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At least 271 records · Page 15Linked to original sources
PubMed1997

Characterization of the basic amphiphilic alpha-helix calmodulin-binding domain of a 61.5 kDa tobacco calmodulin-binding protein.

A 19-amino acid residue peptide, Gly-Trp-Leu-Lys-Ile-Lys-Ala-Ala-Met-Arg-Trp-Gly-Phe-Phe-Val-Arg-Lys-Lys- Ala, corresponding to the basic amphiphilic alpha-helix (BAA) motif at the C-terminus of a recombinant tobacco calmodulin-binding protein, TCB60, was synthesized. The interaction of the synthetic binding domain with calmodulin (CaM) was analyzed by gel mobility shift assays, phosphodiesterase competition assays, and fluorescence, circular dichroism, and nuclear magnetic resonance spectroscopy. Mobility shift assays showed an apparent 2 kDa increase in CaM Mr in presence of synthetic peptide and CaCl2 in 4 M urea polyacrylamide gel electrophoresis. HPLC measurements of hydrolysis of cyclic AMP by CaM-dependent phosphodiesterase indicated the synthetic peptide competitively inhibits (Ki = 15-20 nM) stimulation of phosphodiesterase activity by CaM. Upon binding CaM, the fluorescence emission maximum of the synthetic peptide, which contained two tryptophanyl residues, shifted toward blue and increased in intensity. The circular dichroism spectra indicated the ellipticity of CaM increased at 208 and 222 nm upon complex formation with the synthetic peptide. 1H NMR studies showed that the peptide interacts with the aromatic residues in domains I and III of CaM. Taken together, these data provide direct evidence that the structurally conserved basic amphiphilic alpha-helix CaM-binding domain of the recombinant tobacco CaM-binding protein interacts with CaM at physiologically significant nanomolar concentrations and the microenvironments of both CaM and the synthetic binding domain are modified upon complex formation.

Binding Sites↗
PubMed1999

Calcium-dependent and -independent interactions of the calmodulin-binding domain of cyclic nucleotide phosphodiesterase with calmodulin.

The ubiquitous Ca2+-binding regulatory protein calmodulin (CaM) binds and activates a wide range of regulatory enzymes. The binding is usually dependent on the binding of Ca2+ to CaM; however, some target proteins interact with CaM in a calcium-independent manner. In this work, we have studied the interactions between CaM and a 20-residue synthetic peptide encompassing the major calmodulin-binding domain of cyclic nucleotide phosphodiesterase (PDE1A2). The binding was studied in the absence and presence of Ca2+ by far-UV and near-UV circular dichroism, fluorescence, and infrared spectroscopy. In addition, two-dimensional heteronuclear NMR studies with 13C-methyl-Met-CaM and uniformly 15N-labeled CaM were performed. Competition assays with smooth muscle myosin light chain kinase revealed a Kd of 224 nM for peptide binding to Ca2+-CaM, while binding of the peptide to apo-CaM is weaker. The peptide binds with an alpha-helical structure to both lobes of Ca2+-saturated CaM, and the single Trp residue is firmly anchored into the C-terminal lobe of CaM. In contrast, the Trp residue plays a minor role in the binding to the apo-protein. Moreover, when bound to apo-CaM, the PDE peptide is only partially helical, and it interacts solely with the C-terminal lobe of CaM. These results show that the Ca2+-induced activation of PDE involves a significant change in the structure and positioning of the CaM-bound PDE peptide domain.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

Characterization of the mechanism of regulation of Ca2+/ calmodulin-dependent protein kinase I by calmodulin and by Ca2+/calmodulin-dependent protein kinase kinase.

Ca2+/calmodulin-dependent protein kinase I (CaMKI) is maintained in an autoinhibited state by the interaction of a COOH-terminal helix-loop-helix (Ile286-Met316) regulatory domain with the catalytic core. Activation of the enzyme by calmodulin (CaM) also allows CaMKI to be phosphorylated and activated by a second enzyme, CaMK kinase (CaMKK). To more thoroughly characterize the regulation of CaMKI by CaM and its interrelationship with phosphorylation by CaMKK, we have carried out a detailed structure-function analysis using recombinant wild-type (WT) and mutant forms of CaMKI and CaMKK. CaMKI-WT, in the absence of CaM, or CaMKI-299 and CaMKI-298 were autoinhibited and could not be phosphorylated by CaMKK-433 (a truncated constitutively active form of CaMKK). Removal of Phe298 (CaMK-297) generated a constitutively active form of CaMKI that was also phosphorylated by CaMKK-433. CaMKI-WT was essentially inactive in the absence of CaM (K0.5 for activation by CaM approximately 30 nM). Mutation of Ile294 and Phe298 to alanine (CaMKI-2A) resulted in measurable basal enzyme activity. Additional mutation of Ile286 and Val290 to alanine (CaMKI-4A) increased this basal activity. Mutation of Trp303 (CaMKI-W303S) resulted in a large increase in the K0.5 for CaM ( approximately 100 microM), supporting a role for this residue as an initial target for CaM. Mutation of Phe307 (CaMKI-F307A) resulted in increased basal enzyme activity, supporting a role for this residue in autoinhibition of CaMKI. Together these studies demonstrate the critical role of specific amino acids in the autoinhibition of CaMKI and also in its activation by CaM and phosphorylation by CaMKK.

Amino Acid Sequence↗
PubMed2001

Calmodulin binding properties of peptide analogues and fragments of the calmodulin-binding domain of simian immunodeficiency virus transmembrane glycoprotein 41.

The calcium-regulatory protein calmodulin (CaM) can bind with high affinity to a region in the cytoplasmic C-terminal tail of glycoprotein 41 of simian immunodeficiency virus (SIV). The amino acid sequence of this region is (1)DLWETLRRGGRW(13)ILAIPRRIRQGLELT(28)L. In this work, we have used near- and far-uv CD, and fluorescence spectroscopy, to study the orientation of this peptide with respect to CaM. We have also studied biosynthetically carbon-13 methyl-Met calmodulin by (1)H, (13)C heteronuclear multiple quantum coherence NMR spectroscopy. Two Trp-substituted peptides, SIV-W3F and SIV-W12F, were utilized in addition to the intact SIV peptide. Two half-peptides, SIV-N (residues 1-13) and SIV-C (residues 13-28) were also synthesized and studied. The spectroscopic results obtained with the SIV-W3F and SIV-W12F peptides were generally consistent with those obtained for the native SIV peptide. Like the native peptide, these two analogues bind with an alpha-helical structure as shown by CD spectroscopy. Fluorescence intermolecular quenching studies suggested binding of Trp3 to the C-lobe of CaM. Our NMR results show that SIV-N can bind to both lobes of calcium-CaM, and that it strongly favors binding to the C-terminal hydrophobic region of CaM. The SIV-C peptide binds with relatively low affinity to both halves of the protein. These data reveal that the intact SIV peptide binds with its N-terminal region to the carboxy-terminal region of CaM, and this interaction initiates the binding of the peptide. This orientation is similar to that of most other CaM-binding domains.

Amino Acid Sequence↗
PubMed1998

In vitro generation of an active calmodulin-independent phosphodiesterase from brain calmodulin-dependent phosphodiesterase (PDE1A2) by m-calpain.

In the present study we have shown that bovine brain 60-kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme (CaMPDE - PDE1A2) is proteolyzed by a Ca2+-dependent cysteine protease, m-calpain. The proteolysis of PDE1A2 by m-calpain results in its conversion to a totally calmodulin (CaM)-independent form accompanied by degradation of PDE1A2 into a 45-kDa catalytic fragment and a 15-kDa fragment. The activity of PDE1A2 is unaffected by the presence or absence of CaM during cleavage, suggesting that the interaction between CaM and PDE1A2 does not alter substrate recognition by calpain. Furthermore, we provide evidence, based on the studies of CaM overlay and phosphorylation, that the cleavage site is not present either in the CaM-binding domain or phosphorylation site. N-terminal sequence analysis of the 45-kDa fragment indicated that cleavage occurs between residues 126Gln and 127Ala, and eliminates the CaM-dependent activity of carboxy termini PDE1A2. The present findings suggest that limited proteolysis in the brain through calpains could be an alternate mechanism for activating CaMPDE(s) and for regulating intracellular levels of cAMP.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

Toxicity of cadmium in human trophoblast cells (JAr choriocarcinoma): role of calmodulin and the calmodulin inhibitor, zaldaride maleate.

Cadmium (Cd), the heavy metal, is toxic to the placenta. The objectives of this study were to determine if Cd toxicity is due to inhibition of placental or trophoblast cell proliferation through interactions with the intracellular calcium binding protein, calmodulin (CaM). Cd can replace calcium and thus interfere with CaM's function. Also, CaM inhibitors reverse selected toxic effects of Cd. The CaM inhibitor, zaldaride maleate, was used to determine if Cd inhibits trophoblast cell proliferation through interactions with CaM. JAr choriocarcinoma cells, a neoplastic trophoblast cell line which is similar to early human trophoblast cells, were selected to study this question. Cd (20 and 40 microM) inhibits JAr cell proliferation, as measured by cell number and BrdU incorporation. Zaldaride (10 and 20 microM) inhibits proliferation to a lesser extent; 100 microM is lethal. To determine if zaldaride alters actions of Cd, zaldaride and Cd are added simultaneously. Zaldaride (20 microM) and Cd (20 microM) together inhibit proliferation less than Cd alone, thus partially protecting cells. Metallothionein is induced in cells exposed to Cd, while zaldaride does not cause induction of this cellular defense mechanism protein. To determine if Cd inhibits proliferation through alterations of cell cycle, JAr cells enriched for G0/G1 phase were exposed to 20 microM Cd, 20 microM zaldaride, or 20 microM Cd plus 20 microM zaldaride for 24 hr. Cells remain in G0/G1 following Cd exposure; cells treated with 20 microM zaldaride progress through S phase and into G2. Zaldaride and Cd together allow JAr cells to leave G1 and enter S phase, partially relieving the cycle block produced by Cd. This study demonstrates a role for calmodulin in mediating the toxicity of Cd in trophoblast cell proliferation.

Antidiarrheals↗
PubMed1994

Calcium- and calmodulin-independent modulation of calmodulin-sensitive hypothalamic cyclic nucleotide phosphodiesterase activity by the (11-19) fragment of thymosin beta 4.

A fragment (11-19) of thymosin beta 4 was found to stimulate phosphodiesterase activity even in the absence of calcium and calmodulin. Half-maximal enzyme activation occurred with 10 nM peptide, and was further increased by phospholipids such as phosphatidylserine. The mechanism of stimulation is an increase in the Vmax of cAMP degradation without a substantial change in the Km for the substrate. In the presence of calcium ions and calmodulin the peptide was also stimulatory.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

The inhibitor peptide of the mitochondrial F1.F0-ATPase interacts with calmodulin and stimulates the calmodulin-dependent Ca2+-ATPase of erythrocytes.

The binding of calmodulin to the mitochondrial F1.F0-ATPase has been studied. [125I]Iodoazidocalmodulin binds to the epsilon-subunit and to the endogeneous ATPase inhibitor peptide in a Ca2+-dependent reaction. The effect of the mitochondrial ATPase inhibitor peptide on the purified Ca2+-ATPase of erythrocytes has also been analyzed. The inhibitor peptide stimulates the ATPase when pre-incubated with the enzyme. The activation of the Ca2+-ATPase by calmodulin is not influenced by the inhibitor peptide, indicating that the two mechanisms of activation are different. These in vitro effects of the two regulatory proteins may reflect a common origin of the two ATPases considered and/or of the regulatory proteins.

Adenosine Triphosphatases↗
PubMed1988

Calmodulin antagonists of improved potency and specificity for use in the study of calmodulin biochemistry.

Syntheses are described for a range of N-(omega-aminoalkyl)-5-iodo- and -5-cyanonaphthalene-1-sulphonamides. The selective activity of these compounds as inhibitors for calmodulin-dependent phosphodiesterase (EC 3.1.4.17) is compared with their activity for the calmodulin-independent but calcium-dependent enzymes protein kinase C and transglutaminase (EC 2.3.2.13). The results show a drastic improvement in the selectivity of effect for the 5-iodo-compounds compared with the widely-used drug, W7, N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide.

Calmodulin↗
PubMed1991

Activation mechanism of rabbit skeletal muscle myosin light chain kinase. 5'-p-fluorosulfonylbenzoyl adenosine as a probe of the MgATP-binding site of the calmodulin-bound and calmodulin-free enzyme.

5'-p-fluorosulfonylbenzoyl adenosine (FSBA), an ATP-like affinity labelling reagent, reacted with rabbit skeletal muscle myosin light chain kinase (skMLCK) and its calmodulin complex in a site-specific manner. Reaction was dependent upon the presence of the adenosine moiety of FSBA, saturated with increasing FSBA, was inhibited by MgATP, and was accompanied by stoichiometric incorporation of [14C]FSBA. The kinetic constants describing the reaction were similar for skMLCK and its calmodulin complex: k3 = -0.040 min-1 and -0.038 min-1, and Ki = 0.18 mM and 0.40 mM, respectively. It is concluded that the MgATP-binding site on skMLCK remains accessible at all times and maintains a near constant conformation.

Adenosine↗
PubMed1993

A ubiquityl-calmodulin synthetase that effectively recognizes the Ca(2+)-free form of calmodulin.

Ubiquityl-calmodulin synthetase (uCaM-synthetase) activity as detected in reticulocyte lysate and the crude extracts of rabbit tissues [FEBS Lett. 294 (1991) 229-233] has been well characterized as being essentially Ca(2+)-dependent (-Ca2+/+Ca2+ activity ratio: 0.15-0.2). However, during the purification of this enzyme on ubiquitin-Sepharose the Ca(2+)-dependent activity is lost and an essentially Ca(2+)-independent enzyme (-Ca2+/+Ca2+ activity ratio: 1.0-1.5) is obtained which was purified 90-fold (uCaM-Syn F1) to a final specific activity of 0.32 pkat/mg. During the purification procedure a second protein factor (uCaM-Syn F2) was isolated that has no catalytic activity by itself but restores Ca2+ dependence to the uCaM-Syn F1 fraction (-Ca2+/+Ca2+ activity ratio: 0.1) and enhances the catalytic activity in uCaM-Syn F1 in the presence of Ca2+ over 40-fold. It is concluded that several (possibly interdependent) forms of uCaM-synthetase exist which display different substrate specificities for calmodulin.

Animals↗
PubMed1984

Phosphorylation and activation of calmodulin-sensitive cyclic nucleotide phosphodiesterase by a brain Ca2+, calmodulin-dependent protein kinase.

A Ca2+, calmodulin-dependent protein kinase from rat brain with a MW of 640,000 phosphorylated calmodulin-sensitive phosphodiesterase from the brain cytosol. The Km of the enzyme for the phosphodiesterase was 5.0 microM and the Vmax was 212 nmol/mg/min. The amount of phosphate incorporated into the phosphodiesterase was 0.7 mol/mol subunit. Phosphorylation of the phosphodiesterase enhanced the enzyme activity by about 20% for hydrolysis of a higher concentration of cyclic AMP.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMedDate not supplied

Regulation of expression of calmodulin and calmodulin-related genes by environmental stimuli in plants.

Plants are very sensitive to environmental stimuli and have evolved the ability to adapt to many environmental stresses by altering development. In particular, mechanical stimuli such as touch or wind, result in growth changes that result in plants with greater resistance to such mechanical stimuli. We have initiated a molecular dissection of the pathways that enable perception of and responses to these environmental stimuli in plants. We have discovered five genes--termed the TCH genes--whose expression levels are strongly and rapidly increased in response to stimuli such as touch, wind, rain, wounding and darkness. Three of the TCH genes encode proteins related to calmodulin thereby implicating roles for calcium ions and calmodulin in the transduction of signals from the environment.

Calcium↗
PubMed1993

Functional analysis of the promoters of the human CaMIII calmodulin gene and of the intronless gene coding for a calmodulin-like protein.

More than 1 kb of the 5'-flanking DNA of the human CaMIII calmodulin gene and of the calmodulin-like protein (CLP) gene have been sequenced. Notable features are the absence of a TATA-box and the presence of AGGGA elements in both upstream regions, the presence of several sequences with homology to known regulatory elements (cAMP-, retinoic acid- and interferon-responsive elements) in the CLP gene, and a high G + C-content and several putative Sp1-factor-binding sites in the CaMIII gene. 1 kb of the CaMIII upstream region was driving high-level growth hormone (hGH) reporter gene expression in human teratoma and monkey COS cells. Promoter activity dropped to about 30% and 10% when only 252 bp and 114 bp, respectively, of the CaMIII sequence were present. In contrast to the CaMIII promoter, the CLP gene upstream region was driving hGH expression only in human teratoma, but not in monkey COS cells. Addition of retinoic acid to the transfected cells had minimal effects on both promoters, leading to a 10-30% decrease of activity. The results show that the human CaMIII gene contains a strong and ubiquitously active promoter, whereas the promoter of the intronless CLP gene appears to be regulated in a cell-specific manner.

Animals↗
PubMed1990

Adenylate cyclase activity in cyanobacteria: activation by Ca(2+)-calmodulin and a calmodulin-like activity.

An adenylate cyclase activity was partially characterized in the cyanobacterium Anabaena sp. The enzyme activity is found in soluble cell fractions and shows an apparent molecular weight of about 183,400. This adenylate cyclase is activated by Ca2+ and bovine brain or spinach calmodulin and it is inhibited by EGTA and some phenothiazine derivatives. Furthermore, Anabaena sp. extracts contain a calmodulin-like activity which stimulates bovine brain cyclic AMP phosphodiesterase and the Anabaena adenylate cyclase. EGTA and phenothiazine derivatives block the cyanobacterial modulator effect.

Adenylyl Cyclases↗
PubMed1994

Phosphorylation of the 61-kDa calmodulin-stimulated cyclic nucleotide phosphodiesterase at serine 120 reduces its affinity for calmodulin.

Phosphorylation of the 61-kDa isoform of bovine calmodulin (CaM)-stimulated cyclic nucleotide phosphodiesterase (CaM-PDE) by the catalytic subunit of cyclic AMP-dependent protein kinase A (PKA) results in a decrease in the affinity of the enzyme for calmodulin [Sharma, R. K., & Wang, J. H. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 2603-2607]. In the present study, purified 61-kDa CaM-PDE was phosphorylated in the presence of [gamma-32P]ATP and cleaved with a Lys-C endoproteinase. The resultant phosphopeptides were resolved by reverse-phase HPLC and analyzed by electrospray mass spectrometry and Edman sequencing. Serine residues 120 and 138 were identified as the principal sites of phosphorylation. A cDNA encoding the 61-kDa CaM-PDE [Sonnenburg, W. K., Seger, D., & Beavo, J. A. (1993) J. Biol. Chem. 268, 645-652] was used to generate point mutants in which either or both of these serines were replaced with alanine. The mutants were expressed in COS-7 cells, purified, and phosphorylated. Phosphorylation of the mutant Ser 138-->Ala resulted in a decrease in affinity for CaM that was comparable to that seen with the wild-type enzyme. In contrast, phosphorylation of the mutant Ser 120-->Ala had virtually no effect on CaM affinity. We conclude that phosphorylation of serine 120 by PKA is responsible for the reduction in affinity of the 61-kDa CaM-PDE for CaM.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Drug binding by calmodulin: crystal structure of a calmodulin-trifluoperazine complex.

The crystal structure of calmodulin (CaM) bound to trifluoperazine (TFP) has been determined and refined to a resolution of 2.45 A. Only one TFP is bound to CaM, but that is sufficient to cause distortion of the central alpha-helix and juxtaposition of the N- and C-terminal domains similar to that seen in CaM-polypeptide complexes. The drug makes extensive contacts with residues in the C-terminal domain of CaM but only a few contacts with one residue in the N-terminal domain. The structure suggests that substrate binding to the C-terminal domain is sufficient to cause the conformational changes in calmodulin that lead to activation of its targets.

Animals↗
PubMed1984

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, a hydrophobic, photoreactive probe, labels calmodulin and calmodulin fragments in a Ca2+-dependent way.

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID), a highly hydrophobic, carbene-generating photoreactive probe, labels calmodulin and some of its proteolytic fragments in the Ca2+-bound conformation only. It is assumed that [125I]TID labels hydrophobic sites exposed by the binding of Ca2+. The finding offers a new and powerful means to characterize calmodulin sites that play a role in the interaction with targets.

Amino Acids↗