Protein kinase CK1alpha regulates mRNA binding by heterogeneous nuclear ribonucleoprotein C in response to physiologic levels of hydrogen peroxide.
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At low concentrations, hydrogen peroxide (H(2)O(2)) is a positive endogenous regulator of mammalian cell proliferation and survival; however, the signal transduction pathways involved in these processes are poorly understood. In primary human endothelial cells, low concentrations of H(2)O(2) stimulated the rapid phosphorylation of the acidic C-terminal domain (ACD) of heterogeneous nuclear ribonucleoprotein C (hnRNP-C), a nuclear restricted pre-mRNA-binding protein, at Ser(240) and at Ser(225)-Ser(228). A kinase activity was identified in mouse liver that phosphorylates the ACD of hnRNP-C at Ser(240) and at two sites at Ser(225)-Ser(228). The kinase was purified and identified by tandem mass spectrometry as protein kinase CK1alpha (formerly casein kinase 1alpha). Protein kinase CK1alpha immunoprecipitated from primary human endothelial cell nuclei also phosphorylated the ACD of hnRNP-C at these positions. Pretreatment of endothelial cells with the protein kinase CK1-specific inhibitor IC261 prevented the H(2)O(2)-stimulated phosphorylation of hnRNP-C. Utilizing phosphoserine-mimicking Ser-to-Glu point mutations, the effects of phosphorylation on hnRNP-C function were investigated by quantitative equilibrium fluorescence RNA binding analyses. Wild-type hnRNP-C1 and hnRNP-C1 modified at the basal sites of phosphorylation (S247E and S286E) both avidly bound RNA with similar binding constants. In contrast, hnRNP-C1 that was also modified at the CK1alpha phosphorylation sites exhibited a 14-500-fold decrease in binding affinity, demonstrating that CK1alpha-mediated phosphorylation modulates the mRNA binding ability of hnRNP-C. (+info)
Physiological role for casein kinase 1 in glutamatergic synaptic transmission.
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Casein kinase 1 (CK1) is a highly conserved serine/threonine kinase, present in virtually all cell types, in which it phosphorylates a wide variety of substrates. So far, no role has been found for this ubiquitous protein kinase in the physiology of nerve cells. In the present study, we show that CK1 regulates fast synaptic transmission mediated by glutamate, the major excitatory neurotransmitter in the brain. Through the use of CK1 inhibitors, we present evidence that activation of CK1 decreases NMDA receptor activity in the striatum via a mechanism that involves activation by this kinase of protein phosphatase 1 and/or 2A and resultant increased dephosphorylation of NMDA receptors. Indeed, inhibition of CK1 increases NMDA-mediated EPSCs in medium spiny striatal neurons. This effect is associated with an increased phosphorylation of the NR1 and NR2B subunits of the NMDA receptor and is occluded by the phosphatase inhibitor okadaic acid. The mGluR1, but not mGluR5, subclass of metabotropic glutamate receptors uses CK1 to inhibit NMDA-mediated synaptic currents. These results provide the first evidence for a role of CK1 in the regulation of synaptic transmission in the brain. (+info)
Regulation of p53-MDMX interaction by casein kinase 1 alpha.
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MDMX is a homolog of MDM2 that is critical for regulating p53 function during mouse development. MDMX degradation is regulated by MDM2-mediated ubiquitination. Whether there are other mechanisms of MDMX regulation is largely unknown. We found that MDMX binds to the casein kinase 1 alpha isoform (CK1alpha) and is phosphorylated by CK1alpha. Expression of CK1alpha stimulates the ability of MDMX to bind to p53 and inhibit p53 transcriptional function. Regulation of MDMX-p53 interaction requires CK1alpha binding to the central region of MDMX and phosphorylation of MDMX on serine 289. Inhibition of CK1alpha expression by isoform-specific small interfering RNA (siRNA) activates p53 and further enhances p53 activity after ionizing irradiation. CK1alpha siRNA also cooperates with DNA damage to induce apoptosis. These results suggest that CK1alpha is a functionally relevant MDMX-binding protein and plays an important role in regulating p53 activity in the absence or presence of stress. (+info)
Phosphorylation of FADD at serine 194 by CKIalpha regulates its nonapoptotic activities.
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FADD is essential for death receptor (DR)-induced apoptosis. However, it is also critical for cell cycle progression and proliferation, activities that are regulated by phosphorylation of its C-terminal Ser194, which has also been implicated in sensitizing cancer cells to chemotherapeutic drugs and in regulating FADD's intracellular localization. We now demonstrate that casein kinase Ialpha (CKIalpha) phosphorylates FADD at Ser194 both in vitro and in vivo. FADD-CKIalpha association regulates the subcellular localization of FADD, and phosphorylated FADD was found to colocalize with CKIalpha on the spindle poles in metaphase. Inhibition of CKIalpha diminished FADD phosphorylation, prevented the ability of Taxol to arrest cells in mitosis, and blocked mitogen-induced proliferation of mouse splenocytes. In contrast, a low level of cycling splenocytes from mice expressing FADD with a mutated phosphorylation site was insensitive to CKI inhibition. These data suggest that phosphorylation of FADD by CKI is a crucial event during mitosis. (+info)
Phosphorylation by double-time/CKIepsilon and CKIalpha targets cubitus interruptus for Slimb/beta-TRCP-mediated proteolytic processing.
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Hedgehog (Hh) proteins govern animal development by regulating the Gli/Ci family of transcription factors. In Drosophila, Hh signaling blocks proteolytic processing of full-length Ci to generate a truncated repressor form. Ci processing requires sequential phosphorylation by PKA, GSK3, and a casein kinase I (CKI) family member(s). Here we show that Double-time (DBT)/CKIepsilon and CKIalpha act in conjunction to promote Ci processing. CKI phosphorylates Ci at three clusters of serine residues primed by PKA and GSK3 phosphorylation. CKI phosphorylation of Ci confers binding to the F-box protein Slimb/beta-TRCP, the substrate recognition component of the SCF(Slimb/beta-TRCP) ubiquitin ligase required for Ci processing. CKI phosphorylation sites act cooperatively to promote Ci processing in vivo. Substitution of Ci phosphorylation clusters with a canonical Slimb/beta-TRCP recognition motif in beta-catenin renders Slimb/beta-TRCP binding and Ci processing independent of CKI. We propose that phosphorylation of Ci by CKI creates multiple Slimb/beta-TRCP binding sites that act cooperatively to recruit SCF(Slimb/beta-TRCP). (+info)
Coordination of NF-kappaB and NFAT antagonism by the forkhead transcription factor Foxd1.
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Forkhead transcription factors play critical roles in the maintenance of immune homeostasis. In this study, we demonstrate that this regulation most likely involves intricate interactions between the forkhead family members and inflammatory transcription factors: the forkhead member Foxd1 coordinates the regulation of the activity of two key inflammatory transcription factors, NF-AT and NF-kappaB, with Foxd1 deficiency resulting in multiorgan, systemic inflammation, exaggerated Th cell-derived cytokine production, and T cell proliferation in autologous MLRs. Foxd1-deficient T cells possess increased activity of both NF-AT and NF-kappaB: the former correlates with the ability of Foxd1 to regulate casein kinase 1, an NF-AT inhibitory kinase; the latter with the ability of Foxd1 to regulate Foxj1, which regulates the NF-kappaB inhibitory subunit IkappaB beta. Thus, Foxd1 modulates inflammatory reactions and prevents autoimmunity by directly regulating anti-inflammatory regulators of the NF-AT pathway, and by coordinating the suppression of the NF-kappaB pathway via Foxj1. These findings indicate the presence of a general network of forkhead proteins that enforce T cell quiescence. (+info)
Planar polarity is positively regulated by casein kinase Iepsilon in Drosophila.
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Members of the casein kinase I (CKI) family have been implicated in regulating canonical Wnt/Wingless (Wg) signaling by phosphorylating multiple pathway components. Overexpression of CKI in vertebrate embryos activates Wg signaling, and one target is thought to be the cytoplasmic effector Dishevelled (Dsh), which is an in vitro target of CKI phosphorylation. Phosphorylation of Dsh by CKI has also been suggested to switch its activity from noncanonical to canonical Wingless signaling. However, in vivo loss-of-function experiments have failed to identify a clear role for CKI in positive regulation of Wg signaling. By examining hypomorphic mutations of the Drosophila CKIepsilon homolog discs overgrown (dco)/double-time, we now show that it is an essential component of the noncanonical/planar cell polarity pathway. Genetic interactions indicate that dco acts positively in planar polarity signaling, demonstrating that it does not act as a switch between canonical and noncanonical pathways. Mutations in dco result in a reduced level of Dishevelled phosphorylation in vivo. Furthermore, in these mutants, Dishevelled fails to adopt its characteristic asymmetric subcellular localisation at the distal end of pupal wing cells, and the site of hair outgrowth is disrupted. Finally, we also find that dco function in polarity is partially redundant with CKIalpha. (+info)
Wnt-5a/Ca2+-induced NFAT activity is counteracted by Wnt-5a/Yes-Cdc42-casein kinase 1alpha signaling in human mammary epithelial cells.
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Wnt-5a has been shown to influence the metastatic behavior of human breast cancer cells, and the loss of Wnt-5a expression is associated with metastatic disease. We show here that NFAT1, a transcription factor connected with breast cancer metastasis, is activated by Wnt-5a through a Ca2+ signaling pathway in human breast epithelial cells. This activation was simultaneously counteracted by a Wnt-5a-induced Yes/Cdc42 signaling pathway. The observation that inhibition of the Wnt-5a/Yes/Cdc42 signal prolonged the duration of ionomycin-induced NFAT1 activation revealed the general importance of this pathway. The Wnt-5a-induced inhibition of NFAT1 did not require glycogen synthase kinase 3beta, JNK, or Pak1 activity or modulation of the cytoskeleton. Instead, we observed that Wnt-5a induced a complex formation of NFAT1/casein kinase 1alpha, even upon treatment with ionomycin, which was blocked upon inhibition of the Wnt-5a/Yes/Cdc42 signaling pathway. Our results explain why Wnt-5a/Ca2+-induced NFAT activity is hard to detect and suggest a novel mechanism by which Wnt-5a can suppress tumor-specific, agonist-induced NFAT activity and thus the metastatic behavior of breast cancer cells. (+info)