p21-Activated kinase 6 (PAK6) is associated with enhanced proliferation in lung cells exposed chronically to cigarette smoke
Epidemiological data clearly establishes cigarette smoking as one of the major cause for lung cancer worldwide. There is no standard screening method for lung cancer even in high-risk populations and the overall five-year survival has not changed significantly in the last decade. First-line treatment for lung cancer includes surgical resection, chemotherapy, and radiation. Recently with the advancement of systems biology, targeted therapy has become one of the most preferred modes of treatment for cancer. Though certain targeted therapies such as anti-EGFR are in clinical practice, they have shown limited success in the smokers suffering from lung cancer. This demands the discovery of alternative drug targets through systematic investigation of altered signaling mechanisms. To study dysregulated signaling pathways due to chronic cigarette smzoke exposure, we carried out SILAC-based phosphoproteome analysis of lung cell line H358 chronically exposed to cigarette smoke. We identified 1,812 phosphosites, of which 278 were hyperphosphorylated (≥ 3-fold) in the H358 cells exposed to cigarette smoke. We identified several known and some novel kinases and key signaling molecules that were hyperphosphorylated in response to chronic exposure to cigarette smoke
Sample Processing Protocol
H358 cells were subjected to chronic treatment with 0.1% CSC for 12 months. Cells that were grown in a normal incubator that did not have any cell lines treated with CSC are labeled as control or parental. Post twelve hours of serum starvation, cells were harvested and lysed in urea lysis buffer (20 mM HEPES pH8.0, 9 M urea, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM phosphoglycerophosphate). Protein concentration was determined using BCA assay. Five mg of lysate from treated and untreated conditions were mixed. The mixture was reduced with dithiothreitol at 600C and alkylated using iodoacetamide at room temperature. Concentration of urea was brought to 2M using HEPES buffer and digested overnight at 370C using modified trypsin (Promega). The protein digests were loaded onto Sep-Pak C18 column which was washed with TFA and eluted using 40% ACN with 0.1% TFA. The peptide samples were lyophilized and subjected to basic pH reverse phase chromatography (bRPLC). The lyophilized samples were reconstituted in bRPLC solvent A (7 mM TAEBC, pH 9) and were separated on XBridge BEH C18 Column (Waters, UK) with a linear increase in gradient from 5 to 100% of 7 mM TEABC with 90% acetonitrile (pH 9) over 30 min. and persisting for 10 minutes. For each condition, 24 fractions were collected and dried before LC-MS/MS analysis. The peptides from each fraction were further enriched for phosphopeptides using TiO2-based enrichment method. Briefly, the TiO2 beads were washed in 5% 2, 5-dihydroxybenzoic acid (DHB) for 2 hours on the rotator at room temperature. The bRPLC peptide fractions were redissolved in 5% DHB and incubated with TiO2 beads for 30 minutes on a rotator at room temperature. The phosphopeptide-TiO2 beads were washed several times and eluted thrice with 2% ammonia. The enriched peptides were concentrated by vacuum centrifugation and desalted using C18 StageTips. The enriched and desalted peptide samples were further subjected to mass spectrometry analysis.
Data Processing Protocol
The tandem mass spectrometry data were searched using MASCOT (v 2.2) and SEQUEST search algorithms against a Human RefSeq database (RefSeq 59) supplemented with frequently observed contaminants through the Proteome Discoverer platform (v1.3, Thermo Scientific, Bremen, Germany). For both algorithms, the search parameters included a maximum of 2 missed cleavage; carbamidomethylation at cysteine as a fixed modification, oxidation at methionine, phosphorylation at serine, threonine and tyrosine and SILAC labels 13C6-Lysine; 13C6-Arginine as variable modifications. The MS error tolerance was set at 20 ppm and MS/MS error tolerance to 0.1 Da. The peptides that scored better than the score cut-off for 1% false discovery rate were considered for further analysis. The probability of phosphorylation for each S/T/Y site on each peptide was calculated by the PhosphoRS node (Version 3.0) in the Proteome Discoverer. Peptides with ≥ 75% phosphosites probability were considered for further analysis. Identification of enriched motifs was carried out using motif-X algorithm. Phosphowindow of 15 aa long was used for extracting consensus motif.
Akhilesh Pandey, Johns Hopkins University
Akhilesh Pandey, McKusick-Nathans Institute of Genetic Medicine, Department of Biological Chemistry, Oncology and Pathology, Johns Hopkins University School of Medicine, Baltimore 21205, Maryland, USA ( lab head )
Raja R, Sahasrabuddhe NA, Radhakrishnan A, Syed N, Solanki HS, Puttamallesh VN, Balaji SA, Nanjappa V, Datta KK, Babu N, Renuse S, Patil AH, Izumchenko E, Prasad TS, Chang X, Rangarajan A, Sidransky D, Pandey A, Gowda H, Chatterjee A. Chronic exposure to cigarette smoke leads to activation of p21 (RAC1)-activated kinase 6 (PAK6) in non-small cell lung cancer cells. Oncotarget. 2016 Aug 16 PubMed: 27542207