Phosphorylation of RhoB by CK1 impedes actin stress fiber organization and epidermal growth factor receptor stabilization

INSERM, U563, CPTP, Département Innovation Thérapeutique et Oncologie Moléculaire, Toulouse F-31052, France.
Experimental Cell Research (Impact Factor: 3.25). 07/2008; 314(15):2811-21. DOI: 10.1016/j.yexcr.2008.06.011
Source: PubMed


RhoB is a small GTPase implicated in cytoskeletal organization, EGF receptor trafficking and cell transformation. It is an immediate-early gene, regulated at many levels of its biosynthetic pathway. Herein we show that the serine/threonine protein kinase CK1 phosphorylates RhoB in vitro but not RhoA or RhoC. With the use of specific CK1 inhibitors, IC261 and D4476, we show that the kinase phosphorylates also RhoB in HeLa cells. Mass spectrometry analysis demonstrates that RhoB is monophosphorylated by CK1, in its C-terminal end, on serine 185. The substitution of Ser185 by Ala dramatically inhibited the phosphorylation of RhoB in cultured cells. Lastly we show that the inhibition of CK1 activates RhoB and promotes RhoB dependent actin fiber formation and EGF-R level. Our data provide the first demonstration of RhoB phosphorylation and indicate that this post-translational maturation would be a novel critical mechanism to control the RhoB functions.

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    • "properties in pros - tatic cancer ( Hooi et al . , 2006 ; Chen et al . , 2007 ) . Finally , the biological relevance of NAG - 1 in Rho - mediated pathways was fur - ther demonstrated in vivo in a model of tumorigenesis . We and others recently reported direct cross - regulation within the Rho subclass ( Simpson et al . , 2004 ; Ho et al . , 2008 ; Tillement et al . , 2008 ; Steffan et al . , 2009 ) . As a result , the phenotypic modifications in - duced by RhoC silencing in PC - 3 cells might be due to either the repression of RhoC or the concurrent induction of RhoA or RhoB . Of interest , the use of inducible clones over - expressing RhoA recapitulates most of the effects elicited by silencing RhoC , i"
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    ABSTRACT: RhoGTPases are key signaling molecules regulating main cellular functions such as migration, proliferation, survival, and gene expression through interactions with various effectors. Within the RhoA-related subclass, RhoA and RhoC contribute to several steps of tumor growth, and the regulation of their expression affects cancer progression. Our aim is to investigate their respective contributions to the acquisition of an invasive phenotype by using models of reduced or forced expression. The silencing of RhoC, but not of RhoA, increased the expression of genes encoding tumor suppressors, such as nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1), and decreased migration and the anchorage-independent growth in vitro. In vivo, RhoC small interfering RNA (siRhoC) impaired tumor growth. Of interest, the simultaneous knockdown of RhoC and NAG-1 repressed most of the siRhoC-related effects, demonstrating the central role of NAG-1. In addition of being induced by RhoC silencing, NAG-1 was also largely up-regulated in cells overexpressing RhoA. The silencing of RhoGDP dissociation inhibitor α (RhoGDIα) and the overexpression of a RhoA mutant unable to bind RhoGDIα suggested that the effect of RhoC silencing is indirect and results from the up-regulation of the RhoA level through competition for RhoGDIα. This study demonstrates the dynamic balance inside the RhoGTPase network and illustrates its biological relevance in cancer progression.
    Molecular biology of the cell 07/2011; 22(17):3263-75. DOI:10.1091/mbc.E11-01-0020 · 4.47 Impact Factor
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    • "This assumption was further underlined by time-lapse analyses showing that the CK1δ and ε specific inhibitors IC261 and D4476 induced a collapse and retraction of growing neurites of isolated RGCs, probably due to a destabilization of the microtubule and/or actin cable network. This hypothesis is supported by previous observations showing that CK1 is involved in regulating both microtubule and actin filament dynamics [38], [39], [47], [49], [50], [73]–[75]. "
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    PLoS ONE 06/2011; 6(6):e20857. DOI:10.1371/journal.pone.0020857 · 3.23 Impact Factor
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    • "This is consistent with D4776 blocking phosphorylation at S56, thereby allowing more cap methylation of the viral replicon RNA and subsequent protein translation. Concentrations of 10-100 μM of D4476 are often used in other cell lines to inhibit CK1 (Maclaine et al., 2008; Tillement et al., 2008), but we observed cytotoxicity via a soluble tetrazolium salt cytotoxicity assay at concentrations over 30 μM (data not shown). D4476 is considered specific for CK1, but the observed decrease in viral yield could either be mediated by a CK1-like kinase or could be an indirect effect of CK1 inhibition on YFV. "
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    ABSTRACT: Serine/Threonine phosphorylation of the nonstructural protein 5 (NS5) is a conserved feature of flaviviruses, but the identity and function(s) of the responsible kinase(s) remain unknown. Serine 56 in the methyltransferase domain of NS5 can be phosphorylated intracellularly, is conserved in all flaviviruses, and is a critical residue in the catalytic mechanism. A negative charge at this residue inactivates the 2'-0 methyltransferase activity necessary to form a 5' cap structure of the viral RNA. Here we show pharmacologic inhibition of Casein Kinase 1 (CK1) suppresses yellow fever virus (YFV) production. We also demonstrate the alpha isoform of Casein Kinase 1 (CK1alpha), a kinase previously identified as phosphorylating Hepatitis C Virus NS5A protein, also phosphorylates serine 56 of YFV methyltransferase. Overall these results suggest CK1 activity can influence flaviviral replication.
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