[Show abstract][Hide abstract] ABSTRACT: Protein methylation is a common posttranslational modification that mostly occurs on arginine and lysine residues. Arginine methylation has been reported to regulate RNA processing, gene transcription, DNA damage repair, protein translocation, and signal transduction. Lysine methylation is best known to regulate histone function and is involved in epigenetic regulation of gene transcription. To better study protein methylation, we have developed highly specific antibodies against monomethyl arginine, asymmetric dimethyl arginine, and monomethyl, dimethyl, and trimethyl lysine motifs respectively. These antibodies were used to perform immunoaffinity purification (IAP) of methyl peptides followed by LC-MS/MS analysis to identify and quantify arginine and lysine methylation sites in several model studies. Overall, we identified over 1000 arginine methylation sites in human cell lines and mouse tissues, and approximately 160 lysine methylation sites in human cell line HCT116. The methylation sites that were identified in this study exceed those found in the literature to date. Detailed analysis of arginine-methylated proteins observed in mouse brain compared to those found in mouse embryo shows tissue-specific distribution of arginine methylation, and extends the type of proteins that are known to be arginine methylated to many new protein types. Many arginine-methylated proteins that we identified from the brain including receptors, ion channels, transporters, and vesicle proteins, are involved in synaptic transmission, while the most abundant methylated proteins identified from mouse embryo are transcriptional regulators and RNA processing proteins.
[Show abstract][Hide abstract] ABSTRACT: Activating mutations of FMS-like tyrosine kinase-3 (FLT3) are found in approximately 30% of patients with acute myeloid leukemia (AML). FLT3 is therefore an attractive drug target. However, the molecular mechanisms by which FLT3 mutations lead to cell transformation in AML remain unclear. To develop a better understanding of FLT3 signaling as well as its downstream effectors, we performed detailed phosphoproteomic analysis of FLT3 signaling in human leukemia cells. We identified over 1000 tyrosine phosphorylation sites from about 750 proteins in both AML (wild type and mutant FLT3) and B cell acute lymphoblastic leukemia (normal and amplification of FLT3) cell lines. Furthermore, using stable isotope labeling by amino acids in cell culture (SILAC), we were able to quantified over 400 phosphorylation sites (pTyr, pSer, and pThr) that were responsive to FLT3 inhibition in FLT3 driven human leukemia cell lines. We also extended this phosphoproteomic analysis on bone marrow from primary AML patient samples, and identify over 200 tyrosine and 800 serine/threonine phosphorylation sites in vivo. This study showed that oncogenic FLT3 regulates proteins involving diverse cellular processes and affects multiple signaling pathways in human leukemia that we previously appreciated, such as Fc epsilon RI-mediated signaling, BCR, and CD40 signaling pathways. It provides a valuable resource for investigation of oncogenic FLT3 signaling in human leukemia.
[Show abstract][Hide abstract] ABSTRACT: Mantle cell lymphoma (MCL) is currently an incurable entity, and new therapeutic approaches are needed. We have applied a high-throughput phospho-proteomic technique to MCL cell lines to identify activated pathways and we have then validated our data in both cell lines and tumor tissues.
PhosphoScan analysis was performed on MCL cell lines. Results were validated by flow cytometry and western blotting. Functional validation was performed by blocking the most active pathway in MCL cell lines.
PhosphoScan identified more than 300 tyrosine-phosporylated proteins, among which many protein kinases. The most abundant peptides belonged to proteins connected with B-cell receptor (BCR) signaling. Active BCR signaling was demonstrated by flow cytometry in MCL cells and by western blotting in MCL tumor tissues. Blocking BCR signaling by Syk inhibitor piceatannol induced dose/time-dependent apoptosis in MCL cell lines, as well as several modifications in the phosphorylation status of BCR pathway members and a collapse of cyclin D1 protein levels.
Our data support a pro-survival role of BCR signaling in MCL and suggest that this pathway might be a candidate for therapy. Our findings also suggest that Syk activation patterns might be different in MCL compared to other lymphoma subtypes.
[Show abstract][Hide abstract] ABSTRACT: Cholangiocarcinoma, also known as bile duct cancer, is the second most common primary hepatic carcinoma with a median survival of less than 2 years. The molecular mechanisms underlying the development of this disease are not clear. To survey activated tyrosine kinases signaling in cholangiocarcinoma, we employed immunoaffinity profiling coupled to mass spectrometry and identified DDR1, EPHA2, EGFR, and ROS tyrosine kinases, along with over 1,000 tyrosine phosphorylation sites from about 750 different proteins in primary cholangiocarcinoma patients. Furthermore, we confirmed the presence of ROS kinase fusions in 8.7% (2 out of 23) of cholangiocarcinoma patients. Expression of the ROS fusions in 3T3 cells confers transforming ability both in vitro and in vivo, and is responsive to its kinase inhibitor. Our data demonstrate that ROS kinase is a promising candidate for a therapeutic target and for a diagnostic molecular marker in cholangiocarcinoma. The identification of ROS tyrosine kinase fusions in cholangiocarcinoma, along with the presence of other ROS kinase fusions in lung cancer and glioblastoma, suggests that a more broadly based screen for activated ROS kinase in cancer is warranted.
[Show abstract][Hide abstract] ABSTRACT: Expression and characterization of ROS fusions in either 3T3 cells or BaF3 cells. (A) Expression of FIG-ROS(L) and FIG-ROS(S) in 3T3 cells phosphorylate their downstream substrates, such as STAT3 and AKT. Arrow denote the correct size of FIG-ROS. (B) Expression of SLC34A2-ROS(S) in either 3T3 or BaF3 cells failed to activate its downstream signaling molecules. HCC78, which expresses SLC34A2-ROS(S), was included as a control. (C) Expression of FIG-ROS(L) and FIG-ROS(S) in BaF3 cells either in the presence of absence of IL3. (D) BaF3 cells were stably transfected with different ROS fusions, as well as empty Neo-Myc vector. BaF3 lysates were immunoprecipitated with Myc-tag antibody, and kinase assay was performed as described in experimental procedure. Kinase activity was expressed relative to that of empty Neo-Myc construct. Western blot showed similar amount of ROS proteins were used for kinase reaction.
[Show abstract][Hide abstract] ABSTRACT: Expression of ROS in primary cholangiocarcinoma samples. (A) Detection of ROS expression by Western blot from protein lysates of a liver cancer patient (TC23). Arrows denote truncated forms of ROS. (B) Identification of genomic breakpoints of ROS fusions by sequencing genomic PCR products from two FIG-ROS positive patients (TC23 and TC03). U118MG was used as a control.