins. GSTP1, one of the EGFR-regulated proteins, associated with chemoresistance was analyzed. The results showed that GSTP1 could contribute to paclitaxel resistance in EGF-stimulated CNE2 cells. Furthermore, an EGFR signaling network based on the identified EGFR-regulated phosphoproteins were constructed using Pathway Studio 5.0 software, which includes canonical and novel EGFR-regulated proteins and implicates the possible biological roles for those proteins.

Conclusion: The data not only can extend our knowledge of canonical EGFR signaling, but also will be useful to understand the molecular mechanisms of EGFR in NPC pathogenesis and search therapeutic targets for NPC.

No MeSH data available.


Related in: MedlinePlus