Weis Center for Research

Danville, PA, United States

Weis Center for Research

Danville, PA, United States
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Adilakshmi T.,Weis Center for Research | Ness-Myers J.,Weis Center for Research | Madrid-Aliste C.,Yeshiva University | Fiser A.,Yeshiva University | Tapinos N.,Weis Center for Research
Journal of Neuroscience | Year: 2011

Reciprocal interactions between glia and neurons are essential for the proper organization and function of the nervous system. Recently, the interaction between ErbB receptors (ErbB2 and ErbB3) on the surface of Schwann cells and neuronal Neuregulin-1 (NRG1) has emerged as the pivotal signal that controls Schwann cell development, association with axons, and myelination. To understand the function of NRG1-ErbB2/3 signaling axis in adult Schwann cell biology,weare studying the specific role of ErbB3 receptor tyrosine kinase (RTK) since it is the receptor for NRG1 on the surface of Schwann cells. Here, we show that alternative transcription initiation results in the formation of a nuclear variant of ErbB3 (nuc-ErbB3) in rat primary Schwann cells. nuc-ErbB3 possesses a functional nuclear localization signal sequence and binds to chromatin. Using chromatin immunoprecipitation (ChIP)- chip arrays, we identified the promoters that associate with nuc-ErbB3 and clustered the active promoters in Schwann cell gene expression. nuc-ErbB3 regulates the transcriptional activity of ezrin and HMGB1 promoters, whereas inhibition of nuc-ErbB3 expression results in reduced myelination and altered distribution of ezrin in the nodes of Ranvier. Finally, we reveal that NRG1 regulates the translation of nuc-ErbB3 in rat Schwann cells. For the first time, to our knowledge, we show that alternative transcription initiation from a gene that encodes a RTK is capable to generate a protein variant of the receptor with a distinct role in molecular and cellular regulation. We propose a new concept for the molecular regulation of myelination through the expression and distinct role of nuc-ErbB3. © 2011 the authors.

Chernousov M.A.,Weis Center for Research | Stahl R.C.,Weis Center for Research | Carey D.J.,Weis Center for Research
Journal of Neuroscience Research | Year: 2013

Many studies have shown that tetraspanins play important role in cell-cell and cell-extracellular matrix (ECM) interactions. The repertoire and functions of tetraspanins in Schwann cells, glial cells of the peripheral nervous system have remained largely uncharacterized. This study was undertaken to identify Schwann cell tetraspanins and to elucidate their possible functions. Microarray analysis revealed the expression of numerous tetraspanins in primary culture of Schwann cells. Expression of five of them, CD9, CD63, CD81, CD82, and CD151, and of tetraspanin-associated protein EWI-2 was also confirmed by immunofluorescence. Localization of CD9, CD63, CD81, and EWI-2 was largely confined to paranodes and Schmidt-Lanterman incisures, regions of noncompact myelin. Immunoprecipitation experiments showed that these four proteins form a complex in Schwann cells. siRNA silencing of individual components of the complex did not affect Schwann cell adhesion to ECM proteins or attachment to and alignment with axons. However, suppression of both CD63 and CD81 expression together significantly inhibited extension of Schwann cell processes along axons, without affecting initial attachment of the cells to the axonal surface. Adhesion, spreading, and migration of Schwann cells on ECM proteins also were not affected by double silencing of CD63 and CD81. Suppression of CD63 and CD81 expression did not affect the ability of Schwann cells to myelinate dorsal root ganglion neurons in vitro. These findings strongly suggest that CD63 and CD81 play an important role in Schwann cell spreading along axons but seem to be dispensable for Schwann cell myelination. © 2013 Wiley Periodicals, Inc.

Sudol M.,Weis Center for Research | Sudol M.,Mount Sinai School of Medicine | Shields D.C.,University College Dublin | Farooq A.,University of Miami
Seminars in Cell and Developmental Biology | Year: 2012

YAP (Yes-associated protein) is a potent oncogene and a major effector of the mammalian Hippo tumor suppressor pathway. In this review, our emphasis is on the structural basis of how YAP recognizes its various cellular partners. In particular, we discuss the role of LATS kinase and AMOTL1 junction protein, two key cellular partners of YAP that bind to its WW domain, in mediating cytoplasmic localization of YAP and thereby playing a key role in the regulation of its transcriptional activity. Importantly, the crystal structure of an amino-terminal domain of YAP in complex with the carboxy-terminal domain of TEAD transcription factor was only recently solved at atomic resolution, while the structure of WW domain of YAP in complex with a peptide containing the PPxY motif has been available for more than a decade. We discuss how such structural information may be exploited for the rational development of novel anti-cancer therapeutics harboring greater efficacy coupled with low toxicity. We also embark on a brief discussion of how recent in silico studies led to identification of the cardiac glycoside digitoxin as a potential modulator of WW domain-ligand interactions. Conversely, dobutamine was identified in a screen of known drugs as a compound that promotes cytoplasmic localization of YAP, thereby resulting in growth suppressing activity. Finally, we discuss how a recent study on the dynamics of WW domain folding on a biologically critical time scale may provide a tool to generate repertoires of WW domain variants for regulation of the Hippo pathway toward desired, non-oncogenic outputs. © 2012 Elsevier Ltd.

Grant M.P.,Weis Center for Research | Stepanchick A.,Weis Center for Research | Breitwieser G.E.,Weis Center for Research
Molecular Endocrinology | Year: 2012

Calcium-sensing receptors (CaSRs) regulate systemic Ca2+ homeostasis. Loss-of-function mutations cause familial benign hypocalciuric hypercalcemia (FHH) or neonatal severe hyperparathyroidism (NSHPT). FHH/NSHPT mutations can reduce trafficking of CaSRs to the plasma membrane. CaSR signaling is potentiated by agonist-driven anterograde CaSR trafficking, leading to a new steady state level of plasma membrane CaSR, which is maintained, with minimal functional desensitization, as long as extracellular Ca2+ is elevated. This requirement for CaSR signaling to drive CaSR trafficking to the plasma membrane led us to reconsider the mechanism(s) contributing to dysregulated trafficking of FHH/NSHPT mutants. We simultaneously monitored dynamic changes in plasma membrane levels of CaSR and intracellular Ca2+, using a chimeric CaSR construct, which allowed explicit tracking of plasma membrane levels of mutant or wild-type CaSRs in the presence of nonchimeric partners. Expression of mutants alone revealed severe defects in plasma membrane targeting and Ca2+ signaling, which were substantially rescued by coexpression with wildtype CaSR. Biasing toward heterodimerization of wild-type and FHH/NSHPT mutants revealed that intracellular Ca2+ oscillations were insufficient to rescue plasma membrane targeting. Coexpression of the nonfunctional mutant E297K with the truncation CaSR{increment}868 robustly rescued trafficking and Ca2+ signaling, whereas coexpression of distinct FHH/NSHPT mutants rescued neither trafficking nor signaling. Our study suggests that rescue of FHH/NSHPT mutants requires a steady state intracellular Ca2+ response when extracellular Ca2+ is elevated and argues that Ca2+ signaling by wild-type CaSRs rescues FHH mutant trafficking to the plasma membrane. © 2012 by The Endocrine Society.

Stepanchick A.,Weis Center for Research | Breitwieser G.E.,Weis Center for Research
Biochemical and Biophysical Research Communications | Year: 2010

The calcium sensing receptor (CaSR) is a Family 3/C G protein-coupled receptor with slow and partial targeting to the plasma membrane in both native and heterologous cells. We identified cargo receptor family member p24A in yeast two-hybrid screens with the CaSR carboxyl terminus. Interactions were confirmed by immunoprecipitation of either p24A or CaSR in transiently transfected HEK293 cells. Only the immaturely glycosylated form of CaSR interacts with p24A. Dissociation likely occurs in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) or cis-Golgi, since only the uncleaved form of a CaSR mutant sensitive to the trans-Golgi enzyme furin was co-immunoprecipitated with p24A. p24A and p24A(DGOLD) significantly increased total and plasma membrane CaSR protein but p24A(FF/AA) did not. The CaSR carboxyl terminus distal to T868 is required for differential sensitivity to p24A and its mutants. Interaction with p24A therefore increases CaSR stability in the ER and enhances plasma membrane targeting. Neither wt Sar1p or the T39N mutant increased CaSR maturation or abundance while the H79G mutant increased abundance but prevented maturation of CaSR. These results suggest that p24A is the limiting factor in CaSR trafficking in the early secretory pathway, and that cycling between the ER and ERGIC protects CaSR from degradation. © 2010 Elsevier Inc.

Sudol M.,Weis Center for Research | Sudol M.,Mount Sinai School of Medicine
Oncogene | Year: 2011

Plasminogen activator (PLAU) is a serine protease that converts plasminogen to plasmin, a general protease, which promotes fibrinolysis and degradation of extracellular matrix. PLAU was reported in 1970s as one of the robustly induced enzymatic activities in Rous sarcoma virus (RSV)-transformed chicken cells. More than three decades later, with the completion of the sequencing of the chicken genome and the subsequent availability of Affymetrix GeneChip genome arrays, several laboratories have surveyed the transcriptional program affected by the RSV transformation. Interestingly, the PLAU gene was shown to be the most highly upregulated transcript. The induction of PLAU was a transformation-dependent process because viruses with deleted Src gene did not induce the transcription of the PLAU gene. Both Src and PLAU genes are associated with and contribute to the complex phenotype of human cancer. Although the activity and structures of these two enzymes are well characterized, the precise molecular function of these gene products in signaling networks is still not fully understood. Yet, the knowledge of their association with cancer is already translated into the clinical setting. Src kinase inhibitors are being tested in clinical trials of cancer therapy, and PLAU gene and its inhibitor have been included as biomarkers with strong prognostic and therapeutic predictive values. This vignette reviews the history of PLAU and Src discovery, and illuminates the complexity of their relationship, but also points to their emerging impact on public health. © 2011 Macmillan Publishers Limited All rights reserved.

Adilakshmi T.,Weis Center for Research | Sudol I.,Weis Center for Research | Tapinos N.,Weis Center for Research
PLoS ONE | Year: 2012

Injury response in the peripheral nervous system (PNS) is characterized by rapid alterations in the genetic program of Schwann cells. However, the epigenetic mechanisms modulating these changes remain elusive. Here we show that sciatic nerve injury in mice induces a cohort of 22 miRNAs, which coordinate Schwann cell differentiation and dedifferentiation through a combinatorial modulation of their positive and negative gene regulators. These miRNAs and their targeted mRNAs form functional complexes with the Argonaute-2 protein to mediate post-transcriptional gene silencing. MiR-138 and miR-709 show the highest affinity amongst the cohort, for binding and regulation of Egr2, Sox-2 and c-Jun expression following injury. Moreover, miR-709 participates in the formation of epigenetic silencing complexes with H3K27me3 and Argonaute-1 to induce transcriptional gene silencing of the Egr2 promoter. Collectively, we identified a discrete cohort of miRNAs as the central epigenetic regulators of the transition between differentiation and dedifferentiation during the acute phase of PNS injury. © 2012 Adilakshmi et al.

Sudol M.,Weis Center for Research
Genes and Cancer | Year: 2011

The Hippo tumor suppressor pathway regulates the size of organs by controlling 2 opposing processes: proliferation and apoptosis. The pathway was originally defined in Drosophila, but it is well conserved in mammals. One of the unique features of Hippo signaling is the unusually wide occurrence of WW domains and its cognate PPxY ligand motifs within components of this pathway. Recently, it was proposed that the prevalence of WW domain-mediated complexes in the Hippo signaling pathway should facilitate its molecular analysis and help in the identification of new components of the Hippo-centered network. Indeed, several new members of the Hippo pathway, which form functional complexes with WW domains of YAP and TAZ effectors, were recently described. We focus here on 2 families of such proteins, angiomotins and SMADs, plus 1 regulatory factor, WBP-2, which together shed new light on the rapidly expanding Hippo network. Since the Hippo pathway acts as a tumor suppressor pathway, the complexes described here, which assemble on WW domains of YAP and TAZ, represent potential targets of cancer therapy. © The Author(s) 2011.

Oka T.,Weis Center for Research | Oka T.,Tokyo Medical and Dental University | Schmitt A.P.,Pennsylvania State University | Sudol M.,Weis Center for Research | Sudol M.,Mount Sinai School of Medicine
Oncogene | Year: 2012

YAP (Yes-associated protein) oncogene has been found to form a stable complex with members of the Angiomotin (Amot) family of proteins, which bind WW domains of YAP and sequester the protein in the cytoplasm and junctional complexes. The Amot-mediated retention of YAP in the cytoplasm results in the inhibition of its proliferative function. Using apoptotic read-out of YAP in HEK293 cells, we confirmed the molecular mode by which Amot regulates YAP. We showed that a representative member of the Amot family, AmotL1 (Angiomotin-like-1), uses its PPxY motifs to bind WW domains of YAP and inhibit YAP's nuclear translocation and pro-apoptotic function. Recently we also showed that YAP uses its PDZ-binding motif to interact with zona occludens-2 (ZO-2) protein, which promotes YAP's translocation to the nucleus. We also asked if AmotL1, YAP and ZO-2 signal together. We report here that AmotL1 and ZO-2 form a tripartite complex with YAP and regulate its function in HEK293 cells in opposite directions. AmotL1 inhibits pro-apoptotic function of YAP, whereas ZO-2 enhances it. As YAP is a potent oncogene, the identification and characterization of its regulators is important. AmotL1 and ZO-2 are two candidates that could be harnessed to control the oncogenic function of YAP. © 2012 Macmillan Publishers Limited All rights reserved.

Sudol M.,Weis Center for Research | Sudol M.,Mount Sinai School of Medicine | Harvey K.F.,Peter MacCallum Cancer Center | Harvey K.F.,University of Melbourne
Trends in Biochemical Sciences | Year: 2010

Metazoans have evolved several pathways to regulate the size of organs and ultimately that of organisms. One such pathway is known as Salvador-Warts-Hippo, or simply Hippo. Research on the Hippo pathway has grown exponentially during the past 8 years, revealing a complex signaling network. Intriguingly, within this complexity, there are levels of modularity. One level of modularity is represented by the unusually wide occurrence of the WW module in the Hippo core kinase cassette, the upstream regulatory components and the downstream nuclear proteins. We suggest that the prevalence of WW domain-mediated complexes in the Hippo pathway should facilitate its molecular analysis and aid prediction of new pathway components. © 2010 Elsevier Ltd.

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