RESUMO
Drosophila melanogaster casein kinase II (DmCKII) is composed of catalytic (alpha) and regulatory (beta) subunits associated as an alpha2beta2 heterotetramer. Using the two-hybrid system, we have screened a D. melanogaster embryo cDNA library for proteins that interact with DmCKIIalpha. 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 DmCKIIalpha but not with DmCKIIbeta, suggesting that this interaction is specific for the catalytic subunit of DmCKII. In addition to m7, we demonstrate that DmCKIIalpha 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 DmCKIIalpha 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 DmCKIIalpha, as well as by the alpha2beta2 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 DmCKIIalpha, 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.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/enzimologia , Proteínas de Insetos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Caseína Quinase II , Primers do DNA , DNA Complementar , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Insetos/química , Proteínas de Insetos/genética , Dados de Sequência Molecular , Fosforilação , Plasmídeos , Proteínas Serina-Treonina Quinases/química , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
D. melanogaster CK2 (DmCK2) is a highly conserved protein kinase that is composed of catalytic, alpha, and regulatory, beta, subunits associated as an alpha2beta2 heterotetramer. In order to analyze the functions of CK2 in this metazoan model, we have used the two hybrid approach to identify interacting proteins. One of these cDNAs, DmA24, encodes a novel polypeptide with no homologs in GenBank, and is notable in that it contains a bipartite nuclear localization signal and two sites for phosphorylation by CK2. In situ hybridization to polytene chromosomes indicates that the DmA24 gene is located at the 61 D interval of chromosome II a region that also harbors 3 additional genes with similar structure. DmA24p interacts with DmCK2alpha, but not with DmCK2beta, demonstrating that this interaction is specific for the catalytic subunit of CK2. In addition, the protein is phosphorylated by the holoenzyme purified from Drosophila embryos. These studies identify DmA24p as a potentially new physiological partner of DmCK2. In addition, we also report the results of a large-scale screen that has identified a new set of DmCK2-interacting proteins. Most notable among these are Surf6, a nucleolar protein involved in RNA processing, and Spalt, a homeotic protein.
Assuntos
Drosophila melanogaster/enzimologia , Proteínas Serina-Treonina Quinases/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Caseína Quinase II , DNA Complementar/metabolismo , Proteínas de Drosophila , Proteínas de Homeodomínio/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/metabolismo , Transcrição Gênica , Técnicas do Sistema de Duplo-Híbrido , beta-Galactosidase/metabolismoRESUMO
Drosophila melanogaster protein kinase CK2 (DmCK2) is a Ser/Thr protein kinase composed of catalytic alpha and regulatory beta subunits associated as an alpha2beta2 heterotetramer. Using the two hybrid system, we have screened a Drosophila embryo cDNA library in order to identify proteins that interact with DmCK2alpha. One of these cDNAs encodes a novel previously undescribed zinc-finger protein, which we call ZFP47. ZFP47 interacts with DmCK2alpha but not with DmCK2beta, indicating that this interaction is specific for the catalytic subunit of CK2. In situ hybridization to polytene chromosomes indicates that the corresponding gene is located at the 72A interval of chromosome III. Sequence analysis indicates that ZFP47 contains a consensus site for phosphorylation by CK2, 4 C1H1-type zinc-fingers, and a bipartite nuclear localization signal. Consistent with the prediction of a site for phosphorylation by CK2, we demonstrate that ZFP47 is phosphorylated by CK2 purified from Drosophila embryos. These studies demonstrate that ZFP47 is a new physiological partner and substrate of CK2.
