The casein kinase I family in Wnt signaling.

The canonical Wnt-signaling pathway is critical for many aspects of development, and mutations in components of the Wnt pathway are carcinogenic. Recently, sufficiency tests identified casein kinase Iepsilon (CKIepsilon) as a positive component of the canonical Wnt/beta-catenin pathway, and necessity tests showed that CKIepsilon is required in vertebrates to transduce Wnt signals. In addition to CKIepsilon, the CKI family includes several other isoforms (alpha, beta, gamma, and delta) and their role in Wnt sufficiency tests had not yet been clarified. However, in Caenorhabditis elegans studies, loss-of-function of a CKI isoform most similar to alpha produced the mom phenotype, indicative of loss-of-Wnt signaling. In this report, we examine the ability of the various CKI isoforms to activate Wnt signaling and find that all the wild-type CKI isoforms do so. Dishevelled (Dsh), another positive component of the Wnt pathway, becomes phosphorylated in response to Wnt signals. All the CKI isoforms, with the exception of gamma, increase the phosphorylation of Dsh in vivo. In addition, CKI directly phosphorylates Dsh in vitro. Finally, we find that CKI is required in vivo for the Wnt-dependent phosphorylation of Dsh. These studies advance our understanding of the mechanism of Wnt action and suggest that more than one CKI isoform is capable of transducing Wnt signals in vivo.     

Phosphorylation and regulation of beta-catenin by casein kinase I epsilon

beta-Catenin transduces cytosolic signals to the nucleus in the Wnt pathway. The Wnt ligand stabilizes cytosolic beta-catenin protein, preventing its phosphorylation by inhibiting glycogen synthase kinase 3 (GSK3). Serine-33 and -37 of beta-catenin are GSK3 phosphorylation sites that serve as recognition sites for the beta-TRCP-ubiquitin ligase complex, which ultimately triggers beta-catenin degradation. Mutations at those two sites, as well as in Ser-45, stabilize beta-catenin. Recently, casein kinase I epsilon (CKI epsilon) has been shown to be a positive regulator of the Wnt pathway. Its action mechanism, however, remains unknown. Here I show that Ser-45 is phosphorylated not by GSK3 but by CKI epsilon. Axin, a scaffold protein that binds CKI epsilon and beta-catenin, enhances this CKI epsilon-mediated phosphorylation. Overexpression of CKI epsilon in cells increases the amount of beta-catenin phosphorylated at Ser-45. Ser-45 phosphorylated beta-catenin is a better substrate for GSK3, which suggests that CKI epsilon and GSK3 may co-operate in destabilizing beta-catenin. In spite of the fact that CKI epsilon was found as a positive regulator of the Wnt pathway, mutational analysis suggests that mutation of Ser-45 regulates beta-catenin stability by inhibiting the ability of GSK3 to phosphorylate Ser-33 and -37, thereby disrupting the interaction between beta-catenin, beta-TRCP and Axin. I propose that phosphorylation of Ser-45 by CKI epsilon plays an important role in regulating beta-catenin stability.     

Negative regulation of LRP6 function by casein kinase I epsilon phosphorylation

Wnt signaling acts in part through the low density lipoprotein receptor-related transmembrane proteins LRP5 and LRP6 to regulate embryonic development and stem cell proliferation. Up-regulated signaling is associated with many forms of cancer. Casein kinase I epsilon (CKIepsilon) is a known component of the Wnt-beta-catenin signaling pathway. We find that CKIepsilon binds to LRP5 and LRP6 in vitro and in vivo and identify three CKIepsilon-specific phosphorylation sites in LRP6. Two of the identified phosphorylation sites, Ser1420 and Ser1430, influence Wnt signaling in vivo, since LRP6 with mutation of these sites is a more potent activator of both beta-catenin accumulation and Lef-1 reporter activity. Whereas Wnt3a regulates CKIepsilon kinase activity, LRP6 does not, placing CKIepsilon upstream of LRP6. Mutation of LRP6 Ser1420 and Ser1430 to alanine strengthens its interaction with axin, suggesting a mechanism by which CKIepsilon may negatively regulate Wnt signaling. The role of CKIepsilon is therefore more complex than was previously appreciated. Generation of active CKIepsilon may induce a negative feedback loop by phosphorylation of sites on LRP5/6 that modulate axin binding and hence beta-catenin degradation.     

Identification of inhibitory autophosphorylation sites in casein kinase I epsilon.

Casein kinase I epsilon (CKIepsilon) is a widely expressed protein kinase implicated in the regulation of diverse cellular processes including DNA replication and repair, nuclear trafficking, and circadian rhythm. CKIepsilon and the closely related CKIdelta are regulated in part through autophosphorylation of their carboxyl-terminal extensions, resulting in down-regulation of enzyme activity. Treatment of CKIepsilon with any of several serine/threonine phosphatases causes a marked increase in kinase activity that is self-limited. To identify the sites of inhibitory autophosphorylation, a series of carboxyl-terminal deletion mutants was constructed by site-directed mutagenesis. Truncations that eliminated specific phosphopeptides present in the wild-type kinase were used to guide construction of specific serine/threonine to alanine mutants. Amino acids Ser-323, Thr-325, Thr-334, Thr-337, Ser-368, Ser-405, Thr-407, and Ser-408 in the carboxyl-terminal tail of CKIepsilon were identified as probable in vivo autophosphorylation sites. A recombinant CKIepsilon protein with serine and threonine to alanine mutations eliminating these autophosphorylation sites was 8-fold more active than wild-type CKIepsilon using IkappaBalpha as a substrate. The identified autophosphorylation sites do not conform to CKI substrate motifs identified in peptide substrates.     

Mechanism of regulation of casein kinase I activity by group I metabotropic glutamate receptors

Previously, we reported that (S)-3,5-dihydroxypenylglycine (DHPG), an agonist for group I metabotropic glutamate receptors (mGluRs), stimulates CK1 and Cdk5 kinase activities in neostriatal neurons, leading to enhanced phosphorylation, respectively, of Ser-137 and Thr-75 of DARPP-32 (dopamine and cAMP-regulated phosphoprotein, 32 kDa). We have now investigated the signaling pathway that leads from mGluRs to casein kinase 1 (CK1) activation. In mouse neostriatal slices, the effect of DHPG on phosphorylation of Ser-137 or Thr-75 of DARPP-32 was blocked by the phospholipase Cbeta inhibitor, the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), and the calcineurin inhibitor cyclosporin A. In neuroblastoma N2a cells, the effect of DHPG on the activity of transfected HA-tagged CK1(epsilon) was blocked by BAPTA/AM and cyclosporin A. In neostriatal slices, the effect of DHPG on Cdk5 activity was also abolished by BAPTA/AM and cyclosporin A, presumably through blocking activation of CK1. Metabolic labeling studies and phosphopeptide mapping revealed that a set of C-terminal sites in HA-CK1epsilon were transiently dephosphorylated in N2a cells upon treatment with DHPG, and this was blocked by cyclosporin A. A mutant CK1epsilon with a nonphosphorylatable C-terminal domain was not activated by DHPG. Together, these studies suggest that DHPG activates CK1(epsilon) via Ca(2+)-dependent stimulation of calcineurin and subsequent dephosphorylation of inhibitory C-terminal autophosphorylation sites.     

Cloning and characterization of rat casein kinase 1epsilon

Genes differentially expressed in the subjective day and night in the rat suprachiasmatic nucleus (SCN) were surveyed by differential display. A gene homologous to human casein kinase 1epsilon (CK1epsilon) was isolated, which initially appeared to be expressed in the suprachiasmatic nucleus (SCN) in a circadian manner. We here describe the cDNA cloning of the rat CK1epsilon and characterization of the protein products. The rCK1epsilon is predominantly expressed in the brain including the SCN, binds and phosphorylates mPer1, mPer2, and mPer3 in vitro, and translocates mPer1 and mPer3, but not mPer2, to the cell nucleus depending on its kinase activity when coexpressed with these Per proteins in COS-7 cells.     

Casein kinase I and casein kinase II differentially regulate axin function in Wnt and JNK pathways

Axin uses different combinations of functional domains in down-regulation of the Wnt pathway and activation of the MEKK1/JNK pathway. We are interested in the elucidation of the functional switch of Axin. In the present study, we show that the Wnt activator CKIepsilon, but not CKIIalpha, Frat1, LRP5, or LRP6, inhibited Axin-mediated JNK activation. We also found that both CKIalpha and CKIepsilon interacted with Axin, whereas CKIIalpha did not bind to Axin and had no effect on Axin-mediated JNK activity even though CKIIalpha has also been suggested to be an activator for the Wnt pathway. The COOH-terminal region and the MEKK1-interacting domain of Axin are important for CKIalpha-Axin and CKIepsilon-Axin interaction. We further demonstrated that CKIepsilon and CKIalpha binding to Axin excluded MEKK1 binding, indicating that a competitive physical occupancy may underlie the inhibitory effect. Moreover, our data indicated that CKIepsilon kinase activity plays an additive role in this effect. Taken together, we have demonstrated that CKI and CKII exhibit differential effects on Axin-MEKK1 interaction and Axin-mediated JNK activation. Furthermore, our data suggest that CKI may provide a possible switch mechanism for Axin function in the regulation of Wnt and JNK pathways.     

Modulation of glycogen synthase kinase activity of skeletal and smooth muscle casein kinase I by spermine

Casein kinase I (CK-I) from skeletal muscle was stimulated 2-3 fold by 0.25-1 mM spermine. The polyamine also stimulated the phosphorylation of glycogen synthase by another casein kinase purified from aortic smooth muscle [DiSalvo et al. (1986) Biochem. Biophys. Res. Commun. 136, 789-796]. Phosphopeptide maps and phosphoamino acid analysis of [32P]glycogen synthase revealed that smooth muscle casein kinase phosphorylated glycogen synthase in the same sites that undergo phosphorylation by CK-I. The stimulatory effect of spermine on glycogen synthase kinase activity of CK-I was accompanied by increased phosphorylation of all peptide sites of glycogen synthase. Increased phosphorylation was observed in both seryl and threonyl residues. Higher concentrations (4 mM) of spermine inhibited CK-I activity by about 50%. These results indicate that aortic smooth muscle casein kinase is a CK-I enzyme and that skeletal and smooth muscle CK-I can be modulated by spermine.     

Regulation of arrestin-3 phosphorylation by casein kinase II

Arrestins play an important role in regulating the function of G protein-coupled receptors including receptor desensitization, internalization, down-regulation, and signaling via nonreceptor tyrosine kinases and mitogen-activated protein kinases. Previous studies have revealed that arrestins themselves are also subject to regulation. In the present study, we focused on identifying potential mechanisms involved in regulating the function of arrestin-3. Using metabolic labeling, phosphoamino acid analysis, and mutagenesis studies, we found that arrestin-3 is constitutively phosphorylated at Thr-382 and becomes dephosphorylated upon beta(2)-adrenergic receptor activation in COS-1 cells. Casein kinase II (CKII) appears to be the major kinase mediating arrestin-3 phosphorylation, since 1) Thr-382 is contained within a canonical consensus sequence for CKII phosphorylation and 2) wild type arrestin-3 but not a T382A mutant is phosphorylated by CKII in vitro. Functional analysis reveals that mutants mimicking the phosphorylated (T382E) and dephosphorylated (T382A or T382V) states of arrestin-3 promote beta(2)-adrenergic receptor internalization and bind clathrin, beta-adaptin, and Src to comparable levels as wild type arrestin-3. This suggests that the phosphorylation of arrestin-3 does not directly regulate interaction with endocytic (clathrin, beta-adaptin) or signaling (Src) components and is in contrast to arrestin-2, where phosphorylation appears to regulate interaction with clathrin and Src. However, additional analysis reveals that arrestin-3 phosphorylation may regulate formation of a large arrestin-3-containing protein complex. Differences between the regulatory roles of arrestin-2 and -3 phosphorylation may contribute to the different cellular functions of these proteins in G protein-coupled receptor signaling and regulation.     

Regulation of Wnt signaling during adipogenesis

We have identified Wnt10b as a potent inhibitor of adipogenesis that must be suppressed for preadipocytes to differentiate in vitro. Here, we demonstrate that a specific inhibitor of glycogen synthase kinase 3, CHIR 99021, mimics Wnt signaling in preadipocytes. CHIR 99021 stabilizes free cytosolic beta-catenin and inhibits adipogenesis by blocking induction of CCAAT/enhancer-binding protein alpha and peroxisome proliferator-activated receptor gamma. Preadipocyte differentiation is inhibited when 3T3-L1 cells are exposed to CHIR 99021 for any 24 h period during the first 3 days of adipogenesis. Consistent with this time frame of inhibition, expression of Wnt10b mRNA is suppressed upon induction of differentiation, with a 50% decline by 6 h and complete inhibition by 36 h. Of the agents used to induce differentiation, exposure of 3T3-L1 cells to methyl-isobutylxanthine or cAMP is sufficient to suppress expression of Wnt10b mRNA. Inhibition of adipogenesis by Wnt10b is likely mediated by Wnt receptors, Frizzled 1, 2, and/or 5, and co-receptors low density lipoprotein receptor-related proteins 5 and 6. These receptors, like Wnt10b, are highly expressed in preadipocytes and stromal vascular cells. Finally, we demonstrate that disruption of extracellular Wnt signaling by expression of secreted Frizzled related proteins causes spontaneous adipocyte conversion.     

Regulation of casein kinase 2 by phosphorylation/dephosphorylation.

The effects of various polycation-stimulated (PCS) phosphatases and of the active catalytic subunit of the ATPMg-dependent (AMDc) protein phosphatase on the activity of casein kinase 2 (CK-2) were investigated by using the synthetic peptide substrate Ser-Glu-Glu-Glu-Glu-Glu, whose phosphorylated derivative is entirely insensitive to these protein phosphatases. Previous dephosphorylation of native CK-2 enhances its specific activity 2-3-fold. Such an effect, accounted for by an increase in Vmax, is more readily promoted by the PCS phosphatases than by the AMDc phosphatase. The phosphate incorporated by autophosphorylation could not be removed by the protein phosphatases, suggesting the involvement of phosphorylation site(s) other than the one(s) affected by intramolecular autophosphorylation. The activation of CK-2 by the phosphatase pretreatment is neutralized during the kinase assay; the mechanism of this phenomenon, which is highly dependent on the kinase concentration, is discussed.     

Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I.

Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, we investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing [gamma-32P]ATP. The same glycoprotein was phosphorylated when [32P]GTP was substituted for [32P]ATP in the protein kinase assay. We also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. Immediately upstream from each of the casein kinase II sites was a potential casein kinase I phosphorylation site. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.     

Axin-dependent phosphorylation of the adenomatous polyposis coli protein mediated by casein kinase 1epsilon

Axin and the adenomatous polyposis coli protein (APC) interact to down-regulate the proto-oncogene beta-catenin. We show that transposition of an axin-binding site can confer beta-catenin regulatory activity to a fragment of APC normally lacking this activity. The fragment containing the axin-binding site also underwent hyperphosphorylation when coexpressed with axin. The phosphorylation did not require glycogen synthase kinase 3beta but instead required casein kinase 1epsilon, which bound directly to axin. Mutation of conserved serine residues in the beta-catenin regulatory motifs of APC interfered with both axin-dependent phosphorylation and phosphorylation by CKIepsilon and impaired the ability of APC to regulate beta-catenin. These results suggest that the axin-dependent phosphorylation of APC is mediated in part by CKIepsilon and is involved in the regulation of APC function.     

Subcellular localization of casein kinase I

An anti-yeast CKI antiserum was shown to cross-react with CKI isolated from Krebs II mouse ascites tumour cells. The mammalian CKI showed virtually the same molecular mass (app. 45 kDa) as the yeast enzyme. By immunofluorescence it could be shown that CKI is preferably located in the nucleolus.     

Phosphorylation of the insulin receptor by casein kinase I

Insulin receptor was examined as a substrate for the multipotential protein kinase casein kinase I. Casein kinase I phosphorylated partially purified insulin receptor from human placenta as shown by immunoprecipitation of the complex with antiserum to the insulin receptor. Analysis of the phosphorylated complex by polyacrylamide gel electrophoresis under nonreducing conditions showed a major phosphorylated band at the position of the alpha 2 beta 2 complex. When the phosphorylated receptor was analyzed on polyacrylamide gels under reducing conditions, two phosphorylated bands, Mr 95,000 and Mr 135,000, were observed which corresponded to the alpha and beta subunits. The majority of the phosphate was associated with the beta subunit with minor phosphorylation of the alpha subunit. Phosphoamino acid analysis revealed that casein kinase I phosphorylated only seryl residues. The autophosphorylated alpha 2 beta 2 receptor purified by affinity chromatography on immobilized O-phosphotyrosyl binding antibody was also a substrate for casein kinase I. Reduction of the phosphorylated alpha 2 beta 2 receptor indicated that casein kinase I incorporated phosphate into seryl residues only in the beta subunit.     

Regulation of the Wnt/beta-catenin pathway by redox signaling

Although it is well established that reactive oxygen species (ROS) can function as intracellular messengers, the mechanism of ROS dependent signaling is largely unknown (Rhee et al.,2005). In a recent paper in Nature Cell Biology, Funato et al. (2006) demonstrate that ROS can modulate signaling by the Wnt/beta-catenin pathway. This work provides interesting new insight into cross-talk between redox and Wnt/beta-catenin signaling in normal physiology and cancer.     

Identification of syntaxin-1A sites of phosphorylation by casein kinase I and casein kinase II.

Casein kinases I (CKI) are serine/threonine protein kinases widely expressed in a range of eukaryotes including yeast, mammals and plants. They have been shown to play a role in diverse physiological events including membrane trafficking. CKI alpha is associated with synaptic vesicles and phosphorylates some synaptic vesicle associated proteins including SV2. In this report, we show that syntaxin-1A is phosphorylated in vitro by CKI on Thr21. Casein kinase II (CKII) has been shown previously to phosphorylate syntaxin-1A in vitro and we have identified Ser14 as the CKII phosphorylation site, which is known to be phosphorylated in vivo. As syntaxin-1A plays a key role in the regulation of neurotransmitter release by forming part of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, we propose that CKI may play a role in synaptic vesicle exocytosis.     

Regulation of Wnt signaling by nociceptive input in animal models

Background

Cutaneous peripheral neuropathies have been associated with changes of the sensory fiber innervation in the dermis and epidermis. These changes are mediated in part by the increase in local expression of trophic factors. Increase in target tissue nerve growth factor has been implicated in the promotion of peptidergic afferent and sympathetic efferent sprouting following nerve injury. The primary source of nerve growth factor is cells found in the target tissue, namely the skin. Recent evidence regarding the release and extracellular maturation of nerve growth factor indicate that it is produced in its precursor form and matured in the extracellular space. It is our hypothesis that the precursor form of nerve growth factor should be detectable in those cell types producing it. To date, limitations in available immunohistochemical tools have restricted efforts in obtaining an accurate distribution of nerve growth factor in the skin of na?ve animals and those with neuropathic pain lesions. It is the objective of this study to delineate the distribution of the precursor form of nerve growth factor to those cell types expressing it, as well as to describe its distribution with respect to those nerve fibers responsive to it.

Results

We observed a decrease in peptidergic fiber innervation at 1 week after the application of a chronic constriction injury (CCI) to the sciatic nerve, followed by a recovery, correlating with TrkA protein levels. ProNGF expression in CCI animals was significantly higher than in sham-operated controls from 1-4 weeks post-CCI. ProNGF immunoreactivity was increased in mast cells at 1 week post-CCI and, at later time points, in keratinocytes. P75 expression within the dermis and epidermis was significantly higher in CCI-operated animals than in controls and these changes were localized to neuronal and non-neuronal cell populations using specific markers for each.

Conclusions

We describe proNGF expression by non-neuronal cells over time after nerve injury as well as the association of NGF-responsive fibers to proNGF-expressing target tissues. ProNGF expression increases following nerve injury in those cell types previously suggested to express it.