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    • CK comprises a family of serine threonine protein

    2019-11-07

    CK1 comprises a family of serine/threonine protein kinases, that includes different CK1 isoforms namely α, β, δ, ε and γ their various splice variants [56]. It was reported that the pharmacological inhibitor IC261 shows an order of magnitude of higher selectivity for CK1δ or CK1ε isoforms (IC50 of 1 μM) over other CK1 isoforms (IC50 of 16 μM for CK1α) [50]. Hence the IC261 concentration required to inhibit RhoB phosphorylation suggests that RhoB would be the substrate of a CK1 isoform distinct from CK1δ or CK1ε. The similarity of the ratio of phosphorylated RhoB/total RhoB in CTPB mg transfected with wild-type or constitutively-activated RhoB indicated that the nature of the nucleotide bound, GDP or GTP, to RhoB did not alter its rate of phosphorylation in cellulo, as reported previously for Cdc42 by Src [11]. Moreover the phosphorylation of RhoB was also observed in A549 and COS-7 (data not shown) indicating that it is not restricted to a single cell model and could be a broad-spectrum regulation process of RhoB. Further work need to be performed to determine whether RhoB is constitutively phosphorylated by CK1 as it was shown for Parkinson disease associated αsynuclein [58] or whether its phosphorylation takes place during responses to extracellular stimuli such as for hnRNP in response to H2O2[59] or Dvl in response to Wnt-5a [60]. Nevertheless since RhoB is highly regulated by diverse stimuli such as stress and growth factors, its phosphorylation might probably be a regulated process. The pull-down assay using the Rho Binding Domain (RBD) of Rhotekin that selectively binds to the Rho activated form [46] showed that exposure to CK1 inhibitors, either D4476 or IC261, increased significantly RhoB binding to the RBD. The effects of the CK1 inhibitors in increasing RhoB activity could be due to indirect effects on phosphorylation of other proteins, such as GEFs/GAPs for RhoB, rather than directly via reduced phosphorylation of RhoB itself. Nevertheless the mutant RhoBS185A mutant appears to be more active than wild-type RhoB in this pull-down assay. These observations strongly suggest that RhoB phosphorylation by CK1 impaired the binding of RhoB to its effectors. This could be the result of the reduction of RhoB-GTP following the modulation of its GTPase activity such as for Rab24 [61] or of its interaction with the GDP/GTP cycle regulators e.g, GDI or GEFs such as for CdC42 [11]. But the CK1-mediated phosphorylation of RhoB on Ser185 might also merely alter the binding of RhoB to this effector, without any incidence of GTP loading, as recently described for PKA phosphorylation of RhoA [15]. Accordingly we observed that the unphosphorylated mutant RhoBS185A might recruit its effector PRK1, as well before as after EGF treatment, which is reported to stimulate GTP loading on RhoB [24], [49]. Hence as described previously for RhoA [15], the phosphorylation of RhoB which inhibits its cell functions could serve as an alternative regulation mechanism in addition to GDP/GTP cycle. Our data show that inhibition of CK1 activity induced an important stress fiber formation which is known to be under the control of Rho proteins. The silencing experiments indicated that RhoB expression is critical for IC261 effect on actin cytoskeleton, suggesting that CK1 phosphorylation of RhoB down regulates its regulatory role on actin cytoskeleton organization. Our data also emphasized the role of RhoB in control of actin organization as we previously described [42]. Chardin et al., using C. botulinum, which affects the three Rho proteins, first proposed that they may be involved in stress fiber control [53]. That was subsequently argued by Rho protein overexpression or injection experiments that underlined the role of the three Rho proteins on actin stress fiber formation [62], [63]. Nevertheless an incomplete effect of siRhoA on actin fiber regulation was also reported [64], [65] due to RhoB activity. Herein we showed with RhoB siRNA treatment that RhoB plays a true role in stress fiber organization.