The Notch effector E(spl)M8 is phosphorylated at Ser159 by CK2, a highly conserved Ser/Thr protein kinase. We have used the Gal4-UAS system to assess the role of M8 phosphorylation during bristle and eye morphogenesis by employing a non-phosphorylatable variant (M8SA) or one predicted to mimic the 'constitutively' phosphorylated protein (M8SD). We find that phosphorylation of M8 does not appear to be critical during bristle morphogenesis. In contrast, only M8SD elicits a severe 'reduced eye' phenotype when it is expressed in the morphogenetic furrow of the eye disc. M8SD elicits neural hypoplasia in eye discs, elicits loss of phase-shifted Atonal-positive cells, i.e. the 'founding' R8 photoreceptors, and consequently leads to apoptosis. The ommatidial phenotype of M8SD is similar to that in Nspl/Y; E(spl)D/+ flies. E(spl)D, an allele of m8, encodes a truncated protein known as M8*, which, unlike wild type M8, displays exacerbated antagonism of Atonal via direct protein-protein interactions. In line with this, we find that the M8SD-Atonal interaction appears indistinguishable from that of M8*-Atonal, whereas interaction of M8 or M8SA appears marginal, at best. These results raise the possibility that phosphorylation of M8 (at Ser159) might be required for its ability to mediate 'lateral inhibition' within proneural clusters in the developing retina. This is the first identification of a dominant allele encoding a phosphorylation-site variant of an E(spl) protein. Our studies uncover a novel functional domain that is conserved amongst a subset of E(spl)/Hes repressors in Drosophila and mammals, and suggests a potential role for CK2 during retinal patterning.

Lateral inhibition is critical for cell fate determination and involves the functions of Notch (N) and its effectors, the Enhancer of Split Complex, E(spl)C repressors. Although E(spl) proteins mediate the repressive effects of N in diverse contexts, the role of phosphorylation was unclear. The studies we describe implicate a common role for the highly conserved Ser/Thr protein kinase CK2 during eye and bristle development. Compromising the functions of the catalytic (alpha) subunit of CK2 elicits a rough eye and defects in the interommatidial bristles (IOBs). These phenotypes are exacerbated by mutations in CK2 and suppressed by an increase in the dosage of this protein kinase. The appearance of the rough eye correlates, in time and space, to the specification and refinement of the 'founding' R8 photoreceptor. Consistent with this observation, compromising CK2 elicits supernumerary R8's at the posterior margin of the morphogenetic furrow (MF), a phenotype characteristic of loss of E(spl)C and impaired lateral inhibition. We also show that compromising CK2 elicits ectopic and split bristles. The former reflects the specification of excess bristle SOPs, while the latter suggests roles during asymmetric divisions that drive morphogenesis of this sensory organ. In addition, these phenotypes are exacerbated by mutations in CK2 or E(spl), indicating genetic interactions between these two loci. Given the centrality of E(spl) to the repressive effects of N, our studies suggest conserved roles for this protein kinase during lateral inhibition. Candidates for this regulation are the E(spl) repressors, the terminal effectors of this pathway.

Drosophila melanogaster casein kinase II (DmCKII) is composed of catalytic (␣) and regulatory (␤) subunits associated as an ␣ 2 ␤ 2 heterotetramer. Using the twohybrid system, we have screened a D. melanogaster embryo cDNA library for proteins that interact with Dm-CKII␣. One of the cDNAs isolated in this screen encodes m7, a basic helix-loop-helix (bHLH)-type transcription factor encoded by the Enhancer of split complex (E(spl)C), which regulates neurogenesis. m7 interacts with DmCKII␣ but not with DmCKII␤, suggesting that this interaction is specific for the catalytic subunit of Dm-CKII. In addition to m7, we demonstrate that DmCKII␣ also interacts with two other E(spl)C-derived bHLH proteins, m5 and m8, but not with other members, such as m3 and mC. Consistent with the specificity observed for the interaction of DmCKII␣ with these bHLH proteins, sequence alignment suggests that only m5, m7, and m8 contain a consensus site for phosphorylation by CKII within a subdomain unique to these three proteins. Accordingly, these three proteins are phosphorylated by DmCKII␣, as well as by the ␣ 2 ␤ 2 holoenzyme purified from Drosophila embryos. In line with the prediction of a single consensus site for CKII, replacement of Ser 159 of m8 with either Ala or Asp abolishes phosphorylation, identifying this residue as the site of phosphorylation. We also demonstrate that m8 forms a direct physical complex with purified DmCKII, corroborating the observed two-hybrid interaction between these proteins. Finally, substitution of Ser 159 of m8 with Ala attenuates interaction with DmCKII␣, whereas substitution with Asp abolishes the interaction. These studies constitute the first demonstration that DmCKII interacts with and phosphorylates m5, m7, and m8 and suggest a biochemical and/or structural basis for the functional equivalency of these bHLH proteins that is observed in the context of neurogenesis. Casein kinase II (CKII)1 is a ubiquitous protein kinase that is highly conserved among eukaryotes (1, 2) and is capable of functioning as an oncogene in mammals (3). CKII is composed of catalytic (␣) and regulatory (␤) subunits that combine to form an ␣ 2 ␤ 2 holoenzyme. With the exceptions of Drosophila melanogaster (4), Caenorhabditis elegans (5), and Schizosaccharomyces pombe (6), CKII from most eukaryotic organisms contains two ␣ subunits, ␣ and ␣Ј, that are encoded by distinct genes. In contrast, ␤ subunit heterogeneity has been documented via protein microchemical approaches in Saccharomyces cerevisiae (7) and via molecular/genetic approaches in Arabidopsis thaliana (8) and D. melanogaster (9).CKII preferentially phosphorylates Ser/Thr residues in an hyperacidic context (10), although phosphorylation of Tyr has been documented in at least one case, i.e. yeast Fpr3 (11). Analysis of the phosphorylation of synthetic peptides suggests that the consensus site for phosphorylation by CKII can best be described as (S/T)(D/E)X(D/E) (10). Consistent with this, a number of proteins critical for transcription, cell cycle regulation, and s...

Saccharomyces cerevisiae casein kinase II (CKII) contains two distinct catalytic (alpha and alpha') and regulatory (beta and beta') subunits. We report here the isolation and disruption of the gene, CKB1, encoding the 38-kDa beta subunit. The predicted Ckb1 sequence includes the N-terminal autophosphorylation site, internal acidic domain, and potential metal binding motif (CPX3C-X22-CPXC) present in other beta subunits but is unique in that it contains two additional autophosphorylation sites as well as a 30-amino-acid acidic insert. CKB1 is located on the left arm of chromosome VII, approximately 33 kilobases from the centromere and does not correspond to any previously characterized genetic locus. Haploid and diploid strains lacking either or both beta subunit genes are viable, demonstrating that the regulatory subunit of CKII is dispensable in S. cerevisiae. Such strains exhibit wild type behavior with regard to growth on both fermentable and nonfermentable carbon sources, mating, sporulation, spore germination, and resistance to heatshock and nitrogen starvation, but are salt-sensitive. Salt sensitivity is specific for NaCl and LiCl and is not observed with KCl or agents which increase osmotic pressure alone. These data suggest a role for CKII in ion homeostasis in S. cerevisiae.

A family of baculovirus inhibitor-of-apoptosis (IAP) genes is present in mammals, insects, and baculoviruses, but the mechanism by which they block apoptosis is unknown. We have identified a protein encoded by the Drosophila mod(mdg4) gene which bound to the baculovirus IAPs. This protein induced rapid apoptosis in insect cells, and consequently we have named it Doom. Baculovirus IAPs and P35, an inhibitor of aspartatespecific cysteine proteases, blocked Doom-induced apoptosis. The carboxyl terminus encoded by the 3 exon of the doom cDNA, which distinguishes it from other mod(mdg4) cDNAs, was responsible for induction of apoptosis and engagement of the IAPs. Doom localized to the nucleus, while the IAPs localized to the cytoplasm, but when expressed together, Doom and the IAPs both localized in the nucleus. Thus, IAPs might block apoptosis by interacting with and modifying the behavior of Doom-like proteins that reside in cellular apoptotic pathways.