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.     

Serine-71 phosphorylation of rac1 modulates downstream signaling

The Rho GTPases Rac1 and Cdc42 regulate a variety of cellular functions by signaling to different signal pathways. It is believed that the presence of a specific effector at the location of GTPase activation determines the route of downstream signaling. We previously reported about EGF-induced Ser-71 phosphorylation of Rac1/Cdc42. By using the phosphomimetic S71E-mutants of Rac1 and Cdc42 we investigated the impact of Ser-71 phosphorylation on binding to selected effector proteins. Binding of the constitutively active (Q61L) variants of Rac1 and Cdc42 to their specific interaction partners Sra-1 and N-WASP, respectively, as well as to their common effector protein PAK was abrogated when Ser-71 was exchanged to glutamate as phosphomimetic substitution. Interaction with their common effector proteins IQGAP1/2/3 or MRCK alpha was, however, hardly affected. This ambivalent behaviour was obvious in functional assays. In contrast to Rac1 Q61L, phosphomimetic Rac1 Q61L/S71E was not able to induce increased membrane ruffling. Instead, Rac1 Q61L/S71E allowed filopodia formation, which is in accordance with abrogation of the dominant Sra-1/Wave signalling pathway. In addition, in contrast to Rac1 transfected cells Rac1 S71E failed to activate PAK1/2. On the other hand, Rac1 Q61L/S71E was as effective in activation of NF-kappaB as Rac1 Q61L, illustrating positive signal transduction of phosphorylated Rac1. Together, these data suggest that phosphorylation of Rac1 and Cdc42 at serine-71 represents a reversible mechanism to shift specificity of GTPase/effector coupling, and to preferentially address selected downstream pathways.     

Phosphorylation of rabbit skeletal muscle glycogen synthase by casein kinase 1: Evidence of phosphorylation sites specific for casein kinase 1

Casein kinase 1 phosphorylated rabbit skeletal muscle glycogen synthase at both seryl and threonyl residues. With glycogen synthase phosphorylated up to 7.5 mol phosphate/mol subunit, about 26% of the phosphate was present in the N-terminal cyanogen bromide fragment (CB1) and 74% in the C-terminal fragment (CB2). Both fragments contained phosphothreonine (11 to 14%) in addition to phosphoserine. When ³²P-labeled glycogen synthase was totally digested with trypsin and chromatographed on reversephase high-performance liquid chromatography, seven phosphopeptides were observed. Peptide I eluted in the vicinity of the peptide containing site 1a, peptide II coincided with sites 4 + 5, peptides III and IV eluted in the region corresponding to sites 3a + 3b + 3c, peptide V appeared slightly after the peptide containing site 1b and peptide VII behaved as the peptide containing site 2, whereas peptide VI did not coincide with any of the known phosphopeptides. Limited trypsinization prior to analysis by HPLC led to the disappearance of peaks V and VI without altering peaks I to IV and VII. Only peaks I and VII remained when limited chymotrypsinization was performed prior to HPLC analysis. Chromatography on HPLC of the fragments derived from complete trypsinization of CB2 showed the presence of peaks II to VI. Phosphoamino acid analysis of the different peptides demonstrated the presence of quantitative amounts of phosphothreonine in peptides V, VI, and VII. These results indicate that multiple phosphorylation sites for casein kinase 1 must exist in both the N-terminal and C-terminal regions of glycogen synthase, some of which would only be labeled by casein kinase 1.     

Axin-independent phosphorylation of APC controls beta-catenin signaling via cytoplasmic retention of beta-catenin

It has been shown that accumulation of free beta-catenin leads to mobility shift of adenomatous polyposis coli (APC) protein and that Axin facilitates this process. Here we show that the beta-catenin-mediated mobility shift of APC is due to phosphorylation of two domains of APC by casein kinase 1epsilon/glycogen synthase kinase 3beta and unknown kinase(s), respectively. Interestingly, our results suggest that this process does not require Axin. The phosphorylated APC showed higher affinity to beta-catenin in vivo, and fragments of APC containing the phosphorylated domains can inhibit beta-catenin/Tcf-mediated reporter activity regardless of their ability to reduce the level of beta-catenin. From our data we propose a new role of APC: accumulation of excessive cytoplasmic beta-catenin induces phosphorylation of APC and the phosphorylated APC retains beta-catenin in cytoplasm to prevent excessive beta-catenin signaling. The retained beta-catenin in cytoplasm by APC may be down-regulated by Axin 2, which is induced by beta-catenin/Tcf signaling.     

Wnt signaling controls the phosphorylation status of beta-catenin

At the heart of the canonical Wnt signaling cascade, adenomatous polyposis coli (APC), axin, and GSK3 constitute the so-called destruction complex, which controls the stability of beta-catenin. It is generally believed that four conserved Ser/Thr residues in the N terminus of beta-catenin are the pivotal targets for the constitutively active serine kinase GSK3. In cells that do not receive Wnt signals, glycogen synthase kinase (GSK) is presumed to phosphorylate beta-catenin, thus marking the latter for proteasomal degradation. Wnt signaling inhibits GSK3 activity. As a consequence, beta-catenin would no longer be phosphorylated and accumulate to form nuclear complexes with TCF/LEF factors. Although mutations in or near the N-terminal Ser/Thr residues stabilize beta-catenin in several types of cancer, the hypothesis that Wnt signaling controls phosphorylation of these residues remains unproven. We have generated a monoclonal antibody that recognizes an epitope containing two of the four residues when both are not phosphorylated. The epitope is generated upon Wnt signaling as well as upon pharmacological inhibition of GSK3 by lithium, providing formal proof for the regulated phosphorylation of the Ser/Thr residues of beta-catenin by Wnt signaling. Immunohistochemical analysis of mouse embryos utilizing the antibody visualizes sites that transduce Wnt signals through the canonical Wnt cascade.     

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.     

Regulation of ribosomal protein S6 phosphorylation by casein kinase 1 and protein phosphatase 1

Ribosomal protein S6 (rpS6) is a critical component of the 40 S ribosomal subunit that mediates translation initiation at the 5'-m(7)GpppG cap of mRNA. In response to mitogenic stimuli, rpS6 undergoes ordered C-terminal phosphorylation by p70 S6 kinases and p90 ribosomal S6 kinases on four conserved Ser residues (Ser-235, Ser-236, Ser-240, and Ser-244) whose modification potentiates rpS6 cap binding activity. A fifth site, Ser-247, is also known to be phosphorylated, but its function and regulation are not well characterized. In this study, we employed phospho-specific antibodies to show that Ser-247 is a target of the casein kinase 1 (CK1) family of protein kinases. CK1-dependent phosphorylation of Ser-247 was induced by mitogenic stimuli and required prior phosphorylation of upstream S6 kinase/ribosomal S6 kinase residues. CK1-mediated phosphorylation of Ser-247 also enhanced the phosphorylation of upstream sites, which implies that bidirectional synergy between C-terminal phospho-residues is required to sustain rpS6 phosphorylation. Consistent with this idea, CK1-dependent phosphorylation of rpS6 promotes its association with the mRNA cap-binding complex in vitro. Additionally, we show that protein phosphatase 1 (PP1) antagonizes rpS6 C terminus phosphorylation and cap binding in intact cells. These findings further our understanding of rpS6 phospho-regulation and define a direct link between CK1 and translation initiation.     

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.     

Wnt-3a utilizes a novel low dose and rapid pathway that does not require casein kinase 1-mediated phosphorylation of Dvl to activate beta-catenin

The current view of canonical Wnt signalling is that following Wnt binding to its receptors (Frizzled-Lrp5/6), dishevelled (Dvl) becomes hyperphosphorylated, and the signal is transduced to the APC-GSK3beta-axin-beta-catenin multiprotein complex, which subsequently dissociates. As a result beta-catenin is not phosphorylated, escapes proteosomal degradation and activates its target genes after translocation to the nucleus. Here, we analyzed the importance of the Wnt-3a-induced phosphorylation and shift in electrophoretic migration of Dvl (PS-Dvl) for the activation of beta-catenin. Analysis of Wnt-3a time- and dose-responses in a dopaminergic cell line showed that beta-catenin is activated rapidly (within minutes) and at a low dose of Wnt-3a (1 ng/ml). Surprisingly, PS-Dvl appeared only after 30 min and at greater doses (> or =20 ng/ml) of Wnt-3a. Moreover, we found that a casein kinase 1 inhibitor (D4476) or siRNA for casein kinase 1 delta/epsilon (CK1delta/epsilon) blocked the Wnt-3a-induced PS-Dvl. Interestingly, CK1 inhibition or siRNA for CK1delta/epsilon did not ablate the activation of beta-catenin by Wnt-3a, indicating that there is a PS-Dvl-independent path to activate beta-catenin. The increase in beta-catenin activation by Wnt-3a (PS-Dvl-dependent or -independent) were blocked by Dickkopf1 (Dkk1), suggesting that the effect of Wnt-3a is in both cases mediated by Lrp5/6 receptors. Thus, our results show that Wnt-3a rapidly induce a partial activation of beta-catenin in the absence of PS-Dvl at low doses, while at high doses induce a full activation of beta-catenin in a PS-Dvl-dependent manner.     

Modulation of beta-catenin phosphorylation/degradation by cyclin-dependent kinase 2

beta-Catenin functions as a downstream component of the Wnt/Wingless signal transduction pathway, and inappropriate control of cytosolic beta-catenin is a crucial step in the genesis of several human cancers. Here we demonstrate that cyclin-dependent kinase 2 (CDK2) in association with cyclin A or cyclin E directly binds to beta-catenin. In vivo and in vitro kinase assays with cyclin-CDK2 demonstrate beta-catenin phosphorylation on residues Ser(33), Ser(37), Thr(41), and Ser(45). This phosphorylation promotes rapid degradation of cytosolic beta-catenin via the beta-TrCP-mediated proteasome pathway. Moreover, cyclin E-CDK2 contributes to rapid degradation of cytosolic beta-catenin levels during G(1) phase by regulating beta-catenin phosphorylation and subsequent degradation. In this way, CDK2 may "fine tune" beta-catenin levels over the course of the cell cycle.     

Syndecan-4 modulates focal adhesion kinase phosphorylation

The cell-surface heparan sulfate proteoglycan syndecan-4 acts in conjunction with the alpha(5)beta(1) integrin to promote the formation of actin stress fibers and focal adhesions in fibronectin (FN)-adherent cells. Fibroblasts seeded onto the cell-binding domain (CBD) fragment of FN attach but do not fully spread or form focal adhesions. Activation of Rho, with lysophosphatidic acid (LPA), or protein kinase C, using the phorbol ester phorbol 12-myristate 13-acetate, or clustering of syndecan-4 with antibodies directed against its extracellular domain will stimulate formation of focal adhesions and stress fibers in CBD-adherent fibroblasts. The distinct morphological differences between the cells adherent to the CBD and to full-length FN suggest that syndecan-4 may influence the organization of the focal adhesion or the activation state of the proteins that comprise it. FN-null fibroblasts (which express syndecan-4) exhibit reduced phosphorylation of focal adhesion kinase (FAK) tyrosine 397 (Tyr(397)) when adherent to CBD compared with FN-adherent cells. Treating the CBD-adherent fibroblasts with LPA, to activate Rho, or the tyrosine phosphatase inhibitor sodium vanadate increased the level of phosphorylation of Tyr(397) to match that of cells plated on FN. Treatment of the fibroblasts with PMA did not elicit such an effect. To confirm that this regulatory pathway includes syndecan-4 specifically, we examined fibroblasts derived from syndecan-4-null mice. The phosphorylation levels of FAK Tyr(397) were lower in FN-adherent syndecan-4-null fibroblasts compared with syndecan-4-wild type and these levels were rescued by the addition of LPA or re-expression of syndecan-4. These data indicate that syndecan-4 ligation regulates the phosphorylation of FAK Tyr(397) and that this mechanism is dependent on Rho but not protein kinase C activation. In addition, the data suggest that this pathway includes the negative regulation of a protein-tyrosine phosphatase. Our results implicate syndecan-4 activation in a direct role in focal adhesion regulation.     

Comparison of phosphorylation of elongation factor 1 from different species by casein kinase II

One subunit of EF-1 or EF-1 beta gamma from Artemia salina, wheat germ and rabbit reticulocytes is modified by casein kinase II. The subunit corresponds to the low Mr subunit of EF-1 (26,000-36,000) which functions along with a higher Mr subunit (46,000-48,000), to catalyze the exchange of GDP for GTP on EF-1 alpha. The factor from Artemia and wheat germ is phosphorylated directly on serine by casein kinase II whereas a modulatory compound is required for phosphorylation of EF-1 from reticulocytes. Polylysine increases the rate of phosphorylation of EF-1 from reticulocytes by 24-fold; both serine and threonine are modified. This suggests that polylysine may be substituting for a physiological regulatory compound which modulates phosphorylation in vivo.     

Human casein kinase Idelta phosphorylation of human circadian clock proteins period 1 and 2

Casein kinase Iepsilon (CKIepsilon), a central component of the circadian clock, interacts with and phosphorylates human period protein 1 (hPER1) [Keesler, G.A. et al. (2000) NeuroReport 5, 951-955]. A mutation in CKIepsilon causes a shortened circadian period in Syrian Golden hamster. We have now extended our previous studies to show that human casein kinase Idelta (hCKIdelta), the closest homologue to hCKIepsilon, associates with and phosphorylates hPER1 and causes protein instability. Furthermore, we observed that both hCKIdelta and hCKIepsilon phosphorylated and caused protein instability of human period 2 protein (hPER2). Immunohistochemical staining of rat brains demonstrates that CKIdelta protein is localized in the suprachiasmatic nuclei, the central location of the master clock. These results indicate that CKIdelta may play a role similar to CKIepsilon, suggesting that it may also be involved in regulating circadian rhythmicity by post-translation modification of mammalian clock proteins hPER1 and 2.     

Nerve growth factor-induced phosphorylation of amphiphysin-1 by casein kinase 2 regulates clathrin-amphiphysin interactions

Amphiphysins interact directly with clathrin and have a function in clathrin-mediated synaptic vesicle recycling and clathrin-mediated endocytosis. The neuronal isoform amphiphysin-1 is a serine/threonine phosphoprotein that is dephosphorylated upon stimulation of synaptic vesicle endocytosis. Rephosphorylation was stimulated by nerve growth factor. We analysed the regulation of amphiphysin-clathrin interactions by phosphorylation. The N-terminal domain of clathrin bound to unphosphorylated amphiphysin-1, but not to the phosphorylated protein. A search for possible phosphorylation sites revealed two casein kinase 2 consensus motifs in close proximity to the clathrin binding sites in amphiphysin-1 and -2. We mutagenized these residues (T350 and T387) to glutamate, mimicking a constitutive phosphorylation. The double mutant showed a strong reduction in clathrin binding. The assumption that casein kinase 2 phosphorylates amphiphysin-1 at T350 and T387 was corroborated by experiments showing that: (i) casein kinase 2 phosphorylated these residues directly in vitro, (ii) when expressed in HeLa cells, the glutamate mutant showed reduced phosphorylation, and (iii) casein kinase 2 inhibitors blocked nerve growth factor-induced phosphorylation of endogenous amphiphysin-1 in PC12 cells. These observations are consistent with the hypothesis that, upon activation by nerve growth factor, casein kinase 2 phosphorylates amphiphysin-1 and thereby regulates the endocytosis of clathrin-coated vesicles via the interaction between clathrin and amphiphysin.     

Site-specific phosphorylation of the purified receptor for calcium-channel blockers by cAMP- and cGMP-dependent protein kinases, protein kinase C, calmodulin-dependent protein kinase II and casein kinase II

Five protein kinases were used to study the phosphorylation pattern of the purified skeletal muscle receptor for calcium-channel blockers (CaCB). cAMP kinase, cGMP kinase, protein kinase C, calmodulin kinase II and casein kinase II phosphorylated the 165-kDa and the 55-kDa proteins of the purified CaCB receptor. The 130/28-kDa and the 32-kDa protein of the receptor are not phosphorylated by these protein kinases. Among these protein kinases only cAMP kinase phosphorylated the 165-kDa subunit with 2-3-fold higher initial rate than the 55-kDa subunit. Casein kinase II phosphorylated the 165-kDa and the 55-kDa protein of the receptor with comparable rates. cGMP kinase, protein kinase C and calmodulin kinase II phosphorylated preferentially the 55-kDa protein. The 55-kDa protein is phosphorylated 50 times faster by cGMP kinase and protein kinase C than by calmodulin kinase II or casein kinase II and about 10 times faster by these enzymes than by cAMP kinase. Two-dimensional peptide maps of the 165-kDa subunit yielded a total of 11 phosphopeptides. Four or five peptides are phosphorylated specifically by cAMP kinase, cGMP kinase, casein kinase II and protein kinase C, whereas the other peptides are modified by several kinases. The same kinases phosphorylate 11 peptides in the 55-kDa subunit. Again, some of these peptides are modified specifically by each kinase. These results suggest that the 165-kDa and the 55-kDa subunit contain specific phosphorylation sites for cAMP kinase, cGMP kinase, casein kinase II and protein kinase C. Phosphorylation of these sites may be relevant for the in vivo function of the CaCB receptor.     

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.     

Role of casein kinase 1 in the glucose sensor-mediated signaling pathway in yeast

Background  

In yeast, glucose-dependent degradation of the Mth1 protein, a corepressor of the glucose transporter gene (HXT) repressor Rgt1, is a crucial event enabling expression of several HXT. This event occurs through a signaling pathway that involves the Rgt2 and Snf3 glucose sensors and yeast casein kinase 1 and 2 (Yck1/2). In this study, we examined whether the glucose sensors directly couple with Yck1/2 to convert glucose binding into an intracellular signal that leads to the degradation of Mth1.