Phosphoproteomics Identifies Oncogenic Ras Signaling Targets and Their Involvement in Lung Adenocarcinomas
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.