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.     

Casein kinase II and protein kinase C modulate hepatitis delta virus RNA replication but not empty viral particle assembly.

Hepatitis delta virus (HDV) contains two virus-specific delta antigens (HDAgs), large and small forms, which are identical in sequence except that the large one contains 19 extra amino acids at the C terminus. HDAgs are nuclear phosphoproteins with distinct biological functions; the small form activates HDV RNA replication, whereas the large form suppresses this process but is required for viral particle assembly. In this study, we have characterized the phosphorylative property of HDAg in a human hepatoma cell line (HuH-7) and examined the role of phosphorylation in HDAg function. As demonstrated by in vivo labeling and kinase inhibitor experiments, the phosphorylation levels of both HDAgs were diminished by the inhibitor of casein kinase II (CKII). Nevertheless, phosphorylation of only the small form could be markedly reduced by the protein kinase C (PKC) inhibitor, suggesting different phosphorylation properties between the two HDAgs. When these two kinase inhibitors were added separately to the transient-expression system, HDV RNA replication was profoundly suppressed. In contrast, the inhibitors did not affect the assembly of empty HDAg particle from HDAgs and hepatitis B virus surface antigen. To further examine the role of phosphorylation in HDAg function, two conservative CKII recognition sites at Ser-2 and Ser-123 of both HDAgs and one potential PKC recognition site at Ser-210 of the large HDAg were altered to alanine by site-directed mutagenesis. Transfection experiments indicated that mutation at Ser-2, but not Ser-123, significantly impaired the activity of the small HDAg in assisting HDV RNA replication. This property is in accordance with our observation that Ser-2, not Ser-123, was the predominant CKII phosphorylation site in the small HDAg. Our studies also excluded the possibility that the phosphorylation of Ser-2, Ser-123, or Ser-210, had roles in the trans-suppression activity of the large HDAg, in the assembly of empty virus-like HDAg particle, and in the nuclear transport of HDAgs. In conclusion, our results indicate that both CKII and PKC positively modulate HDV RNA replication but not the assembly of empty HDAg particle. The role of CKII in HDV replication may at least in part be accounted for by the phosphorylation of Ser-2 in the small HDAg. The effect of PKC on HDV RNA replication is, however, not to mediate the phosphorylation of the conservative Ser-210 in the large HDAg but rather to act on as-yet-unidentified Ser or Thr residues in the small HDAg or cellular factors. These findings provide the first insight into the roles of phosphorylation of the two HDAgs in the HDV replication cycle.     

Casein kinase II from Rana temporaria oocytes. Intracellular localization and activity during progesterone-induced maturation

A homogeneous preparation of casein kinase II has been isolated from the ribosome-free extracts of Rana temporaria oocytes by means of chromatography on heparin-Sepharose, phosphocellulose and mono Q. The enzyme consists of three subunits with molecular mass of 43 kDa, 41 kDa and 29 kDa. The protein kinase was labelled with radioactive iodine and injected back into oocytes. As shown by histoautoradiography the enzyme forms a diffuse ring around the nucleus in the oocyte cytoplasm. A part of casein kinase II is found in informosomes. During oocytes maturation casein kinase II activity increases 7 h after progesterone administration and at the final stages of maturation (20-23 h). Cycloheximide blocks the second augmentation of kinase activity and does not influence the first one.     

Casein kinase II is a predominantly nuclear enzyme

Casein kinase II (CK II) has been implicated in regulating multiple processes related to cell growth, proliferation, and differentiation. To better understand the function(s) and regulation of this ubiquitous kinase, it is important to know its subcellular distribution. However, this issue has been the subject of contradictory reports. In this study, we have used indirect immunofluorescence microscopy and cell fractionation to study the subcellular distribution of all three subunits of chicken CK II, alpha, alpha', and beta. We examined primary chick embryo fibroblasts, virally transformed chicken hepatoma cells, as well as HeLa cells transiently transfected with cDNAs encoding chicken CK II subunits. We found that each of the three CK II subunits was located predominantly in the cell nucleus, irrespective of the cell type analyzed or the procedure used for cell fixation. No major differences were detected in the subcellular distributions of individual CK II subunits, and no evidence was obtained for subunit redistributions during interphase of the cell cycle. During mitosis, the bulk of the enzyme was dispersed throughout the cell, though a fraction of all three subunits was associated with the mitotic spindle. Biochemical studies based on mechanical enucleation of chicken cells confirmed the predominantly nuclear location of all three CK II subunits. Finally, immunoblotting experiments were carried out to study the expression of CK II subunits. A survey of different adult chicken tissues revealed substantial tissue-specific differences in the levels of CK II protein, but no evidence was obtained for pronounced tissue specificity in the expression of individual CK II subunits. These results strongly suggest that CK II functions primarily in regulating nuclear activities, and that the two catalytic subunits, alpha and alpha', may carry out overlapping functions.     

Casein kinase II in signal transduction and cell cycle regulation

Molecular and Cellular Biochemistry - Casein kinase II is a protein serine/threonine kinase that is ubiquitously distributed in eukaryotes. Molecular cloning studies and protein sequence analysis...     

Casein kinase II phosphorylation of caldesmon downregulates myosin-caldesmon interactions

It is well-known that caldesmon (CaD) is a substrate for casein kinase II (CKII), and the phosphorylation of CaD by CKII regulates the interaction of CaD with myosin. However, the functionally relevant CKII phosphorylation site(s) on CaD and the precise role of CaD phosphorylation by CKII in mediating CaD's function have remained elusive. In this study, we demonstrate that Ser-26 is the major CKII phosphorylation site on CaD, while Ser-73 is of relatively minor importance. Moreover, the phosphorylation of Ser-26 and Ser-73 reduced CaD's ability to bind myosin by 45% and 27%, respectively, suggesting that the interaction of CaD with myosin is downregulated, at least in part, by the phosphorylation of these serine residues by CKII. Our results also demonstrate that there are at least four myosin-binding motifs within the amino-terminal region of CaD, located between residues 1-23, 34-43, 44-53, and 86-115, respectively. The myosin-binding motif between residues 44-53 contributes to strong myosin binding, while the three other myosin-binding motifs are responsible for weak myosin binding. The sequences between residues 24-33 and 54-85 on CaD are not required for the binding of CaD to myosin; thus, both Ser-26 and Ser-73 are located outside of the myosin-binding motifs. It is therefore likely that the downregulation of myosin-CaD interactions by CKII phosphorylation is due to phosphorylation-induced conformational changes in the adjacent myosin-binding motifs on CaD, rather than by the direct modification of these myosin-binding motifs by CKII.     

A diurnally regulated dehydrin from Avicennia marina that shows nucleo-cytoplasmic localization and is phosphorylated by Casein kinase II in vitro

Dehydrins have a key role in protecting plants from dehydration stress. We report here the isolation of two cDNAs coding for the same dehydrin, AmDHN1 and AmDHN1a from salt stressed leaves of Avicennia marina (Forsk.) Vierh. by EST library screening. AmDHN1 was found to contain a retained intron that was absent in AmDHN1a. AmDHN1 expression in the context of various environmental stresses was investigated. In leaves, AmDHN1 shows a diurnal pattern of regulation and is induced only by mannitol application. In roots, AmDHN1 is rapidly induced by salinity (NaCl) and dehydration stress (PEG and mannitol). A fragment of 795 bp corresponding to the 5′ upstream region of AmDHN1 was isolated by TAIL-PCR. In silico analysis of this sequence reveals the presence of putative stress regulatory elements (ABRE, DRE, MYB and MYC binding sequences). Putative phosphorylation sites for Casein kinase II were identified in the AmDHN1a ORF. In vitro phosphorylation of Escherichia coli expressed Trx-AmDHN1a by Casein kinase II was observed that was reversed by Shrimp Alkaline Phosphatase treatment. A putative nuclear targeting domain was identified in the translated AmDHN1a ORF and stably transformed AmDHNIa-GFP was found to show nucleo-cytoplasmic localization in tobacco guard cells. As observed for maize Rab17, the phosphorylation of AmDHN1a may contribute to its nuclear localization.     

Casein kinase type II during lens fiber differentiation in amphibians

cAMP-independent protein kinase activity of casein type was found in Rana temporaria eye lens. The highest activity was observed in "cortex" lens fibres, and decreased two-fold in lens epithelium. Minimum activity was found in lens "nucleus" fibres. Thus, protein kinase activity is characteristic of metabolically active differentiating lens cells. Enzyme fraction showed almost complete binding to the immobilized RNA. The enzyme was inhibited by heparine, phosphorylated casein (but not histones). It could use either ATP or GTP as a source of phosphate, and caused modification of serine and threonine residues in casein molecule. The protein kinase from lens epithelium and cortex was purified 6,000-7,000-fold and was identified as a type II casein kinase.     

Casein kinase II stimulates rat liver mitochondrial glycerophosphate acyltransferase activity

Rat liver mitochondrial glycerophosphate acyltransferase (mtGAT) possesses 14 consensus sites for casein kinase II (CKII) phosphorylation. To study the functional relevance of phosphorylation to the activity of mtGAT, we treated isolated rat liver mitochondria with CKII and found that CKII stimulated mtGAT activity approximately 2-fold. Protein phosphatase-lambda treatment reversed the stimulation of mtGAT by CKII. Labeling of both solubilized and non-solubilized mitochondria with CKII and [gamma-32P]ATP resulted in a 32P-labeled protein of 85kDa, the molecular weight of mtGAT. Our findings suggest that CKII stimulates mtGAT activity by phosphorylation of the acyltransferase. The significance of this observation with respect to hormonal control of the enzyme is discussed.     

Casein kinase II activity is required for transferrin receptor endocytosis.

The effect of protein kinase inhibitors on transferrin receptor (TR) internalization was examined in HeLa, A431, 3T3-L1 cells, and primary chicken embryo fibroblasts. We show that TR endocytosis is not affected by tyrosine kinase or protein kinase C inhibitors, but is inhibited by one serine/threonine kinase inhibitor, H-89. Inhibition occurred within 15 min, was completely reversible after H-89 withdrawal, and was specific for endocytosis rather than pinocytosis since a TR mutant lacking an internalization signal was not affected. Interestingly, H-89 also inhibited the internalization of a TR chimera containing the major histocompatibility complex class II invariant chain cytoplasmic tail, indicating that the effect was not specific for the TR. Since H-89 inhibits a number of kinases, we employed a permeabilized cell endocytosis assay to further characterize the kinase. In permeabilized 3T3-L1 cells, addition of pseudosubstrate inhibitor peptides of casein kinase II (CKII) blocked TR internalization by more than 50%, whereas pseudosubstrates of cyclic AMP-dependent kinase A, protein kinase C, and casein kinase I had no effect. Furthermore, addition of purified CKII to the cell-free reactions containing CKII pseudosubstrates reversed the endocytosis block, suggesting that CKII or a CKII-like activity is required for constitutive endocytosis.     

Casein kinase II phosphorylates DNA-polymerase-alpha--DNA-primase without affecting its basic enzymic properties

Immunoaffinity-purified DNA-polymerase-alpha--DNA-primase complex from calf thymus was phosphorylated in vitro by highly purified casein kinase II from the same tissue. Specific phosphorylation of the DNA-polymerizing alpha subunit and the primase-associated gamma subunit was observed. About 1 mol phosphate/mol polymerase--primase was incorporated. Despite this effect, neither the DNA polymerase nor the DNA primase activity were changed after phosphorylation by casein kinase II. Furthermore, dephosphorylation of polymerase--primase with alkaline phosphatase did not change the polymerase or the primase activity to a significant extent. Moreover, both alkaline phosphatase and casein kinase II had no effect on the processivity of DNA synthesis and on the lengths and amounts of primers formed by the DNA primase. Because DNA polymerase alpha maintained all its basic properties even after extensive treatment with alkaline phosphatase, it is unlikely that phosphorylation has a direct influence on the activities of the DNA-polymerase-alpha--DNA-primase complex. The possible influence of post-translational phosphorylation on the formation of a complex of polymerase alpha and its accessory proteins is discussed.     

Casein kinase II phosphorylates the eukaryote-specific C-terminal domain of topoisomerase II in vivo.

The decatenation activity of DNA topoisomerase II is essential for viability as eukaryotic cells traverse mitosis. Phosphorylation has been shown to stimulate topoisomerase II activity in vitro. Here we show that topoisomerase II is a phosphoprotein in yeast and that the level of incorporated phosphate is significantly higher at mitosis than in G1. Comparison of tryptic phosphopeptide maps reveals that the major phosphorylation sites in vivo are targets for casein kinase II. Incorporation of phosphate into topoisomerase II is nearly undetectable at the non-permissive temperature in a conditional casein kinase II mutant. The sites modified by casein kinase II are located in the extreme C-terminal domain of topoisomerase II. This domain is absent in prokaryotic and highly divergent among eukaryotic type II topoisomerases, and may serve to regulate functions of topoisomerase II that are unique to eukaryotic cells.     

Casein kinase II phosphorylates lens connexin 45.6 and is involved in its degradation

Connexin (Cx) 45.6, an avian counterpart of rodent Cx50, is phosphorylated in vivo, but the sites and function of the phosphorylation have not been elucidated. Our peptide mapping experiments showed that the Ser(363) site in the carboxyl (COOH) terminus of Cx45.6 was phosphorylated and that this site is within casein kinase (CK) II consensus sequence, although showing some similarity to CKI sequence. The peptide containing Ser(363) could be phosphorylated in vitro by CKII, but not by CKI. Furthermore, CKII phosphorylated Cx45.6 in embryonic lens membrane and the fusion protein containing the COOH terminus of Cx45.6. Two-dimensional peptide mapping experiments showed that one of the Cx45.6 peptides phosphorylated in vivo migrated to the same spot as one of those phosphorylated by CKII in vitro. Furthermore, CKII activity could be detected in lens lysates. To assess the function of this phosphorylation event, exogenous wild type and mutant Cx45.6 (Ser(363) --> Ala) were expressed in lens primary cultures by retroviral infection. The mutant Cx45.6 was shown to be more stable having a longer half-life compared with wild type Cx45.6. Together, the evidence suggests that CKII is likely a kinase responsible for the Ser(363) phosphorylation, leading to the destablization and degradation of Cx45.6. The connexin degradation induced by phosphorylation has a broad functional significance in the regulation of gap junctions in vivo.     

Casein kinase II accumulation in the nucleolus and its role in nucleolar phosphorylation

A rabbit antiserum against highly purified casein kinase II from mouse tumor cells was used for immunolocalization of the enzyme in fixed, permeabilized mouse cells. Casein kinase II was highly accumulated in nucleoli compared to the extra-nucleolar space of the nucleus or to the cytoplasma. Casein kinase II samples highly purified from the cytoplasma, from the extra-nucleolar fraction of the nucleus or from nucleoli exhibited no differences with respect to structure and function. All samples originally had an alpha 2 beta 2 structure (alpha, 42 kDa; beta, 24 kDa) showing formation of the alpha'-chain (36 kDa) only in the late steps of purification. The isoelectric point of the alpha-chain of all three samples was pH 7.7 and that of the beta-chain was pH 6.4-6.6. Using ATP or GTP, all three casein kinase II samples gave the same results of maximum phosphorylation of purified nucleolar marker phosphoproteins pp105/C23, pp135 and B23, yielding pp135 as one of the most highly phosphorylated proteins with an incorporation of about 75 phosphate groups per molecule pp135. Studies on optimum conditions of phosphorylation of nucleolar phosphoproteins by casein kinase II revealed that each of the protein substrates individually responded to alterations of assay parameters such as pH, magnesium ion and sodium chloride concentrations indicating that predominantly individual structural criteria were responsible for optimum phosphorylation. The determination of the apparent Km of casein kinase II for purified nucleolar phosphoproteins yielded values of 0.15 microM (pp105/C23), 0.1 microM (pp135) and 1.0 microM (B23) identifying them as high-affinity substrates of casein kinase II.     

Casein kinase II is elevated in solid human tumours and rapidly proliferating non-neoplastic tissue

Protein kinase CKII (i.e. casein kinase II, CKII, NII) is expressed at a higher level in rapidly proliferating tissues and in solid human tumours (e.g. colorectal carcinomas) when compared to the corresponding non-neoplastic colorectal mucosa. This could be shown by (a) Western blotting of cellular extracts from solid tumours followed by immunostaining with an anti-CKII polyclonal antibody, (b) immunohistochemical staining of cells from tissue sections and (c) by activity measurements using the CKII-specific synthetic peptide (RRRDDDSDDD). The maximum observed activity in the colorectal carcinomas investigated was up to eightfold higher in the tumour specimens than in the non-neoplastic tissue (i.e. colorectal mucosa). The activity range was between 33-350 U/mg protein and in the case of colorectal mucosa 13-106 U/mg protein. The amount of CKII determined in the individual tumours was in the range 0.4-1.6 nmol/g tissue.     

Casein kinase 2- and protein kinase A-regulated adenomatous polyposis coli and beta-catenin cellular localization is dependent on p38 MAPK

Skin cancer is the most common form of malignancy in the world with epidemic proportions. Identifying the biochemical and molecular mechanisms underlying the events leading to tumors is paramount to designing new and effective treatments that may aid in treating and/or preventing skin cancers. Herein we identify p38 MAPK, along with its positive modulator, Gadd45a, as important regulators of nucleocytoplasmic shuttling of the adenomatous polyposis coli (APC) tumor suppressor. APC normally functions to block beta-catenin from promoting cell proliferation and migration/invasion. Keratinocytes lacking proper p38 MAPK activation, either due to lack of Gadd45a or through the use of p38 MAPK-specific inhibitors, are unable to effectively transport APC into the nucleus. We also show that p38 MAPK is able to directly associate with and modulate both casein kinase 2 (CK2) and protein kinase A (PKA), which promote and block APC nuclear import, respectively. We demonstrate that p38 MAPK is able to not only enhance CK2 kinase activity but also suppress PKA kinase activity. Moreover, lack of normal p38 MAPK activity in either Gadd45a-null keratinocytes or in p38 MAPK inhibitor treated keratinocytes leads to decreased CK2 activity and increased PKA activity. In either case, disruption of APC nuclear import results in elevated levels of free cellular, and potentially oncogenic, beta-catenin. Numerous tumors, including skin cancers, are associated with high levels of beta-catenin, and our data indicate that p38 MAPK signaling, along with Gadd45a, may provide tumor suppressor-like functions in part by promoting APC nuclear localization and effective beta-catenin regulation.     

Casein kinase II inhibitors isolated from two Brazilian plants Hymenaea parvifolia and Wulffia baccata.

Two dihydroflavonol rhamnosides (1 and 2) isolated from the bark of Hymenaea parvifolia and two pentacyclic triterpenoids (3 and 6) obtained from the leaves of Wulffia baccata have been found to exhibit inhibitory effects of casein kinase II (CK-II) dose-dependently, suggesting that at higher doses more than 10 microM, these four compounds may act as potent CK-II suppressors of the CK-II-mediated activation of 60S acidic ribosomal P proteins in vitro.