Calmodulin

Protein Interactions (part 3/3)

Miscellaneous interactions

Studies in Aplysia and Drosophila have suggested that Ca2+/calmodulin-sensitive adenylyl cyclase may act as a site of convergence for the cellular representations of the conditioned stimulus (Ca2+ influx) and unconditioned stimulus (facilitatory transmitter) during elementary associative learning. This hypothesis predicts that the rise in intracellular free Ca2+ concentration produced by spike activity during the conditioned stimulus will cause an increase in the activity of adenylyl cyclase. However, published values for the Ca2+ sensitivity of Ca2+/calmodulin-sensitive adenylyl cyclase in mammals and in Drosophila vary widely. The difficulty in evaluating whether adenylyl cyclase would be activated by physiological elevations in intracellular Ca2+ levels is in part a consequence of the use of Ca2+/EGTA buffers, which are prone to several types of errors. Using a procedure that minimizes these errors, the Ca2+ sensitivity of adenylyl cyclase in membranes from Aplysia, Drosophila, and rat brain has been quantitified with purified species-specific calmodulins. In all three species, adenylyl cyclase is activated by an increase in free Ca2+ concentration in the range caused by spike activity. Ca2+ sensitivity is dependent on both calmodulin concentration and Mg2+ concentration. Mg2+ raises the threshold for adenylyl cyclase activation by Ca2+ but also acts synergistically with Ca2+ to activate maximally adenylyl cyclase (Yovell, 1992).

For more information on the interaction of Calmodulin with adenylyl cyclase, see rutabaga.

Nitric oxide (NO) is an intercellular messenger involved in various aspects of mammalian physiology ranging from vasodilation and macrophage cytotoxicity to neuronal transmission. NO is synthesized from L-arginine by NO synthase (NOS). A Drosophila NOS gene, dNOS, located at cytological position 32B encodes a protein of 152 kDa, with 43% amino acid sequence identity to rat neuronal NOS. Like mammalian NOSs, dNOS protein contains putative binding sites for Calmodulin, FMN, FAD, and NADPH. dNOS activity is Ca2+/Calmodulin dependent when expressed in cell culture. An alternative RNA splicing pattern also exists for dNOS, which is identical to that for vertebrate neuronal NOS. These structural and functional observations demonstrate remarkable conservation of NOS between vertebrates and invertebrates (Regulski, 1995).

Genomic clones containing the full coding sequences of the two subunits of the Ca2+/Calmodulin-stimulated protein phosphatase, calcineurin, were isolated from a Drosophila genomic library using highly conserved human cDNA probes. Three clones encoded a 19.3-kDa protein whose sequence is 88% identical to that of human calcineurin B, the Ca(2+)-binding regulatory subunit of calcineurin. The coding sequences of the Drosophila and human calcineurin B genes are 69% identical. Drosophila calcineurin B is the product of a single intron-less gene located at position 4F on the X chromosome. Drosophila genomic clones encoding a highly conserved region of calcineurin A, the catalytic subunit of calcineurin, were used to locate the calcineurin A gene at position 21 EF on the second chromosome of Drosophila and to isolate calcineurin A cDNA clones from a Drosophila embryonic cDNA library. The structure of the calcineurin A gene was determined by comparison of the genomic and cDNA sequences. Twelve exons, spread over a total of 6.6 kilobases, were found to encode a 64.6-kDa protein 73% identical to either human calcineurin A alpha or beta. At the nucleotide level Drosophila calcineurin A cDNA is (respectively) 67% and 65% identical to human calcineurin A alpha and beta cDNAs. Major differences between human and Drosophila calcineurins A are restricted to the amino and carboxyl termini, including two stretches of repetitive sequences in the carboxyl-terminal third of the Drosophila molecule. Motifs characteristic of the putative catalytic centers of protein phosphatase-1 and -2A and calcineurin are almost perfectly conserved. The Calmodulin-binding and auto-inhibitory domains, characteristic of all mammalian calcineurin As, are also conserved. A remarkable feature of the calcineurin A gene is the location of the intron/exon junctions at the boundaries of the functional domains and the apparent conservation of the intron/exon junctions from Drosophila to man (Guerini, 1992).

The Drosophila Cactus and Dorsal proteins are required for the development of embryonic dorso-ventral polarity and most likelyfor the innate immune response of the insect as well. Like their mammalian counterparts (the cytoplasmic anchor protein I kappa B and the rel/NF kappa B transcription factors) cactus and dorsal are regulated at the level of nuclear localization. Increased intra-cellular calcium levels induced by the ionophore ionomycin can activate dorsal/cactus complexes in the Drosophila cell line SL2. In a cell line (SLDL) in which dorsal is expressed constitutively, ionomycin induces a rapid destruction of Cactus and dephosphorylation of Dorsal. These results suggest a role for the protein phosphatase calcineurin in calcium mediated activation of dorsal/cactus complexes. They also indicate that in the resting cell, constitutive phosphorylation of Dorsal is in equilibrium with calcium dependent dephosphorylation (Kubota, 1995).

Partial and total loss of function mutant alleles of a putative Drosophila homolog (DPhK-gamma) of the vertebrate phosphorylase kinase gamma-subunit gene have been isolated. DPhK-gamma is required in early embryonic processes, such as gastrulation and mesoderm formation; however, defects in these processes are seen only when both the maternal and zygotic components of DPhK-gamma expression are eliminated. Loss of zygotic expression alone does not appear to affect normal embryonic and larval development; some pupal lethality is observed but the majority of mutant animals eclose as adults. Many of these adults show defects in their leg musculature (e.g. missing and degenerating muscles), in addition to exhibiting melanised "tumours" on their leg joints. Loss of only the maternal component has no obvious phenotypic consequences. The DPhK-gamma gene has been cloned and sequenced. It has an open reading frame (ORF) of 1680 bp encoding a 560 amino acid protein. The predicted amino acid sequence of DPhK-gamma has two conserved domains, the catalytic kinase and Calmodulin-binding domains, separated by a linker sequence. The amino acid sequence of DPhK-gamma is homologous to that of mammalian PhK-gamma proteins but differs in the length and amino acid composition of its linker sequence. The expression of DPhK-gamma mRNA is developmentally regulated (Bahri, 1994).

GAP-43 (growth-associated protein, 43 x 10(3) M[r]) is an essential, membrane-associated, neuronal phosphoprotein in vertebrates. The protein is abundantly produced in the growth cones of developing and regenerating neurons, and it is phosphorylated upon induction of long-term potentiation (LTP). Prior work has identified GAP-43-like proteins only in chordates. In this paper, a nervous system-specific gene from Drosophila melanogaster is described that encodes two proteins sharing biochemical activities and sequence homology with GAP-43. The region of homology encompasses the Calmodulin-binding domain and protein kinase C (PKC) phosphorylation site of GAP-43. The fly proteins are shown to bind Drosophila Calmodulin (CaM), and are phosphorylated by purified PKC after a fashion predicted from prior work with vertebrate GAP-43. GAP-43 is modified by palmitoylation. An amino-terminal myristoylation site is described for the Drosophila protein, which may play a similar role in membrane association in the fly. While a small family of GAP-43-related genes has been recognized in vertebrates, only a single gene appears to be present in the fly. Since the Drosophila gene encodes two proteins, each with multiple Calmodulin-binding domains and repeated sites for PKC phosphorylation, it may illuminate functions carried out by the family of vertebrate genes (Neel, 1994).

A 3.3 kb cDNA encoding the complete amino acid sequence of a calcium/Calmodulin regulated protein phosphatase has been isolated from a Drosophila eye disc cDNA library. The predicted protein of 560 amino acids (molecular mass 62 kDa) is 73-78% identical to human PP2B isoforms. The cDNA hybridizes to the X-chromosome at cytological position 14D1-4. Two transcripts of 3.5 kb and 3.0 kb are expressed during embryonic development, their levels being highest in the early embryo. The larger transcript was also clearly present in adult females. This pattern of expression indicates a role for calcium/Calmodulin regulated protein phosphatase in embryonic development (Brown, 1994).

Drosophila A kinase anchor protein 200 (Akap200), is predicted to be involved in routing, mediating, and integrating signals carried by cAMP, Ca²⁺, and diacylglycerol. Experiments designed to assess this hypothesis establish (1) the function, boundaries and identity of critical amino acids of the protein kinase AII (PKAII) tethering site of Akap200; (2) demonstrate that residues 119-148 mediate binding with Ca²⁺-calmodulin and F-actin; (3) show that a polybasic region of Akap200 is a substrate for protein kinase C; (4) reveal that phosphorylation of the polybasic domain regulates affinity for F-actin and Ca²⁺-calmodulin, and (5) indicate that Akap200 is myristoylated and that this modification promotes targeting of Akap200 to plasma membrane. DAkap200, a second product of the Akap200 gene, cannot tether PKAII. However, DAkap200 is myristoylated and contains a phosphorylation site domain that binds Ca²⁺-calmodulin and F-actin. An atypical amino acid composition, a high level of negative charge, exceptional thermostability, unusual hydrodynamic properties, properties of the phosphorylation site domain, and a calculated M_r of 38,000 suggest that DAkap200 is a new member of the myristoylated alanine-rich C kinase substrate protein family. Akap200 is a potentially mobile, chimeric A kinase anchor protein-myristoylated alanine-rich C kinase substrate protein that may facilitate localized reception and targeted transmission of signals carried by cAMP, Ca²⁺, and diacylglycerol (Rossi, 1999).

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