Interactive Fly, Drosophila

Miscellaneous targets of mammalian CkII

The dishevelled (dsh) gene family encodes cytoplasmic proteins that have been implicated in Wnt/Wingless (Wg) signaling. To demonstrate functional conservation of Dsh family proteins, two mouse homologs of Drosophila Dsh, Dvl-1 and Dvl-2, have been biochemically characterized in mouse and Drosophila cell culture systems. Treatment with a soluble Wnt-3A leads to hyperphosphorylation of Dvl proteins and a concomitant elevation of the cytoplasmic beta-catenin levels in mouse NIH3T3, L, and C57MG cells. This coincides well with the finding in a Drosophila wing disc cell line, clone-8, that Wg treatment induces hyperphosphorylation of Dsh. Mouse Dvl proteins affect downstream components of Drosophila Wg signaling as Dsh does; overexpression of Dvl proteins in clone-8 cells results in elevation of Armadillo (Drosophila homolog of beta-catenin) and Drosophila E-cadherin levels, hyperphosphorylation of the Dvl proteins themselves, and inhibition of Zeste-White3 kinase-mediated phosphorylation of a microtubule-binding protein, Tau. In addition, casein kinase II coimmunoprecipitates with Dvl proteins, and Dvl proteins are phosphorylated in these immune complexes. These results are direct evidence that Dsh family proteins mediate a set of conserved biochemical processes in the Wnt/Wg signaling pathway (Lee, 1999).

Beta-catenin, a member of the Armadillo repeat protein family, binds directly to the cytoplasmic domain of E-cadherin, linking it via alpha-catenin to the actin cytoskeleton. A 30-amino acid region within the cytoplasmic domain of E-cadherin, conserved among all classical cadherins, has been shown to be essential for beta-catenin binding. This region harbors several putative casein kinase II (CKII) and glycogen synthase kinase-3beta (GSK-3beta) phosphorylation sites and is highly phosphorylated. In vitro this region is indeed phosphorylated by CKII and GSK-3beta, which results in an increased binding of beta-catenin to E-cadherin. Additionally, in mouse NIH3T3 fibroblasts expression of E-cadherin with mutations in putative CKII sites results in reduced cell-cell contacts. Thus, phosphorylation of the E-cadherin cytoplasmic domain by CKII and GSK-3beta appears to modulate the affinity between beta-catenin and E-cadherin, ultimately modifying the strength of cell-cell adhesion (Lickert, 2000).

Several second-messenger-regulated protein kinases have been implicated in the regulation of N-methyl-D-aspartate (NMDA) channel function. Yet the role of calcium and cyclic-nucleotide-independent kinases, such as casein kinase II (CKII), has remained unexplored. CKII is identified as an endogenous Ser/Thr protein kinase that potently regulates NMDA channel function and mediates intracellular actions of spermine on the channel. The activity of NMDA channels in cell-attached and inside-out recordings is enhanced by CKII or spermine and is decreased by selective inhibition of CKII. In hippocampal slices, inhibitors of CKII reduce synaptic transmission mediated by NMDA but not AMPA receptors. The dependence of NMDA receptor channel activity on tonically active CKII thus permits changes in intracellular spermine levels or phosphatase activities to effectively control channel function (Lieberman, 1999).

The alpha catalytic subunit of casein kinase II (CkII) associates with and phosphorylates IkappaBalpha. Deletion mutants of IkappaBalpha localize phosphorylation to the C-terminal PEST domain of IkappaBalpha. Point mutation of residues T-291, S-283, and T-299 dramatically reduces phosphorylation of IkappaBalpha by the kinase in vitro. Cells that stably expresses wild-type IkappaBalpha (wtIkappaB), double-point-mutated IkappaBalpha (T291A, S283A), or triple-point-mutated IkappaBalpha (T291A, S283A, T299A) under the control of the tetracycline-responsive promoter were generated. Constitutive phosphorylation of the triple point mutant is eliminated in vivo. Mutation of the CkII sites in IkappaBalpha result in a protein with increased intrinsic stability. Together with results demonstrating a role for N-terminal sites in inducer-mediated phosphorylation and degradation of IkappaBalpha, these studies indicate that CkII sites in the C-terminal PEST domain are important for constitutive phosphorylation and intrinsic stability of IkappaBalpha (Lin, 1996).

In vivo IkappaB alpha is a stronger inhibitor of NF-kappaB than is IkappaB beta. This difference is directly correlated with their varying abilities to inhibit NF-kappaB binding to DNA in vitro and in vivo. Moreover, IkappaB alpha, but not IkappaB beta, can remove NF-kappaB from functional preinitiation complexes in in vitro transcription experiments. Both IkappaBs function in vivo not only in the cytoplasm but also in the nucleus, where they inhibit NF-kappaB binding to DNA. The inhibitory activity of IkappaB beta, but not that of IkappaB alpha, is facilitated by phosphorylation of the C-terminal PEST sequence by casein kinase II and/or by the interaction of NF-kappaB with high-mobility group protein I (HMG I) on selected promoters. The unphosphorylated form of IkappaB beta forms stable ternary complexes with NF-kappaB on the DNA either in vitro or in vivo. These experiments suggest that IkappaB alpha works as a postinduction repressor of NF-kappaB independently of HMG I, whereas IkappaB beta functions preferentially in promoters regulated by the NF-kappaB/HMG I complexes (Tran, 1997).

Sp1 (see Drosophila Buttonhead), a ubiquitous zinc finger transcription factor, is phosphorylated during terminal differentiation in the whole animal; this results in decreased DNA binding activity. Casein kinase II (CkII) is able to phosphorylate the C terminus of Sp1 and results in a decrease in DNA binding activity. This suggests that CkII may be responsible for the observed regulation of Sp1. Mutation of a consensus CkII site at amino acid 579, within the second zinc finger, eliminates phosphorylation of this site and the CkII-mediated inhibition of Sp1 binding. Phosphopeptide analysis confirms the presence of a CkII site at Thr-579 as well as additional sites within the C terminus. No gross changes in CkII subunit levels were seen during de-differentiation associated with liver regeneration. The serine/threonine phosphatase PP1 is identified as the endogenous liver nuclear protein able to dephosphorylate Sp1 but again no gross changes in activity are observed in the regenerating liver. Okadaic acid treatment of K562 cells increases Sp1 phosphorylation and inhibits its DNA binding activity suggesting that steady state levels of Sp1 phosphorylation are established by a balance between kinase and phosphatase activities (Armstrong, 1997).

The Drosophila and mammalian Cut homeodomain proteins contain, in addition to the homeodomain, three other DNA binding regions called Cut repeats. Cut-related proteins thus belong to a distinct class of homeodomain proteins with multiple DNA binding domains. Using nuclear extracts from mammalian cells, Cut-specific DNA binding is increased following phosphatase treatment, suggesting that endogenous Cut proteins are phosphorylated in vivo. Sequence analysis of Cut repeats reveals the presence of sequences that match the consensus phosphorylation site for casein kinase II (CKII). Therefore, an investigation was carried out to determine whether CKII can modulate the activity of mammalian Cut proteins. In vitro, a purified preparation of CKII efficiently phosphorylated Cut repeats, causing an inhibition of DNA binding. In vivo, overexpression of the CKII alpha and beta causes a decrease in DNA binding by Cut. The CKII phosphorylation sites within the murine Cut (mCut) protein have been identified by in vitro mutagenesis as residues Ser400, Ser789, and Ser972 within Cut repeats 1, 2, and 3, respectively. Cut homeodomain proteins are known to function as transcriptional repressors. Overexpression of CKII reduces transcriptional repression by mCut, whereas a mutant mCut protein containing alanine substitutions at these sites is not affected. Altogether these results indicate that the transcriptional activity of Cut proteins is modulated by CKII (Coqueret, 1998).

Csx/Nkx2.5 (see Drosophila Tinman) serves critical developmental functions in heart formation in vertebrates and nonvertebrates. The putative nuclear localization signal (NLS) of Csx/Nkx2.5 has been identified by site-directed mutagenesis to the amino terminus of the homeodomain, which is conserved in almost all homeodomain proteins. When the putative NLS of Csx/Nkx2.5 is mutated, a significant amount of the cytoplasmically localized Csx/Nkx2.5 is unphosphorylated. This is in contrast to the Csx/Nkx2.5 that is localized to the nucleus; that is serine- and threonine-phosphorylated, and suggests that Csx/Nkx2.5 phosphorylation is regulated, at least in part, by intracellular localization. Tryptic phosphopeptide mapping indicates that Csx/Nkx2.5 has at least five phosphorylation sites. Using in-gel kinase assays, a Csx/Nkx2.5 kinase has been identified whose molecular mass is approximately 40 kDa in both cytoplasmic and nuclear extracts. Mutational analysis and in vitro kinase assays suggest that this 40-kDa Csx/Nkx2.5 kinase is a catalytic subunit of casein kinase II (CKII) that phosphorylates the serine residue between the first and second helix of the homeodomain. This CKII site is phosphorylated in vivo. CKII-dependent phosphorylation of the homeodomain increases the DNA binding activity of Csx/Nkx2.5. Serine-to-alanine mutation at the CKII phosphorylation site reduces transcriptional activity when the carboxyl-terminal repressor domain is deleted. Although the precise biological function of Csx/Nkx2.5 phosphorylation by CKII remains to be determined, it may play an important role, since this CKII phosphorylation site within the homeodomain is fully conserved in all known members of the NK2 family of the homeobox genes (Kasahara, 1999).

Casein kinase II (CkII) is a ubiquitous and highly conserved serine/threonine protein kinase found in the nucleus and cytoplasm of most cells. Using a combined biochemical and genetic approach in the yeast Saccharomyces cerevisiae, the role of CkII was assessed in specific transcription by RNA polymerases I, II, and III. CkII is not required for basal transcription by RNA polymerases I and II but is important for polymerase III transcription. Polymerase III transcription is high in extracts with normal CkII activity but low in extracts from a temperature-sensitive mutant that has decreased CkII activity due to a lesion in the enzyme's catalytic alpha' subunit. Polymerase III transcription of 5S rRNA and tRNA templates in the temperature-sensitive extract is rescued by purified, wild-type CkII. An inhibitor of CkII represses polymerase III transcription in wild-type extract, and this repression is partly overcome by supplementing reaction mixtures with active CkII. Polymerase III transcription in vivo is impaired when CkII is inactivated. These results demonstrate that CkII, an oncogenic protein kinase previously implicated in cell cycle and growth control, is required for high-level transcription by RNA polymerase III (Hockman, 1996).

Eukaryotic DNA topoisomerase II is an abundant nuclear enzyme that is essential for cell proliferation. This homodimeric enzyme catalyzes the cleavage and re-ligation of double-stranded DNA required to separate replicated sister chromatids. Both biochemical and genetic studies show that its catalytic activity is required for chromosome condensation and segregation, and that its decatenation activity can be stimulated by a variety of protein kinases in vitro. In budding yeast, topoisomerase II is most highly phosphorylated in metaphase, and casein kinase II (CkII) has been shown shown to be the major kinase modifying topoisomerase II. The effects of phosphorylation of yeast topoisomerase II by CkII were investigated in vitro. The phosphorylation of the C terminus of topoisomerase II by CkII appears to increase the stability of the complex formed with linear DNA fragments, while dephosphorylation has the opposite effect. Rephosphorylation of phosphatase-treated topoisomerase II by chicken casein kinase II restores a stable protein-DNA complex using a linear DNA fragment. The enhanced stability of the topoisomerase II-DNA complex is also observed with relaxed circular DNA, but not with supercoiled minicircles, in agreement with published results using topoisomerase II from Drosophila. Limited proteolysis and probing with domain-specific antibodies shows that, with the exception of a weakly modified residue between amino acid residues 660 and 1250, all residues modified by casein kinase II are in the last 180 amino acid residues of yeast topoisomerase II (Dang, 1994).

A single double-stranded DNA (dsDNA) break will cause yeast cells to arrest in G2/M at the DNA damage checkpoint. If the dsDNA break cannot be repaired, cells will eventually override (that is, adapt to) this checkpoint, even though the damage that elicited the arrest is still present. Two adaptation-defective mutants have been identified that remain permanently arrested as large-budded cells when faced with an irreparable dsDNA break in a nonessential chromosome. This adaptation-defective phenotype is entirely relieved by deletion of RAD9, a gene required for the G2/M DNA damage checkpoint arrest. One mutation resides in CDC5, which encodes a polo-like kinase (see Drosophila Polo), whereas a second, less penetrant, adaptation-defective mutant is affected at the CKB2 locus, which encodes a nonessential specificity subunit of casein kinase II. It is likely that Cdc5p promotes checkpoint adaptation by inhibiting or bypassing the checkpoint pathway. The Cdc5p polo-like kinase has a role in activating Cdc25C, a conserved tyrosine phosphatase that removes an inhibitory phosphate on Cdc2. It may be that CKII acts to inhibit some part of the cell cycle arrest machinery that is not essential for maintaining the checkpoint arrest but is extremely important for maintaining viability during arrest (Toczyski, 1997).

The presence of fibroblast growth factor-2 (Fgf-2) in the nucleus has now been reported both in vitro and in vivo, but its nuclear functions are unknown. Fgf-2 added to nuclear extract binds to protein kinase Ck2 and nucleolin, a Ck2 natural substrate. Added to baculovirus-infected cell extracts overexpressing Ck2 or its isolated subunits, Fgf-2 binds to the enzyme through its regulatory beta subunit. Using purified proteins, Fgf-2 is shown to directly interact with Ck2 and to stimulate Ck2 activity toward nucleolin. A mitogenic-deficient Fgf-2 mutant protein has an impaired ability to interact with Ck2 and to stimulate Ck2 activity using nucleolin as substrate. It is proposed that in growing cells, one function of nuclear Fgf-2 is to modulate Ck2 activity through binding to its regulatory beta subunit (Bonnet, 1996).

A 27-kDa protein was isolated that binds to cytoplasmic dynein. Microsequencing of a 17-amino acid peptide of this polypeptide yields a sequence that completely matches the predicted sequence of the beta subunit of casein kinase II, a highly conserved serine/threonine kinase. Affinity chromatography using a dynein column indicates that both the alpha and beta subunits of casein kinase II are retained by the column from rat brain cytosol. Although dynactin is also bound to the column, casein kinase II is not a dynactin subunit. Casein kinase II does not co-immunoprecipitate with dynactin, and it binds to a dynein intermediate chain column which has been preblocked with excess p150(Glued), a treatment that inhibits the binding of dynactin from cytosol. Bacterially expressed and purified rat dynein intermediate chain can be phosphorylated by casein kinase II in vitro. Native cytoplasmic dynein purified from rat brain can also be phosphorylated by casein kinase II in vitro. It is propose that CkII may be involved in the regulation of dynein function possibly by altering its cargo specificity or its ability to interact with dynactin (Karki, 1997).

FKBP52 (HSP56, p59, HBI) is the 59-kDa immunosuppressant FK506-binding protein; it possesses peptidyl prolyl isomerase activity and produces a chaperone-like activity in vitro. FKBP52 associates with the heat shock protein HSP90 and is included in the steroid hormone receptor complexes in vivo. FKBP52 possesses a well conserved phosphorylation site for casein kinase II (CK2) that was previously shown to be associated with HSP90. An examination was performed to determine if FKBP52 is phosphorylated by CK2 both in vivo and in vitro. Recombinant rabbit FKBP52 was phosphorylated by purified CK2. Deletion mutants of FKBP52 were expressed to determine the site(s) phosphorylated by CK2. Thr-143 in the hinge I region has been identified as the major phosphorylation site for CK2. A synthetic peptide corresponding to this region is phosphorylated by CK2, and the peptide competitively inhibits the phosphorylation of other substrates by CK2. The [32P]phosphate labeling of FKBP52-expressing cells reveals that the same site is also phosphorylated in vivo. FK506 binding to FKBP52 does not affect the phosphorylation by CK2 and, conversely, the FK506-binding activity of FKBP52 is not affected by the phosphorylation. Most important, CK2-phosphorylated FKBP52 does not bind to HSP90. These results indicate that CK2 phosphorylates FKBP52 both in vitro and in vivo and thus may regulate the protein composition of chaperone-containing complexes such as those of steroid receptors and certain protein kinases (Miyata,1997).

The high mobility group (HMG) 1 and 2 proteins are the most abundant non-histone components of chromosomes. HMG1 proteins are abundant components of chromatin. A subfamily of the HMG proteins containing the HMG1 box domain (HMG1-BD) is widely distributed in eukaryotic cells from yeast to man. The members of this group are thought to have various functions related to modulation of transcription, DNA integration, and recombination. Since these proteins have an ability to induce strong bends and unwind DNA, they are called architectural components of chromatin. The most abundant of the HMG1 box proteins are the HMG1 and HMG2 proteins. They are composed of one or two HMG1-BDs, which are primarily responsible for contacts with DNA. HMG1-BDs are amino- and/or carboxyl-terminally flanked by stretches of positively or negatively charged residues. These regions modulate the binding affinity of HMG1-BDs, but do not influence the extent of DNA distortion. Deletion of these regions, in particular those of the negatively charged carboxyl-terminal tails, alters binding specificity of the HMG1 proteins. Moreover, the C-terminal portion of the HMG1 proteins is important for stimulation of transcription and nuclear retention (Wisniewski, 1999 and references).

Essentially the entire pool of HMG1 proteins in Drosophila embryos and Chironomus cultured cells is phosphorylated at multiple serine residues located within acidic tails of these proteins. The phosphorylation sites match the consensus phosphorylation site of casein kinase II. Electrospray ionization mass spectroscopic analyses reveal that Drosophila HMGD and Chironomus HMG1a and HMG1b are double-phosphorylated and that Drosophila HMGZ is triple-phosphorylated. The importance of this post-translational modification was studied by comparing some properties of the native and in vitro dephosphorylated proteins. It was found that dephosphorylation affects the conformation of the proteins and decreases their conformational and metabolic stability. Moreover, dephosphorylation weakens binding of the proteins to four-way junction DNA by 2 orders of magnitude, whereas the strength of binding to linear DNA remains unchanged. Based on these observations, it has been proposed that the detected phosphorylation is important for the proper function and turnover rates of these proteins. Since the occurrence of acidic tails containing canonical casein kinase II phosphorylation sites is common to diverse HMG and other chromosomal proteins, these results are probably of general significance (Wisniewski, 1999).

NF-κB is activated in response to proinflammatory stimuli, infections, and physical stress. While activation of NF-κB by many stimuli depends on the IκB kinase (IKK) complex, which phosphorylates IκBs at N-terminal sites, the mechanism of NF-κB activation by ultraviolet (UV) radiation remained enigmatic, since it is IKK independent. UV-induced NF-κB activation has been shown to depend on phosphorylation of IκBα at a cluster of C-terminal sites that are recognized by CK2 (formerly casein kinase II). Furthermore, CK2 activity toward IκB is UV inducible through a mechanism that depends on activation of p38 MAP kinase. Inhibition of this pathway prevents UV-induced IκBα degradation and increases UV-induced cell death. Thus, the p38-CK2-NF-κB axis is an important component of the mammalian UV response (Kato, 2003).

Back to Evolutionary homologs part 1/2 |

Casein kinase II: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

The Interactive Fly resides on the
Society for Developmental Biology's Web server.