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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Multiple endogenous substrates phosphorylated by four distinct protein kinases were identified in particulate and cytosolic fractions from the larval prothoracic gland of the tobacco hornworm, Manduca sexta. Three prominent particulate-associated phosphoprotein substrates (19, 21, and 34 kDa) were of particular interest. The in vitro phosphorylation of the 19 and 21 kDa peptides was markedly enhanced by cAMP, Ca2+/calmodulin, as well as Ca2+/phospholipids, presumably via cAMP-dependent protein kinase (cAMP-PK), Ca2+/calmodulin-dependent protein kinase (Ca2+/CaM-PK), and protein kinase C (PKC), respectively. The polyamine spermine markedly inhibits both PKC- and cAMP-PK-mediated phosphorylation of the 19 and 21 kDa peptides but had no effect on the Ca2+/CaMP-PK-mediated phosphorylation. Spermine also inhibits the phosphorylation of the 34 kDa peptide via cAMP-PK but does not affect PKC-promoted phosphorylation. In contrast to this differential inhibition of phosphorylation by a polyamine, four cytosolic and three particulate-associated peptides from the prothoracic glands undergo enhanced phosphorylation in the presence of spermine, presumably by stimulating casein kinase II activity. Therefore, polyamines appear to have multiple effects on protein phosphorylation pathways in this important endocrine gland, perhaps representing an important new regulatory control mechanism.
Mol Cell Endocrinol 1992 Jan
PMID:Polyamines modulate multiple protein phosphorylation pathways in the insect prothoracic gland. 155 68

The crystal structure of a sarcoplasmic Ca(2+)-binding protein (SCP) from the sandworm Nereis diversicolor has been determined and refined at 2.0 A resolution using restrained least-squares techniques. The two molecules in the crystallographic asymmetric unit, which are related by a non-crystallographic 2-fold axis, were refined independently. The refined model includes all 174 residues and three calcium ions for each molecule, as well as 213 water molecules. The root-mean-square difference in co-ordinates for backbone atoms and calcium ions of the two molecules is 0.51 A. The final crystallographic R-factor, based on 18,959 reflections in the range 2.0 A less than or equal to d less than or equal to 7.0 A, with intensities exceeding 2.0 sigma, is 0.182. Bond lengths and bond angles in the molecules have root-mean-square deviations from ideal values of 0.013 A and 2.2 degrees, respectively. SCP has four distinct domains with the typical helix-loop-helix (EF-hand) Ca(2+)-binding motif, although the second Ca(2+)-binding domain is not functional due to amino acid changes in the loop. The structure shows several unique features compared to other Ca(2+)-binding proteins with four EF-hand domains. The overall structure is highly compact and globular with a predominant hydrophobic core, unlike the extended dumbbell-shaped structure of calmodulin or troponin C. A hydrophobic tail at the COOH terminus adds to the structural stability by packing against a hydrophobic pocket created by the folding of the NH2 and COOH-terminal Ca(2+)-binding domain pairs. The first and second domains show different helix-packing arrangements from any previously described for Ca(2+)-binding proteins.
J Mol Biol 1992 Mar 20
PMID:Structure of a sarcoplasmic calcium-binding protein from Nereis diversicolor refined at 2.0 A resolution. 156 Apr 59

The present study was undertaken to systematically purify calcium binding proteins (CaBPs) from homogenates of Trypanosoma brucei. This work is important since CaBPs either serve as intracellular calcium buffers or mediate cellular response to calcium signals. Disruption of either process should be lethal to trypanosomes. We report that the 45Ca-gel overlay assay can be used to detect CaBPs following fractionation on DE-52, phenyl-Sepharose, Mono-Q, and Superose 12. Specific CaBPs of 22, 24, and 38 kDa were purified. Each of these proteins associated with 45Ca under denaturing and non-denaturing conditions. An approximate Kd for calcium of 8 microM was calculated for 22-kDa CaBP. None of the trypanosome CaBPs were related to known calcium binding protein families. They did not associate with hydrophobic interaction columns or cellular membranes in a calcium-dependent way, nor cross-react with 2 separate antibodies against annexin consensus sequences. A synthetic peptide corresponding to amino terminal residues 16-30 of 22-kDa CaBP was used to generate polyclonal antibodies. Immunoblots identified 22-kDa CaBP in African trypanosomes but not in other Kinetoplastidae or mammalian cells. Nonetheless, significant homology (58%) was observed between the amino terminal 37 residues of 22-kDa CaBP and the amino terminus of translationally controlled p21 from mammalian tumor cells. The present study is the first to apply systemic fractionation techniques to identify the complement of CaBPs in T. brucei. We conclude that novel CaBPs other than calmodulin and annexin family members contribute towards calcium pathways in these organisms.
Mol Biochem Parasitol 1992 Mar
PMID:Purification of novel calcium binding proteins from Trypanosoma brucei: properties of 22-, 24- and 38-kilodalton proteins. 156 42

A novel gene fusion approach which may be of more general use has been developed for investigating the function of calmodulin in the budding yeast Saccharomyces cerevisiae. By fusing a portion of the Staphylococcus aureus spa gene (encoding protein A) to CMD1, the S. cerevisiae gene encoding calmodulin, we have generated a yeast calmodulin with an affinity tag able to bind immunoglobulins. The chimaeric protein A-calmodulin (ProtA-CaM) polypeptide functions in vivo and shows Ca(2+)-dependent binding to calmodulin target proteins. The spa-CMD1 fusion has been used (i) to prepare (by affinity chromatography) a fraction of yeast proteins which interact with calmodulin, (ii) to isolate genes encoding calmodulin target proteins by direct screening of an expression library, and (iii) to visualize calmodulin-binding proteins in crude extracts by Western blot analysis.
Mol Microbiol 1992 Mar
PMID:Protein A-calmodulin fusions: a novel approach for investigating calmodulin function in yeast. 157 99

A cDNA sequence encoding a Schistosoma mansoni egg antigen SmE16 was cloned in Escherichia coli. The 16-kDa polypeptide deduced from the nucleotide sequence is related to the calmodulin and troponin C gene families of calcium-binding proteins, and the most significant homology is displayed around the four calcium-binding sites. The antigen was expressed as a hybrid protein of the bacteriophage MS2 polymerase. The MS2-SmE16 fusion protein binds calcium, as demonstrated via ligand blotting with 45Calcium. The detection of antibodies to the purified recombinant egg antigen in sera of schistosomiasis patients opens up the possibility that it may be a useful candidate for the development of serodiagnostic assays. The function of the protein in the egg is presently unclear.
Mol Biochem Parasitol 1992 Apr
PMID:A stage-specific calcium-binding protein expressed in eggs of Schistosoma mansoni. 157 81

Calcium and calmodulin have been widely implicated in the control of cell proliferation. We have created a strain of the genetically tractable filamentous fungus, Aspergillus nidulans, that is conditional for calmodulin expression. This was accomplished by replacing the unique endogenous calmodulin gene with one regulated by the inducible alcohol dehydrogenase (alcA) gene promoter by homologous recombination. This strain cannot grow when the cells are incubated in medium containing a carbon source that represses the alcA promoter. Characterization of the arrested cells shows that 83% are blocked in the G2 phase of the cell cycle. The block is due to very low levels of calmodulin and is fully reversible upon changing to medium that contains an inducer of the alcA promoter. The rate of cell proliferation in this strain is dependent upon both the intracellular calmodulin and extracellular Ca2+ concentrations. Raising the calmodulin concentration by inducing the alcA promoter not only causes the cells to enter the proliferative cycle more quickly and to grow faster, but also decreases the concentration of extracellular Ca2+ required to support growth by 10-fold, as compared with cells grown in noninducing medium. Thus both the intracellular calmodulin and extracellular Ca2+ concentrations are important and interactive factors in regulating the nuclear division cycle of Aspergillus nidulans.
Mol Endocrinol 1992 Mar
PMID:Cooperative regulation of cell proliferation by calcium and calmodulin in Aspergillus nidulans. 158 13

We previously proposed a molecular mechanism for the activation of smooth muscle myosin light chain kinase (smMLCK) by calmodulin (CaM). According to this model, smMLCK is autoinhibited in the absence of Ca2+/CaM due to the interaction of a pseudosubstrate prototope, contained within the CaM binding/regulatory region, with the active site of the enzyme. Binding of Ca2+/CaM releases the autoinhibition and allows access of the protein substrate to the active site of the enzyme, resulting in phosphorylation of the myosin light chains. We now provide direct experimental evidence that the pseudosubstrate prototope can associate with the active site. We constructed a smMLCK mutant in which the five-amino acid phosphorylation site of the myosin light chain substrate was inserted into the pseudosubstrate sequence of the CaM binding domain without disrupting the ability of the enzyme to bind Ca2+/CaM. We demonstrate that this mutant undergoes intramolecular autophosphorylation at the appropriate inserted serine residue in the absence of CaM and that this autophosphorylation activates the enzyme. Binding of Ca2+/CaM to the mutant enzyme stimulated myosin light chain substrate phosphorylation but strongly inhibited autophosphorylation, presumably by removing the pseudosubstrate from the active site. These results confirm that the pseudosubstrate sequence has access to the catalytic site and that the activation of the enzyme is accompanied by its removal from this position due to Ca2+/CaM binding as predicted by the model.
Mol Endocrinol 1992 Apr
PMID:Intrasteric regulation of myosin light chain kinase: the pseudosubstrate prototope binds to the active site. 158 24

In the first report in this series we described the relationships and evolution of 152 individual proteins of the EF-hand subfamilies. Here we add 66 additional proteins and define eight (CDC, TPNV, CLNB, LPS, DGK, 1F8, VIS, TCBP) new subfamilies and seven (CAL, SQUD, CDPK, EFH5, TPP, LAV, CRGP) new unique proteins, which we assume represent new subfamilies. The main focus of this study is the classification of individual EF-hand domains. Five subfamilies--calmodulin, troponin C, essential light chain, regulatory light chain, CDC31/caltractin--and three uniques--call, squidulin, and calcium-dependent protein kinase--are congruent in that all evolved from a common four-domain precursor. In contrast calpain and sarcoplasmic calcium-binding protein (SARC) each evolved from its own one-domain precursor. The remaining 19 subfamilies and uniques appear to have evolved by translocation and splicing of genes encoding the EF-hand domains that were precursors to the congruent eight and to calpain and to SARC. The rates of evolution of the EF-hand domains are slower following formation of the subfamilies and establishment of their functions. Subfamilies are not readily classified by patterns of calcium coordination, interdomain linker stability, and glycine and proline distribution. There are many homoplasies indicating that similar variants of the EF-hand evolved by independent pathways.
J Mol Evol 1992 May
PMID:Evolution of EF-hand calcium-modulated proteins. II. Domains of several subfamilies have diverse evolutionary histories. 160 95

We identified several open reading frames between the regions encoding calmodulin and ubiquitin-EP52/1 in the genome of Trypanosoma brucei. One of these, EFH5, encodes a protein 192 amino acids long. The EFH5 transcript is present in poly(A)+ mRNA and is present at similar levels in the mammalian bloodstream form and the insect procyclic form. EFH5 contains four EF-hand homolog domains, two of which are inferred to bind Ca2+ ions. We expressed EFH5 as a fusion protein in Escherichia coli and demonstrated calcium-binding activity of the fusion protein using the 45Ca-overlay technique. The function of EFH5 remains unknown; however, as the fourth EF-hand homolog identified in trypanosomes, it attests to the broad range of functions assumed by calcium functioning as a second messenger. EFH5, which is most closely related to LAV1-2 from Physarum, represents a distinct subfamily among the EF-hand-containing proteins.
Mol Gen Genet 1992 May
PMID:Identification of a new EF-hand superfamily member from Trypanosoma brucei. 160 64

Genomic and cDNA sequences encoding a calmodulin (CaM) gene from Arabidopsis (ACaM-3) have been isolated and characterized. ACaM-3 represents a sequence distinct from two previously isolated Arabidopsis CaM cDNA clones. A 2.3 kb Eco RI restriction fragment was sequenced and found to encode a complete CaM-coding sequence interrupted by a single 491 bp intron, together with 750 bp and 600 bp of 5' and 3' flanking sequences, respectively. The polypeptide encoded by ACaM-3 is identical to that encoded by ACaM-2 and it differs from the one encoded by ACaM-1 by four of 148 residues. The putative promoter of ACaM-3 was atypical of CaM genes previously isolated from animals in that it contained consensus TATA and CAAT box sequences and lacked GC-rich regions. Two DNA sequence elements closely resembling cyclic AMP regulatory elements, which have been identified in animal CaM genes, were located in the 5' flanking region of ACaM-3. Northern blot and polymerase chain reaction amplification assays confirmed that each of the three ACaM mRNAs were expressed in similar but distinct patterns in different organs. ACaM-1 mRNA was the only species detectable in root RNA fractions, and ACaM-3 mRNA could not be detected in floral stalks. Accumulation of the three CaM mRNAs in leaves was induced by a touch stimulus, but the kinetics and extent of the induction varied among the three mRNA species. Run-on transcription assays indicated that a portion of the differences in accumulation of ACaM-1, 2, and 3 mRNAs in leaves and siliques was attributable to differences in their net rates of transcription.
Plant Mol Biol 1992 Jul
PMID:Structure and expression of the Arabidopsis CaM-3 calmodulin gene. 162 78


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