A Chemical Inhibitor Reveals the Role of Raf Kinase Inhibitor Protein in Cell Migration

Zhu, Shoutian; Henry, Kevin T. Mc; Lane, William S.; Fenteany, Gabriel

doi:10.1016/j.chembiol.2005.07.007

Cited by 102 publications

(136 citation statements)

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“…Since the discovery of the Raf kinase inhibitory protein (RKIP) 26 years ago by Jones and colleagues (1), and later by Bernier and colleagues (2), there has been considerable amount of published data linking RKIP to various intracellular signaling networks that control cellular growth (3)(4)(5)(6), motility (4,7,8), epithelial to mesenchymal transition (EMT; refs. 9, 10), and differentiation (11)(12)(13).…”

Section: Introductionmentioning

confidence: 99%

Raf Kinase Inhibitor Protein RKIP Enhances Signaling by Glycogen Synthase Kinase-3β

et al. 2011

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Raf kinase inhibitory protein (RKIP) is a physiologic inhibitor of c-RAF kinase and nuclear factor kB signaling that represses tumor invasion and metastasis. Glycogen synthase kinase-3b (GSK3b) suppresses tumor progression by downregulating multiple oncogenic pathways including Wnt signaling and cyclin D1 activation. Here, we show that RKIP binds GSK3 proteins and maintains GSK3b protein levels and its active form. Depletion of RKIP augments oxidative stress-mediated activation of the p38 mitogen activated protein kinase, which, in turn, inactivates GSK3b by phosphorylating it at the inhibitory T390 residue. This pathway de-represses GSK3b inhibition of oncogenic substrates causing stabilization of cyclin D, which induces cell-cycle progression and b-catenin, SNAIL, and SLUG, which promote epithelial to mesenchymal transition. RKIP levels in human colorectal cancer positively correlate with GSK3b expression. These findings reveal the RKIP/GSK3 axis as both a potential therapeutic target and a prognosis-based predictor of cancer progression. Cancer Res; 71(4); 1334-43. Ó2011 AACR.

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Section: Introductionmentioning

confidence: 99%

Raf Kinase Inhibitor Protein RKIP Enhances Signaling by Glycogen Synthase Kinase-3β

et al. 2011

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“…Liquid chromatography-tandem mass spectrometry was performed using a 75-m reverse-phase microcolumn terminating in a PicoView nanoelectrospray source (New Objective) directly coupled to a Thermo Scientific LTQ Orbitrap linear quadrupole ion trap mass spectrometer. These procedures and the subsequent analyses have been described elsewhere (16,18).…”

Section: Methodsmentioning

confidence: 99%

“…The siRNA target sequences were synthesized as short hairpin RNA (shRNA) molecules and ligated into an shRNA expression vector under the direction of the U6 promoter. This shRNA expression vector has been described previously (12); it contains a GFP expression cassette to aid in isolation of stable transfectants and was modified from an earlier vector we designed (16). The constructs were transfected into MDCK cells with Lipofectamine (Invitrogen) according to the manufacturer's protocol.…”

Section: Preparation Of Mdck Cells Expressing Green Fluorescent Protementioning

confidence: 99%

“…We have exploited the classical scratch-wound closure assay for screening chemical * This work was supported, in whole or in part, by National Institutes of Health libraries to discover new compounds that inhibit or accelerate cell migration. We have identified a number of new inhibitors of cell migration, including locostatin (15)(16)(17) and the tetrahydroisoquinoline DX-52-1, a semisynthetic derivative of quinocarmycin (also known as quinocarcin) (18). These small molecules target Raf kinase inhibitor protein (16) and the membrane-actin cytoskeleton linker protein radixin (18), respectively.…”

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confidence: 99%

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Analogs of Tetrahydroisoquinoline Natural Products That Inhibit Cell Migration and Target Galectin-3 Outside of Its Carbohydrate-binding Site

Kahsai

Cui

Kaniskan

et al. 2008

Journal of Biological Chemistry

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Cell migration is central to a number of normal and disease processes. Small organic molecules that inhibit cell migration have potential as both research probes and therapeutic agents. We have identified two tetrahydroisoquinoline natural product analogs with antimigratory activities on Madin-Darby canine kidney epithelial cells: a semisynthetic derivative of quinocarmycin (also known as quinocarcin), DX-52-1, and a more complex synthetic molecule, HUK-921, related to the naphthyridinomycin family. It has been assumed that the cellular effects of reactive tetrahydroisoquinolines result from the alkylation of DNA. We have reported previously that the primary target of DX-52-1 relevant to cell migration appears to be the membranecytoskeleton linker protein radixin. Here we extend the analysis of the protein targets of DX-52-1, reporting that the multifunctional carbohydrate-binding protein galectin-3 is a secondary target of DX-52-1 that may also be relevant to the antimigratory effects of both DX-52-1 and HUK-921. All known inhibitors of galectin-3 target its ␤-galactoside-binding site in the carbohydrate recognition domain. However, we found that DX-52-1 and HUK-921 bind galectin-3 outside of its ␤-galactoside-binding site. Intriguingly HUK-921, although a less potent inhibitor of cell migration than DX-52-1, had far greater selectivity for galectin-3 over radixin, exhibiting little binding to radixin, both in vitro and in cells. Overexpression of galectin-3 in cells led to a dramatic increase in cell adhesion on different extracellular matrix substrata as well as changes in cell-cell adhesion and cell motility. Galectin-3-overexpressing cells had greatly reduced sensitivity to DX-52-1 and HUK-921, and these compounds caused a change in localization of the overexpressed galectin-3 and reversion of the cells to a more normal morphology. The converse manipulation, RNA interference-based silencing of galectin-3 expression, resulted in reduced cell-matrix adhesion and cell migration. In aggregate, the data suggest that DX-52-1 and HUK-921 inhibit a carbohydrate binding-independent function of galectin-3 that is involved in cell migration.

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“…At a cellular level, locostatin prevents cell migration by binding to PEBP1, as demonstrated by a chemical genetics approach [22,23]. The unique observation of the in vitro anti-migration effect of locostatin, together with the anti-metastasis effect of its target protein, is the basis for the study of the hPEBP1-locostatin complex in vitro with the aim to obtain valuable information for the design of new molecules as potential antimetastatic leads.…”

Section: Biological Significancementioning

confidence: 99%

SSPaQ: A Subtractive Segmentation Approach for the Exhaustive Parallel Quantification of the Extent of Protein Modification at Every Possible Site

Gabant

Boyer

Cadène

2016

J. Am. Soc. Mass Spectrom.

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Abstract. Protein modifications, whether chemically induced or post-translational (PTMs), play an essential role for the biological activity of proteins. Understanding biological processes and alterations thereof will rely on the quantification of these modifications on individual residues. Here we present SSPaQ, a subtractive method for the parallel quantification of the extent of modification at each possible site of a protein. The method combines uniform isotopic labeling and proteolysis with MS, followed by a segmentation approach, a powerful tool to refine the quantification of the degree of modification of a peptide to a segment containing a single modifiable amino acid. The strength of this strategy resides in: (1) quantification of all modifiable sites in a protein without prior knowledge of the type(s) of modified residues; (2) insensitivity to changes in the solubility and ionization efficiency of peptides upon modification; and (3) detection of missed cleavages caused by the modification for mitigation. The SSPaQ method was applied to quantify modifications resulting from the interaction of human phosphatidyl ethanolamine binding protein 1 (hPEBP1), a metastasis suppressor gene product, with locostatin, a covalent ligand and antimigratory compound with demonstrated activity towards hPEBP1. Locostatin is shown to react with several residues of the protein. SSPaQ can more generally be applied to induced modification in the context of drugs that covalently bind their target protein. With an alternate front-end protocol, it could also be applied to the quantification of protein PTMs, provided a removal tool is available for that PTM.

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A Chemical Inhibitor Reveals the Role of Raf Kinase Inhibitor Protein in Cell Migration

Cited by 102 publications

References 48 publications

Raf Kinase Inhibitor Protein RKIP Enhances Signaling by Glycogen Synthase Kinase-3β

Raf Kinase Inhibitor Protein RKIP Enhances Signaling by Glycogen Synthase Kinase-3β

Analogs of Tetrahydroisoquinoline Natural Products That Inhibit Cell Migration and Target Galectin-3 Outside of Its Carbohydrate-binding Site

SSPaQ: A Subtractive Segmentation Approach for the Exhaustive Parallel Quantification of the Extent of Protein Modification at Every Possible Site

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