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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

Validation of EGFR-regulated phosphoproteins by IP-Western blotting. A, IP-Western blotting analysis showing that EGF induces phosphorylation of three identified proteins (ANXA3, KRT8 and KRT18). Try, anti-phosphotyrosine antibody B, IP-Western blotting analysis showing that phospho-EGFR interacts with GSTP1 and GRB2. CBB gel staining serves as loading control (IgG heavy chain).
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Figure 3: Validation of EGFR-regulated phosphoproteins by IP-Western blotting. A, IP-Western blotting analysis showing that EGF induces phosphorylation of three identified proteins (ANXA3, KRT8 and KRT18). Try, anti-phosphotyrosine antibody B, IP-Western blotting analysis showing that phospho-EGFR interacts with GSTP1 and GRB2. CBB gel staining serves as loading control (IgG heavy chain).

Mentions: To confirm the results of phosphoproteomics, we detected the phosphorylated levels of three identified proteins (ANXA3, KRT8, and KRT18) by IP-Western blotting. Following immunoprecipitation (IP) of ANXA3, KRT8, and KRT18 from total cellular proteins, immunocomplexes were analyzed by Western blotting using anti-phosphotyrosine antibody. As shown in Figure 3A, the levels of phosphotyrosine of ANXA3, KRT8, and KRT18 were significantly higher in the 30 ng/mL EGF-stimulated CNE2 cells than in EGF-unstimulated CNE2 cells, and tyrosine phosphorylation of ANXA3, KRT8, and KRT18 could be blocked by the pretreatment of the cells with 1 μm EGFR inhibitor PD153035. The results indicate that EGFR activation can induce phosphorylation of ANXA3, KRT8, and KRT18, which is consistent with results of phosphoproteomics.


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)

Validation of EGFR-regulated phosphoproteins by IP-Western blotting. A, IP-Western blotting analysis showing that EGF induces phosphorylation of three identified proteins (ANXA3, KRT8 and KRT18). Try, anti-phosphotyrosine antibody B, IP-Western blotting analysis showing that phospho-EGFR interacts with GSTP1 and GRB2. CBB gel staining serves as loading control (IgG heavy chain).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Validation of EGFR-regulated phosphoproteins by IP-Western blotting. A, IP-Western blotting analysis showing that EGF induces phosphorylation of three identified proteins (ANXA3, KRT8 and KRT18). Try, anti-phosphotyrosine antibody B, IP-Western blotting analysis showing that phospho-EGFR interacts with GSTP1 and GRB2. CBB gel staining serves as loading control (IgG heavy chain).
Mentions: To confirm the results of phosphoproteomics, we detected the phosphorylated levels of three identified proteins (ANXA3, KRT8, and KRT18) by IP-Western blotting. Following immunoprecipitation (IP) of ANXA3, KRT8, and KRT18 from total cellular proteins, immunocomplexes were analyzed by Western blotting using anti-phosphotyrosine antibody. As shown in Figure 3A, the levels of phosphotyrosine of ANXA3, KRT8, and KRT18 were significantly higher in the 30 ng/mL EGF-stimulated CNE2 cells than in EGF-unstimulated CNE2 cells, and tyrosine phosphorylation of ANXA3, KRT8, and KRT18 could be blocked by the pretreatment of the cells with 1 μm EGFR inhibitor PD153035. The results indicate that EGFR activation can induce phosphorylation of ANXA3, KRT8, and KRT18, which is consistent with results of phosphoproteomics.

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