Project description:Akt is a Ser/Thr protein kinase that regulates cell growth, metabolism and is considered a therapeutic target for cancer. Regulation of Akt by membrane recruitment and post-translational modifications (PTMs) has been extensively studied. The most well-established mechanism for cellular Akt activation involves phosphorylation on its activation loop on Thr308 by PDK1 and on its C-terminal tail on Ser473 by mTORC2. Other C-terminal tail PTMs have been identified, but their functional impacts have not been well-characterized. We use expressed protein ligation (EPL) as a tool to produce semisynthetic Akt proteins to dissect the enzymatic functions of these PTMs. We performed kinase assays employing human protein microarrays to investigate global substrate specificity of Akt, comparing phosphorylated vs. O-GlcNAcylated Ser473 forms.
Project description:Human antibody responses to AM/LAM are heterogenous and knowledge of reactivity to specific glycan epitopes at the monoclonal level is limited. Using novel glycan arrays, we characterized very high affinity monoclonal antibodies to AM/LAM, determined these mAbs are non-competing, and recognized distinct glycan epitopes. distinct from other anti-AM/LAM mAbs reported.
Project description:To identify cytokines secreted by pancreatic cancer cells, we compared the cytokine profiles of conditioned medium from pancreatic cancer cell Su.86.86 treated with or without JAG1 siRNA, using a Human Antibody Arrays 507 (L-507, Raybiotech)
Project description:Protein tyrosine kinase 6 (PTK6; also called Brk) is overexpressed in 86% of breast cancer patients; high PTK6 expression predicts poor outcome. We reported PTK6 induction by HIF/GR complexes in response to either cellular or host stress. However, PTK6-driven signaling events in the context of TNBC remain undefined. In a mouse model of TNBC, manipulation of PTK6 levels (i.e. via knock-out or add-back) had little effect on primary tumor volume but altered lung metastasis. To delineate the mechanisms of PTK6 downstream signaling, we created kinase-dead (KM) and kinase-intact domain structure mutants of PTK6 via in frame deletions of the N-terminal SH3 or SH2 domains. While the PTK6 kinase domain contributed to soft-agar colony formation, PTK6 kinase activity was entirely dispensable for cell migration. Specifically, TNBC models expressing a PTK6 variant lacking the SH2 domain (SH2-del PTK6) were unresponsive to growth factor-stimulated cell motility relative to SH3-del, KM or wild-type PTK6 controls. Reverse phase protein array (RPPA) revealed that while intact PTK6 mediates spheroid formation via p38 MAPK signaling, the SH2 domain of PTK6 limits this biology, and instead mediates TNBC cell motility via activation of the RhoA and/or AhR signaling pathways. Inhibition of RhoA and/or AhR blocked TNBC cell migration as well as the branching/invasive morphology of PTK6+/AhR+ primary breast tumor tissue organoids. Inhibition of RhoA also enhanced paclitaxel cytotoxicity in TNBC cells, including in a taxane-refractory TNBC model. Together, these studies reveal that the SH2-domain of PTK6 is a potent effector of advanced cancer phenotypes in TNBC and identify RhoA and AhR as novel therapeutic targets in PTK6+ breast tumors.
Project description:The calcitonin receptor (CTR) signaling axis has been implicated in growth regulation in Glioblastoma (GBM). This study examines the molecular and signaling effects of salmon calcitonin (sCT) treatment in GBM using proteomic, cellular, in vivo, and computational approaches. Reverse Phase Protein Array (RPPA) analysis was performed to assess changes in protein expression in glioma cells following sCT treatment and to identify signaling pathways associated with CTR activation. Complementary biochemical and functional assays were used to characterize CTR-mediated regulation of the Hippo pathway. CTR expression was found to be elevated in human and murine glioma stem-like cells in comparison to their differentiated counterparts, and intranasal sCT treatment lead to decrease in the GSC-initiated tumor growth in orthotopic mouse glioma model. In parallel, microsecond-scale all-atom molecular dynamics simulations were conducted to examine structural features of wild-type and patient-derived mutant CTR variants and their interactions with calcitonin and Gα subunits. Together, this work reveals mechanism of CT/CTR axis, therapeutic implications of sCT, and impact of structural alterations in patient-derived CTR variants in GBM.
Project description:β-cell specific IFT88 knock-out mice recapitulate human diabetes with impaired insulin secretion and altered islet hormone paracrine regulation. To examine the signaling pathways regulating islet cell function, we subjected protein lysates of whole islets from control and IFT88 knockout mice to a commercial signaling-protein array analysis (Full Moon Bio, Inc). Samples were probed against 1358 antibodies with 2 replicates per antibody on 76 x 25 x 1mm glass slides.
Project description:Human serum samples from Multiple Sclerosis (MS) subjects and healthy control subjects were probed onto human protein microarrays in order to identify differentially expressed autoantibody biomarkers that could be used as diagnostic indicators. Other neurodegenerative and non-neurodegenerative diseases were also used to help measure the specificity of the selected biomarkers.
Project description:Temproral networks of (phospho)-proteins are constructed and analyzed to infer differential interactions under insulin and IGF1 stimulation.
Project description:The identification of target antigens recognized by monoclonal antibodies that have been derived from oligoclonal band IgG of CSF samples from multiple sclerosis patients.
Project description:Ewing Sarcoma is caused by a pathognomonic genomic translocation that places an N-terminal EWSR1 gene in approximation with one of several ETS genes (typically FLI1). This aberration, in turn, alters the transcriptional regulation of more than five hundred genes and perturbs a number of critical pathways that promote oncogenesis, cell growth, invasion, and metastasis. Among them, translocation-mediated up-regulation of the insulin-like growth factor receptor 1 (IGF-1R) and mammalian target of rapamycin (mTOR) are of particular importance since they work in concert to facilitate IGF-1R expression and ligand-induced activation, respectively, of proven importance in ES transformation. When used as a single agent in Ewing sarcoma therapy, IGF-1R or mTOR inhibition leads to rapid counter-regulatory effects that blunt the intended therapeutic purpose. Therefore, identify new mechanisms of resistance that are used by Ewing sarcoma to evade cell death to single-agent IGF-1R or mTOR inhibition might suggest a number of therapeutic combinations that could improve their clinical activity.