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.     

A phosphatidylinositol 4,5-bisphosphate-sensitive casein kinase I alpha associates with synaptic vesicles and phosphorylates a subset of vesicle proteins

In interphase cells, alpha-casein kinase I (alpha-CKI) is found associated with cytosolic vesicular structures, the centrosome, and within the nucleus. To identify the specific vesicular structures with which alpha-CKI is associated, established cell lines and primary rat neurons were immunofluorescently labeled with an antibody raised to alpha-CKI. In nonneuronal cells, alpha-CKI colocalizes with vesicular structures which align with microtubules and are partially coincident with both Golgi and endoplasmic reticulum markers. In neurons, alpha- CKI colocalizes with synaptic vesicle markers. When synaptic vesicles were purified from rat brain, they were highly enriched in a CKI, based on activity and immunoreactivity. The synaptic vesicle-associated CKI is an extrinsic kinase and was eluted from synaptic vesicles and purified. This purified CKI has properties most similar to alpha-CKI. When the activities of casein kinase I or II were specifically inhibited on isolated synaptic vesicles, CKI was shown to phosphorylate a specific subset of vesicle proteins, one of which was identified as the synaptic vesicle-specific protein SV2. As with alpha-CKI, the synaptic vesicle CKI is inhibited by phosphatidylinositol 4,5- bisphosphate (PIP2). However, synthesis of PIP2 was detected only in plasma membrane-containing fractions. Therefore, PIP2 may spatially regulate CKI. Since PIP2 synthesis is required for secretion, this inhibition of CKI may be important for the regulation of secretion.     

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.     

Localization of Sites in the Tail Domain of the Middle Molecular Mass Neurofilament Subunit Phosphorylated by a Neurofilament-Associated Kinase and by Casein Kinase I

Abstract: We have shown previously that a neurofilament (NF)-associated kinase (NFAK) extracted from chicken NF preparations phosphorylates selectively the middle molecular mass NF subunit (NF-M). Here we show that the major kinase activity in NFAK is indistinguishable from enzymes of the casein kinase I (CKI) family based on the following criteria: (1) inhibition of NFAK phosphorylation by the selective CKI inhibitor CKI-7, (2) the similarity in substrate specificity of NFAK and authentic CKI, (3) the correspondence of two-dimensional phosphopeptide maps of NF-M phosphorylated in vitro by NFAK with those generated by CKI under similar conditions, and (4) immunological cross-reactivity of NFAK with an antibody raised against CKI. We have also identified Ser⁵⁰², Ser⁵²⁸, and Ser⁵³⁶ as phosphorylation sites by NFAK/CKI in vitro, each of which is also phosphorylated in vivo. All three serines are found in peptides with CKI phosphorylation consensus sequences, and Ser⁵²⁸ and Ser⁵³⁶ and flanking amino acids are highly conserved in higher vertebrate NF-M sequences. Neither Ser⁵⁰² nor Ser⁵³⁶ has been identified previously as NF-M phosphorylation sites.     

CKI isoforms α and ε regulate Star-PAP target messages by controlling Star-PAP poly(A) polymerase activity and phosphoinositide stimulation

Star-PAP is a non-canonical, nuclear poly(A) polymerase (PAP) that is regulated by the lipid signaling molecule phosphatidylinositol 4,5 bisphosphate (PI4,5P(2)), and is required for the expression of a select set of mRNAs. It was previously reported that a PI4,5P(2) sensitive CKI isoform, CKIα associates with and phosphorylates Star-PAP in its catalytic domain. Here, we show that the oxidative stress-induced by tBHQ treatment stimulates the CKI mediated phosphorylation of Star-PAP, which is critical for both its polyadenylation activity and stimulation by PI4,5P(2). CKI activity was required for the expression and efficient 3'-end processing of its target mRNAs in vivo as well as the polyadenylation activity of Star-PAP in vitro. Specific CKI activity inhibitors (IC261 and CKI7) block in vivo Star-PAP activity, but the knockdown of CKIα did not equivalently inhibit the expression of Star-PAP targets. We show that in addition to CKIα, Star-PAP associates with another CKI isoform, CKIε in the Star-PAP complex that phosphorylates Star-PAP and complements the loss of CKIα. Knockdown of both CKI isoforms (α and ε) resulted in the loss of expression and the 3'-end processing of Star-PAP targets similar to the CKI activity inhibitors. Our results demonstrate that CKI isoforms α and ε modulate Star-PAP activity and regulates Star-PAP target messages.     

Multiple isoforms of Arabidopsis casein kinase I combine conserved catalytic domains with variable carboxyl-terminal extensions.

Three cDNA clones encoding isoforms of casein kinase I (CKI) were isolated from Arabidopsis thaliana. One full-length clone, designated CKI1, contained an open reading frame of 1371 bp encoding a protein of 51,949 D with an isoelectric point of 9.7. In addition to the highly conserved catalytic domain (of about 300 amino acids), the Arabidopsis CKI isoforms contain 150 to 180 amino acid carboxyl-terminal extensions, which show among themselves a lower level of sequence conservation. These extensions do not show any sequence similarity to nonplant CKI isoforms, such as rat testis CKI delta, which is their closest isolated homolog, or to yeast CKI isoforms. Three additional isoforms of Arabidopsis CKI were found in the data bases of expressed sequence tags and/or were isolated serendipitously in nonspecific screening procedures by others. One of them also shows a carboxyl-terminal extension, but of only 80 amino acids. Casein kinase activity was detected in the soluble fraction of Escherichia coli strains expressing the CKI1 protein. This activity showed the crucial properties of CKI, including the ability to phosphorylate the D4 peptide, a specific substrate of CKI, and inhibition by N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide, a specific CKI inhibitor. Like several recombinant CKI isoforms from yeast, CKI1 was able to phosphorylate tyrosine-containing acidic polymers.     

Casein Kinase I: Spatial Organization and Positioning of a Multifunctional Protein Kinase Family

The casein kinase I family of serine/threonine protein kinases is highly conserved from yeast to humans. Until only recently, both the function and regulation of these enzymes remained poorly uncharacterised in that they appeared to be constitutively active and were capable of phosphorylating an untold number of other proteins. While relatively little was known regarding the exact function of the higher eukaryotic isoforms, the casein kinase I (CKI) isoforms from yeast have been genetically linked to vesicular trafficking, DNA repair, cell cycle progression and cytokinesis. All five S. cerevisiae isoforms are known to be associated with discrete cellular compartments and this localization has been shown to be absolutely essential for their respective functions. New evidence now suggests that the CKI isoforms in more complex systems also exhibit non-homogeneous subcellular distributions that may prove vital to defining the function and regulation of these enzymes. In particular, CKI, the most-characterized vertebrate isoform, is associated with cytosolic vesicles, the mitotic spindle and structures within the nucleus. Functions associated with these localizations coincide with those previously reported in yeast, suggesting a conservation of function. Other reports have indicated that each of the remaining CKI isoforms have the capacity to make associations with components of several signal transduction pathways, thereby channeling CKI function toward specific regulatory events. This review will examine what is now known about the higher eukaryotic CKI family members from the perspective localization as a means of gaining a better understanding of the function and regulation of these kinases.     

Casein kinase I epsilon associates with and phosphorylates the tight junction protein occludin

Occludin is an integral-membrane protein that contributes to tight junction function. We have identified casein kinase I epsilon (CKI epsilon) as a binding partner for the C-terminal cytoplasmic domain of occludin by yeast two-hybrid screening. CKI epsilon phosphorylated occludin and co-localised and co-immunoprecipitated with occludin from human endothelial cells. Amino acids 265-318 of occludin were sufficient for CKI epsilon binding and phosphorylation. Deletion of the C-terminal 48 amino acids of occludin increased CKI epsilon binding and phosphorylation, suggesting that this region inhibits CKI epsilon binding. These data identify CKI epsilon as a novel occludin kinase that may be important for the regulation of occludin.     

Casein kinase 1δ activates human recombinant deoxycytidine kinase by Ser-74 phosphorylation, but is not involved in the in vivo regulation of its activity

Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxynucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We recently showed that dCK was activated in vivo by phosphorylation of Ser-74. However, the protein kinase responsible was not identified. Ser-74 is located downstream a Glu-rich region, presenting similarity with the consensus phosphorylation motif of casein kinase 1 (CKI), and particularly of CKI δ. We showed that recombinant CKI δ phosphorylated several residues of bacterially overexpressed dCK: Ser-74, but also Ser-11, Ser-15, and Thr-72. Phosphorylation of dCK by CKI δ correlated with increased activity reaching at least 4-fold. Site-directed mutagenesis demonstrated that only Ser-74 phosphorylation was involved in dCK activation by CKI δ, strengthening the key role of this residue in the control of dCK activity. However, neither CKI δ inhibitors nor CKI δ siRNA-mediated knock-down modified Ser-74 phosphorylation or dCK activity in cultured cells. Moreover, these approaches did not prevent dCK activation induced by treatments enhancing Ser-74 phosphorylation. Taken together, the data preclude a role of CKI δ in the regulation of dCK activity in vivo. Nevertheless, phosphorylation of dCK by CKI δ could be a useful tool for elucidating the influence of Ser-74 phosphorylation on the structure-activity relationships in the enzyme.     

Compound Kushen Injection intervenes metabolic reprogramming and epithelial-mesenchymal transition of HCC via regulating β-catenin/c-Myc signaling

BackgroundHepatocellular carcinoma (HCC) is one of the most extensive and most deadly cancers worldwide. The invasion and metastasis characteristics of HCC dramatically affect the prognosis and survival of HCC patients. Compound Kushen Injection (CKI) is a GMP produced, proverbially applied traditional Chinese medicine formula in China to treat cancer-associated pains, and used as an adjunctive therapy for HCC. Until so far, whether CKI could suppress the metastasis of HCC through regulation of epithelial-mesenchymal transition or metabolic reprogramming is still ambiguous.PurposeIn this study, the anti-metastasis effects of CKI were clarified and its pharmacological mechanisms were systematically explored.MethodsCell invasion and cell adhesion assay were performed in SMMC-7721 cells to assess the anti-metastasis role of CKI, and the histopathological evaluation and biochemical detection were utilized in DEN-induced HCC rats to verify the anti-HCC effect of CKI. Serum and liver samples were analyzed with ¹H NMR metabolomics approach to screen the differential metabolites and further target quantification the content of key metabolites. Finally, western blotting and immunofluorescence assay were applied to verify the crucial signaling pathway involved in metabolites.ResultsCKI markedly repressed the invasion and adhesion in SMMC-7721 cells and significantly improved the liver function of DEN-induced HCC rats. CKI significantly regulated the expression of epithelial-mesenchymal transition (EMT) markers (Vimentin and E-cadherin). Metabolomics results showed that CKI regulated the metabolic reprogramming of HCC by inhibiting the key metabolites (citrate and lactate) and enzymes (HK and PK) in glycolysis process. Importantly, we found that c-Myc mediates the inhibitory effect of CKI on glycolysis. We further demonstrated that CKI inhibits c-Myc expression through modulating Wnt/β-catenin pathway in SMMC-7721 cells and DEN-induced HCC rats. Furthermore, through activating Wnt/β-catenin pathway with LiCl, the inhibitory effects of CKI on HCC were diminished.ConclusionTogether, this study reveals that CKI intervenes metabolic reprogramming and epithelial-mesenchymal transition of HCC via regulating β-catenin/c-Myc signaling pathway. Our research provides a new understanding of the mechanism of CKI against invasion and metastasis of HCC from the perspective of metabolic reprogramming.     

Two genes in Saccharomyces cerevisiae encode a membrane-bound form of casein kinase-1.

Two cDNAs encoding casein kinase-1 have been isolated from a yeast cDNA library and termed CKI1 and CKI2. Each clone encodes a protein of approximately 62,000 Da containing a highly conserved protein kinase domain surrounded by variable amino- and carboxy-terminal domains. The proteins also contain two conserved carboxy-terminal cysteine residues that comprise a consensus sequence for prenylation. Consistent with this posttranslational modification, cell fractionation experiments demonstrate that intact CKI1 is found exclusively in yeast cell membranes. Gene disruption experiments reveal that, although neither of the two CKI genes is essential by itself, at least one CKI gene is required for yeast cell viability. Spores deficient in both CKI1 and CKI2 fail to grow and, therefore, either fail to germinate or arrest as small cells before bud emergence. These results suggest that casein kinase-1, which is distributed widely in nature, plays a pivotal role in eukaryotic cell regulation.     

Characterization of Additional Casein Kinase I Sites in the C-Terminal "Tail" Region of Chicken and Rat Neurofilament-M

Abstract: In previous studies we have identified Ser⁵⁰², Ser⁵²⁸, and Ser⁵³⁴ as target sites in chicken neurofilament middle molecular mass protein (NF-M) for casein kinase I (CKI) in vitro and have shown that these sites are also phosphorylated in vivo. We now make use of a combination of molecular biological and protein chemical techniques to show that two additional in vivo phosphorylation sites in chicken NF-M, Ser⁴⁶⁴ and Ser⁴⁷¹, can also be phosphorylated by CKI in vitro. These two sites are conserved in higher vertebrate NF-M molecules, and recombinant protein constructs containing the homologous rat NF-M peptides can be phosphorylated by CKI in vitro, suggesting that phosphorylation of these sites is conserved at least in higher vertebrates. The two new sites are adjacent to a conserved peptide sequence (VEE-IIEET-V) found once in higher vertebrate NF-M molecules and twice in lamprey NF-180. Variants of this sequence are also found in neurofilament low and high molecular mass proteins (NF-L and NF-H) and α-internexin, and in mammalian NF-L are known to be associated with in vivo phosphorylation sites. We speculate that CKI phosphorylation in general, and these sites in particular, may be important in neurofilament function.     

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.     

Casein Kinase I epsilon positively regulates the Akt pathway in breast cancer cell lines

The Akt pathway is very important in both development and cancer. Here we show that, expression of Casein Kinase I epsilon (CKIε) causes up-regulation of the Akt pathway despite normal protein expression of the pathway inhibitor phosphate and tensin homologue deleted on chromosome ten (PTEN). Conversely, we show that a CKIε/δ-specific inhibitor can inhibit Akt phosphorylation at both Thr308 and Ser473 and drastically reduce phosphorylation of the Akt target Glycogen Synthase Kinase 3β (GSK3β). These conclusions were confirmed between MCF7 cells transiently transfected with CKIε and Hs578T cells which already express endogenous CKIε. The results suggest that CKIε is a new positive regulator of the Akt pathway. Here we propose that, rather than inhibiting PTEN function, CKIε positively regulates Akt possibly by inhibiting Protein Phosphatase 2A (PP2A).     

The Histidine Kinases CYTOKININ-INDEPENDENT1 and ARABIDOPSIS HISTIDINE KINASE2 and 3 Regulate Vascular Tissue Development in Arabidopsis Shoots

The development and activity of the procambium and cambium, which ensure vascular tissue formation, is critical for overall plant architecture and growth. However, little is known about the molecular factors affecting the activity of vascular meristems and vascular tissue formation. Here, we show that the His kinase CYTOKININ-INDEPENDENT1 (CKI1) and the cytokinin receptors ARABIOPSIS HISTIDINE KINASE2 (AHK2) and AHK3 are important regulators of vascular tissue development in Arabidopsis thaliana shoots. Genetic modifications of CKI1 activity in Arabidopsis cause dysfunction of the two-component signaling pathway and defects in procambial cell maintenance. CKI1 overexpression in protoplasts leads to cytokinin-independent activation of the two-component phosphorelay, and intracellular domains are responsible for the cytokinin-independent activity of CKI1. CKI1 expression is observed in vascular tissues of inflorescence stems, and CKI1 forms homodimers both in vitro and in planta. Loss-of-function ahk2 and ahk3 mutants and plants with reduced levels of endogenous cytokinins show defects in procambium proliferation and an absence of secondary growth. CKI1 overexpression partially rescues ahk2 ahk3 phenotypes in vascular tissue, while the negative mutation CKI1^H405Q further accentuates mutant phenotypes. These results indicate that the cytokinin-independent activity of CKI1 and cytokinin-induced AHK2 and AHK3 are important for vascular bundle formation in Arabidopsis.     

Recombinant rabbit muscle casein kinase I alpha is inhibited by heparin and activated by polylysine.

The casein kinase I (CKI) family consists of widely distributed monomeric Ser/Thr protein kinases that have a preference for acidic substrates. Four mammalian isoforms are known. A full length cDNA encoding the CKI alpha isoform was cloned from a rabbit skeletal muscle cDNA library and was utilized to construct a bacterial expression vector. Active CKI alpha was expressed in Escherichia coli as a polypeptide of Mr 36,000. The protein kinase phosphorylated casein, phosvitin and a specific peptide substrate (D4). The enzyme was inhibited by the isoquinolinesulfonamide CKI-7, half-maximally at 70 microM. Heparin inhibited phosphorylation of the D4 peptide or phosvitin by CKI alpha. Polylysine activated when the D4 peptide was the substrate but had no effect on phosvitin phosphorylation. It is becoming clear that the individual CKI isoforms have different kinetic properties and hence could have quite distinct cellular functions.     

The putative sensor histidine kinase CKI1 is involved in female gametophyte development in<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Embryo sac formation is a fundamental step in sexual reproduction in plants. However, the key players involved in the development of the female gametophyte remain elusive. We present data indicating that a two-component sensor histidine kinase, CKI1, originally implicated in cytokinin perception, is required for completion of megagametogenesis in Arabidopsis. We isolated a loss-of-function mutation in CKI1 resulting from an insertion of the En-1 transposon into the CKI1 coding sequence. Genetic analysis revealed that the mutant allele, cki1-i, could not be transmitted through the female germ line. Confocal laser scanning microscopy identified a block in megagametogenesis, characterized by the abortion of the central vacuole in mutant embryo sacs, and degradation of the developing female gametophyte after completion of all mitotic divisions. The recovery of two independent stable alleles and one revertant wild-type allele resulting from En-1 excision confirmed unambiguously the causal link between the cki1-i mutation and the abnormal phenotype. In situ localization of CKI1 mRNA and histochemical analysis of stable transformants harboring the uidA gene under the control of CKI1 promoter revealed that expression of CKI1 starts at the very beginning of female gametophyte development, and continues until fertilization. This suggests that the developing embryo sac may remain sensitive to signals recognized by CKI1 throughout megagametogenesis. Furthermore, expression of the paternally transmitted CKI1 was detected early after fertilization. The results indicate a role for a two-component signaling system during female gametophyte development, and provide the first evidence that gametophytic expression of a sensor-like molecule is essential for specific processes during megagametogenesis.     

Casein kinase I is anchored on axonemal doublet microtubules and regulates flagellar dynein phosphorylation and activity

Flagellar dynein activity is regulated by phosphorylation. One critical phosphoprotein substrate in Chlamydomonas is the 138-kDa intermediate chain (IC138) of the inner arm dyneins (Habermacher, G., and Sale, W. S. (1997) J. Cell Biol. 136, 167-176). In this study, several approaches were used to determine that casein kinase I (CKI) is physically anchored in the flagellar axoneme and regulates IC138 phosphorylation and dynein activity. First, using a videomicroscopic motility assay, selective CKI inhibitors rescued dynein-driven microtubule sliding in axonemes isolated from paralyzed flagellar mutants lacking radial spokes. Rescue of dynein activity failed in axonemes isolated from these mutant cells lacking IC138. Second, CKI was unequivocally identified in salt extracts from isolated axonemes, whereas casein kinase II was excluded from the flagellar compartment. Third, Western blots indicate that within flagella, CKI is anchored exclusively to the axoneme. Analysis of multiple Chlamydomonas motility mutants suggests that the axonemal CKI is located on the outer doublet microtubules. Finally, CKI inhibitors that rescued dynein activity blocked phosphorylation of IC138. We propose that CKI is anchored on the outer doublet microtubules in position to regulate flagellar dynein.