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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

Comparative phosphoproteomics analysis of EGF-stimulated and unstimulated NPC cells by 2D-DIGE and MS. A, Representative 2D-DIGE gel images. Internal standard labeled with Cy2 (a, blue), phosphoproteins from EGF-unstimulated cells labeled with Cy3 (b, green), phosphoproteins from EGF-stimulated cells labeled with Cy5 (c, red), and overlaying Cy3 and Cy5 images (d, yellow). B, (Left) the 38 differential phosphoprotein spots detected by Decyder software; (Right) a close-up of the region of 2D-DIGE gel images and a 3D simulation of spots 22 and 33. C, MALDI-TOF-MS analysis of differential protein spot 22. Mass spectrum of spot 22 identified as GSTP1 according to the matched peaks is shown (top). Protein sequence of GSTP1 is shown, and matched peptides are labeled in red letters (bottom).
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Figure 2: Comparative phosphoproteomics analysis of EGF-stimulated and unstimulated NPC cells by 2D-DIGE and MS. A, Representative 2D-DIGE gel images. Internal standard labeled with Cy2 (a, blue), phosphoproteins from EGF-unstimulated cells labeled with Cy3 (b, green), phosphoproteins from EGF-stimulated cells labeled with Cy5 (c, red), and overlaying Cy3 and Cy5 images (d, yellow). B, (Left) the 38 differential phosphoprotein spots detected by Decyder software; (Right) a close-up of the region of 2D-DIGE gel images and a 3D simulation of spots 22 and 33. C, MALDI-TOF-MS analysis of differential protein spot 22. Mass spectrum of spot 22 identified as GSTP1 according to the matched peaks is shown (top). Protein sequence of GSTP1 is shown, and matched peptides are labeled in red letters (bottom).

Mentions: 2D-DIGE and MS analysis were performed to identify differential phosphoproteins in EGF-stimulated and unstimulated (control) CNE2 cells. As shown in Figure 2A, phosphoproteins were labeled with either Cy3 (EGF-stimulated cells) or Cy5 (control) fluorescent dyes, while internal standards were labeled with Cy2. The interchangeable use of either Cy3 or Cy5 for each experiment has already been established. After 2D-DIGE, the Cy2, Cy3 and Cy5 images were scanned and analyzed using DeCyder software. 38 protein spots were differentially expressed in all nine protein-spot maps (Figure 2B left); 33 nonredundant proteins were identified by MS (Table 1); among them, five proteins are known EGFR-regulated proteins (KRT8, hnRNPK, KRT18, GRB2, Stathmin), and the other twenty-eight proteins have not been reported as EGFR-regulated proteins. A close-up of the region of 2D-DIGE gel images and a three-dimensional (3D) simulation of spots 22 and 33 significantly up-regulated in EGF-stimulated cells compared with control are shown in Figure 2B (right). MALDI-TOF-MS analysis and database matching identified spot 22 as Glutathione S-transferase P 1(GSTP1) with high sequence coverage and mass accuracy (Figure 2C).


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)

Comparative phosphoproteomics analysis of EGF-stimulated and unstimulated NPC cells by 2D-DIGE and MS. A, Representative 2D-DIGE gel images. Internal standard labeled with Cy2 (a, blue), phosphoproteins from EGF-unstimulated cells labeled with Cy3 (b, green), phosphoproteins from EGF-stimulated cells labeled with Cy5 (c, red), and overlaying Cy3 and Cy5 images (d, yellow). B, (Left) the 38 differential phosphoprotein spots detected by Decyder software; (Right) a close-up of the region of 2D-DIGE gel images and a 3D simulation of spots 22 and 33. C, MALDI-TOF-MS analysis of differential protein spot 22. Mass spectrum of spot 22 identified as GSTP1 according to the matched peaks is shown (top). Protein sequence of GSTP1 is shown, and matched peptides are labeled in red letters (bottom).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Comparative phosphoproteomics analysis of EGF-stimulated and unstimulated NPC cells by 2D-DIGE and MS. A, Representative 2D-DIGE gel images. Internal standard labeled with Cy2 (a, blue), phosphoproteins from EGF-unstimulated cells labeled with Cy3 (b, green), phosphoproteins from EGF-stimulated cells labeled with Cy5 (c, red), and overlaying Cy3 and Cy5 images (d, yellow). B, (Left) the 38 differential phosphoprotein spots detected by Decyder software; (Right) a close-up of the region of 2D-DIGE gel images and a 3D simulation of spots 22 and 33. C, MALDI-TOF-MS analysis of differential protein spot 22. Mass spectrum of spot 22 identified as GSTP1 according to the matched peaks is shown (top). Protein sequence of GSTP1 is shown, and matched peptides are labeled in red letters (bottom).
Mentions: 2D-DIGE and MS analysis were performed to identify differential phosphoproteins in EGF-stimulated and unstimulated (control) CNE2 cells. As shown in Figure 2A, phosphoproteins were labeled with either Cy3 (EGF-stimulated cells) or Cy5 (control) fluorescent dyes, while internal standards were labeled with Cy2. The interchangeable use of either Cy3 or Cy5 for each experiment has already been established. After 2D-DIGE, the Cy2, Cy3 and Cy5 images were scanned and analyzed using DeCyder software. 38 protein spots were differentially expressed in all nine protein-spot maps (Figure 2B left); 33 nonredundant proteins were identified by MS (Table 1); among them, five proteins are known EGFR-regulated proteins (KRT8, hnRNPK, KRT18, GRB2, Stathmin), and the other twenty-eight proteins have not been reported as EGFR-regulated proteins. A close-up of the region of 2D-DIGE gel images and a three-dimensional (3D) simulation of spots 22 and 33 significantly up-regulated in EGF-stimulated cells compared with control are shown in Figure 2B (right). MALDI-TOF-MS analysis and database matching identified spot 22 as Glutathione S-transferase P 1(GSTP1) with high sequence coverage and mass accuracy (Figure 2C).

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