The Drosophila melanogaster DmCK2β transcription unit encodes for functionally non-redundant protein isoforms

Gene 374 (2006) 142 – 152 www.elsevier.com/locate/gene

The Drosophila melanogaster DmCK2β transcription unit encodes for functionally non-redundant protein isoforms Eike Jauch, Heike Wecklein, Felix Stark, Mandy Jauch, Thomas Raabe ⁎ University of Wuerzburg, Institut fuer Medizinische Strahlenkunde und Zellforschung, Versbacherstrasse 5, 97078 Wuerzburg, Germany Received 4 November 2005; received in revised form 23 January 2006; accepted 27 January 2006 Available online 13 March 2006

Abstract Genes encoding for the two evolutionary highly conserved subunits of a heterotetrameric protein kinase CK2 holoenzyme are present in all examined eukaryotic genomes. Depending on the organism, multiple transcription units encoding for a catalytically active CK2α subunit and/or a regulatory CK2β subunit may exist. The phosphotransferase activity of members of the protein kinase CK2α family is thought to be independent of second messengers but is modulated by interaction with CK2β-like proteins. In the genome of Drosophila melanogaster, one gene encoding for a CK2α subunit and three genes encoding for CK2β-like proteins are present. The X-linked DmCK2β transcription unit encodes for several CK2β protein isoforms due to alternative splicing of its primary transcript. We addressed the question whether CK2β-like proteins are redundant in function. Our in vivo experiments show that variations of the very C-terminal tail of CK2β isoforms encoded by the X-linked DmCK2β transcription unit influence their functional properties. In addition, we find that CK2β-like proteins encoded by the autosomal D. melanogaster genes CK2βtes and CK2β′ cannot fully substitute for a loss of CK2β isoforms encoded by DmCK2β. © 2006 Elsevier B.V. All rights reserved. Keywords: Protein kinase CK2; CK2β isoforms; CK2β′; CK2βtes; Mushroom bodies undersized; Andante

1. Introduction Protein kinase CK2β-like proteins have been isolated and characterized from a large number of organisms and are most likely encoded by all eukaryotic genomes. No proteins homologous to members of the CK2β protein family are present in prokaryotes. For a long time, CK2β function was only defined by its interaction with the serine/threonine protein kinase CK2α in a heterotetrameric holoenzyme (α2β2): CK2β modulates

Abbreviations: a.e., after eclosion; cDNA, DNA complementary to RNA; CHK1, checkpoint kinase 1; CHK2, checkpoint kinase 2; CK2, casein kinase 2; Dm, Drosophila melanogaster; ds, double-stranded; GT, gene trap; h, hour; HA, hemagglutinin; HEK, human embryonic kidney; mbu, mushroom bodies undersized; mRNA, messenger RNA; PAGE, polyacrylamid gel electrophoresis; PCR, polymerase chain reaction; P[hsP-Gal4 ], P[heat-shock promotor-Gal4]; P[tubP-Gal4], P[tubulin promotor-Gal4]; ry, rosy; SAP, shrimps alkaline phosphatase; SDS, sodium dodecyl sulfate; Ste, Stellate; Su(Ste), Suppressor-ofStellate; UAS, upstream activator sequence; V, volume; w, white; wt, wild-type; y, yellow. ⁎ Corresponding author. Tel.: +49 931 20145841; fax: +49 931 20145835. E-mail address: [email protected] (T. Raabe). 0378-1119/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2006.01.026

CK2α substrate specificity, its phosphotransferase activity and confers stability to the holoenzyme. More recently, additional functions of CK2β have emerged: an association-dependent inhibitory effect of Xenopus laevis CK2β on the c-MOS serine/ threonine protein kinase, a stimulatory effect of Mus musculus CK2β on A-RAF kinase activity, an activating effect of Homo sapiens CK2β on checkpoint kinase CHK1 and a reduction of checkpoint kinase CHK2 phosphotransferase activity by CK2β were documented (Bibby and Litchfield, 2005; BjorlingPoulsen et al., 2005). In Caenorhabditis elegans and M. musculus one CK2β-encoding gene was identified. In both organisms, CK2β function is essential for viability (Fraser et al., 2000; Buchou et al., 2003; Blond et al., 2005). Multiple genes encoding for CK2β-related proteins have so far been identified in organisms as diverse as Saccharomyces cerevisiae (Bidwai et al., 1995), Arabidopsis thaliana (Klimczak et al., 1995) and Drosophila melanogaster (Bidwai et al., 1999). The X-chromosomal DmCK2β gene of Drosophila can be mutated to lethality. At least three protein isoforms are translated from alternatively spliced mRNAs (Jauch et al., 2002). Functional analysis established a role of DmCK2β in the development of the mushroom bodies, a paired central brain

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neuropil involved in olfactory learning and memory processes (Jauch et al., 2002), and in the circadian oscillator (Akten et al., 2003). A DmCK2β allele characterized by two P-element insertions into the first non-coding DmCK2β exon is called mushroom bodies undersized (DmCK2βmbuP1). It causes a severe loss of intrinsic mushroom body neurons (Kenyon cells) that results in a reduced size of all mushroom body neuropil substructures. However, it is not known whether the phenotype is caused by a defect in proliferation of the neuronal stem cells (neuroblasts) or by a defect in Kenyon cell differentiation or survival (Jauch et al., 2002). Another DmCK2β allele named Andante (DmCK2βAndante ) lengthens circadian period for both locomotor activity and adult eclosion rhythms. In Andante flies, the clock proteins PERIOD and TIMELESS accumulate to abnormally high levels and the nuclear translocation of both proteins is delayed (Akten et al., 2003). Conversely, wild-type flies carrying two extra copies of a genomic DmCK2β transgene show a short-period behaviour (Akten et al., 2003). As mutations in the D. melanogaster CK2α (Lin et al., 2002) result in a defective circadian oscillator, CK2β is likely to fulfil its circadian clock function by regulating CK2α phosphotransferase activity. A duplicated and translocated CK2β gene derived from the X-chromosomal DmCK2β transcription unit is considered to be the ancestor of the CK2βtes gene locus on the second chromosome that subsequently gave rise to the paralogous X- and Y-linked Stellate (Ste) and Suppressor-of-Stellate (Su(Ste)) clusters (Kalmykova et al., 1997). The removal of the Su(Ste) repeats, for example in XO males, causes Ste hyperexpression in the testis that results in sterility due to the formation of needle- or star-shaped STELLATE-containing proteinaceous crystals in primary spermatocytes (Bozzetti et al., 1995). Stellate repeats are silenced by dsRNA generated by the transcription of both strands of Su(Ste) repeats (Aravin et al., 2001). The 2nd chromosomal CK2β′ gene encodes for a CK2β-like protein that is more similar to CK2β than CK2βtes (Bidwai et al., 1999), implicating that during evolution of D. melanogaster, a second DmCK2β duplication and translocation took place. The CK2β′ transcription units seems to be preferentially transcribed in adult Drosophila in a testis-specific manner (Kalmykova et al., 2002). CK2βtes is also a male-specific gene (Karandikar et al., 2003), but its expression occurs across a wider developmental window than that of CK2β′. However, nothing is known about the in vivo function of either gene. So far, only few studies addressed the question of functional specialization of CK2β-related proteins. In S. cerevisiae, two transcription units encoding for CK2β-like proteins were identified, CKB1 and CKB2. Deletion of either or both CKB genes results in viable haploid and diploid strains that show a specific sensitivity to high concentrations of Na+ or Li+ ions. Their gene products function in a pathway regulating ion homeostasis. This salt-sensitive phenotype was used as an assay to explore the functional relationship between CKB1 and CKB2. Overexpression of CKB2 yielded in a partial suppression of the NaCl sensitivity of a ckb1 strain, but the NaCl sensitivity of a ckb2 strain could not be suppressed by overexpression of CKB1.

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Therefore, the two S. cerevisiae CK2β proteins that are identical in 45% of their amino acids are not identical in function. Overexpression of either yeast CK2β subunit in a wild-type strain did not result in any detectable phenotype (Bidwai et al., 1995). All known CK2β isoforms encoded by alternatively spliced transcripts of the X-linked D. melanogaster DmCK2β gene differ in their very C-terminal tail but have the typical structural properties of a regulatory CK2 subunit: In addition to Nterminal phosphoacceptor sites that are phosphorylated by the CK2 α2β2 holoenzyme, both an acidic loop that binds polyamines and a zinc finger essential for CK2β subunit dimerization are present (Jauch et al., 2002). Does this high degree of structural identity imply that all CK2β isoforms are functionally redundant? If so, we assumed that they should produce the same phenotype upon ectopic expression in a wild-type background and that they should equally be able to rescue the lethality associated with a DmCK2β null allele. We show that in these genetic test systems, only some CK2β isoforms behave identical, an observation that can be only explained by different functional properties of the very C-terminal tail of the CK2β isoforms. Like the different CK2β isoforms, the proteins encoded by CK2β′ and CK2βtes possess a zinc finger domain. Both CK2β′ and CK2βtes are considered to be tissue-specific regulatory subunits of CK2 (Bidwai et al., 1999; Kalmykova et al., 2002; Karandikar et al., 2003). Do these proteins have the potential to complement for a loss of DmCK2β function? We find that, in contrast to some CK2β isoforms, neither CK2β′ nor CK2βtes possess the functional properties to compensate for the lack of DmCK2β gene products. 2. Materials and methods 2.1. Fly culture and strains Flies were reared in a 12/12 h light/dark cycle at 60% relative humidity and 25 °C on a cornmeal/molasses medium supplemented with yeast. DmCK2βmbuP1, DmCK2βmbuΔA26-2L and the transgenic lines P[UAS:CK2β-VIIa] (corresponding to P[UAS:CK2βIe-VIIa]) and P[CK2β-gDNA] were described previously (Jauch et al., 2002). DmCK2βmbuΔA, DmCK2βP[GT1]Δ6, DmCK2βP[GT1]Δ8 and transgenic lines P[UAS:CK2β′], P[UAS: CK2βtes], P[UAS:CK2β-VIIb], P[UAS:CK2β-VIIc], P[UAS: CK2β-VIId-VI], P[UAS:CK2β-VIId] and P[UAS:CK2βM166I] were generated in this work. DmCK2βAndante was a kind gift of Bikem Akten (Akten et al., 2003). DmCK2βP[GT1] and the transgenic strains P[tubP-Gal4] and P[hsP-Gal4] were obtained from the Bloomington stock center. The wild-type Berlin stock was obtained from the Institute of Genetics and Neurobiology, Wuerzburg, Germany. 2.2. Genetics, histology and neuropil volume determination Excision alleles of DmCK2βmbuP1 and DmCK2βP[GT1] were generated using the P[ry+,Δ2–3] transposase. Recombination of the y− marker to the DmCK2βmbuΔA26-2L , w− chromosome was

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performed following standard genetics. Rescue experiments were done by crossing DmCK2βmbuΔA26-2L , w− , y−/FM7a; P[tubP-Gal4]/+ virgins with w−/Y; P[UAS:DmCK2β cDNA]/ balancer males. Rescued males were detected by the y− marker of the DmCK2βmbuΔA26-2L, w−, y− chromosome. For ubiquitous overexpression, w−/w−; P[UAS:DmCK2β cDNA]/balancer virgins were crossed with FM7a/Y; P[tubP-Gal4]/balancer males. Progeny male flies overexpressing the transgene were detected by the absence of balancer chromosomes. The volumes of mushroom body calyces and fanshape bodies of flies were calculated by planimetric measurements from autofluorescent frontal 7μm paraffin sections (Heisenberg et al., 1995). Individual normalized calyx volumes were determined by calculation of [V(left calyx) + V(right calyx)] / 2 * V (fanshape body) values. To show differences in the normalized calyx volume of experimental groups, mean normalized calyx volume values were compared. 2.3. Molecular biology and generation of transgenic lines Complementary DNA clones GH28737, AT31207, RE31047, AT15417, AT09746 and AT25555 were generated by the Berkeley Drosophila Genome Project (Rubin et al., 2000) and obtained from ResGen™ Invitrogen Corporation. bs09g07 was generated by Brian Oliver and obtained from Medical Research Council gene service. The QuickChange™ Site-Directed Mutagenesis kit (Stratagene) was used for in vitro mutagenesis. The oligonucleotides 5′-GCCACTACGAGCGTTTACAACG GG-3′ and 5′-CCCGTTGTAAACGCTCGTAGT GGC-3′ were used to delete exon VIIa sequences on cDNA clone RE31047 to generate a cDNA encoding for CK2β-VIIb (RE31047VIIb) according to Bidwai et al. (2000) and the oligonucleotides 5′-CGGCACTGGATTTCCACACATACTCTTCATGGTG CATCCCG-3′ and 5′-CGGGATGCACCATGAAGAGTATGT GTGGAAATCCAGTGCCG-3′ were used to introduce the amino acid substitution M166I in DmA15-12zap which encodes isoform CK2β-VIIa (Jauch et al., 2002). RE31047VIIb encoding for CK2β-VIIb and cDNAs GH28737, AT31207, bs09g07 encoding for CK2β-VIIc, CK2β-VIId and CK2β-VIId-VI, respectively, were cloned as EcoRI-XbaI, AT09746 encoding for CK2β′ and AT25555 encoding for CK2βtes were cloned as NotI-Asp718I fragments and the mutated DmA15-12zap cDNA was cloned as a EcoRI-XhoI fragment into the pP[UAST] transformation vector (Brand and Perrimon, 1993). Transgenic lines were generated by injecting Qiagen-purified plasmid DNA (pP[UAST]/pUChsπΔ2–3) into w1118 embryos. Plasmid vectors for transient expression of N-terminal tagged CK2α and CK2β isoforms in HEK293 cells were constructed on the basis of modified pcDNA3 vectors that either contain a 1×HA (pcDNA3-HA1, Invitrogen) or a 6×MYC (pcDNA3MYC) epitope sequence. CK2β open reading frames of cDNA clones RE31047, RE31047 VIIb , GH28737, AT31207 and bs09g07 were amplified by linker-PCR and cloned as BamH1EcoRI fragments into pcDNA3-HA1. The MYC-tagged CK2α construct was generated by cloning the open reading frame of AT15417 amplified by linker-PCR as a BamHI-XhoI fragment into pcDNA3-MYC.

2.4. Cell culture, immunoprecipitation, phosphatase treatment and Western blot analysis Human embryonic kidney (HEK) 293 cells were grown at 37°C in 5% CO2 and cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) containing 10% fetal calf serum. For transient expression of proteins, 6 × 105 HEK293 cells were seeded in six-well plates and transfected 24h later with the indicated DNA constructs using the PolyFect reagent (Qiagen). Five hours prior to harvesting, the proteasome inhibitor MG132 (Calbiochem) was added to the medium at a final concentration of 20 μM. Cells were harvested 48 h after transfection in PBS and lysed at 4 °C for 40 min in 320 μl lysis buffer (25 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 2 mM DTT, 10% glycerol, 0.1% Nonidet NP-40 supplemented with 5μg/mL Antipain, 10 μg/mL Aprotinin, 0.5μg/mL Leupeptin, 0.7μg/mL Pepstatin A and 200 μg/mL phenylmethylsulfonyl fluoride). 500μg of protein lysate was supplemented with lysis buffer to a final volume of 500 μl and incubated for 30 min at 4°C with 0.8μg monoclonal anti-HA (12CA5, Roche) antibody. Protein complexes were precipitated over night at 4 °C using protein-Gagarose (Roche). The bead fraction was washed four times with lysis buffer. To control expression of proteins prior to immunoprecipitation, 50 μg protein lysate was used for Western blot analysis. The proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane and probed with polyclonal rabbit anti-MYC (A-14, 1 : 1000, Santa Cruz) and monoclonal mouse anti-HA (12CA5, 1 : 1000, Roche) antibodies. To induce expression of P[UAS:CK2β]-transgenes by the P[heat shock-promotor (hsP)-Gal4] driver, flies were given a 37°C heat shock for 1 h and were subsequently kept at room temperature for additional 5h prior to homogenization, SDSPAGE and Western blot analysis. To test for phosphorylation of CK2β isoforms in vivo, flies were homogenized in 10μl phosphatase buffer per fly (0.05 M Tris–HCl, 5 mM MgCl2, pH 8.5 supplemented with a protease inhibitor cocktail (complete protein inhibitor cocktail tablets, Roche) at a final concentration of 2×). Homogenates were centrifuged at 13,000 rpm at 4 °C. Shrimps alkaline phosphatase (SAP), or — for control experiments — the equivalent volume of storage buffer (25 mM Tris–HCl, 1 mM MgCl2, 0.1mM ZnCl2, 50% glycerol (v/v), pH 7.6), was added to the supernatant at a final concentration of 0.2 U/μl. After 30 min of incubation at 30°C, homogenates of 2 virgin and 3 male flies were separated by SDS-PAGE. A peptide (PEDELEDNPLQSDMT) corresponding to amino acids 58–72 of all CK2β isoforms was synthesized and used to immunize rabbits by a commercial supplier. To analyse the CK2β isoform expression pattern in wild-type and mutant flies, protein lysates of three male flies were analysed by probing blots with the affinity-purified anti-CK2β serum (1 : 100). The monoclonal mouse antibody nc46 (1 : 1000) was used to detect SAP47 as a loading control (Reichmuth et al., 1995). Primary antibodies were detected using HRP-coupled secondary antibodies and the ECL detection reagent (Amersham Biosciences).

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3. Results 3.1. The Drosophila DmCK2β transcription unit encodes for five protein isoforms We have previously reported that the DmCK2β gene encodes for at least three protein isoforms that are translated from alternatively spliced mRNAs (Jauch et al., 2002). The lethality of a DmCK2β null allele (Fig. 1A, CK2βmbuΔA26-2L ) can be rescued by a genomic DmCK2β transgene (Fig. 1A, P[CK2β-gDNA]) that does not encompass alternative exon VIIc of the DmCK2β transcription unit. Both male and female DmCK2βmbuΔA26-2L flies rescued by the DmCK2β transgene are fertile (unpublished observations). Furthermore, this DmCK2β transgene rescued the mushroom body phenotype associated with the DmCK2β allele mushroom bodies undersized (Fig. 1A, CK2βmbuP1, Jauch et al., 2002) and

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the circadian rhythm phenotype of the Andante allele (Fig. 1A, CK2βAndante, Akten et al., 2003). Taken together, these observations suggested that the CK2β isoform (Fig. 1A, CK2β-VIIc) that is not fully encoded by the genomic DmCK2β transgene is redundant with regard to all known DmCK2β functions. The P-element induced DmCK2βmbuP1 allele causes a partial loss of male and female fertility that is reverted by P-element excision (unpublished observations). In an attempt to characterize the molecular basis for the partial loss of fertility in mushroom bodies undersized flies, we characterized two DmCK2β cDNAs derived from testis cDNA libraries: AT31207 and bs09g07 (Fig. 1A). In AT31207, the alternative exon VIId is spliced to the coding exons II–VI. In bs09g07, exon VIId is spliced to the coding exons II–V (Fig. 1A). These cDNAs encode for two so far unknown CK2β isoforms (Fig. 1B). The corresponding gene products were named CK2β-VIId and CK2βVIId-VI, respectively. bs09g07 is the first DmCK2β cDNA

Fig. 1. Alternative spliced DmCK2β mRNAs encode for five CK2β isoforms. (A) At the top the genomic region spanning the DmCK2β gene locus is depicted. BamHI (B) and EcoRI (E) restriction sites are shown. The P-element insertion sites of the DmCK2β mbuP1 and the DmCK2β P[GT1] alleles are indicated by triangles. An asterisk indicates the point mutation of the DmCK2β Andante allele. P[CK2β-gDNA] is the genomic rescue fragment. The approximate break points of the DmCK2β mbuΔA26-2L null allele are shown in light grey. DmCK2β exons I to VII are indicated above the cDNAs. cDNA and CK2β isoform nomenclatures as well as the predicted molecular polypeptide weights are shown in front and behind each cDNA. Open reading frames are indicated in medium grey. The ATG codon is shown by an arrow, arrowheads depict stop codons. CG4139 and CG4147 are neighboring genes. Black bar: 1 kb. (B) C-terminal amino acid sequence alignment of the five CK2β isoforms. Amino acid positions are indicated.

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clone that provides evidence that exon VI is not always used to generate a CK2β open reading frame. As deduced from all characterized DmCK2β cDNA clones, the DmCK2β transcription unit should encode for at least five different isoforms that differ in their very C-terminal tail (Fig. 1B). To examine the expression of CK2β isoforms in flies, we generated a polyclonal rabbit antiserum directed against a peptide shared by all predicted D. melanogaster CK2β isoforms. On Western blots of wild-type male and virgin female protein extracts, this antiserum detects six polypeptides of a molecular weight predicted for CK2β isoforms (Fig. 2A, B). In order to assign these polypeptides to the five CK2β isoforms encoded by DmCK2β mRNA splice variants we generated transgenic flies in which DmCK2β cDNA transgene expression can be induced by the UAS/GAL4 system (Brand and Perrimon, 1993). We used a P[heat-shock promotor (hsP)-Gal4]driver to induce ubiquitous transcription of the P[UAS:DmCK2β cDNA] transgenes and compared the ectopically expressed CK2β isoforms with the protein pattern detected in wild-type flies using the polyclonal CK2β antiserum. We observed that most overexpressed CK2β polypeptides had a slightly lower molecular weight than their putative counterparts in wild-type flies (Fig. 2A) and assumed that due to a lack of free CK2α subunits, ectopically expressed CK2β isoforms are not readily integrated into a holoenzyme and therefore represent non-phosphorylated CK2β polypeptides. Treatment of whole fly protein lysates with shrimps alkaline phosphatase (SAP) confirmed that with the exception of CK2β isoform CK2β-VIId-VI all CK2β isoforms are phosphorylated in vivo (Fig. 2B). On the basis of this observation, we were able to assign each of the five known DmCK2β splice variants to a defined CK2β protein isoform. CK2β isoforms CK2β-VIId and CK2β-VIId-VI encoded by the cDNAs derived from testis cDNA libraries can also be detected in unfertilized females. These proteins are therefore not

specifically expressed in male testis. Aged virgin females (48 h after eclosion (a.e.)) that already produce unfertilized eggs express much higher levels of isoforms CK2β-VIId-VI and CK2βVIIa than their newly eclosed virgin counterparts (1 h a.e). In addition, we found that isoform CK2β-VIIb is expressed in aged virgin females but neither in newly eclosed virgin females nor in male flies. This indicates that isoforms CK2β-VIIa, CK2β-VIIb and CK2β-VIId-VI are expressed during oogenesis. The nature of one protein (indicated by an asterisk in Fig. 2) that can be detected in both sexes by the polyclonal CK2β antiserum and does not show a mobility shift after SAP-treatment remained obscure. In summary, we could show that the DmCK2β transcription unit encodes for five CK2β isoforms that can be distinguished by their molecular weight and that can be assigned to specific DmCK2β cDNA clones. D. melanogaster is the first organism in which alternative splicing could be documented as a mechanism to generate CK2β polypeptide diversity. 3.2. All CK2β isoforms interact with CK2α We next investigated, whether C-terminal variability of CK2β isoforms influences the interaction with CK2α. We coexpressed MYC-tagged CK2α together with HA-tagged CK2β isoforms in human embryonic kidney cells (HEK293) and performed co-immunoprecipitation experiments (Fig. 3). The results of these experiments showed that CK2α interacts equally well with all CK2β isoforms. All HA-tagged CK2β isoforms appeared as double bands in whole cell lysates and in immunoprecipitates when HA-CK2β isoforms were co-expressed with MYC-CK2α (Fig. 3). In contrast, single bands were observed when the different CK2β isoforms were expressed in the absence of CK2α (data not shown), indicating that cotransfected CK2α is able to phosphorylate all CK2β isoforms.

Fig. 2. Drosophila melanogaster expresses five CK2β isoforms. CK2β isoforms were detected with a polyclonal rabbit antiserum directed against Drosophila CK2β. Molecular weight markers are indicated on the left of panel (A). (A) Ectopically expressed single CK2β isoforms CK2β-VIIc, CK2β-VIIb, CK2β-VIId and CK2βVIIa migrate slightly faster than their corresponding endogenous counterparts, indicating that they are unphosphorylated. With the exception of CK2β-VIIb, wild-type male flies express all CK2β isoforms. (B) All CK2β isoforms can be detected in aged virgin females (48 h after eclosion). In comparison to their newly eclosed counterparts (1h after eclosion), expression of isoforms CK2β-VIIa and CK2β-VIId-VI is strongly enhanced. Shrimps alkaline phosphatase (SAP) treatment (+) results in a faster migration of isoforms CK2β-VIIc, CK2β-VIIb, CK2β-VIId and CK2β-VIIa compared to their non-treated (−) counterparts. In contrast, isoform CK2β-VIId-VI seems not to be phosphorylated. An asterisk indicates a polypeptide that cannot be assigned to a specific CK2β isoform.

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In summary, these experiments showed that C-terminal amino acid variations of CK2β isoforms and the lack of amino acids encoded by DmCK2β exon VI do not interfere with binding to CK2α. 3.3. DmCK2β alleles differentially affect CK2β isoform expression On the basis of our analysis of the DmCK2β transcription unit at both the mRNA and the protein level, we asked whether allelic variants of this gene might have specific effects on the expression of one or more CK2β isoforms. We compared the CK2β isoform expression pattern of wild-type flies with that of three fly strains mutant for DmCK2β. Our previous analysis showed that DmCK2βmbuP1 (Fig. 1A) is a P-element induced hypomorphic DmCK2β allele (Jauch et al., 2002). P-element remobilization in male DmCK2βmbuP1 flies resulted in the imprecise jump-out allele DmCK2βmbuΔA in which only 46 bp of P-element DNA is still present. As shown by Western blot analysis, the DmCK2βmbuP1 allele causes a reduced expression of all identified CK2β isoforms (Fig. 4). Such a general effect on the expression of all CK2β isoforms is in good agreement with the assumption that the P-element insertion into the 5′ non-coding region of the DmCK2β trans-

Fig. 3. Drosophila CK2α interacts with all CK2β isoforms. HEK293 cells were transiently transfected with MYC-CK2α in combination with the indicated HAtagged Drosophila CK2β isoforms. Expression of MYC-CK2α and HA-tagged CK2β isoforms was confirmed by Western blot analysis of cell lysates using anti-MYC or anti-HA antibodies, respectively. Co-immunoprecipitation (CoIP) experiments were performed by incubating cell lysates with an anti-HA antibody. MYC-CK2α bound to all precipitated HA-tagged CK2β isoforms. All CK2β isoforms were detected as double bands, indicating that they become phosphorylated by CK2α.

Fig. 4. DmCK2β mutations differentially affect CK2β isoform expression. CK2β isoforms were detected on Western blots with a polyclonal anti-CK2β serum. An asterisk marks a band that cannot be assigned to a specific CK2β isoform. The monoclonal nc46 antibody was used to detect SAP47 as a loading control. In comparison to wild-type Berlin (wt) and revertant DmCK2β mbuΔA (mbuΔA) flies, CK2β isoform levels are reduced in DmCK2β mbuP1 (mbuP1) flies. In DmCK2β Andante (Andante) flies, isoforms CK2β-VIId and CK2β-VIIa are reduced when compared to the wild-type (wt) control. DmCK2β P[GT1] (P[GT1]) and DmCK2β P[GT1]Δ6 (P[GT1]Δ6) flies lack expression of isoforms CK2β-VIId and CK2β-VIIc that is restored in DmCK2β P[GT1]Δ8 (P[GT1]Δ8) revertant flies. All flies express isoform CK2β-VIId-VI at a low level.

cription unit results in the formation of reduced amounts of the primary DmCK2β transcript. Consistent with this, DmCK2βmbuΔA jump-out flies express all CK2β isoforms at wild-type levels (Fig. 4). It is noteworthy that the expression level of the one protein detected by the CK2β antiserum that could not be assigned to a specific DmCK2β splice variant (indicated by an asterisk in Figs. 2 and 4) is not affected by the DmCK2βmbuP1 allele. Therefore, it is likely that this polypeptide does not represent a CK2β isoform but a cross-reacting protein. Sequencing of the Andante allele identified a point mutation in the DmCK2β open reading frame (Fig. 1A) that results in a substitution of methionine 166 to isoleucine (Akten et al., 2003). This M166I substitution occurs within an α-helical domain (αF or α6) that is identical between humans and flies and is shared by all identified CK2β subunit isoforms. Structural analysis implicates that αF is important for both CK2β dimerization and CK2α/CK2β interactions (Niefind et al., 2001) and therefore the M166I substitution may destabilize the CK2 holoenzyme. Degradation of free CK2β isoform subunits may explain why in DmCK2βAndante mutant flies the protein levels of CK2β isoforms CK2β-VIId, CK2β-VIIa in males (Fig. 4) and, in addition, CK2β-VIIb in aged virgin females (data not shown) are reduced (Akten et al., 2003). However, such an explanation cannot explain our finding that the DmCK2βAndante allele does not result in a reduction of the protein level of CK2β isoforms CK2β-VIIc and CK2β-VIId-VI (Fig. 4). DmCK2βP[GT1] is a so far uncharacterized allele of DmCK2β caused by the insertion of a gene trap P[GT1] element in the 3′ region of the DmCK2β transcription unit (Fig. 1A). We generated the two excision alleles DmCK2βP[GT1]Δ6 and DmCK2βP[GT1]Δ8 by remobilizing the P[GT] element in male flies. All three alleles are without obvious external phenotypes. Both the DmCK2βP[GT1] and the DmCK2βP[GT1]Δ6 excision

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allele cause a loss of CK2β isoforms CK2β-VIIc and CK2βVIId (Fig. 4), but express the remaining isoforms at wild-type levels. In contrast, all isoforms are expressed at wild-type levels in DmCK2βP[GT1]Δ8 flies (Fig. 4), an observation that shows that the P[GT1] element of the DmCK2βP[GT1] allele is the reason for the loss of CK2β isoforms and that these isoforms are dispensable for viability. In summary, we have examined the influence of three independent DmCK2β alleles on the expression of all known CK2β isoforms. DmCK2βmbuP1 equally lowers the expression of all five CK2β isoforms. The DmCK2βAndante allele reduces the expression of three CK2β isoforms. Finally, the DmCK2βP[GT1] allele results in a specific loss of two CK2β isoforms. As indicated by three fly strains allelic for DmCK2β (DmCK2βmbuΔA, wild-type Berlin and DmCK2βP[GT1]Δ8), the genetic background has no significant influence on the expression level of CK2β isoforms. 3.4. CK2β isoforms are not redundant in function The existence of alternatively spliced DmCK2β mRNAs could reflect a mechanism that permits the generation of cell type specific regulatory subunits that differ in their functional properties due to variations of the very C-terminal tail. Alternatively, all CK2β isoforms could be functionally redundant and their existence would rather reflect the availability of cell type specific splice factors than the cell type specific need of functionally different regulatory subunits. Limited information about tissue specific expression of Drosophila CK2β isoforms can be deduced from the origin of the corresponding cDNAs. As mentioned above, cDNA clones isolated from testis-specific libraries encode for CK2β-VIId-VI and CK2β-VIId, whereas all so far characterized cDNA clones that correspond to CK2βVIIa and CK2β-VIIb (Bidwai et al., 2000) are derived from embryonic cDNA libraries. cDNA clones encoding for CK2βVIIc have so far only been isolated from an adult head library (Jauch et al., 2002). No mutant flies are available that lack only a single CK2β isoform and it seems to be unlikely that such a goal can be achieved by an RNAi approach as some of the alternative seventh exons overlap in sequence. We have therefore chosen both an overexpression and a rescue approach to gather information about functional differences among CK2β isoforms. We also extended this approach to the two CK2β-like proteins CK2βtes and CK2β′. It is reasonable to assume that in the case of functional identity ubiquitous overexpression of any of the five CK2β isoforms should have the same consequences on the development of the fly. We used a P[tubulin promotor (tubP)-Gal4] driver line to test the effects of ubiquitously expressed CK2β isoforms in flies wild-type for endogenous DmCK2β. As the transcriptional rate of a UAS-transgene might depend on its chromosomal insertion site, always six to eight independent insertion lines were tested. In this in vivo assay, ubiquitous overexpression of CK2β-VIIa, CK2β-VIIc and CK2β-VIId-VI did not interfere with normal development. In contrast, ubiquitous overexpression of CK2β-VIId from all and ubiquitous overexpression of CK2β-VIIb from five out of eight tested transgenic lines induced lethality (Table 1). Therefore, the C-

Table 1 In vivo properties of CK2β′, CK2βtes and CK2β isoforms

CK2β-VIIa CK2β-VIIb CK2β-VIIc CK2β-VIId CK2β-VIId-VI CK2β′ CK2βtes

Rescue of CK2β null

Ectopic expression lethal

+ + + − − − −

− + − + − + +

Upon ubiquitous expression in a DmCK2βmbuΔA26-2L mutant background, some CK2β isoforms can rescue the lethality of this allele (+), others cannot (−). Ubiquitous ectopic expression of CK2β isoforms and CK2β-like proteins in a wild-type background is either lethal (+) or does not interfere with the viability of the fly (−).

terminal tail of isoforms CK2β-VIIb and CK2β-VIId confers functional properties to these CK2β isoforms that can disrupt cellular processes essential for normal development. As overexpression of isoforms CK2β-VIIb and CK2β-VIId from two copies of a genomic DmCK2β transgene in a wild-type background does not induce lethality, their endogenous expression pattern seems to be tissue specific rather than ubiquitous. The genomic DmCK2β transgene that encodes — with the exception of CK2β-VIIc — for all isoforms rescues the lethality of a DmCK2β loss of function allele (DmCK2βmbuΔA26-2L, Jauch et al., 2002). Which of the five CK2β isoforms possesses the functional properties to compensate for a loss of DmCK2β function? We found that upon P[tubP-Gal4] driven ubiquitous expression, isoforms CK2β-VIIa, CK2β-VIIb and CK2β-VIIc rescued the lethality of the DmCK2βmbuΔA26-2L allele. In the case of CK2β-VIIb, not all of the independent insertion lines rescued. In contrast, no rescued flies could be obtained by ubiquitous expression of isoforms CK2β-VIId-VI and CK2βVIId in a DmCK2β null background (Table 1). In summary, the functional properties of isoforms CK2βVIIa and CK2β-VIIc were indistinguishable in the two test systems used. CK2β-VIId-VI can neither substitute for a loss of DmCK2β function nor does it induce lethality upon ectopic expression. CK2β-VIId overexpression induces lethality and its functional properties are insufficient to rescue the lethality of DmCK2βmbuΔA26-2L mutant flies. CK2β-VIIb can substitute for a loss of DmCK2β function but induces lethality upon ectopic expression in a wild-type background. 3.5. CK2βtes and CK2β′ cannot substitute for a loss of CK2β function With regard to CK2β-VIIa, CK2β′ is identical in 58.4% and similar in 71.9% of its amino acid sequence. The amino acid sequence of CK2βtes is less conserved. 42.6% of its amino acids are identical and 58.3% of its amino acids are similar to CK2β-VIIa. In both proteins, structural motifs are conserved that are needed for β-subunit dimerization and CK2α interaction. The interaction of CK2α with CK2β′ and CK2βtes has been shown (Karandikar et al., 2003). To address the question whether CK2β′ and CK2βtes can regulate CK2α, Karandikar et al. (2003) made use of the defects in ion-

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Fig. 5. DmCK2β mbuP1 reduces mushroom body calyx size. A schematic drawing of a frontal Drosophila brain section is shown in (A). The neural cell bodies form a cellular cortex that is shown in light grey in (A) and appears yellowish in (B-D) and (F–H). These figures show frontal 7μm paraffin section series arranged from posterior to anterior of DmCK2β mbuP1 (B–D) or wild-type Berlin (F–H) flies at the same magnification. Neuropils are formed by dendritic and axonal components and are dark grey or highlighted in black in (A). They appear dark greenish in (B–D) and (F–H). Note that the mushroom body calyces are located posterior to the fanshape body of the central complex that is therefore never visible in a section that cuts the calyces. In (E), the morphology of Kenyon cells with branched axons is shown. The Kenyon cell bodies are part of the cellular cortex, dendrites form the calyx and axons form the peduncle and branch to project to a medial and dorsal lobe. The mushroom body calyces of a DmCK2β mbuP1 fly (B–D) appear more rounded in shape and are reduced in size when compared to those of a wild-type Berlin fly (F–H).

homeostasis typical for ckb1 or ckb2 mutant S. cerevisiae strains. They found that expression of Drosophila CK2β (CK2β-VIIa) and CK2βtes, but not of CK2β′ transgenes, elicited similar suppression of the Na+ sensitivity in these strains. It is known that loss of either yeast CK2β subunit results in downregulated transcription of ENA1 that encodes for a Na+-ATPase that mediates Na+ export in yeast (Tenney and Glover, 1999). As both Drosophila CK2β-VIIa and CK2βtes regulate yeast CK2α subunits in a similar manner that makes CKB mutant yeast strains less sensitive to Na+, functional redundancy of these proteins in regulating yeast ion-homeostasis was concluded. We tested this observation the same way as we examined the possible functional redundancy of CK2β isoforms. Each eight independent UAS-transgenes encoding for CK2β′ or CK2βtes were ectopically expressed in a wild-type background using a P[tubP-Gal4] driver. Ectopic expression induced lethality in both cases (Table 1). In addition, none of the tested P[UAS:CK2β′] or P[UAS:CK2βtes] transgene insertions could rescue the lethality of DmCK2βmbuΔA26-2L mutant flies upon ubiquitous expression (Table 1). This lack of rescue cannot be explained by the lethal effect of ectopically expressed CK2β′ or CK2βtes in a wild-type background, as CK2β isoform CK2β-VIIb induces lethality upon ectopic expression in a wild-type background but nevertheless can substitute for a loss of DmCK2β function (Table 1). Our results obtained for CK2β′ or CK2βtes are similar to the results obtained with CK2β isoform CK2β-VIId and support the conclusion of Karandikar et al. (2003) that CK2β-VIIa and CK2β′ are functionally non-redundant. However, in our test systems, also CK2β-VIIa and CK2βtes behave differently, what makes them unlikely to be functionally redundant. 3.6. Two CK2β isoforms are dispensable for mushroom body development The three independent DmCK2β alleles DmCK2βmbuP1, DmCK2βAndante and DmCK2βP[GT1] (Fig. 1A) affect CK2β

isoform expression in a distinct manner (Fig. 4). As we previously observed that the DmCK2βmbuP1 allele affects the development of the mushroom bodies (Jauch et al., 2002), we asked whether DmCK2βAndante or DmCK2βP[GT1] mutant flies do also have defects in this brain structure. The aim of this comparative approach was to figure out which CK2β isoforms have to be expressed at wild-type levels to allow for normal mushroom body development. The intrinsic neurons of the mushroom bodies, the Kenyon cells (Fig. 5E), are generated by symmetric cell divisions of ganglion mother cells that are formed by continuous asymmetric divisions of four neural stem cells (mushroom body neuroblasts) per brain hemisphere, a process which begins during embryogenesis and ends in the pupa (Lee et al., 1999). In the adult, three different types of Kenyon cells contribute to the mushroom body neuropil that can be roughly divided into four substructures. The Kenyon cells of each of the two mushroom bodies project their dendrites to a structure called calyx that is embedded in the cellular cortex of the brain (Fig. 5A). The axons of the Kenyon cells enter the central brain neuropil and form the peduncle. At the end of the peduncle, the axons of a subset of Kenyon cells bifurcate and send one axonal branch in dorsal, the other branch in medial direction (Fig. 5E). Other Kenyon cells project their unbranched axon only in medial direction. Thereby, a medial and dorsal lobe system is formed. Analysis of mutation specific effects on the size of the mushroom bodies is mainly hampered by the variability of the total brain size and all its substructures. This variability may depend on fly strain specific variations of the genetic background, different culture conditions of the flies examined and natural variability. In addition, differences in handling during the fixation procedure cause considerable differences in the absolute size of the brain (compare Fig. 5B–D with Fig. 5F– H). We therefore analysed mushroom body specific effects of DmCK2β alleles by setting the measured absolute volume of the mushroom body calyx in relation to the absolute volume of another central brain structure, the fanshape body (Fig. 5A)

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that is not affected by known mutations in DmCK2β and therefore served as an internal brain size control. DmCK2βmbuP1 causes a reduction of Kenyon cell number, a phenotype that can be detected in frontal paraffin sections by the reduced size of the mushroom body calyx (Fig. 5B–D), a smaller diameter of the peduncle and a thinner dorsal and medial lobe system (Jauch et al., 2002). As expected for a hypomorphic DmCK2β allele that acts by reducing the expression of all CK2β isoforms (Fig. 4), the mushroom body defect is enhanced in flies that are transheterozygous for DmCK2βmbuP1 and DmCK2βmbuΔA26-2L (Fig. 6). Conversely, revertant DmCK2βmbuΔA flies develop normal mushroom bodies (Fig. 6). This result fits with our observation that this allele does not affect the expression of any CK2β isoform when compared to wild-type flies (Fig. 4). A role for CK2β isoforms CK2β-VIId and CK2β-VIIc in the formation of normal mushroom bodies could be excluded by analysis of DmCK2βP[GT1] flies that do not express these isoforms (Fig. 4). Homozygous DmCK2βP[GT1] flies have normal mushroom bodies (Fig. 6). Homozygous DmCK2βAndante flies display a strong mushroom body phenotype (Fig. 6). A second site mutation outside

the DmCK2β transcription unit that might be responsible for the mushroom body phenotype was excluded by complementation analysis with DmCK2βmbuΔA26-2L and rescue of mushroom body phenotype with two copies of the genomic DmCK2β transgene (Fig. 6). To test whether the M166I substitution caused by the Andante allele is responsible for the mushroom body defect, we rescued homozygous DmCK2βmbuΔA26-2L flies by expression of a CK2β-VIIa isoform carrying the M166I substitution (P[UAS:CK2βM166I ]) using the P[tubP-Gal4] driver. Rescued flies did not show the marked mushroom body defect seen in homozygous Andante flies (Fig. 6). Although Andante is by all genetic criteria a DmCK2β allele, the M166I amino acid exchange of the Andante CK2β-VIIa isoform seems not to alter its in vivo properties, at least not during mushroom body development. This confirms the results of Rasmussen et al. (2005) who found on the biochemical level that a conservative M166I amino acid exchange of recombinant CK2β does neither affect β/β association nor β/α interaction. We assume that one of the intronic polymorphisms detected in Andante (Akten et al., 2003) is likely to cause both changes of Andante CK2β isoform levels (Fig. 4) and its mushroom body phenotype (Fig. 6). As the DmCK2βAndante allele does not reduce the expression of CK2β isoforms CK2β-VIId-VI and CK2β-VIIc (Fig. 4), and normal mushroom bodies can be formed in the absence of isoform CK2β-VIId (as revealed by the DmCK2βP[GT1] allele), the mushroom body phenotype observed in DmCK2βAndante flies is likely to be caused by reduced expression of isoforms CK2β-VIIa and/or CK2β-VIIb (Fig. 4). In summary, our molecular and genetic analysis of the DmCK2βmbuP1 allele showed that the formation of normal mushroom bodies depends on DmCK2β gene dosage. As revealed by examination of DmCK2βP[GT1] mutant flies, this brain structure develops normal in the absence of CK2β isoforms CK2β-VIId and CK2β-VIIc. Additional analysis of DmCK2βAndante mutant flies indicated that isoforms CK2β-VIIa and/or CK2β-VIIb are necessary for normal mushroom body development. 3.7. Single CK2β isoforms are sufficient for mushroom body development

Fig. 6. Effects of DmCK2β alleles on mushroom body calyx size. The mean calyx volumes and the mean fanshape body volumes of flies with the indicated DmCK2β genotypes (x-axis) were determined and used to calculate a value (y-axis) that represents the normalized calyx size and sets the mean calyx volume in relation to the mean fanshape body volume. At the bottom of each column the number of flies used in the experiment is indicated. The normalized calyx size is nearly identical in wild-type Berlin (CK2β-wt/CK2β-wt), DmCK2β mbuΔA (mbuΔA/mbuΔA), DmCK2β P[GT1] (P[GT1]/P[GT1]) and DmCK2β Andante flies (And/And; g resc) that in addition carry two copies of the genomic DmCK2β rescue construct P[CK2β-gDNA]. Flies transheterozygous for DmCK2β mbuP1 and DmCK2β mbuΔA26-2L (mbuP1/ΔA26-2L) have smaller mushroom bodies than DmCK2β mbuP1 flies (mbuP1/mbuP1). Both DmCK2β Andante flies (And/And) and flies transheterozygous for DmCK2β Andante and DmCK2β mbuΔA26-2L (And/ ΔA26-2L) have small mushroom bodies. Homozygous DmCK2β mbuΔA26-2L flies rescued by a P[tubP-Gal4]driven P[UAS:CK2β M166I]) transgene (ΔA26-2L/ ΔA26-2L; M166I c resc) have mushroom bodies of almost wid-type size.

Our analysis of the CK2β isoform protein expression pattern (Fig. 4) in combination with the mushroom body phenotype of DmCK2β mutant flies (Fig. 6) did not allow us to examine whether Drosophila mushroom body development depends on a specific CK2β isoform. As the formation of mushroom bodies is not a prerequisite for the viability of a fly, the expression of a single CK2β isoform in a DmCK2βmbuΔA26-2L mutant background could in principle result in viable flies that lack this neuropil. We therefore examined the mushroom bodies of DmCK2βmbuΔA26-2L mutant male flies rescued by P[tubP-Gal4] driven expression of a P[UAS:CK2β-VIIa], a P[UAS:CK2βVIIb] or a P[UAS:CK2β-VIIc] transgene. CK2β isoforms CK2β-VIIa and CK2β-VIIc promote mushroom body development equally well (Fig. 7) and therefore behave functionally identical in this process. In male flies rescued by a P[tubPGal4] driven P[UAS:CK2β-VIIb] transgene, we observed

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Fig. 7. Single CK2β isoforms are sufficient for mushroom body formation. The normalized calyx size of wild-type Berlin (CK2β-wt/Y) and DmCK2β mbuΔA26-2L flies rescued by P[tubP-Gal4] driven expression of P[UAS:CK2β] transgenes encoding for isoforms CK2β-VIIa (ΔA26-2L/Y; CK2β-VIIa), CK2β-VIIb (ΔA26-2L/Y; CK2β-VIIb) and CK2β-VIIc (ΔA26-2L/Y; CK2β-VIIc), respectively, were calculated as described in the legend of Fig. 6. The number of flies analysed is indicated at the bottom of each column. DmCK2β mbuΔA26-2L flies rescued by ubiquitous expression of isoform CK2β-VIIb (ΔA26-2L/Y; CK2βVIIb) develop mushroom bodies that appear proportionally larger than those of wild-type Berlin (CK2β-wt/Y) controls.

mushroom body calyces that were proportionally larger than those of wild-type male flies (Fig. 7). These experiments show that normal mushroom body development does not rely on the co-expression of two or more CK2β isoforms and indicate that in wild-type flies, only one CK2β isoform could be needed for mushroom body development. Taken together, the presence of one of the three CK2β isoforms that can rescue DmCK2βmbuΔA26-2L mutant flies upon ubiquitous expression is sufficient for mushroom body development. As revealed by analysis of the DmCK2βP[GT1] allele, isoforms CK2β-VIIc and CK2β-VIId are dispensable for the development of this neuropil. In addition, isoform CK2βVIId-VI expression is not reduced in DmCK2βAndante flies. As DmCK2βmbuΔA26-2L flies rescued by CK2β-VIIa or CK2β-VIIb possess normal mushroom bodies, the development of this brain structure is dependant on one or both isoforms. 4. Discussion Despite more than 50 years of research on CK2, in vivo regulation of this pleiotropic protein kinase remains enigmatic. Biochemical experiments suggested that CK2 holoenzyme activity depends on N-terminal phosphorylation of its regulatory subunits (Lin et al., 1994). In addition, binding of polyamines to the acidic loop of the β-subunit was found to modulate CK2

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holoenzyme phosphotransferase activity in vitro (Leroy et al., 1999). It remains an unanswered question whether these modulating processes work in vivo. The identification of the two Drosophila CK2β-like proteins CK2β′ and CK2βtes that interact with CK2α pointed towards a mechanism to regulate CK2α in vivo by the usage of tissue specific regulatory subunits that may differ in their regulatory properties, but the restricted expression pattern of these two CK2β-like proteins would limit this assumed regulatory mechanism to the male testis. This limitation could be overcome by the usage of CK2β isoforms generated by the translation of alternatively spliced DmCK2β mRNAs. All of the identified CK2β variants interact with CK2α, a prerequisite for a differential regulation of this serine/threonine kinase. However, the existence of different CK2β-like proteins in Drosophila does not rule out the possibility of functional redundancy as it was observed for a CK2β isoform and CK2βtes in ion-homeostasis rescue experiments in CKB mutant yeast strains (Karandikar et al., 2003). Based on our ectopic expression experiments and the experiments designed to rescue the lethality of a DmCK2β null allele we could clearly rule out full functional redundancy among CK2β isoforms on the one and CK2βtes and CK2β′ on the other hand. This does not exclude that these CK2β-like proteins have some overlapping functions, but the assumed common functions are not sufficient to confer the same properties to these evolutionary closely related members of the Drosophila CK2β-like protein family. Furthermore, in our overexpression and rescue experiments we observed full functional redundancy only between two CK2β isoforms, namely CK2βVIIa and CK2β-VIIc. Taken together, our experiments point to specific functions of different CK2β isoforms that might be employed to fine-tune the CK2 holoenzyme. The very C-terminal tail of human CK2β is disordered in the crystal structure of recombinant human CK2. Furthermore, truncation of these amino acids has no influence on the biochemical properties of recombinant CK2. Therefore, the conclusion was drawn that these amino acid residues are not important for holoenzyme function and formation (Niefind et al., 2001). As only the very C-terminal tail varies amongst Drosophila CK2β isoforms that are functionally non-redundant, this CK2β domain is likely to have a so far not recognized in vivo function. It might be used as a domain that allows integration of interacting proteins to the CK2 holoenzyme in a CK2β isoform specific manner. Thereby, the catalytic activity or substrate specificity and the localization of the Drosophila holoenzyme could be influenced. Alternatively, variations in the C-terminal tail might influence the formation of higher-order structures of the CK2 holoenzyme. In the crystals of human CK2, trimeric rings of the CK2 holoenzyme have been observed (Niefind and Issinger, 2005). It remains to be determined whether other higher eukaryotes also generate CK2β isoform diversity by alternative splicing. Acknowledgements We would like to thank Martin Heisenberg for continuous support. This work was supported by grants from the Deutsche Forschungsgemeinschaft to T.R. (Ra561/6-1; SFB581/B14).

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