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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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Related in: MedlinePlus

Characterization of the differentially phosphorylated events in Ras-transformed HBECs and NSCLC cells.(A) The kinases targeting regulated phosphosites were derived using NetworKIN analysis. According to their target sequences, the upstream kinases were grouped into subsets of proline-directed, basophilic, acidophilic, and other kinases. Their frequencies are shown for each cell line. (B) Western blot analysis of ERK activation in HBECs and NSCLC cells. (C) Phosphorylation of lamin-A/C and cortactin in HBECs. Western blot analysis was performed to examine the phosphorylation of lamin-A/C at S392 using site-specific antibody. Phosphorylation of cortactin (CTTN) was determined by immunoprecipitation of cortactin in cell lysates using anti-cortactin antibody followed by immunoblot analysis using antibody specific for phosphorylated Ser/Thr. (D) Western blot analysis of lamin-A/C phosphorylation in KRAS knockdown NSCLC cell lines. Lung cancer A549, H322 and H1299 cells were infected with Lentivirus harboring shRNAs targeting at luciferase or KRAS overnight. Two individual shRNAs targeting at KRAS were employed. Cells were grown in serum-free medium for an additional 24 hrs prior to western blot analysis of designated proteins. The status of lamin-A/C pS392 was detected by site-specific antibody. β-actin was used as protein loading control. (E) Ras-regulated phosphorylation events observed in Ras-transformed HBECs and NSCLC cells. The Ras-regulated phosphorylation events, including 49 upregulated and 28 downregulated events, identified in Ras-transformed HBECs were classified as proline-directed and non-proline-directed phosphosites according to their consensus sequences. The conservation of Ras-regulated phosphorylation events in NSCLC A549, H322, and H1299 cells is shown.
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pone-0020199-g003: Characterization of the differentially phosphorylated events in Ras-transformed HBECs and NSCLC cells.(A) The kinases targeting regulated phosphosites were derived using NetworKIN analysis. According to their target sequences, the upstream kinases were grouped into subsets of proline-directed, basophilic, acidophilic, and other kinases. Their frequencies are shown for each cell line. (B) Western blot analysis of ERK activation in HBECs and NSCLC cells. (C) Phosphorylation of lamin-A/C and cortactin in HBECs. Western blot analysis was performed to examine the phosphorylation of lamin-A/C at S392 using site-specific antibody. Phosphorylation of cortactin (CTTN) was determined by immunoprecipitation of cortactin in cell lysates using anti-cortactin antibody followed by immunoblot analysis using antibody specific for phosphorylated Ser/Thr. (D) Western blot analysis of lamin-A/C phosphorylation in KRAS knockdown NSCLC cell lines. Lung cancer A549, H322 and H1299 cells were infected with Lentivirus harboring shRNAs targeting at luciferase or KRAS overnight. Two individual shRNAs targeting at KRAS were employed. Cells were grown in serum-free medium for an additional 24 hrs prior to western blot analysis of designated proteins. The status of lamin-A/C pS392 was detected by site-specific antibody. β-actin was used as protein loading control. (E) Ras-regulated phosphorylation events observed in Ras-transformed HBECs and NSCLC cells. The Ras-regulated phosphorylation events, including 49 upregulated and 28 downregulated events, identified in Ras-transformed HBECs were classified as proline-directed and non-proline-directed phosphosites according to their consensus sequences. The conservation of Ras-regulated phosphorylation events in NSCLC A549, H322, and H1299 cells is shown.

Mentions: Given that the regulated phosphopeptides consisted of motif sequences with distinctive features, we further explored the potential upstream kinases accountable for the regulated phosphorylation events by NetworKIN analysis. The NetworKIN algorithm predicts in vivo kinase-substrate relationships based on not only motif sequences but also various contextual factors [20]–[22]. The site-specific upstream kinases of regulated phosphorylation events identified by NetworKIN (Table S2) were further arranged into four subgroups according to their target motif sequences (Table S3 and Figure 3A). In Ras-transformed HBECs, kinases targeting proline-directed phosphosites represented the major subset in the phosphorylation network. A similar pattern was observed in AD cells (A549 and H322), but not in LCC H1299 cells (Figure 3A). Through a homogeneity test, the AD A549 and H322 cells appeared to share an undistinguishable pattern of upstream kinomes (p = 0.716). In contrast, the LCC H1299 cells displayed a more unique pattern, significantly different from that of the AD cells (p<0.0001), in which subsets of basophilic kinases and proline-directed kinases played equally important roles in the upstream kinomes (Figure 3A).


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)

Characterization of the differentially phosphorylated events in Ras-transformed HBECs and NSCLC cells.(A) The kinases targeting regulated phosphosites were derived using NetworKIN analysis. According to their target sequences, the upstream kinases were grouped into subsets of proline-directed, basophilic, acidophilic, and other kinases. Their frequencies are shown for each cell line. (B) Western blot analysis of ERK activation in HBECs and NSCLC cells. (C) Phosphorylation of lamin-A/C and cortactin in HBECs. Western blot analysis was performed to examine the phosphorylation of lamin-A/C at S392 using site-specific antibody. Phosphorylation of cortactin (CTTN) was determined by immunoprecipitation of cortactin in cell lysates using anti-cortactin antibody followed by immunoblot analysis using antibody specific for phosphorylated Ser/Thr. (D) Western blot analysis of lamin-A/C phosphorylation in KRAS knockdown NSCLC cell lines. Lung cancer A549, H322 and H1299 cells were infected with Lentivirus harboring shRNAs targeting at luciferase or KRAS overnight. Two individual shRNAs targeting at KRAS were employed. Cells were grown in serum-free medium for an additional 24 hrs prior to western blot analysis of designated proteins. The status of lamin-A/C pS392 was detected by site-specific antibody. β-actin was used as protein loading control. (E) Ras-regulated phosphorylation events observed in Ras-transformed HBECs and NSCLC cells. The Ras-regulated phosphorylation events, including 49 upregulated and 28 downregulated events, identified in Ras-transformed HBECs were classified as proline-directed and non-proline-directed phosphosites according to their consensus sequences. The conservation of Ras-regulated phosphorylation events in NSCLC A549, H322, and H1299 cells is shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102680&req=5

pone-0020199-g003: Characterization of the differentially phosphorylated events in Ras-transformed HBECs and NSCLC cells.(A) The kinases targeting regulated phosphosites were derived using NetworKIN analysis. According to their target sequences, the upstream kinases were grouped into subsets of proline-directed, basophilic, acidophilic, and other kinases. Their frequencies are shown for each cell line. (B) Western blot analysis of ERK activation in HBECs and NSCLC cells. (C) Phosphorylation of lamin-A/C and cortactin in HBECs. Western blot analysis was performed to examine the phosphorylation of lamin-A/C at S392 using site-specific antibody. Phosphorylation of cortactin (CTTN) was determined by immunoprecipitation of cortactin in cell lysates using anti-cortactin antibody followed by immunoblot analysis using antibody specific for phosphorylated Ser/Thr. (D) Western blot analysis of lamin-A/C phosphorylation in KRAS knockdown NSCLC cell lines. Lung cancer A549, H322 and H1299 cells were infected with Lentivirus harboring shRNAs targeting at luciferase or KRAS overnight. Two individual shRNAs targeting at KRAS were employed. Cells were grown in serum-free medium for an additional 24 hrs prior to western blot analysis of designated proteins. The status of lamin-A/C pS392 was detected by site-specific antibody. β-actin was used as protein loading control. (E) Ras-regulated phosphorylation events observed in Ras-transformed HBECs and NSCLC cells. The Ras-regulated phosphorylation events, including 49 upregulated and 28 downregulated events, identified in Ras-transformed HBECs were classified as proline-directed and non-proline-directed phosphosites according to their consensus sequences. The conservation of Ras-regulated phosphorylation events in NSCLC A549, H322, and H1299 cells is shown.
Mentions: Given that the regulated phosphopeptides consisted of motif sequences with distinctive features, we further explored the potential upstream kinases accountable for the regulated phosphorylation events by NetworKIN analysis. The NetworKIN algorithm predicts in vivo kinase-substrate relationships based on not only motif sequences but also various contextual factors [20]–[22]. The site-specific upstream kinases of regulated phosphorylation events identified by NetworKIN (Table S2) were further arranged into four subgroups according to their target motif sequences (Table S3 and Figure 3A). In Ras-transformed HBECs, kinases targeting proline-directed phosphosites represented the major subset in the phosphorylation network. A similar pattern was observed in AD cells (A549 and H322), but not in LCC H1299 cells (Figure 3A). Through a homogeneity test, the AD A549 and H322 cells appeared to share an undistinguishable pattern of upstream kinomes (p = 0.716). In contrast, the LCC H1299 cells displayed a more unique pattern, significantly different from that of the AD cells (p<0.0001), in which subsets of basophilic kinases and proline-directed kinases played equally important roles in the upstream kinomes (Figure 3A).

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