Jyoti Srivastava

A human MAP kinase interactome

Mitogen-activated protein kinase (MAPK) pathways form the backbone of signal transduction in the mammalian cell. Here we applied a systematic experimental and computational approach to map 2,269 interactions between human MAPK-related proteins and other cellular machinery and to assemble these data into functional modules. Multiple lines of evidence including conservation with yeast supported a core network of 641 interactions. Using small interfering RNA knockdowns, we observed that approximately one-third of MAPK-interacting proteins modulated MAPK-mediated signaling. We uncovered the Na-H exchanger NHE1 as a potential MAPK scaffold, found links between HSP90 chaperones and MAPK pathways and identified MUC12 as the human analog to the yeast signaling mucin Msb2. This study makes available a large resource of MAPK interactions and clone libraries, and it illustrates a methodology for probing signaling networks based on functional refinement of experimentally derived protein-interaction maps.

Dynamic actin remodeling during epithelial–mesenchymal transition depends on increased moesin expression

LifeAct-GFP, a fluorescent reporter for actin filaments, is used to uncover the dynamics of actin cytoskeleton remodeling in real time during TGF-β–induced EMT. Efficient actin filament remodeling and complete transition to a mesenchymal phenotype depend on an increase in expression of the ERM protein moesin.

The Nck-interacting kinase phosphorylates ERM proteins for formation of lamellipodium by growth factors

The mammalian Ste20-like Nck-interacting kinase (NIK) and its orthologs Misshapen in Drosophila and Mig-15 in Caenorhabditis elegans have a conserved function in regulating cell morphology, although through poorly understood mechanisms. We report two previously unrecognized actions of NIK: regulation of lamellipodium formation by growth factors and phosphorylation of the ERM proteins ezrin, radixin, and moesin. ERM proteins regulate cell morphology and plasma membrane dynamics by reversibly anchoring actin filaments to integral plasma membrane proteins. In vitro assays show that NIK interacts directly with ERM proteins, binding their N termini and phosphorylating a conserved C-terminal threonine. In cells, NIK and phosphorylated ERM proteins localize at the distal margins of lamellipodia, and NIK activity is necessary for phosphorylation of ERM proteins induced by EGF and PDGF, but not by thrombin. Lamellipodium extension in response to growth factors is inhibited in cells expressing a kinase-inactive NIK, suppressed for NIK expression with siRNA oligonucleotides, or expressing ezrin T567A that cannot be phosphorylated. These data suggest that direct phosphorylation of ERM proteins by NIK constitutes a signaling mechanism controlling growth factor-induced membrane protrusion and cell morphology.

Structural model and functional significance of pH-dependent talin–actin binding for focal adhesion remodeling

Actin filament binding by the focal adhesion (FA)-associated protein talin stabilizes cell-substrate adhesions and is thought to be rate-limiting in cell migration. Although F-actin binding by talin is known to be pH-sensitive in vitro, with lower affinity at higher pH, the functional significance of this pH dependence is unknown. Because increased intracellular pH (pHi) promotes cell migration and is a hallmark of metastatic carcinomas, we asked whether it increases FA remodeling through lower-affinity talin–actin binding. Talin contains several actin binding sites, but we found that only the COOH-terminal USH-I/LWEQ module showed pH-dependent actin binding, with lower affinity and decreased maximal binding at higher pH. Molecular dynamics simulations and NMR of this module revealed a structural mechanism for pH-dependent actin binding. A cluster of titratable amino acids with upshifted pKa values, including His-2418, was identified at one end of the five-helix bundle distal from the actin binding site. Protonation of His-2418 induces changes in the conformation and dynamics of the remote actin binding site. Structural analyses of a mutant talin-H2418F at pH 6.0 and 8.0 suggested changes different from the WT protein, and we confirmed that actin binding by talin-H2418F was relatively pH-insensitive. In motile fibroblasts, increasing pHi decreased FA lifetime and increased the migratory rate. However, expression of talin-H2418F increased lifetime 2-fold and decreased the migratory rate. These data identify a molecular mechanism for pH-sensitive actin binding by talin and suggest that FA turnover is pH-dependent and in part mediated by pH-dependent affinity of talin for binding actin.

Nuclear-localized Calcineurin Homologous Protein CHP1 Interacts with Upstream Binding Factor and Inhibits Ribosomal RNA Synthesis

Calcineurin homologous protein 1 (CHP1) is a widely expressed, 22-kDa myristoylated EF-hand Ca2+-binding protein that shares a high degree of similarity with the regulatory B subunit of calcineurin (65%) and with calmodulin (59%). CHP1 localizes to the plasma membrane, the Golgi apparatus, and the nucleus and functions to regulate trafficking of early secretory vesicles, activation of T cells, and expression and transport of the Na-H exchanger NHE1. Although CHP1 contains nuclear export signals, whether its nuclear and cytoplasmic localization is regulated and has distinct functions remain unknown. We show that CHP1 is predominantly in the nucleus in quiescent fibrobasts, is translocated to cytoplasmic compartments with growth medium, and that translocation is inhibited by mutations in the nuclear export motifs. In a screen for proteins co-precipitating with CHP1 in quiescent cells we identified the upstream binding factor UBF, a DNA-binding protein and component of the RNA polymerase I complex regulating RNA synthesis. The CHP1-UBF interaction is restricted to the nucleus and inhibited by Ca2+. Nuclear retention of CHP1 attenuates the abundance of UBF in the nucleolus and inhibits RNA synthesis when quiescent cells are transferred to growth medium. These data show UBF as a newly identified CHP1-binding protein and regulation of RNA synthesis as a newly identified function for nuclear-localized CHP1, which is distinct from CHP1 functions in the cytosol.

Hypoxia increases the abundance but not the assembly of extracellular fibronectin during epithelial cell transdifferentiation.

ABSTRACT Increased production and assembly of extracellular matrix proteins during transdifferentiation of epithelial cells to a mesenchymal phenotype contributes to diseases such as renal and pulmonary fibrosis. TGF-&bgr; and hypoxia, two cues that initiate injury-induced fibrosis, caused human kidney cells to develop a mesenchymal phenotype, including increased fibronectin expression and secretion. However, upon hypoxia, assembled extracellular fibronectin fibrils were mostly absent, whereas treatment with TGF-&bgr; led to abundant fibrils. Fibrillogenesis required cell-generated force and tension. TGF-&bgr;, but not hypoxia, increased cell contractility, as determined by phosphorylation of myosin light chain and quantifying force and tension generated by cells plated on engineered elastomeric microposts. Additionally, TGF-&bgr;, but not hypoxia, increased the activation of integrins. However, experimentally activating integrins markedly increased the levels of phosphorylated myosin light chain and fibronectin fibril assembly upon hypoxia. Our findings show that deficient integrin activation and subsequent lack of cell contractility are mechanisms that mediate a lack of fibrillogenesis upon hypoxia and they challenge current views on oxygen deprivation being sufficient for fibrosis.

Astrocyte elevated gene-1 (AEG-1) regulates lipid homeostasis

Background: The physiological function of the oncogene astrocyte elevated gene-1 (AEG-1) was analyzed using a knock-out mouse (AEG-1KO). Results: The AEG-1KO mouse shows a lean phenotype, which may be due to decreased intestinal fat absorption because of hyperactivation of LXR and PPARα. Conclusion: A novel role of AEG-1 is identified, regulating lipid metabolism. Significance: AEG-1 may play a role in regulating obesity and its associated disorders. Astrocyte elevated gene-1 (AEG-1), also known as MTDH (metadherin) or LYRIC, is an established oncogene. However, the physiological function of AEG-1 is not known. To address this question, we generated an AEG-1 knock-out mouse (AEG-1KO) and characterized it. Although AEG-1KO mice were viable and fertile, they were significantly leaner with prominently less body fat and lived significantly longer compared with wild type (WT). When fed a high fat and cholesterol diet (HFD), WT mice rapidly gained weight, whereas AEG-1KO mice did not gain weight at all. This phenotype of AEG-1KO mice is due to decreased fat absorption from the intestines, not because of decreased fat synthesis or increased fat consumption. AEG-1 interacts with retinoid X receptor (RXR) and inhibits RXR function. In enterocytes of AEG-1KO mice, we observed increased activity of RXR heterodimer partners, liver X receptor and peroxisome proliferator-activated receptor-α, key inhibitors of intestinal fat absorption. Inhibition of fat absorption in AEG-1KO mice was further augmented when fed an HFD providing ligands to liver X receptor and peroxisome proliferator-activated receptor-α. Our studies reveal a novel role of AEG-1 in regulating nuclear receptors controlling lipid metabolism. AEG-1 may significantly modulate the effects of HFD and thereby function as a unique determinant of obesity.

Astrocyte Elevated Gene-1 (AEG-1) Contributes to Non-thyroidal Illness Syndrome (NTIS) Associated with Hepatocellular Carcinoma (HCC)

Background: Astrocyte elevated gene-1 (AEG-1) inhibits retinoid X receptor (RXR) function and is overexpressed in human hepatocellular carcinoma (HCC), which is associated with non-thyroidal illness syndrome (NTIS). Results: AEG-1 inhibits thyroid hormone (T3) function by down-regulating type I 5′-deiodinase (DIO1) thus contributing to NTIS. Conclusion: A novel role of AEG-1 is identified regulating cancer-associated NTIS. Significance: AEG-1 inhibition might alleviate cancer-associated debilitating disorders. Non-thyroidal illness syndrome (NTIS), characterized by low serum 3,5,3′-triiodothyronine (T3) with normal l-thyroxine (T4) levels, is associated with malignancy. Decreased activity of type I 5′-deiodinase (DIO1), which converts T4 to T3, contributes to NTIS. T3 binds to thyroid hormone receptor, which heterodimerizes with retinoid X receptor (RXR) and regulates transcription of target genes, such as DIO1. NF-κB activation by inflammatory cytokines inhibits DIO1 expression. The oncogene astrocyte elevated gene-1 (AEG-1) inhibits RXR-dependent transcription and activates NF-κB. Here, we interrogated the role of AEG-1 in NTIS in the context of hepatocellular carcinoma (HCC). T3-mediated gene regulation was analyzed in human HCC cells, with overexpression or knockdown of AEG-1, and primary hepatocytes from AEG-1 transgenic (Alb/AEG-1) and AEG-1 knock-out (AEG-1KO) mice. Serum T3 and T4 levels were checked in Alb/AEG-1 mice and human HCC patients. AEG-1 and DIO1 levels in human HCC samples were analyzed by immunohistochemistry. AEG-1 inhibited T3-mediated gene regulation in human HCC cells and mouse hepatocytes. AEG-1 overexpression repressed and AEG-1 knockdown induced DIO1 expression. An inverse correlation was observed between AEG-1 and DIO1 levels in human HCC patients. Low T3 with normal T4 was observed in the sera of HCC patients and Alb/AEG-1 mice. Inhibition of co-activator recruitment to RXR and activation of NF-κB were identified to play a role in AEG-1-mediated down-regulation of DIO1. AEG-1 thus might play a role in NTIS associated with HCC and other cancers.

Actin co-sedimentation assay; for the analysis of protein binding to F-actin.

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape. Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling. A number of proteins can bind to the actin cytoskeleton. The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin. The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin. Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Abstract 5400: A novel combinatorial therapy for hepatocellular carcinoma (HCC)

The incidence of hepatocellular carcinoma (HCC) is rising in the US with parallel increase in mortality rate. Lack of effective therapy for advanced HCC mandates development of novel targeted therapies to counteract this fatal malady. Astrocyte elevated gene-1 (AEG-1), also known as metadherin (MTDH) and LYRIC, plays a pivotal role in hepatocarcinogenesis and serves as an ideal target for anti-HCC therapy. AEG-1 interacts with retinoid X receptor (RXR) and inhibits retinoic acid-induced gene expression and cell death. Retinoic acid has been evaluated for HCC treatment without promising result. Overexpression of AEG-1 might underlie the poor performance of retinoic acid in HCC clinical trials. We documented that combination of a lentivirus expressing AEG-1 shRNA (lenti.shAEG-1) and all-trans retinoic acid (ATRA) profoundly and synergistically inhibited subcutaneous human HCC xenografts in nude mice. We now have developed liver-targeted nanoplexes by conjugating poly(amidoamine) (PAMAM) dendrimers with polyethylene glycol (PEG) and lactobionic acid (PAMAM-PEG-Gal) which were complexed with AEG-1 siRNA (PAMAM-AEG-1si). PAMAM has a net positive charge that complexes with the net negative charge of the siRNA backbone, PEG increases stability of the nanoplexes and increases circulation time and the galactose binds to asialoglycoprotein receptors that are upregulated on liver cells. The polymer conjugate was characterized by 1H-NMR and optimal nanoplex formulations were characterized for surface charge and size using a zetasizer Nano ZS. We established orthotopic xenografts of human HCC cell QGY-7703 expressing luciferase (QGY-luc) in the livers of athymic nude mice and monitored tumor development by bioluminescence imaging (BLI). One week after tumor establishment mice were treated with PAMAM-siCon, PAMAM-siCon+ATRA, PAMAM-AEG-1si and PAMAM-AEG-1si+ATRA by 8 i.v. injections over 4 weeks, and sacrificed 2 weeks after the last injection. In the control group the tumor developed aggressively. ATRA showed little effect due to high AEG-1 levels in QGY-luc cells. PAMAM-AEG-1si showed significant reduction in tumor growth and the combination of PAMAM-AEG-1si+ATRA showed profound and synergistic inhibition so that the tumors were almost undetectable by BLI. Measurement of liver weight at the end of the experiment corroborated these findings. Analysis of AEG-1 mRNA in tumor samples showed a marked decrease in AEG-1 level by PAMAM-AEG-1si indicating efficacy of in vivo knockdown. The group treated with PAMAM-AEG-1si+ATRA nanoplexes showed increased necrosis, inhibition of proliferation and increased apoptosis when compared to other groups. Liver is an ideal organ for RNAi therapy and ATRA is an approved anti-cancer agent. Our exciting observations suggest that the combinatorial approach might be an effective way to combat HCC and need to be evaluated stringently in endogenous mouse models of HCC for a potential Phase I/II clinical trial. Citation Format: Jyoti Srivastava, Devaraja Rajasekaran, Ayesha Siddiq, Rachel Gredler, Chadia L. Robertson, Maaged A. Akiel, Xue-Ning Shen, Kareem A.N. Ebeid, Aliasger K. Salem, Paul B. Fisher, Devanand Sarkar. A novel combinatorial therapy for hepatocellular carcinoma (HCC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5400. doi:10.1158/1538-7445.AM2015-5400