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Phosphoproteomics identifies oncogenic Ras signaling targets and their involvement in lung adenocarcinomas.

Sudhir PR, Hsu CL, Wang MJ, Wang YT, Chen YJ, Sung TY, Hsu WL, Yang UC, Chen JY - PLoS ONE (2011)

Bottom Line: By integrating the phosphorylated signatures into the Pathway Interaction Database, we further inferred Ras-regulated pathways, including MAPK signaling and other novel cascades, in governing diverse functions such as gene expression, apoptosis, cell growth, and RNA processing.Comparisons of Ras-regulated phosphorylation events, pathways, and related kinases in lung cancer-derived cells supported a role of oncogenic Ras signaling in lung adenocarcinoma A549 and H322 cells, but not in large cell carcinoma H1299 cells.This study reveals phosphorylation events, signaling networks, and molecular functions that are regulated by oncogenic Ras.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.

ABSTRACT

Background: Ras is frequently mutated in a variety of human cancers, including lung cancer, leading to constitutive activation of MAPK signaling. Despite decades of research focused on the Ras oncogene, Ras-targeted phosphorylation events and signaling pathways have not been described on a proteome-wide scale.

Methodology/principal findings: By functional phosphoproteomics, we studied the molecular mechanics of oncogenic Ras signaling using a pathway-based approach. We identified Ras-regulated phosphorylation events (n = 77) using label-free comparative proteomics analysis of immortalized human bronchial epithelial cells with and without the expression of oncogenic Ras. Many were newly identified as potential targets of the Ras signaling pathway. A majority (∼60%) of the Ras-targeted events consisted of a [pSer/Thr]-Pro motif, indicating the involvement of proline-directed kinases. By integrating the phosphorylated signatures into the Pathway Interaction Database, we further inferred Ras-regulated pathways, including MAPK signaling and other novel cascades, in governing diverse functions such as gene expression, apoptosis, cell growth, and RNA processing. Comparisons of Ras-regulated phosphorylation events, pathways, and related kinases in lung cancer-derived cells supported a role of oncogenic Ras signaling in lung adenocarcinoma A549 and H322 cells, but not in large cell carcinoma H1299 cells.

Conclusions/significance: This study reveals phosphorylation events, signaling networks, and molecular functions that are regulated by oncogenic Ras. The results observed in this study may aid to extend our knowledge on Ras signaling in lung cancer.

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Inferring pathway activity in Ras-transformed HBECs and NSCLC cells.(A) The levels of regulated phosphoprotein signatures obtained from quantitative proteomics analysis were subjected to pathway activity analysis. Equations for summarizing the levels of phosphorylated proteins and for analyzing pathway activity are shown. xi,j and ρi,j are the mean and standard deviation, respectively, of the value of the phosphoprotein j in class i, and k is the number of proteins in a given pathway. ap is the activity score for pathway p between classes c1 and c2, and  and  are the numbers of replicate experiments for the respective classes. In our case,  and are each 3. (B) Inferred activities of MAPK and Aurora B signaling pathways in Ras-transformed HBECs and NSCLC cells in comparison with HBEC-3KT are shown as relative pathway activity scores (t-scores). (C) The involvement of the Ras/Raf/MEK/MAPK signaling pathway and its cross-talk with basophilic kinases in NSCLC cells. Kinases identified as being upstream of the regulated phosphosites by NetworKIN analysis in this study are highlighted in bold font. Kinases targeting proline-directed and basophilic S/T sites are labeled in green and blue letters, respectively. The frequencies of the individual kinases identified in A549, H322, and H1299 cells in comparison with HBEC-3KT are shown in parentheses.
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pone-0020199-g004: Inferring pathway activity in Ras-transformed HBECs and NSCLC cells.(A) The levels of regulated phosphoprotein signatures obtained from quantitative proteomics analysis were subjected to pathway activity analysis. Equations for summarizing the levels of phosphorylated proteins and for analyzing pathway activity are shown. xi,j and ρi,j are the mean and standard deviation, respectively, of the value of the phosphoprotein j in class i, and k is the number of proteins in a given pathway. ap is the activity score for pathway p between classes c1 and c2, and and are the numbers of replicate experiments for the respective classes. In our case, and are each 3. (B) Inferred activities of MAPK and Aurora B signaling pathways in Ras-transformed HBECs and NSCLC cells in comparison with HBEC-3KT are shown as relative pathway activity scores (t-scores). (C) The involvement of the Ras/Raf/MEK/MAPK signaling pathway and its cross-talk with basophilic kinases in NSCLC cells. Kinases identified as being upstream of the regulated phosphosites by NetworKIN analysis in this study are highlighted in bold font. Kinases targeting proline-directed and basophilic S/T sites are labeled in green and blue letters, respectively. The frequencies of the individual kinases identified in A549, H322, and H1299 cells in comparison with HBEC-3KT are shown in parentheses.

Mentions: Our data showed that Ras-regulated phosphorylation events are conserved in AD cells, and thus we further inferred the activation status of pathways, including MAPK signaling, in these cells by integrating the levels of the regulated phosphorylation events into individual signaling pathways according to the NCI-Nature Pathway Interaction Database (PID). For each signaling pathway, an activity level was summarized from the phosphorylation levels of the subset of proteins in the pathway, following the method reported by Lee et al. with modifications [15]. Figure 4A shows a schematic representation and equations for the pathway activity analysis. Pathways were included in the activity analysis only when they consisted of five or more identified phosphoproteins. The pathway activity signatures identified in each pair of cell lines are listed in Table S5. We observed 23 upregulated and two downregulated pathways downstream of oncogenic Ras in HBECs. The upregulated pathways included growth factor-mediated signaling pathways from HGF, FGF, and VEGF. The computation of pathway activities revealed the upregulation of proline-directed kinase MAPK signaling in 3KTR and AD cells (A549 and H322), but not in LCC H1299 cells (Figure 4B). In contrast, higher activity of the basophilic kinase Aurora B-mediated signaling pathway was observed in H1299 cells compared with A549 and H322 cells (Figure 4B).


Phosphoproteomics identifies oncogenic Ras signaling targets and their involvement in lung adenocarcinomas.

Sudhir PR, Hsu CL, Wang MJ, Wang YT, Chen YJ, Sung TY, Hsu WL, Yang UC, Chen JY - PLoS ONE (2011)

Inferring pathway activity in Ras-transformed HBECs and NSCLC cells.(A) The levels of regulated phosphoprotein signatures obtained from quantitative proteomics analysis were subjected to pathway activity analysis. Equations for summarizing the levels of phosphorylated proteins and for analyzing pathway activity are shown. xi,j and ρi,j are the mean and standard deviation, respectively, of the value of the phosphoprotein j in class i, and k is the number of proteins in a given pathway. ap is the activity score for pathway p between classes c1 and c2, and  and  are the numbers of replicate experiments for the respective classes. In our case,  and are each 3. (B) Inferred activities of MAPK and Aurora B signaling pathways in Ras-transformed HBECs and NSCLC cells in comparison with HBEC-3KT are shown as relative pathway activity scores (t-scores). (C) The involvement of the Ras/Raf/MEK/MAPK signaling pathway and its cross-talk with basophilic kinases in NSCLC cells. Kinases identified as being upstream of the regulated phosphosites by NetworKIN analysis in this study are highlighted in bold font. Kinases targeting proline-directed and basophilic S/T sites are labeled in green and blue letters, respectively. The frequencies of the individual kinases identified in A549, H322, and H1299 cells in comparison with HBEC-3KT are shown in parentheses.
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Related In: Results  -  Collection

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pone-0020199-g004: Inferring pathway activity in Ras-transformed HBECs and NSCLC cells.(A) The levels of regulated phosphoprotein signatures obtained from quantitative proteomics analysis were subjected to pathway activity analysis. Equations for summarizing the levels of phosphorylated proteins and for analyzing pathway activity are shown. xi,j and ρi,j are the mean and standard deviation, respectively, of the value of the phosphoprotein j in class i, and k is the number of proteins in a given pathway. ap is the activity score for pathway p between classes c1 and c2, and and are the numbers of replicate experiments for the respective classes. In our case, and are each 3. (B) Inferred activities of MAPK and Aurora B signaling pathways in Ras-transformed HBECs and NSCLC cells in comparison with HBEC-3KT are shown as relative pathway activity scores (t-scores). (C) The involvement of the Ras/Raf/MEK/MAPK signaling pathway and its cross-talk with basophilic kinases in NSCLC cells. Kinases identified as being upstream of the regulated phosphosites by NetworKIN analysis in this study are highlighted in bold font. Kinases targeting proline-directed and basophilic S/T sites are labeled in green and blue letters, respectively. The frequencies of the individual kinases identified in A549, H322, and H1299 cells in comparison with HBEC-3KT are shown in parentheses.
Mentions: Our data showed that Ras-regulated phosphorylation events are conserved in AD cells, and thus we further inferred the activation status of pathways, including MAPK signaling, in these cells by integrating the levels of the regulated phosphorylation events into individual signaling pathways according to the NCI-Nature Pathway Interaction Database (PID). For each signaling pathway, an activity level was summarized from the phosphorylation levels of the subset of proteins in the pathway, following the method reported by Lee et al. with modifications [15]. Figure 4A shows a schematic representation and equations for the pathway activity analysis. Pathways were included in the activity analysis only when they consisted of five or more identified phosphoproteins. The pathway activity signatures identified in each pair of cell lines are listed in Table S5. We observed 23 upregulated and two downregulated pathways downstream of oncogenic Ras in HBECs. The upregulated pathways included growth factor-mediated signaling pathways from HGF, FGF, and VEGF. The computation of pathway activities revealed the upregulation of proline-directed kinase MAPK signaling in 3KTR and AD cells (A549 and H322), but not in LCC H1299 cells (Figure 4B). In contrast, higher activity of the basophilic kinase Aurora B-mediated signaling pathway was observed in H1299 cells compared with A549 and H322 cells (Figure 4B).

Bottom Line: By integrating the phosphorylated signatures into the Pathway Interaction Database, we further inferred Ras-regulated pathways, including MAPK signaling and other novel cascades, in governing diverse functions such as gene expression, apoptosis, cell growth, and RNA processing.Comparisons of Ras-regulated phosphorylation events, pathways, and related kinases in lung cancer-derived cells supported a role of oncogenic Ras signaling in lung adenocarcinoma A549 and H322 cells, but not in large cell carcinoma H1299 cells.This study reveals phosphorylation events, signaling networks, and molecular functions that are regulated by oncogenic Ras.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.

ABSTRACT

Background: Ras is frequently mutated in a variety of human cancers, including lung cancer, leading to constitutive activation of MAPK signaling. Despite decades of research focused on the Ras oncogene, Ras-targeted phosphorylation events and signaling pathways have not been described on a proteome-wide scale.

Methodology/principal findings: By functional phosphoproteomics, we studied the molecular mechanics of oncogenic Ras signaling using a pathway-based approach. We identified Ras-regulated phosphorylation events (n = 77) using label-free comparative proteomics analysis of immortalized human bronchial epithelial cells with and without the expression of oncogenic Ras. Many were newly identified as potential targets of the Ras signaling pathway. A majority (∼60%) of the Ras-targeted events consisted of a [pSer/Thr]-Pro motif, indicating the involvement of proline-directed kinases. By integrating the phosphorylated signatures into the Pathway Interaction Database, we further inferred Ras-regulated pathways, including MAPK signaling and other novel cascades, in governing diverse functions such as gene expression, apoptosis, cell growth, and RNA processing. Comparisons of Ras-regulated phosphorylation events, pathways, and related kinases in lung cancer-derived cells supported a role of oncogenic Ras signaling in lung adenocarcinoma A549 and H322 cells, but not in large cell carcinoma H1299 cells.

Conclusions/significance: This study reveals phosphorylation events, signaling networks, and molecular functions that are regulated by oncogenic Ras. The results observed in this study may aid to extend our knowledge on Ras signaling in lung cancer.

Show MeSH
Related in: MedlinePlus