Kathy Beckingham

Calmodulin regulation of Drosophila light-activated channels and receptor function mediates termination of the light response in vivo.

Calmodulin (CAM) participates in a variety of intracellular transduction processes by modulating signaling molecules in response to calcium changes. We report the characterization of Drosophila Cam mutants and the role of CAM in photoreceptor cell function. Contrary to current models of excitation and TRP channel function, we demonstrate that the transient phenotype of trp mutants can be explained by CAM regulation of the TRPL channel rather than by the loss of a store-operated conductance leading to depletion of the internal stores. We also analyzed light responses in a variety of mutant and transgenic backgrounds and demonstrate the importance of calmodulin in mediating calcium-dependent negative regulation of phototransduction. Our results show that CAM coordinates termination of the light response by modulating receptor and ion channel activity.

Physiological calcium concentrations regulate calmodulin binding and catalysis of adenylyl cyclase exotoxins

Edema factor (EF) and CyaA are calmodulin (CaM)‐activated adenylyl cyclase exotoxins involved in the pathogenesis of anthrax and whooping cough, respectively. Using spectroscopic, enzyme kinetic and surface plasmon resonance spectroscopy analyses, we show that low Ca2+ concentrations increase the affinity of CaM for EF and CyaA causing their activation, but higher Ca2+ concentrations directly inhibit catalysis. Both events occur in a physiologically relevant range of Ca2+ concentrations. Despite the similarity in Ca2+ sensitivity, EF and CyaA have substantial differences in CaM binding and activation. CyaA has 100‐fold higher affinity for CaM than EF. CaM has N‐ and C‐terminal globular domains, each binding two Ca2+ ions. CyaA can be fully activated by CaM mutants with one defective C‐terminal Ca2+‐binding site or by either terminal domain of CaM while EF cannot. EF consists of a catalytic core and a helical domain, and both are required for CaM activation of EF. Mutations that decrease the interaction of the helical domain with the catalytic core create an enzyme with higher sensitivity to Ca2+–CaM activation. However, CyaA is fully activated by CaM without the domain corresponding to the helical domain of EF.

An extended conformation of calmodulin induces interactions between the structural domains of adenylyl cyclase from Bacillus anthracis to promote catalysis.

The edema factor exotoxin produced byBacillus anthracis is an adenylyl cyclase that is activated by calmodulin (CaM) at resting state calcium concentrations in infected cells. A C-terminal 60-kDa fragment corresponding to the catalytic domain of edema factor (EF3) was cloned, overexpressed inEscherichia coli, and purified. The N-terminal 43-kDa domain (EF3-N) of EF3, the sole domain of edema factor homologous to adenylyl cyclases from Bordetella pertussis andPseudomonas aeruginosa, is highly resistant to protease digestion. The C-terminal 160-amino acid domain (EF3-C) of EF3 is sensitive to proteolysis in the absence of CaM. The addition of CaM protects EF3-C from being digested by proteases. EF3-N and EF3-C were expressed separately, and both fragments were required to reconstitute full CaM-sensitive enzyme activity. Fluorescence resonance energy transfer experiments using a double-labeled CaM molecule were performed and indicated that CaM adopts an extended conformation upon binding to EF3. This contrasts sharply with the compact conformation adopted by CaM upon binding myosin light chain kinase and CaM-dependent protein kinase type II. Mutations in each of the four calcium binding sites of CaM were examined for their effect on EF3 activation. Sites 3 and 4 were found critical for the activation, and neither the N- nor the C-terminal domain of CaM alone was capable of activating EF3. A genetic screen probing loss-of-function mutations of EF3 and site-directed mutations based on the homology of the edema factor family revealed a conserved pair of aspartate residues and an arginine that are important for catalysis. Similar residues are essential for di-metal-mediated catalysis in mammalian adenylyl cyclases and a family of DNA polymerases and nucleotidyltransferases. This suggests that edema factor may utilize a similar catalytic mechanism.

Alternative splicing of Drosophila calcium/calmodulin-dependent protein kinase II regulates substrate specificity and activation.

Drosophila calcium/calmodulin-dependent protein kinase II is alternatively spliced to generate multiple isoforms that vary only in a region between the calmodulin-binding domain and the association domain. This variation has been shown to modulate activation of the enzyme by calmodulin. In this study we examine the ability of seven of the Drosophila isoforms to phosphorylate purified protein substrates and to be inhibited by a substrate analog, and the response of six of the isoforms to a mutant form of calmodulin (V91G) that was isolated in a genetic screen. Significant variation in Kms for Eag, a potassium channel, and Adf-1, a transcription factor, were found. In the case of the a peptide inhibitor, AC3I, there were significant variations in Ki between isoforms. Kact for V91G calmodulin was increased for all of the isoforms. In addition, one isoform, RI, exhibited a lower Vmax when assayed with this mutant CaM. These results indicate that the variable domain of calcium/calmodulin-dependent protein kinase II is capable of altering the substrate specificity of the catalytic domain and the activation response to calmodulin.

Functional Diversity of Alternatively Spliced Isoforms of Drosophila Ca2+/Calmodulin-dependent Protein Kinase II A ROLE FOR THE VARIABLE DOMAIN IN ACTIVATION

Isoforms of calcium/calmodulin-dependent protein kinase II from Drosophila (R1-R6 and R3A) showed differential activation by two series of mutant calmodulins, B1K-B4K and B1Q-B4Q. These mutant calmodulins were generated by changing a glutamic acid in each of the four calcium binding sites to either glutamine or lysine, altering their calcium binding properties. All mutations produced activation defects, with the binding site 4 and B1Q mutants the most severe. Activation differed substantially between isoforms. R4, R5, and R6 were the least sensitive to mutations in calmodulin, while R1, R3, and R3A were the most sensitive. Activation of R1 and R2 by B4K and activation of R3 and R3A by B2K and B2Q produced significant (6-fold and almost 3-fold, respectively) differences in Kact between isoforms that differ structurally by a single amino acid. These differences could not be accounted for by differential binding, as all isoforms showed almost identical binding patterns with the mutants. High binding affinity did not always correlate with ability to increase enzyme activity, implying that activation occurs in at least two steps. The isoform-specific differences seen in this study reflect a role for the COOH-terminal variable region in activation of CaM kinase II.

Under-replication of intron+ rDNA cistrons in polyploid nurse cell nuclei of Calliphora erythrocephala

We have examined the possibility that the intron-containing (intron+) rDNA cistrons of Dipteran flies are active in the germ-line derived polyploid nurse cell nuclei of the ovarian follicles. Using the organism, Calliphora erythrocephala, we describe here a procedure which yields very pure nurse cell nuclei and compare the intron-free (intron−) and intron+ rDNA cistron contents of nurse cell nuclei prepared by this procedure to those of 2–18 h embryo nuclei and 3 day pupal nuclei. DNA from three preparations of each nuclear type was examined and the intron− and intron+ cistron contents quantitated using a Southern transfer procedure. The number of intron− and intron+ rDNA cistrons per haploid genome in the presumed diploid 2–18 h embryo DNA was first established, and then the intron− and intron+ cistron contents of nurse cell nuclear DNA and 3 day pupal DNA were determined relative to these values.The intron− cistron content of nurse cell nuclear DNA was indistinguishable from that of embryonic DNA but the intron+ cistrons showed an 8-fold under-replication relative to the presumed diploid DNA. A slight under-representation of the intron− cistrons and 3-fold under-replication of the intron+ cistrons were demonstrated for 3 day pupal DNA. These findings strongly suggest that intron+ rDNA cistrons are non-functional in nurse cell nuclei and substantiate the generality of this implication for the whole organism during early pupal life.

Calmodulin transcription is limited to the nervous system during Drosophila embryogenesis

We have examined the RNA expression pattern for the Drosophila calmodulin gene during embryogenesis by in situ hybridization to transcripts in whole embryos. Our results indicate that maternally derived calmodulin mRNA is homogeneously distributed throughout the early embryo, but that these maternal transcripts are lost by maximal germ band extension. Zygotic transcription of the gene in mid- to late-stage embryos is restricted to neural cell precursors and their progeny in both the central and peripheral nervous systems. Thus, activation of calmodulin transcription during embryonic development appears to mark a commitment to a neural fate. Northern blot analysis revealed that the two transcripts from the calmodulin gene are differentially expressed during embryogenesis. Comparison of Northern blot and in situ hybridization data indicates that the longer calmodulin mRNA is a nervous tissue-specific transcript. This suggests that neural-specific regulation of polyadenylation site usage occurs. We have also examined calmodulin expression in embryos homozygous for mutations in four loci which are known to affect nervous system development: numb, the achaete-scute complex, daughterless, and mastermind. The calmodulin transcription pattern is altered in embryos mutant for each of these loci, suggesting that regulation by these genes, either directly or indirectly, is taking place.

Mutations in the predicted aspartyl tRNA synthetase of Drosophila are lethal and function as dosage-sensitive maternal modifiers of the sex determination gene Sex-lethal

Abstract Stable activation of the Drosophila sex determination gene Sex-lethal in the female embryo is a multistep process. Early in embryogenesis Sex-lethal is regulated at the level of transcription, and then later in embryogenesis Sex-lethal regulation switches to an autoregulatory RNA splicing mechanism. Previous studies have shown that successful activation of Sxl requires both maternally and zygotically provided gene products, many of which are essential for viability and have other, non-sex specific functions. Using a screen for dosage-sensitive modifiers we identified a new maternally expressed gene, l(2)49Db, as a likely participant in Sxl activation. We show that the establishment of the Sxl autoregulatory splicing loop, but not the earlier steps in Sxl activation, is sensitive to the maternal dosage of l(2)49Db. We further demonstrate that l(2)49Db encodes an aspartyl tRNA synthetase. Finally we present evidence that this effect is indirect, by demonstrating that mutations in tryptophanyl tRNA synthetase are also dosage-sensitive maternal modifiers of Sex-lethal. These data suggest that stable activation of Sex-lethal in the embryo may be particularly sensitive to perturbation of the translational machinery.

Calcium-binding proteins and development

The known roles for calcium-binding proteins in developmental signaling pathways are reviewed. Current information on the calcium-binding characteristics of three classes of cell-surface developmental signaling proteins (EGF-domain proteins, cadherins and integrins) is presented together with an overview of the intra-cellular pathways downstream of these surface receptors. The developmental roles delineated to date for the universal intracellular calcium sensor, calmodulin, and its targets, and for calcium-binding regulators of the cytoskeleton are also reviewed.© Kluwer Academic Publishers

Androcam, a Drosophila calmodulin-related protein, is expressed specifically in the testis and decorates loop kl-3 of the Y chromosome.

The Drosophila genome encodes a protein that is 68% identical to Drosophila calmodulin (Cam). We show here that this Cam-related gene is specifically expressed in the germ-line of the testis, leading to the name Androcam (Acam). Early in spermatogenesis Acam accumulates on one of the chromatin loops of the Y chromosome, kl-3. This association with kl-3 may indicate an RNA processing-related role for Acam and/or could reflect an unusual storage/assembly function hypothesized for the Y loops. After meiosis Acam is detectable in developing sperm tail cytoplasm, where at least some of the protein is not tightly associated with tubulin. Late in spermiogenesis, some Acam staining overlaps the periphery of the investment cones, actin-containing structures hypothesized to support the motor function for cytoplasmic stripping of the tail. Acam cannot be detected in mature sperm by immunolocalization, but immunoblotting established that Acam is present in sperm stored in mated females, suggesting epitope masking during final maturation. Proteins more related to Acam than Cam are present in the testes of other Drosophila species and a mammalian species, the mouse.