Article

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: 3.37). 07/2008; 314(15):2811-21. DOI: 10.1016/j.yexcr.2008.06.011
Source: PubMed

ABSTRACT 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.

2 Followers
 · 
67 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Here we report that targeting casein kinase 1 (CSNK1α1) is a potential novel treatment strategy in multiple myeloma (MM) therapy distinct from proteasome inhibition. CSNK1α1 is expressed in all the tested MM cell lines and patient MM cells, and is not altered during bortezomib-triggered cytotoxicity. Inhibition of CSNK1α1 kinase activity in MM cells with targeted therapy D4476 or shRNAs triggers cell G0/G1 phase arrest, prolonged G2/M phase, and apoptosis. D4476 also induced cytotoxicity in bortezomib-resistant MM cells and enhances bortezomib-triggered cytotoxicity. CSNK1α1 signaling pathways include CDKN1B, P53 and FADD; gene signatures involved included interferon alpha (IFNα), tumor necrosis factor alpha (TNFα), and LIN9. Additionally, reduction of Csnk1α1 prevents cMYC/KRAS12V transformation of BaF3 cells independent of IL3. Impartially, reducing Csnk1α1 prevented development of cMYC/KRAS12V-induced plasmacytomas (PCTs) in mice, suggesting that CSNK1α1 may be involved in MM initiation and progression. Our data suggest that targeting CSNK1α1, alone or combined with bortezomib, is a potential novel therapeutic strategy in MM. Moreover, inhibition of CSNK1α1 may prevent the progression of monoclonal gammopathy of undetermined significance (MGUS) to MM.Leukemia accepted article preview online, 25 June 2014; doi:10.1038/leu.2014.202.
    Leukemia 06/2014; 29(2). DOI:10.1038/leu.2014.202 · 9.38 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Determining the cellular level of activated form of RhoGTPases is of key importance to understand their regulatory functions in cell physiopathology. We previously reported scFvC1, that selectively bind to the GTP-bound form of RhoA, RhoB and RhoC. In this present study we generate, by molecular evolution, a new phage library to isolate scFvs displaying high affinity and selectivity to RhoA and RhoB. Using phage display affinity maturation against the GTP-locked mutant RhoAL63, we isolated scFvs against RhoA active conformation that display Kd values at the nanomolar range, which corresponded to an increase of affinity of three orders of magnitude compared to scFvC1. Although a majority of these evolved scFvs remained selective towards the active conformation of RhoA, RhoB and RhoC, we identified some scFvs that bind to RhoA and RhoC but not to RhoB activated form. Alternatively, we performed a substractive panning towards RhoB, and isolated the scFvE3 exhibiting a 10 times higher affinity for RhoB than RhoA activated forms. We showed the peculiar ability of scFvE3 to detect RhoB but not RhoA GTP-bound form in cell extracts overexpressing Guanine nucleotide Exchange Factor XPLN as well as in EGF stimulated HeLa cells. Our results demonstrated the ability of scFvs to distinguish RhoB from RhoA GTP-bound form and provide new selective tools to analyze the cell biology of RhoB GTPase regulation.
    PLoS ONE 11/2014; 9(11):e111034. DOI:10.1371/journal.pone.0111034 · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Members of the highly conserved and ubiquitously expressed pleiotropic CK1 family play major regulatory roles in many cellular processes including DNA-processing and repair, proliferation, cytoskeleton dynamics, vesicular trafficking, apoptosis, and cell differentiation. As a consequence of cellular stress conditions, interaction of CK1 with the mitotic spindle is manifold increased pointing to regulatory functions at the mitotic checkpoint. Furthermore, CK1 is able to alter the activity of key proteins in signal transduction and signal integration molecules. In line with this notion, CK1 is tightly connected to the regulation and degradation of β-catenin, p53, and MDM2. Considering the importance of CK1 for accurate cell division and regulation of tumor suppressor functions, it is not surprising that mutations and alterations in the expression and/or activity of CK1 isoforms are often detected in various tumor entities including cancer of the kidney, choriocarcinomas, breast carcinomas, oral cancer, adenocarcinomas of the pancreas, and ovarian cancer. Therefore, scientific effort has enormously increased (i) to understand the regulation of CK1 and its involvement in tumorigenesis- and tumor progression-related signal transduction pathways and (ii) to develop CK1-specific inhibitors for the use in personalized therapy concepts. In this review, we summarize the current knowledge regarding CK1 regulation, function, and interaction with cellular proteins playing central roles in cellular stress-responses and carcinogenesis.