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K Beckingham

Publications and source records attributed to K Beckingham.

At least 19 recordsLinked to original sources
PubMed2000

bicaudal encodes the Drosophila beta NAC homolog, a component of the ribosomal translational machinery*.

bicaudal was the first Drosophila mutation identified as producing mirror-image pattern duplications along the anteroposterior axis of the embryo. However the mutation has been little studied due to its low penetrance and suppressibility. We undertook cloning of the bicaudal locus together with studies of the mutation's effects on key elements of the posterior embryonic patterning pathway. Our mapping studies place the bicaudal mutation within a approximately 2 kb region, 3' to the protein coding sequence of the Drosophila homolog of beta NAC, a subunit of Nascent polypeptide Associated Complex (NAC). Genomic DNA encoding beta NAC completely rescues the bicaudal phenotype. The lethal phenotype of Enhancer of Bicaudal, E(Bic), a mutation hypothesized to affect the bicaudal locus, is also completely rescued by the beta NAC locus. We further demonstrate that the E(Bic) mutation is caused by a P element insertion into the transcribed region of the beta NAC gene. NAC is among the first ribosome-associated entities to bind the nascent polypeptide after peptide bond formation. In contrast to other bicaudal-embryo-producing mutations, bicaudal leads to ectopic translation of mRNA for the posterior determinant nanos, without affecting the localization of mRNA for its upstream regulator, oskar, in the embryo. These findings suggest that repression of nanos mRNA translation occurs on the ribosome and involves a role for beta NAC.

Amino Acid Sequence↗
PubMed1999

Autonomous control of cell and organ size by CHICO, a Drosophila homolog of vertebrate IRS1-4.

The control of growth is fundamental to the developing metazoan. Here, we show that CHICO, a Drosophila homolog of vertebrate IRS1-4, plays an essential role in the control of cell size and growth. Animals mutant for chico are less than half the size of wild-type flies, owing to fewer and smaller cells. In mosaic animals, chico homozygous cells grow slower than their heterozygous siblings, show an autonomous reduction in cell size, and form organs of reduced size. Although chico flies are smaller, they show an almost 2-fold increase in lipid levels. The similarities of the growth defects caused by mutations in chico and the insulin receptor gene in Drosophila and by perturbations of the insulin/IGF1 signaling pathway in vertebrates suggest that this pathway plays a conserved role in the regulation of overall growth by controling cell size, cell number, and metabolism.

Amino Acid Sequence↗
PubMed1999

Calmodulin mediates calcium-dependent activation of the intermediate conductance KCa channel, IKCa1.

Small and intermediate conductance Ca2+-activated K+ channels play a crucial role in hyperpolarizing the membrane potential of excitable and nonexcitable cells. These channels are exquisitely sensitive to cytoplasmic Ca2+, yet their protein-coding regions do not contain consensus Ca2+-binding motifs. We investigated the involvement of an accessory protein in the Ca2+-dependent gating of hIKCa1, a human intermediate conductance channel expressed in peripheral tissues. Cal- modulin was found to interact strongly with the cytoplasmic carboxyl (C)-tail of hIKCa1 in a yeast two-hybrid system. Deletion analyses defined a requirement for the first 62 amino acids of the C-tail, and the binding of calmodulin to this region did not require Ca2+. The C-tail of hSKCa3, a human neuronal small conductance channel, also bound calmodulin, whereas that of a voltage-gated K+ channel, mKv1.3, did not. Calmodulin co-precipitated with the channel in cell lines transfected with hIKCa1, but not with mKv1. 3-transfected lines. A mutant calmodulin, defective in Ca2+ sensing but retaining binding to the channel, dramatically reduced current amplitudes when co-expressed with hIKCa1 in mammalian cells. Co-expression with varying amounts of wild-type and mutant calmodulin resulted in a dominant-negative suppression of current, consistent with four calmodulin molecules being associated with the channel. Taken together, our results suggest that Ca2+-calmodulin-induced conformational changes in all four subunits are necessary for the channel to open.

Amino Acid Sequence↗
PubMed1999

Conformational and metal-binding properties of androcam, a testis-specific, calmodulin-related protein from Drosophila.

Androcam is a testis-specific protein of Drosophila melanogaster, with 67% sequence identity to calmodulin and four potential EF-hand calcium-binding sites. Spectroscopic monitoring of the thermal unfolding of recombinant calcium-free androcam shows a biphasic process characteristic of a two-domain protein, with the apo-N-domain less stable than the apo-C-domain. The two EF hands of the C-domain of androcam bind calcium cooperatively with 40-fold higher average affinity than the corresponding calmodulin sites. Magnesium competes with calcium binding [Ka(Mg) approximately 3 x 10(3) M(-1)]. Weak calcium binding is also detected at one or more N-domain sites. Compared to apo-calmodulin, apo-androcam has a smaller conformational response to calcium and a lower alpha-helical content over a range of experimental conditions. Unlike calmodulin, a tryptic cleavage site in the N-domain of apo-androcam remains trypsin sensitive in the presence of calcium, suggesting an altered calcium-dependent conformational change in this domain. The affinity of model target peptides for androcam is 10(3)-10(5) times lower than for calmodulin, and interaction of the N-domain of androcam with these peptides is significantly reduced. Thus, androcam shows calcium-induced conformational responses typical of a calcium sensor, but its properties indicate calcium sensitivity and target interactions significantly different from those of calmodulin. From the sequence differences and the altered calcium-binding properties it is likely that androcam differs from calmodulin in the conformation of residues in the second calcium-binding loop. Molecular modeling supports the deduction that there are significant conformational differences in the N-domain of androcam compared to calmodulin, and that these could affect the surface, conferring a different specificity on androcam in target interactions related to testis-specific calcium signaling functions.

Amino Acid Sequence↗
PubMed1998

Calmodulin binding to myosin light chain kinase begins at substoichiometric Ca2+ concentrations: a small-angle scattering study of binding and conformational transitions.

We have used small-angle scattering to study the calcium dependence of the interactions between calmodulin (CaM) and skeletal muscle myosin light chain kinase (MLCK), as well as the conformations of the complexes that form. Scattering data were measured from equimolar mixtures of a functional MLCK and CaM or a mutated CaM (B12QCaM) incompetent to bind Ca2+ in its N-terminal domain, with increasing Ca2+ concentrations. To evaluate differences between CaM-enzyme versus CaM-peptide interactions, similar Ca2+ titration experiments were performed using synthetic peptides based on the CaM-binding sequence from MLCK (MLCK-I). Our data show there are different determinants for CaM binding the isolated peptide sequence compared to CaM binding to the same sequences within the enzyme. For example, binding of either CaM or B12QCaM to the MLCK-I peptide is observed even in the presence of EGTA, whereas binding of CaM to the enzyme requires Ca2+. The peptide studies also show that the conformational collapse of CaM requires both the N and C domains of CaM to be competent for Ca2+ binding as well as interactions with each end of MLCK-I, and it occurs at approximately 2 mol of Ca2+/mol of CaM. We show that CaM binding to the MLCK enzyme begins at substoichiometric concentrations of Ca2+ (< or = 2 mol of Ca2+/mol of CaM), but that the final compact structure of CaM with the enzyme requires saturating Ca2+. In addition, MLCK enzyme does bind to 2Ca2+ x B12QCaM, although this complex is more extended than the complex with native CaM. Our results support the hypothesis that CaM regulation of MLCK involves an initial binding step at less than saturating Ca2+ concentrations and a subsequent activation step at higher Ca2+ concentrations.

Amino Acid Sequence↗
PubMed1998

High affinity Ca2+ binding sites of calmodulin are critical for the regulation of myosin Ibeta motor function.

We coexpressed myosin Ibeta heavy chain with three different calmodulin mutants in which the two Ca2+-binding sites of the two N-terminal domain (E12Q), C-terminal domain (E34Q), or all four sites (E1234Q) are mutated in order to define the importance of these Ca2+ binding sites to the regulation of myosin Ibeta. The calmodulin mutated at the two Ca2+ binding sites in N-terminal domain and C-terminal domain lost its lower affinity Ca2+ binding site and higher affinity Ca2+ binding site, respectively. We found that, based upon the change in the actin-activated ATPase activities and actin translocating activities, myosin Ibeta with E12Q calmodulin has the regulatory characteristics similar to myosin Ibeta containing wild-type calmodulin, while myosin Ibeta with E34Q or E1234Q calmodulin lose all Ca2+ regulation. While the increase in myosin Ibeta ATPase activity paralleled the dissociation of 1 mol of calmodulin from myosin Ibeta heavy chain for both wild type (above pCa 5) and E12Q calmodulin (above pCa 6), the Ca2+ level required for the inhibition of actin-translocating activity of myosin Ibeta was lower than that required for dissociation of calmodulin, suggesting that the conformational change induced by the binding of Ca2+ at the high affinity site but not the dissociation of calmodulin is critical for the inhibition of the motor activity. Our results suggest that the regulation of unconventional myosins by Ca2+ is directly mediated by the Ca2+ binding to calmodulin, and that the C-terminal pair of Ca2+-binding sites are critical for this regulation.

Actins↗
PubMed1998

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 intracellular 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.

Animals↗
PubMed1998

Increased transmitter release and aberrant synapse morphology in a Drosophila calmodulin mutant.

The ubiquitous calcium-binding protein calmodulin (CaM) has been implicated in the development and function of the nervous system in a variety of eukaryotic organisms. We have generated mutations in the single Drosophila Calmodulin (Cam) gene and examined the effects of these mutations on behavior, synaptic transmission at the larval neuromuscular junction, and structure of the larval motor nerve terminal. Flies hemizygous for Cam3c1, a mutation in the first Ca2+-binding site, exhibit behavioral, neurophysiological, and neuroanatomical abnormalities. In particular, adults exhibit defects in locomotion, coordination, and flight. Larvae exhibit increased neurotransmitter release from the motor nerve terminal at low [Ca2+] in the presence of the K+ channel-blocking drug quinidine. In addition, synaptic bouton structure at motor nerve terminals is altered. These effects are distinct from those produced by altering the activity of the CaM target enzymes CaM-activated kinase II (CaMKII) and CaM-activated adenylyl cyclase (CaMAC). Furthermore, previous in vitro studies of mutant Cam3c1 demonstrated that although its Ca2+ affinity is decreased, Cam3c1 protein can activate CaMKII, CaMAC, and CaM-activated phosphatase calcineurin in a manner similar to wild-type CaM. Thus, the Cam3c1 mutation might affect Ca2+ buffering or interfere with the activation or inhibition of a CaM target distinct from CaMKII or CaMAC.

Amino Acid Sequence↗
PubMed1997

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.

Animals↗
PubMed1997

Calcium binding sites of calmodulin and electron transfer by neuronal nitric oxide synthase.

Although binding of calmodulin (CaM) to neuronal nitric oxide synthase (nNOS) has been demonstrated to act as the trigger necessary for electron transfer and catalytic activity, the exact manner in which this is achieved is unclear. By using a series of single point mutants of Drosophila melanogaster CaM, the role that each Ca2+ binding site plays in the transfer of electrons within nNOS has been examined. In these mutants, the bidentate glutamic acid (E) residue which coordinates Ca2+ at the -Z position in each site has been mutated to a glutamine (Q), preventing Ca2+ binding at that site. The results demonstrate that Ca2+ binding at site I of CaM is critical for all electron transfer reactions. All nNOS activities measured (citrulline formation, NADPH oxidation, and cytochrome c reduction) in the presence of the site I CaM mutant (denoted B1Q) were only 2% of the nNOS activity with wild-type CaM. The B2Q and B4Q mutants activated nNOS to similar levels. These two mutants, however, affected nNOS heme-dependent activities to a greater extent than they affected activities independent of the nNOS heme. The site III CaM mutant (B3Q) activated nNOS to levels similar to activities measured with wild-type CaM. Rates of formation of the ferrous-CO complex were also obtained with each of the mutant CaMs. The relative binding affinities of these mutants do not correlate with the observed differences in electron transfer rates. These results demonstrate that, although binding of CaM to nNOS is necessary for catalysis, specific interactions between the two proteins exist which are required for efficient electron transfer.

Amino Acid Sequence↗
PubMed1997

Calmodulin point mutations affect Drosophila development and behavior.

Calmodulin (CAM) is recognized as a major intermediary in intracellular calcium signaling, but as yet little is known of its role in developmental and behavioral processes. We have generated and studied mutations to the endogenous Cam gene of Drosophila melanogaster that change single amino acids within the protein coding region. One of these mutations produces a striking pupal lethal phenotype involving failure of head eversion. Various mutant combinations produce specific patterns of ectopic wing vein formation or melanotic scabs on the cuticle. Anaphase chromosome bridging is also seen as a maternal effect during the early embryonic nuclear divisions. In addition, specific behavioral defects such as poor climbing and flightlessness are detected among these mutants. Comparisons with other Drosophila mutant phenotypes suggests potential CAM targets that may mediate these developmental and behavioral effects, and analysis of the CAM crystal structure suggests the structural consequences of the individual mutations.

Alleles↗
PubMed1996

Characterization of Eu(III) binding to a series of calmodulin binding site mutants using laser-induced Eu(III) luminescence spectroscopy.

Laser-induced luminescence techniques were used in a rigorous evaluation of the Eu(3+)-binding behavior of a recombinant (Drosophila melanogaster) calmodulin and a series of calmodulin binding site mutants in which the bidentate glutamic acid residue in position 12 of each metal ion binding loop is systematically replaced with lysine. For the range of Ca2+ concentrations at which calmodulin functions (10(-5)-10(-6) M), Ca2+ binding is effectively eliminated at the mutated site; however, the luminescence studies show that the Eu3+ ion binds to the modified site with reduced affinity. The mutations do not significantly change the intermetal ion distances from their wild type values. These were determined by Eu3+-->Nd3+ Förster-type non-radiative energy transfer experiments. Consistent with the results of Ca(2+)-binding studies, mutation of sites II and IV in the N- and C-terminal domains, respectively, produces a larger alteration in the Eu(3+)-luminescence and Eu(3+)-binding behavior than does mutation of sites I and III. Modification of either of the sites in the C-terminus (III or IV, numbered from the amino terminus) causes two additional H2O molecules (four H2O molecules total) to bind to the Eu3+ ion in order to compensate for the loss of the bidentate glutamic acid residue. Consequently, the partner site in the domain loses an H2O molecule, thereby coordinating another ligand from the protein. Mutation of either of the high-affinity Ln(3+)-binding sites (I or II) has global effects on the Eu(3+)-binding behavior of the protein molecule.

Animals↗
PubMed1996

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.

Alternative Splicing↗
PubMed1996

Spontaneous avoidance behavior in Drosophila null for calmodulin expression.

The regulatory protein calmodulin is a major mediator of calcium-induced changes in cellular activity. To analyze the roles of calmodulin in an intact animal, we have generated a calmodulin null mutation in Drosophila melanogaster. Maternal calmodulin supports calmodulin null individuals throughout embryogenesis, but they die within 2 days of hatching as first instar larvae. We have detected two pronounced behavioral abnormalities specific to the loss of calmodulin in these larvae. Swinging of the head and anterior body, which occurs in the presence of food, is three times more frequent in the null animals. More strikingly, most locomotion in calmodulin null larvae is spontaneous backward movement. This is in marked contrast to the wild-type situation where backward locomotion is seen only as a stimulus-elicited avoidance response. Our finding of spontaneous avoidance behavior has striking similarities to the enhanced avoidance responses produced by some calmodulin mutations in Paramecium. Thus our results suggest evolutionary conservation of a role for calmodulin in membrane excitability and linked behavioral responses.

Animals↗
PubMed1996

Interlobe communication in multiple calcium-binding site mutants of Drosophila calmodulin.

We have generated mutants of Drosophila calmodulin in which pairs of calcium-binding sites are mutated so as to prevent calcium binding. In all sites, the mutation involves replacement of the -Z position glutamate residue with glutamine. Mutants inactivated in both N-terminal sites (B12Q) or both C-terminal sites (B34Q), and two mutants with one N- and one C-terminal site inactivated (B13Q and B24Q) were generated. The quadruple mutant with all four sites mutated was also studied. UV-difference spectroscopy and near-UV CD were used to examine the influence of these mutations upon the single tyrosine (Tyr-138) of the protein. These studies uncovered four situations in which Tyr-138 in the C-terminal lobe responds to a change to the calcium-binding properties of the N-terminal lobe. Further, they suggest that N-terminal calcium-binding events contribute strongly to the aberrant behavior of Tyr-138 seen in mutants with a single functional C-terminal calcium-binding site. The data also indicate that loss of calcium binding at site 1 adjusts the aberrant conformation of Tyr-138 produced by mutation of site 3 toward the wild-type structure. However, activation studies for skeletal muscle myosin light chain kinase (SK-MLCK) established that all of the multiple binding site mutants are poor activators of SK-MLCK. Thus, globally, the calcium-induced conformation of B13Q is not closer to wild type than that of either the site 1 or the site 3 mutant. The positioning of Tyr-138 within the crystal structure of calmodulin suggests that effects of the N-terminal lobe on this residue may be mediated via changes to the central linker region of the protein.

Animals↗
PubMed1995

Recovery of native structure by calcium binding site mutants of calmodulin upon binding of sk-MLCK target peptides.

The calcium-dependent binding of two synthetic 18-residue peptides derived from the calmodulin binding region of skeletal myosin light chain kinase to wild-type Drosophila melanogaster calmodulin and four calcium binding site calmodulin mutants has been investigated using optical spectroscopy. The WFF peptide (with W4 and F17) and the FFW peptide (with F4 and W17) both bind to wild-type calmodulin with 1:1 stoichiometry and Kd values of < or = 0.2 and 1.6 nM, respectively. Near-UV CD spectra of the protein-peptide complexes suggest that both peptides bind in the same orientation, with the side chain of residue 4 interacting with the C-domain of calmodulin and that of residue 17 with the N-domain [as in the structure of the calmodulin-M13 peptide complex determined by Ikura et al. [Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, C. B., & Bax, A. (1992) Science 256, 632-638]]. Both peptides have lower affinities for all the mutant calmodulins than for the wild-type protein. Fluorescence measurements suggest that mutation of calcium binding site 2 in the N-domain does not affect the interaction of the W4 side chain of the WFF peptide with the C-domain of calmodulin. However, the E67Q (B2Q) but not the E67K (B2K) mutation (site 2, N-domain) alters the interaction of W17 of the FFW peptide with the protein. In contrast, the E140K (B4K) mutation has a much greater effect than the E140Q (B4Q) mutation (site 4, C-domain) on the interaction of calmodulin with both peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Activation of four enzymes by two series of calmodulin mutants with point mutations in individual Ca2+ binding sites.

Activation of four target enzymes by two series of calmodulin Ca2+ binding site mutants has been examined. In each mutant, the conserved bidentate glutamate of one of the Ca2+ binding sites is mutated to glutamine or lysine. The enzymes studied were smooth and skeletal muscle myosin light chain kinases, adenylylcyclase, and plasma membrane Ca(2+)-ATPase. For the first three enzymes, the activation patterns with the two mutant series were very similar: mutation of site 4 was most deleterious, then site 2, site 3, and site 1. This ranking was observed previously in Ca2+ binding and Ca(2+)-induced conformational studies of these mutants. Thus the response of these enzymes is probably determined by the extent to which each mutant's competence to interact with target binding regions has been compromised. In contrast, for Ca(2+)-ATPase, mutants of sites 3 and 4 were much poorer activators than those of sites 1 and 2. Events beyond calmodulin binding and related to enzyme activation probably dictate this unusual activation pattern and also the anomalously poor activation of skeletal muscle myosin light chain kinase by site 1 mutant B1Q. Site 1 mutant B1K showed wild type activation of all four enzymes suggesting that in site 1, the lysine substitution can evoke the conformational changes associated with Ca2+ binding.

Adenylyl Cyclases↗