Calmodulin

Transcriptional Regulation

Calmodulin expression was examined in embryos homozygous for mutations in four loci that 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 (Kovalick, 1992).

Protein Interactions (part 1/3)

This index of zygotically transcribed genes lists over 30 calcium binding and calcium dependent enzymes and proteins.

Interaction of Calmodulin with Calcineurin

Calcineurin is a Ca²⁺-calmodulin-activated, Ser-Thr protein phosphatase that is essential for the translation of Ca²⁺ signals into changes in cell function and development. A dominant modifier screen was carried out in the Drosophila eye using an activated form of Calcineurin A1 (FlyBase name: Protein phosphatase 2B at 14D), the catalytic subunit, to identify new targets, regulators, and functions of calcineurin. An examination of 70,000 mutagenized flies yielded nine specific complementation groups, four that enhanced and five that suppressed the activated calcineurin phenotype. The gene canB2, which encodes the essential regulatory subunit of calcineurin, was identified as a suppressor group, demonstrating that the screen was capable of identifying genes relevant to calcineurin function. A second suppressor group was sprouty, a negative regulator of receptor tyrosine kinase signaling. Wing and eye phenotypes of ectopic activated calcineurin and genetic interactions with components of signaling pathways have suggested a role for calcineurin in repressing Egf receptor/Ras signal transduction. On the basis of these results, it is proposed that calcineurin, upon activation by Ca²⁺-calmodulin, cooperates with other factors to negatively regulate Egf receptor signaling at the level of Sprouty and the GTPase-activating protein Gap1 (Sullivan, 2002).

Calcineurin is activated by a sustained increase in intracellular Ca²⁺ levels that can result from the opening of intracellular Ca²⁺ channels in response to phosphoinositide (PI) signaling. PI signaling is initiated by the activation of a phosphatidylinositol-specific phospholipase C, either PLCß by G-protein-coupled receptors (GPCR) or PLCgamma by receptor tyrosine kinases (RTK). PI-PLCs cleave phosphatidylinositol 4,5-bisphosphate (PIP₂) to yield inositol 1,4,5-trisphosphate (InsP₃), which then activates the InsP₃ receptor Ca²⁺ channel (Sullivan, 2002).

An activated form of Pp2B-14D, canA^act, was made by deleting the autoinhibitory and calmodulin-binding domains. The canA^act construct was expressed in Drosophila under the control of glass response elements, which induce transcription uniformly in cells posterior to the morphogenetic furrow in the eye imaginal disc (Sullivan, 2002).

Flies carrying one copy of the canA^act.gl transgene have mild rough eyes compared to wild type, and the eyes of flies carrying two copies exhibit a stronger phenotype. Consistent with observations in other systems, neither full-length CanA nor activated canA without a functional CanB-binding domain causes any detectable phenotypes when expressed throughout development (Sullivan, 2002).

Interaction of Calmodulin with CP309 (Pericentrin-like protein)

The centrosome in animal cells provides a major microtubule-nucleating site that regulates the microtubule cytoskeleton temporally and spatially throughout the cell cycle. A large coiled-coil centrosome protein identified in Drosophila can bind to calmodulin. Biochemical studies reveal that this novel centrosome protein, centrosome protein of 309 kDa (Cp309), cofractionates with the gamma-tubulin ring complex and the centrosome-complementing activity. CP309 is required for microtubule nucleation mediated by centrosomes and it interacts with the gamma-tubulin small complex. These findings suggest that the microtubule-nucleating activity of the centrosome requires the function of CP309 (Kawaguchi, 2004).

Because CP309 contains a CaM-binding motif, whether CP309 can bind to CaM was examined along with whether this binding is Ca2+ dependent. CaM-agarose beads were incubated with Drosophila embryo extracts in the presence of 2.5 mM Ca2+ or 5 mM EGTA (EGTA was used to chelate the Ca2+ in the extracts). Proteins bound to the CaM agarose beads were analyzed by Western blotting, probing with antibodies against CP309. It was found that significantly more CP309 in the extracts bound to CaM-agarose beads in the presence of Ca2+ than in the presence EGTA. Next, it was asked whether Ca2+ facilitates CaM-free CP309 binding to CaM. CaM-free CP309 was prepared by eluting CP309 from CaM-agarose beads with EGTA and then used in the same binding assay as described above. It was found that the CaM-free CP309 also binds to CaM-agarose beads more efficiently in the presence of Ca2+ than in the presence of EGTA. Therefore, although CP309 can bind to CaM in the absence of Ca2+, Ca2+ significantly enhances the binding (Kawaguchi, 2004).

Interaction of Calmodulin with kinases

For information on the interaction of Calmodulin with Calcium/calmodulin dependent protein kinase II, see CaMKII.

For information on the interaction of Calmodulin with the Regulatory light chain of myosin II (known in Drosophila as Spaghetti squash, or MRLC).

Calcium/calmodulin-dependent protein kinases (CaM kinases) have been reported to be involved in neuroplasticity. A new Drosophila CaM kinase gene has been cloned, named caki. The caki gene is extremely large; comparison of the genomic and cDNA sequences reveals that the caki transcription unit is at least 150 kb. The catalytic domain of this new CaM kinase protein shares homology (41%) with type II CaM kinases, while the C-terminal part is divergent. Constitutively expressed Caki protein is enzymatically active since it causes a 3-fold increase in the level of the Rous sarcoma virus long terminal repeat (RSV LTR) promoter in a co-transfusion assay. In situ hybridization shows that during embryogenesis, larval and pupal life, transcription of caki is restricted almost exclusively to the central nervous system. In the adult head, immunohistochemistry reveals Caki protein in the lamina, the neuropil of the medulla, lobula, lobula plate and in the central brain. Mutant caki flies show reduced walking speed in 'Buridan's paradigm' (Martin, 1996).

Casein kinase II (CKII) is composed of a catalytic subunit (alpha) and a regulatory subunit (beta) that combine to form an alpha 2 beta 2 holoenzyme. The alpha-subunit monomer is enzymatically active, albeit kinetically attenuated relative to the holoenzyme; the addition of purified beta subunit stimulates its activity against casein. A kinetic analysis was performed of the phosphorylation of various protein and peptide substrates by the alpha subunit and the holoenzyme of Drosophila CKII. The alpha subunit, like the holoenzyme, is competent to phosphorylate typical physiological substrates such as the regulatory (RII) subunit of cAMP-dependent protein kinase (cAMPdPK), as well as artificial substrates such as alpha-casein and the synthetic peptide RRREEETEEE. The Km of the alpha subunit in each case is similar to that of the holoenzyme, whereas the Vmax is 5- to 60-fold lower. In contrast, Calmodulin, a protein that is significantly phosphorylated by the holoenzyme only in the presence of polybasic compounds, is readily phosphorylated by the alpha subunit alone. While the Km values of the alpha subunit and the holoenzyme for Calmodulin are similar, the Vmax of the alpha subunit is at least 10-fold higher than that of the holoenzyme. These results suggest that while the alpha subunit contains the necessary determinants for CKII substrate specificity, the beta subunit can either inhibit or activate it, in a substrate-dependent manner. Polybasic compounds stimulate not only the holoenzyme but, to a lesser extent, the alpha subunit as well (Bidwai, 1993).

Calcium/calmodulin-dependent protein kinase II (CaMKII) is abundant in the CNS and is crucial for cellular and behavioral plasticity. It is thought that the ability of CaMKII to autophosphorylate and become Ca2+ independent allows it to act as a molecular memory switch. Inhibition of Drosophila CaMKII leads to impaired performance in the courtship conditioning associative memory assay, but it was unknown whether the constitutive form of the kinase had a special role in learning. In this study, a tripartite transgenic system combining GAL4/UAS with the tetracycline-off system was used to spatially and temporally manipulate levels of Ca2+-independent CaMKII activity in Drosophila. An enhancement of information processing during the training period was found with Ca2+-independent, but not Ca2+-dependent, CaMKII. During training, control animals have a lag before active suppression of courtship begins. Animals expressing Ca2+-independent CaMKII have no lag, implying that there is a threshold level of Ca2+-independent activity that must be present to suppress courtship. This is the first demonstration, in any organism, of enhanced behavioral plasticity with overexpression of constitutively active CaMKII. Anatomical studies indicate that transgene expression in antennal lobes and extrinsic mushroom body neurons drives this behavioral enhancement. Interestingly, immediate memory was unaffected by expression of T287D CaMKII in mushroom bodies, although previous studies have shown that CaMKII activity is required in this brain region for memory formation. These results suggest that the biochemical mechanisms of CaMKII-dependent memory formation are threshold based in only a subset of neurons (Mehren, 2004; full text or article).

Interaction of Calmodulin with ion channels

Two putative light-sensitive ion channels have been isolated from Drosophila, encoded by the transient-receptor-potential (trp) and transient-receptor-potential-like (trpl ) genes. The cDNA encoding the Trpl protein was initially isolated on the basis that the expressed protein binds Calmodulin. Two Calmodulin-binding sites are present in the C-terminal domain of the Trpl protein: CBS-1 and CBS-2. CBS-1 binds Calmodulin in a Ca2+-dependent fashion, requiring Ca2+ concentrations above 0.3-0.5 microM for Calmodulin binding. In contrast, CBS-2 binds the Ca2+-free form of Calmodulin, with dissociation occurring at Ca2+ concentrations between 5 and 25 microM. Phosphorylation of a serine residue within a peptide encompassing CBS-1 by cyclic AMP-dependent protein kinase (PKA) abolishes Calmodulin binding, and phosphorylation of the adjacent serine by protein kinase C appears to modulate this phosphorylation by PKA. Interpretation of these findings provides a novel model for ion-channel gating and modulation in response to changing levels of intracellular Ca2+ (Warr, 1996).

The effects of expression of the Drosophila Trpl protein, which is thought to encode a putative Ca2+ channel, on divalent cation inflow in Xenopus laevis oocytes were investigated. The addition of extracellular Ca2+ to oocytes injected with TRPL cRNA and to mock-injected controls, both loaded with the fluorescent Ca2+ indicator fluo-3, induces a rapid initial and a slower sustained rate of increase in fluorescence, which are respectively designated the initial and sustained rates of Ca2+ inflow. Compared with mock-injected oocytes, TRPL-cRNA-injected oocytes exhibit a higher resting cytoplasmic free Ca2+ concentration, and higher initial and sustained rates of Ca2+ inflow in the basal (no agonist) states. The basal rate of Ca2+ inflow in TRPL-cRNA-injected oocytes increases with (1) an increase in the time elapsed between injection of TRPL cRNA and the measurement of Ca2+ inflow, (2) an increase in the amount of TRPL cRNA injected and (3) an increase in Ca2+. A GTP antagonist inhibits the trpl cRNA-induced basal rate of Ca2+ inflow. Expression of TRPL cRNA also causes an increase in the basal rate of Mn2+ inflow. The increases in resting Ca2+ and in the basal rate of Ca2+ inflow induced by expression of TRPL cRNA are inhibited by the Calmodulin inhibitors W13, calmodazolium and peptide (amino acids 281-309) of CaM kinase II. A low concentration of exogenous Calmodulin (introduced by microinjection) activates, and a higher concentration inhibits, the TRPL cRNA-induced increase in basal rate of Ca2+ inflow. The action of the high concentration of exogenous Calmodulin is reversed by W13 and calmodazolium. When rates of Ca2+ inflow in TRPL-cRNA-injected oocytes are compared with those in mock-injected oocytes, the guanosine 5'-[beta-thio]diphosphate-stimulated rate is greater, the onset of thapsigargin-stimulated initial rate somewhat delayed and the inositol 1,4,5-trisphosphate-stimulated initial rate markedly inhibited. It is concluded that (1) the divalent cation channel activity of the Drosophila Trpl protein can be detected in Xenopus oocytes; (2) in the environment of the Xenopus oocyte the Trpl channel admits some Mn2+ as well as Ca2+, and is activated by cytoplasmic free Ca2+ (through endogenous Calmodulin) and by a trimeric GTP-binding regulatory protein, but does not appear to be activated by depletion of Ca2+ in the endoplasmic reticulum, and (3) expression of the Trpl protein inhibits the process by which the release of Ca2+ from intracellular stores activates endogenous store-activated Ca2+ channels (Lan, 1996).

The role of the ether a go-go (eag) gene was examined in modulation of K+ currents. The possibility that its protein product Eag is a subunit in the heteromultimeric assembly of K+ channels was examined by voltage-clamp analysis of larval muscle membrane currents. Previous DNA sequence studies indicate that the eag gene codes for a polypeptide homologous to, but distinct from, the Shaker (Sh) K+ channel subunits, and electrophysiological recordings reveal allele-specific effects of eag on four identified K+ currents in Drosophila larval muscles. Further studies of eag alleles indicate that none of the eag mutations, including alleles producing truncated mRNA messages, eliminate any of the four K+ currents, and that the mutational effects exhibit strong temperature dependence. Both W7, an antagonist of Ca2+/Calmodulin, and cGMP analogs modulate K+ currents; their actions are altered or even abolished by eag mutations. These results suggest a role of eag in modulation of K+ currents that may subserve integration of signals at a converging site of the two independent modulatory pathways. The Sh locus is known to encode certain subunits of the IA channel in larval muscle. The existence of multiple eag and Sh alleles enables an independent test of the idea of Eag as a K+ channel subunit by studying IA in different double-mutant combinations. An array of allele-specific interactions between eag and Sh has been observed, reflecting a close association between the Sh and eag subunits within the IA channel. Taken together, these data strengthen the possibility that the eag locus provides a subunit common to different K+ channels. The role of the eag subunit for modulating channels, as opposed to that of Sh subunits required for gating, selectivity, and conductance of the channel, suggest a combinatorial genetic framework for generating diversified K+ channels (Zhong, 1993).

Calmodulin interactions in the Drosophila retina

Continued: see Calmodulin Protein interactions part 2/3 | part 3/3

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.