Figure 1.
Schematic of the experimental design.
Whole cell extracts were prepared from Cdk4/hTERT-immortalized human bronchial epithelial cells (HBEC3-KT or 3KT), KRASV12-transformed 3KT (3KTR), and NSCLC A549, H322, and H1299 cells, then mixed with β-casein as an internal standard, and subjected to trypsin digestion. Phosphopeptides were enriched from each cell extract using IMAC and analyzed three times by LC-MS/MS. Peptides were identified by Mascot search, and phosphorylation changes were quantified in pairs across the five cell lines using the IDEAL-Q label-free system. The interpretation of phosphoproteomics data was performed by means of in silico analysis. Mt, mutant; AD, adenocarcinoma; LCC, large cell carcinoma; IMAC, immobilized metal affinity chromatography.
Figure 2.
Motifs or consensus sequences of regulated phosphorylation events identified in Ras-transformed HBECs and NSCLC cells.
(A) The numbers of comparable phosphopeptides subjected to quantitative proteomics analysis and the upregulated and downregulated phosphopeptides identified in each pair of cell lines are shown. (B) Representative motif sequences were extracted from the regulated phosphopeptides using motif-x. The motifs with significance of p<10−10 are shown.
Figure 3.
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.
Table 1.
Identification of oncogenic Ras-regulated phosphorylation events that are concurrently regulated in adenocarcinoma A549 and H322 cells, but not in large cell carcinoma H1299 cells.
Figure 4.
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.