Drosophila Casein Kinase Research Articles

Transcriptomic Analysis of Drosophila Mushroom Body Neurons Lacking Amyloid-β Precursor-Like Protein Activity.

The amyloid-β protein precursor (AβPP) is subjected to sequential intramembrane proteolysis by α-, β-, andγ-secretases, producing secreted amyloid-β (Aβ) peptides and a cytoplasmically released AβPP 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, we performed a transcriptomic identification of the Drosophila genome that is regulated by the fly AβPP orthologue in fly mushroom body neurons, which control learning- and memory-based behaviors. We find significant changes 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 our Drosophila microarray analyses that have also emerged as putative AβPP targets in similar mammalian transcriptomic studies. Our 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. Our 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.

Drosophila casein kinase I alpha regulates homolog pairing and genome organization by modulating condensin II subunit Cap-H2 levels.

The spatial organization of chromosomes within interphase nuclei is important for gene expression and epigenetic inheritance. Although the extent of physical interaction between chromosomes and their degree of compaction varies during development and between different cell-types, it is unclear how regulation of chromosome interactions and compaction relate to spatial organization of genomes. Drosophila is an excellent model system for studying chromosomal interactions including homolog pairing. Recent work has shown that condensin II governs both interphase chromosome compaction and homolog pairing and condensin II activity is controlled by the turnover of its regulatory subunit Cap-H2. Specifically, Cap-H2 is a target of the SCFSlimb E3 ubiquitin-ligase which down-regulates Cap-H2 in order to maintain homologous chromosome pairing, chromosome length and proper nuclear organization. Here, we identify Casein Kinase I alpha (CK1α) as an additional negative-regulator of Cap-H2. CK1α-depletion stabilizes Cap-H2 protein and results in an accumulation of Cap-H2 on chromosomes. Similar to Slimb mutation, CK1α depletion in cultured cells, larval salivary gland, and nurse cells results in several condensin II-dependent phenotypes including dispersal of centromeres, interphase chromosome compaction, and chromosome unpairing. Moreover, CK1α loss-of-function mutations dominantly suppress condensin II mutant phenotypes in vivo. Thus, CK1α facilitates Cap-H2 destruction and modulates nuclear organization by attenuating chromatin localized Cap-H2 protein.

Signal transduction by the Fat cytoplasmic domain

The large atypical cadherin Fat is a receptor for both Hippo and planar cell polarity (PCP) pathways. Here we investigate the molecular basis for signal transduction downstream of Fat by creating targeted alterations within a genomic construct that contains the entire fat locus, and by monitoring and manipulating the membrane localization of the Fat pathway component Dachs. We establish that the human Fat homolog FAT4 lacks the ability to transduce Hippo signaling in Drosophila, but can transduce Drosophila PCP signaling. Targeted deletion of conserved motifs identifies a four amino acid C-terminal motif that is essential for aspects of Fat-mediated PCP, and other internal motifs that contribute to Fat-Hippo signaling. Fat-Hippo signaling requires the Drosophila Casein kinase 1ϵ encoded by discs overgrown (Dco), and we characterize candidate Dco phosphorylation sites in the Fat intracellular domain (ICD), the mutation of which impairs Fat-Hippo signaling. Through characterization of Dachs localization and directed membrane targeting of Dachs, we show that localization of Dachs influences both the Hippo and PCP pathways. Our results identify a conservation of Fat-PCP signaling mechanisms, establish distinct functions for different regions of the Fat ICD, support the correlation of Fat ICD phosphorylation with Fat-Hippo signaling, and confirm the importance of Dachs membrane localization to downstream signaling pathways.

Signal transduction by the Fat cytoplasmic domain

The large atypical cadherin Fat is a receptor for both Hippo and planar cell polarity (PCP) pathways. Here we investigate the molecular basis for signal transduction downstream of Fat by creating targeted alterations within a genomic construct that contains the entire fat locus, and by monitoring and manipulating the membrane localization of the Fat pathway component Dachs. We establish that the human Fat homolog FAT4 lacks the ability to transduce Hippo signaling in Drosophila, but can transduce Drosophila PCP signaling. Targeted deletion of conserved motifs identifies a four amino acid C-terminal motif that is essential for aspects of Fat-mediated PCP, and other internal motifs that contribute to Fat-Hippo signaling. Fat-Hippo signaling requires the Drosophila Casein kinase 1ε encoded by discs overgrown (Dco), and we characterize candidate Dco phosphorylation sites in the Fat intracellular domain (ICD), the mutation of which impairs Fat-Hippo signaling. Through characterization of Dachs localization and directed membrane targeting of Dachs, we show that localization of Dachs influences both the Hippo and PCP pathways. Our results identify a conservation of Fat-PCP signaling mechanisms, establish distinct functions for different regions of the Fat ICD, support the correlation of Fat ICD phosphorylation with Fat-Hippo signaling, and confirm the importance of Dachs membrane localization to downstream signaling pathways.

The Drosophila casein kinase Iε/δ Discs overgrown promotes cell survival via activation of DIAP1 expression

The proper number of cells in developing tissues is achieved by coordinating cell division with apoptosis. In Drosophila, the adult wing is derived from wing imaginal discs, which undergo a period of growth and proliferation during larval stages without much programmed cell death. In this report, we demonstrate that the Drosophila casein kinase Iε/δ, known as Discs overgrown (Dco), is required for maintaining this low level of apoptosis. Expression of dco can suppress the apoptotic activity of Head involution defective (Hid) in the developing eye. Loss of dco in the wing disc results in a dramatic reduction in expression of the caspase inhibitor DIAP1 and a concomitant activation of caspases. The regulation of DIAP1 by Dco occurs by a post-transcriptional mechanism that is independent of hid. Mutant clones of dco are considerably smaller than controls even when apoptosis is inhibited, suggesting that Dco promotes cell division/growth in addition to its role in cell survival. The dco phenotype cannot be explained by defects Wingless (Wg) signaling. We propose that Dco coordinates tissue size by stimulating cell division/growth and blocking apoptosis via activation of DIAP1 expression.

Protein kinase CK2: a new view of an old molecular complex.

Protein kinase CK2 (formerly known as casein kinase II) has been viewed traditionally as a stable heterotetrameric complex, but new analytical techniques are bringing a different picture into focus. The transient nature of this complex has been highlighted by the elucidation of its structure. Furthermore, analysis of the spatiotemporal organization of individual CK2 subunits in living cells has shown that they are dynamic and that they integrate into different multimolecular assemblies. These new studies give an additional dimension to the challenge of determining the cellular regulation of this protein kinase.

A role for casein kinase 2alpha in the Drosophila circadian clock.

Circadian clocks drive rhythmic behaviour in animals and are regulated by transcriptional feedback loops. For example, the Drosophila proteins Clock (Clk) and Cycle (Cyc) activate transcription of period (per) and timeless (tim). Per and Tim then associate, translocate to the nucleus, and repress the activity of Clk and Cyc. However, post-translational modifications are also critical to proper timing. Per and Tim undergo rhythmic changes in phosphorylation, and evidence supports roles for two kinases in this process: Doubletime (Dbt) phosphorylates Per, whereas Shaggy (Sgg) phosphorylates Tim. Yet Sgg and Dbt often require a phosphoserine in their target site, and analysis of Per phosphorylation in dbt mutants suggests a role for other kinases. Here we show that the catalytic subunit of Drosophila casein kinase 2 (CK2alpha) is expressed predominantly in the cytoplasm of key circadian pacemaker neurons. CK2alpha mutant flies show lengthened circadian period, decreased CK2 activity, and delayed nuclear entry of Per. These effects are probably direct, as CK2alpha specifically phosphorylates Per in vitro. We propose that CK2 is an evolutionary link between the divergent circadian systems of animals, plants and fungi.

Casein kinase II β-subunit inhibits the activity of the catalytic α-subunit in the absence of salt

Casein kinase II (CKII) has a subunit structure α 2 β 2 where α is the catalytic subunit. Recombinant Drosophila casein kinase 11 α-subunit expressed in insect cells is inhibited by NaCl, thermally labile and inactivated by binding to plastic. In the presence of detergent (Tween 80) recombinant α-subunit has a k cat of 249 −1 ( K m 170 μM) for the peptide substrate RRRDDDSDDDNH 2, compared to recombinant Drosophila CKII with a k cat of 71 min −1 (per mol α) ( K m 42 μM) and bovine CKII with a k cat of 123 min −1 (per mol α) ( K m 45 μM) when measured in the absence of NaCl. The k cat values of bovine CKII and recombinant Drosophila CKII measured in the presence of 150 mM NaCl were 429 min −1 (per mol α) ( K m 82 μM) and 204 min −1 (per mol α) ( K m 51 μM), respectively. Since the k cat for the Drosophila α-subunit is approx. 3-fold greater than the Drosophila CKII measured in the absence of added salt these results indicate that the β-subunit acts primarily as an inhibitory subunit.

Purification and characterization of a casein kinase 2-type protein kinase from pea nuclei.

Almost all the polyamine-stimulated protein kinase activity associated with the chromatin fraction of nuclei purified from etiolated pea (Pisum sativum L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.35 molar NaCl. This protein kinase can be further purified over 2000-fold by salt fractionation and anion-exchange and casein-agarose column chromatography, after which it is more than 90% pure. The purified kinase has a specific activity of about 650 nanomoles per minute per milligram protein in the absence of polyamines, with either ATP or GTP as phosphoryl donor. Spermidine can stimulate its activity fourfold, with half-maximal activation at about 2 millimolar. Spermine and putrescine also stimulate activity, although somewhat less effectively. This kinase has a tetrameric alpha 2 beta 2 structure with a native molecular weight of 130,000, and subunit molecular weights of 36,000 for the catalytic subunit (alpha) and 29,000 for the regulatory subunit (beta). In western blot analyses, only the alpha subunit reacts strongly with polyclonal antibodies to a Drosophila casein kinase II. The pea kinase can use casein and phosvitin as artificial substrates, phosphorylating both the serine and threonine residues of casein. It has a pH optimum near 8.0, a Vmax of 1.5 micromoles per minute per milligram protein, and a Km for ATP of approximately 75 micromolar. Its activity can be almost completely inhibited by heparin at 5 micrograms per milliliter, but is relatively insensitive to concentrations of staurosporine, K252a, and chlorpromazine that strongly antagonize Ca(2+) -regulated protein kinases. These results are discussed in relation to recent findings that casein kinase 2-type kinases may phosphorylate trans-acting factors that bind to light-regulated promoters in plants.

Expression and purification of the α and β subunits of Drosophila casein kinase II using a baculovirus vector

Two recombinant baculoviruses that express the alpha and beta subunits of Drosophila melanogaster casein kinase II, respectively, have been constructed. The expressed proteins are similar to the authentic Drosophila subunits in size and are recognized by antisera raised against the Drosophila holoenzyme. Extracts derived from cells infected with the alpha subunit-expressing virus display elevated casein kinase II activity in vitro. This activity is markedly enhanced in extracts of cells infected with both viruses, or when alpha and beta subunit-containing extracts are mixed in vitro following lysis. Recombinant holoenzyme and the alpha subunit were purified to near homogeneity using phosphocellulose column chromatography. The specific activity of the purified recombinant holoenzyme was very similar to that of the native enzyme, and was fivefold higher than that of the purified free alpha subunit. The Stokes radius of the recombinant holoenzyme was estimated to be 50 A, a value similar to that reported for the native enzyme, whereas the alpha subunit demonstrated a Stokes radius of 26.5 A. Studies using sucrose density gradient centrifugation showed that, under conditions of high ionic strength, the quaternary structure of the purified holoenzyme was tetrameric (like the native enzyme), whereas the structure of the alpha subunit was monomeric. At lower ionic strength the recombinant holoenzyme had a significantly higher sedimentation coefficient, characteristic of the formation of filaments found for the native enzyme. Interestingly, the purified catalytic subunit also displayed a higher S value under conditions of low ionic strength, revealing the formation of alpha subunit aggregates.

Isolation, sequencing, and disruption of the yeast CKA2 gene: casein kinase II is essential for viability in Saccharomyces cerevisiae.

Casein kinase II of Saccharomyces cerevisiae contains two distinct catalytic subunits, alpha and alpha', which are encoded by the CKA1 and CKA2 genes, respectively. Null mutations in the CKA1 gene do not confer a detectable phenotype (J. L.-P. Chen-Wu, R. Padmanabha, and C. V. C. Glover, Mol. Cell. Biol. 8:4981-4990, 1988), presumably because of the presence of the CKA2 gene. We report here the cloning, sequencing, and disruption of the CKA2 gene. The alpha' subunit encoded by the CKA2 gene is 60% identical to the CKA1-encoded alpha subunit and 55% identical to the Drosophila alpha subunit (A. Saxena, R. Padmanabha, and C. V. C. Glover, Mol. Cell. Biol. 7:3409-3417, 1987). Deletions of the CKA2 gene were constructed by gene replacement techniques. Haploid cells in which the CKA2 gene alone is disrupted show no detectable phenotype, but haploid cells carrying disruptions in both the CKA1 and CKA2 genes are inviable. Cells in which casein kinase II activity is depleted increase substantially in size prior to growth arrest, and a significant fraction of the arrested cells exhibit a pseudomycelial morphology. Disruption of the activity also results in flocculation. Yeast strains lacking both endogenous catalytic subunit genes can be rescued by expression of the alpha and beta subunits of Drosophila casein kinase II or by expression of the Drosophila alpha subunit alone, suggesting that casein kinase II function has been conserved through evolution.

Isolation, Sequencing, and Disruption of the Yeast CKA2 Gene: Casein Kinase II Is Essential for Viability in Saccharomyces cerevisiae

Casein kinase II of Saccharomyces cerevisiae contains two distinct catalytic subunits, alpha and alpha', which are encoded by the CKA1 and CKA2 genes, respectively. Null mutations in the CKA1 gene do not confer a detectable phenotype (J. L.-P. Chen-Wu, R. Padmanabha, and C. V. C. Glover, Mol. Cell. Biol. 8:4981-4990, 1988), presumably because of the presence of the CKA2 gene. We report here the cloning, sequencing, and disruption of the CKA2 gene. The alpha' subunit encoded by the CKA2 gene is 60% identical to the CKA1-encoded alpha subunit and 55% identical to the Drosophila alpha subunit (A. Saxena, R. Padmanabha, and C. V. C. Glover, Mol. Cell. Biol. 7:3409-3417, 1987). Deletions of the CKA2 gene were constructed by gene replacement techniques. Haploid cells in which the CKA2 gene alone is disrupted show no detectable phenotype, but haploid cells carrying disruptions in both the CKA1 and CKA2 genes are inviable. Cells in which casein kinase II activity is depleted increase substantially in size prior to growth arrest, and a significant fraction of the arrested cells exhibit a pseudomycelial morphology. Disruption of the activity also results in flocculation. Yeast strains lacking both endogenous catalytic subunit genes can be rescued by expression of the alpha and beta subunits of Drosophila casein kinase II or by expression of the Drosophila alpha subunit alone, suggesting that casein kinase II function has been conserved through evolution.

Molecular cloning of the human casein kinase II alpha subunit.

A human cDNA encoding the alpha subunit of casein kinase II and a partial cDNA encoding the rat homologue were isolated by using a Drosophila casein kinase II cDNA probe. The 2.2-kb human cDNA contains a 1.2-kb open reading frame, 150 nucleotides of 5' leader, and 850 nucleotides of 3' noncoding region. Except for the first 7 deduced amino acids that are missing in the rat cDNA, the 328 amino acids beginning with the amino terminus are identical between human and rat. The Drosophila enzyme sequence is 90% identical with the human casein kinase II sequence, and there is only a single amino acid difference between the published partial bovine sequence and the human sequence. In addition, the C-terminus of the human cDNA has an extra 53 amino acids not present in Drosophila. Northern analysis of rat and human RNA showed predominant bands of 5.5, 3.1, and 1.8 kb. In rat tissues, brain and spleen had the highest levels of casein kinase II alpha subunit specific RNA, while skeletal muscle showed the lowest. Southern analysis of human cultured cell and tissue genomic DNA using the full-length cDNA probe revealed two bands with restriction enzymes that have no recognition sites within the cDNA and three to six bands with enzymes having single internal sites. These results are consistent with the possibility that two genes encode the alpha subunits.

Casein kinase II of yeast contains two distinct alpha polypeptides and an unusually large beta subunit.

Casein kinase II of yeast has been purified to near homogeneity by a procedure which includes affinity chromatography on heparin-agarose. The purified enzyme consists of four polypeptides with molecular weights of 42,000, 41,000, 35,000, and 32,000. The 42,000- and 35,000-Da polypeptides are immunologically related and exhibit cross-reactivity with the alpha subunits of calf and Drosophila casein kinase II. Amino-terminal sequencing reveals that the two subunits are distinct but homologous polypeptides and that both sequences share 40-50% homology with the Drosophila alpha subunit. These results demonstrate that yeast contains two distinct alpha subunits which must be encoded by separate genes. The 41,000- and 32,000-Da polypeptides both incorporate phosphate during autophosphorylation, a characteristic of the beta subunit in all type II casein kinases studied to date. The 41,000-Da subunit also exhibits immunological cross-reactivity with the beta subunit of Drosophila casein kinase II. These results identify the 41,000-Da polypeptide as an unusually large beta subunit. The possibility that the 32,000-Da polypeptide may be a beta' subunit is currently under investigation. The interpretation of the subunit structure of yeast casein kinase II reported here differs significantly from previous reports (Rigobello, M. P., Jori, E., Carignani, G., and Pinna, L. A. (1982) FEBS Lett. 144, 354-358; Kudlicki, W. N., Szyszka, R., and Gasior, E. (1984) Biochim. Biophys. Acta 784, 102-107).