Based on these motifs, EGFR-Thr654 matched the R-X-X-S/T consensus sequence and EGFR-Ser1046 matched the R-X-S/T consensus sequence of AURKA (Determine 4D)

Based on these motifs, EGFR-Thr654 matched the R-X-X-S/T consensus sequence and EGFR-Ser1046 matched the R-X-S/T consensus sequence of AURKA (Determine 4D). GUID:?8EB14490-FD2B-4377-9729-A37364F70F4A Physique S4: The calculation of PLA signals). The PLA signals, which were calculated by BlobFinder, were outlined with reddish dots. (A) The PLA signals of pEGFR-Ser1046. (B) The enlarged images of (A). Green: nuclear.(TIF) pone.0055657.s004.tif (1.9M) GUID:?C5959AC9-D211-45B7-B2E8-05DFF05AD509 Figure S5: The phosphorylation kinetics of EGFR-Tyr1068, EGFR-Thr654, and EGFR-Ser1046 under the EGF (10 ng/ml) stimulation in H1299- PLA images were shown in the Figure S2. Images from FR 167653 free base each slide with PLA sample were acquired at 5 different fields with 2 z-axis images.(DOC) pone.0055657.s006.doc (40K) GUID:?E65FCD37-31A5-4FF1-A48C-766E6BB97A61 Table S2: The antibody list for this study. (DOC) pone.0055657.s007.doc (62K) GUID:?2C5D3154-CDC0-4EF5-B1D2-4F34AF2A0656 Abstract The epidermal growth factor receptor (EGFR), which is up-regulated in lung malignancy, involves the activation of mitogenic signals and triggers multiple signaling cascades. To dissect these EGFR cascades, we used 14 different phospho-EGFR antibodies COL1A1 to quantify protein phosphorylation using an proximity ligation assay (PLA). Phosphorylation at EGFR-Thr654 and -Ser1046 was EGF-dependent in the wild-type (WT) receptor but EGF-independent in a cell collection transporting the EGFR-L858R mutation. Using a ProtoAarray? made up of 5000 recombinant proteins on the protein chip, we found that AURKA interacted with the EGFR-L861Q mutant. Moreover, overexpression of EGFR could form a complex with AURKA, and the inhibitors of AURKA and EGFR decreased EGFR-Thr654 and -Ser1046 phosphorylation. Immunohistochemical staining of stage I lung adenocarcinoma tissues demonstrated a positive correlation between AURKA expression and phosphorylation of EGFR at Thr654 and Ser1046 in mutations. Introduction Lung malignancy is the most common cause of cancer deaths worldwide, and the five-year relative survival rate of lung malignancy patients is usually less than 15% [1]. You will find two main types of lung cancers: small-cell lung malignancy (SCLC, approximately 20% of lung cancers) and non-small-cell lung cancers (NSCLC, approximately 80% of lung cancers) [2], [3]. Epidermal growth factor receptor (EGFR), which is a receptor tyrosine kinase (RTK), initiates multiple signaling pathways related to malignancy progression, such as those involved in cell proliferation, migration/invasion and the cell cycle [4]C[7]. Overexpression of EGFR is usually observed in approximately 50% of NSCLCs and is also associated with poor prognosis and a more aggressive disease course [8], [9]. mutations are frequently detected in NSCLC patients (10C40%) [10], [11]. Approximately 50% of mutations consist of deletions in exon 19, whereas 35C45% consist of the L858R mutation and 5% consist of insertions in exon 20 or the L861Q mutation [10]C[12]. Gefitinib (Iressa) and Erlotinib (Tarceva) are EGFR inhibitors that are used clinically for the treatment of advanced NSCLC, primarily that with mutations in the tyrosine kinase domains [13]C[16]. EGFR is usually activated by the binding of its cognate ligands, such as EGF and TGF. Ligand binding to wild-type (WT) EGFR results in receptor dimerization and activation of the intrinsic kinase domain name, followed by phosphorylation of specific tyrosine residues around the cytoplasmic tail [17]C[19]. The dysregulation of EGFR-activated pathways may result from mutations that cause ligand-independent receptor dimerization, activation and downstream signaling [16], [20]. Upon EGF activation, EGFR tyrosine phosphorylation is an early event, whereas EGFR serine/threonine phosphorylation, e.g. Ser967, occurs with a time delay [21], [22]. The phosphorylation of EGFR at many tyrosine sites after ligand activation initiates downstream signaling cascades, and the phosphorylation of EGFR at serine/threonine has been reported to attenuate these signals through negative opinions [23]C[25]. Many serine and threonine phosphorylation sites are present FR 167653 free base in EGFR, but their function remains unclear. Moreover, the signaling end result induced by the phosphorylation of different sites on EGFR is usually complicated and remains to be elucidated for the development of therapeutic FR 167653 free base applications. The AURKA protein kinase has drawn attention because its overexpression has been found in numerous epithelial malignant tumors [26], [27], such as breast [28], colon [29], ovarian [30] and lung cancers [31], as the result of gene amplification, transcriptional deregulation or defects in protein stability and the control of kinase activity [32]. Dysregulation of AURKA and EGFR is usually observed in different types of malignancy and is an important indication of prognosis in malignancy development [33]. A previous study exhibited that EGF-induced recruitment of nuclear EGFR and STAT5 to the AURKA promoter further increased AURKA gene expression [34]. Moreover, EGFR increases the protein expression of AURKA by activating the translational machinery via the ERK and AKT pathways [35]. These findings raise the possibility that.