T Interactive Fly, Drosophila Casein kinase II: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References


Gene name - Casein kinase II (alpha subunit and beta subunit )

Synonyms - CK2, Ck-2

Cytological map positions - 80A1--80A4 and 10E1--10E2

Function - Kinase

Keywords - Signal transduction protein

Symbol - CkIIalpha and CkIIbeta

FlyBase ID: FBgn0264492 and FBgn0000259

Genetic map positions - 3-[47]. and 1-[36].

Classification - Casein kinase II catalytic and regulatory subunits

Cellular location - presumed to be nuclear and cytoplasmic



CkIIalpha NCBI links: Precomputed BLAST | Entrez Gene


CKIIbeta NCBI links: Precomputed BLAST | Entrez Gene | UniGene
BIOLOGICAL OVERVIEW

Recent literature
Khanna, M. R. and Fortini, M. E. (2015). Transcriptomic analysis of Drosophila mushroom body neurons lacking Amyloid-beta precursor-like protein activity. J Alzheimers Dis 46: 913-928. PubMed ID: 26402626
Summary:
The amyloid-&beta: protein precursor (AbetaPP; see Drosophila Appl) is subjected to sequential intramembrane proteolysis by α-, &beta:-, and γ-secretases, producing secreted amyloid-&beta: (A&beta:) peptides and a cytoplasmically released A&beta:PP Intracellular Domain (AICD). AICD complexes with transcription factors in the nucleus, suggesting that this AβPP fragment serves as an active signaling effector that regulates downstream genes, although its nuclear targets are poorly defined. To further understand this potential signaling mechanism mediated by AβPP, a transcriptomic identification of the Drosophila genome that is regulated by the fly AβPP orthologue was performed in fly mushroom body neurons, which control learning- and memory-based behaviors. Significant changes were found in expression of 245 genes, representing approximately 1.6% of the Drosophila genome, with the changes ranging from +6 fold to -40 fold. The largest class of responsive targets corresponds to non-protein coding genes and includes microRNAs that have been previously implicated in Alzheimer's disease pathophysiology. Several genes were identified in the Drosophila microarray analyses that have also emerged as putative AβPP targets in similar mammalian transcriptomic studies. These results also indicate a role for AβPP in cellular pathways involving the regulation of Drosophila Casein Kinase II, mitochondrial oxidative phosphorylation, RNA processing, and innate immunity. These findings provide insights into the intracellular events that are regulated by AβPP activity in healthy neurons and that might become dysregulated as a result of abnormal AβPP proteolysis in AD.

Casein kinase II (CkII) is a heterotetramer composed of two types of subunits: an alpha subunit that is catalytically active and a beta subunit that serves as a regulatory subunit. In many species, including higher vertebrates, the alpha subunit consists of two forms: alpha and alpha1. Alpha1 subunits have not been detected in either Drosophila or Xenopus. The beta subunit is inactive by itself but it stimulates the catalytic activity of alpha 5 to 10 fold. In addition, beta causes stabilization of alpha against heat denaturation and proteolysis and can change the specificity of alpha for its interaction with substrates and inhibitors. CkII can be moderately activated by polyamines such as spermine, spermidine and by polylysine. The most potent inhibitor is heparin. CkII can use either ATP or GTP, and the enzyme targets serines and threonines immersed in acidic motifs of proteins and peptides (Allende, 1995).

The list of CkII's targets is long and varied (Allende, 1995). It includes

Known targets in Drosophila include Cactus (Packman, 1997), Engrailed (Bourbon, 1995), Dishevelled (Willert, 1997), Antennapedia (Jaffe, 1997), Drosophila Max (see Myc) (Gallant, 1996), and Tropoisomerase II (Ackerman, 1988).

The regulation of CkII is enigmatic; CkII does not seem to be regulated by many of the well-known second messenger molecules. Nevertheless, CkII activity is often increased in cells that are actively proliferating and embryonic tissues or organs contain higher concentrations of CkII than do their adult counterparts. It has also been shown that CkIIalpha is phosphorylated by cdc2 kinase in cells arrested in mitosis and that p24cdc2 kinase can be phosphorylated by CkII during G1. This suggests that the cell cycle is regulates and is regulated by CkII. The biological function served by the beta subunit in the regulation of the alpha subunit activity is unknown (Allende, 1995).

One example of the role of CkII in modification of a Drosophila protein is found in the interaction of CkII with Antennapedia. The in vivo activity of this HOX protein is modified by phosphorylation due to CkII. Antp has four putative CkII target sites. Sites 1 and 2 are found in the amino-terminal portion of the protein, whereas sites 3 and 4 are clustered close to the homeodomain in the C-terminal tail. Antp with alanine substitutions at its CkII target sites produces altered thoracic and abdominal development. Ubiquitous expression of Antp in flies produces an inhibition of head involution, the elimination of dorsal head structures, a transformation of T1 into a second thoracic segment (T2), and the appearance of one to two partial T2 denticle belts in the head segment. Embryos that express Antp with altered CkII target sites (alanine replacing serine or threonine) exhibit additional phenotypes including an absence of Keilin's organs, shortened denticle belts, and a failure of germ-band retraction. Embryos that express altered Antp show a disorganized CNS with irregularly spaced or fused horizonal commissures and gaps in the longitudinal commissures. CkII sites 1 and 4 appear to be the most important in terms of the altered phenotypes produced (Jaffe, 1997).

The novel functions that result from mutationally removing CkII sites suggest that altered Antp is not suppressed phenotypically by the more posterior homeotic proteins. In contrast, the in vivo activity of a form of Antp that contains acidic amino acid substitutions at its CkII target sites is greatly reduced, mimicking a constitutively phosphorylated Antp protein. This hypoactive form of Antp, but not the alanine-substituted form, is also reduced in its ability to bind to DNA cooperatively with the homeodomain protein Extradenticle. These results suggest that phosphorylation of Antp by CkII is important for preventing inappropriate activities of this homeotic protein during embryogenesis. The information provided however does not address the mechanism by which phosphorylation alters Antp's properties. Thus phosphorylation appears to modulate Antp's properties, restricting its activity to an appropriate level (Jaffe, 1997).

Dishevelled protein is also targeted by CkII. Immunoprecipitated Dsh protein is associated with Casein Kinase II. Tryptic phosphopeptide mapping indicates that identical peptides are phosphorylated by CkII in vitro and in vivo, suggesting that CkII is at least one of the kinases that phosphorylates Dsh. Overexpression of frizzled2, a Wingless receptor, also stimulates phosphorylation of Dsh, Dsh-associated kinase activity, and association of CkII with Dsh. It is not known whether association of CkII with unphosphorylated Dsh occurs first, or whether phosphorylation of Dsh promotes association with CkII. Unphosphorylated Dsh clearly has some affinity for CkII, however, in vivo phosphorylated Dsh is associated with more CkII than is underphosphorylated Dsh. This suggests a model in which CkII can bind with low affinity to underphosphorylated Dsh and effect its phosphorylation. The phosphorylated Dsh then has a higher affinity for CkII, leading to an increase in the amount of Dsh-CkII complex. Phosphorylation of Dsh in response to the Wg signal, leads to the phosphorylation of Dsh but this is insufficient for the transduction of the Wg signal to Armadillo. Thus the function of the phosphorylation of Dsh by CkII is unknown (Willert, 1997).

Clearly CkII plays an important metabolic role in the regulation of enzyme activity, perhaps linked to the cell cycle. Nevertheless, an understanding of the regulatory inputs into CkII and an understanding of the global role of CkII in regulation of cell function awaits future work, especially the induction of CkII mutation.


PROTEIN STRUCTURE

Amino Acids - 336 and 215 for alpha and beta subunits respectively

Structural Domains

Cloned cDNAs encoding both subunits of Drosophila Casein kinase II have been isolated by immunological screening of lambda gt11 expression libraries, and the complete amino acid sequence of both polypeptides has been deduced by DNA sequencing. The alpha cDNA contains an open reading frame of 336 amino acid residues, yielding a predicted molecular weight for the alpha polypeptide of 39,833. The alpha sequence contains the expected semi-invariant residues present in the catalytic domain of previously sequenced protein kinases, confirming that it is the catalytic subunit of the enzyme. Pairwise homology comparisons between the alpha sequence and the sequences of a variety of vertebrate protein kinase suggest that Casein kinase II is a distantly related member of the protein kinase family. The beta subunit is derived from an open reading frame of 215 amino acid residues and is predicted to have a molecular weight of 24,700. The beta subunit exhibits no extensive homology to other proteins whose sequences are currently known (Saxena, 1987).

There are several notable aspects to CkII structure. Conserved region I is involved in the binding of ATP. A basic stretch is present between conserved regions II and III, which has a high affinity for heparin and possesses a nuclear localization region. Atypical amino acids in conserved regions II and VII differ from those found in more than 95% of other known protein kinases. These regions have both been implicated in the interaction of kinases with ATP. A region in CkII contains putative sites for p24cdc2 phosphorylation. CkII can autophosphorylate the catalytic subunit in a reaction enhanced by the beta subunit. The beta subunit is also autophosphorylated. Deletion of the last 44 amino acids of the C-terminal end of beta eliminates the capacity of truncated beta to form tetramers with alpha and to stimulate alpha activity, but deletion of the last 34 amino acids yield an active beta subunit that has a lower affinity for CkIIalpha. Acidic residues from amino acids 55 to 70 are involved in the interaction of beta with alpha and in the modulation of alpha activity. The beta subunit contains four cysteins that may form a metal binding finger. A 'destruction box' is present in the beta subunit (Allende, 1995).

A useful analogy can be made tetrameric CkII and the heterodimeric cyclin/cyclin dependent kinases (See CyclinA). The beta subunit of CkII contains a 'destruction' box similar to that of cyclins. One caveat to this analogy is the finding that the beta subunit is synthesized in large excess to alpha (Allende, 1995 and references).

The relationship between the structure and the activity of Casein kinase II was examined with regard to its previously reported property to self-aggregate in vitro. Sedimentation velocity and electron microscopy studies show that the purified kinase exhibits four major different oligomeric forms in aqueous solution. This self-polymerization is a reproducible and fully reversible process, highly dependent upon the ionic strength of the medium, suggesting that electrostatic interactions are mostly involved. At high salt concentrations (e.g. 0.5 M NaCl), CkII appears as spherical moieties with a 18.7 +/- 1.6 nm average diameter, roughly corresponding to the alpha 2 beta 2 protomer, as deduced by measurements of the Stokes radius and by light scattering studies. At lower ionic strength (e.g. 0.2 M NaCl), the protomers associate to form ring-like structures with a diameter (averaging 36.6 +/- 2.1 nm) and Stokes radius indicating that they are most likely made of four circularly associated alpha 2 beta 2 protomers. At 0.1 M NaCl, two additional polymeric structures are visualized: thin filaments (16.4 +/- 1.4 nm average), as long as 1 to 5 microns, and thicker and shorter filaments (28.5 +/- 1.6 nm average). Examination of the molecular organization of CkII under different catalytic conditions reveal that the ring-like structure is the favored conformation adopted by the enzyme in the presence of saturating concentrations of substrates and cofactors. During catalysis, well-known cofactors like MgCl2 or spermine are the main factors governing the stabilization of the active ring-like structure. In contrast, inhibitory high salt concentrations promote the dissociation of the active ring-like structure into protomers. Such observations suggest a strong correlation between the ring-like conformation of the enzyme and optimal specific activity. Thus, CkII may be considered as an associating-dissociating enzyme, and this remarkable property supports the hypothesis of a cooperative and allosteric regulation of the kinase in response to appropriate regulatory ligands possibly taking place in intact cells (Valero, 1995).

The noncatalytic beta-subunit of protein kinase CkII has been shown to display various and in some respects antagonistic effects on the catalytic alpha-subunit. The ability of peptides encompassing the N- and C-terminal regions of the beta-subunit [beta(1-77) and beta(155-215)] to mimic the functions of the whole-length beta-subunit were examined. Peptide beta(155-215) possesses only the positive features of the beta-subunit in that it prevents thermal inactivation and stimulates basal activity of the alpha-subunit, while it does not inhibit but rather stimulates calmodulin phosphorylation. In sharp contrast, peptide beta(1-77) neither protects the alpha-subunit nor stimulates its basal activity, while acting as a powerful and specific inhibitor of calmodulin phosphorylation. Peptide beta(155-215), but not peptide beta(1-77), stably interacts with alpha-subunit and also displays remarkable self-associating properties. A shorter derivative of beta(155-215), beta(170-215), displaying weaker stimulatory properties and fails to stably interact with the alpha-subunit and to give rise to dimeric/multimeric forms. These data show that the elements responsible for the negative regulation are concentrated in the N-terminal moiety of the beta-subunit, whereas the C-terminal region retains the beneficial properties of the beta-subunit and is capable of self-association and binding of the alpha-subunit. Residues between 155 and 170 are necessary for the latter functions (Marin, 1997).

Casein kinase II is one of only a few protein kinases that effectively utilize ATP and GTP in the phosphotransferase reaction. Two residues conserved in the ATP binding domain of other protein kinases are unique to the catalytic (alpha) subunit of Casein kinase II. Val-66 is present in subdomain II and Trp-176 in subdomain VII, while greater than 95% of the other protein kinases contain alanine and phenylalanine, respectively, in their subdomains. The residues in the alpha subunit of Casein kinase II were changed to the conserved residues via single and double mutations by site-directed mutagenesis. These mutations enhance the utilization of ATP over GTP by altering the K(m) values of the alpha subunit for ATP and GTP. Following reconstitution of the catalytic subunit with the regulatory (beta) subunit, both the K(m) and Vmax values of the reconstituted alpha 2 beta 2 holoenzyme are altered. Interestingly, the mutations also reduce or eliminate the 4- to 5-fold increase in catalytic activity observed with the holoenzyme over that of the alpha subunit alone. This is due to changes in secondary structure of the holoenzyme as shown by UV circular dichroism spectroscopy. Taken together, the data indicate that utilization of both ATP and GTP can be directly correlated with stimulation of catalytic activity by the regulatory subunit and suggest a co-evolution of these separate functions (Jakobi, 1995a).

Two residues unique to the catalytic subunit of Casein kinase II, Val66 and Trp176, were mutated to the conserved residues, Ala66 and Phe176, respectively. In this study, the mutations have been shown to affect the catalytic activity of the reconstituted holoenzyme by changing both Km and Vmax values. The Vmax for ATP is reduced by the mutation of Trp176 to phenylalanine, but no change is observed with GTP. The Val66 to alanine and Val66/Trp176 to alanine/phenylalanine mutations reduce the Vmax values for ATP and GTP to levels comparable to those of the catalytic subunits alone, indicating that changes in the stimulation of activity by the beta subunit are due to changes in Vmax. Structural studies using ultraviolet CD spectroscopy show that changes in stimulation of Vmax by the beta subunit are correlated with changes in the secondary structure; the extent of these changes is reduced by both mutations. Correlation of changes in secondary structure and stimulation of activity by the beta subunit indicate that the formation of the wild-type holoenzyme causes conformational changes in the active site, leading to an increased rate of reaction. As shown by the mutations, Val66 and Trp176 are involved both in the conformational changes and in the selectivity of ATP and GTP (Jakobi, 1995b).


Casein kinase II: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 2 August 97  

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