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PubMed1997

Oocyte maturation in rabbits: effects of calmodulin inhibitors.

Oocyte maturation in mammals follows a highly conserved pattern of release from arrest through to the extrusion of the first polar body and formation of the second metaphase spindle. Oscillations in cytoplasmic calcium concentration precede the events of maturation in many species. These calcium ions interact with and activate calcium-binding proteins, including calmodulin, within the cell. Thus, it was of interest to us to examine whether calcium acted through calmodulin in the initial stages of maturation in rabbit oocytes or whether calmodulin was required for continuation through metaphase I no to metaphase II. Using the calmodulin inhibitor W-7 we found a significant (p < 0.05) decrease in the percentage of oocytes that underwent germinal vesicle breakdown. Calmidazolium did not prevent germinal vesicle breakdown; however, it caused a significant (p < 0.05) decrease in the proportion of oocytes with fully elaborated spindles and taxol-induced cytoplasmic asters. Both inhibitors caused a significant (p < 0.05) reduction in the proportion of oocytes that extruded their first polar bodies. The kinase inhibitor 6-DMAP caused a significant reduction in the proportion of oocytes with spindles and condensed chromatin, indicating the necessity for phosphorylation events in the resumption of meiosis. In rabbit oocytes calmodulin may play a role in the release from prophase arrest, and it is necessary for spindle preservation and continuation through metaphase I to metaphase II. The varying effects of the two inhibitor stems from their different binding sites on the calmodulin molecule thus causing a differential effect on its downstream effectors.

Adenine↗

PubMed1983

Purification of a novel calmodulin binding protein from bovine cerebral cortex membranes.

A new calmodulin (CaM) binding protein, designated P-57, has been purified to apparent homogeneity from bovine cerebral cortex membranes. In contrast to other calmodulin binding proteins, P-57 has higher affinity for calmodulin in the absence of bound Ca2+ than in its presence. The protein was purified by DEAE-Sephacel chromatography and two CaM-Sepharose affinity column steps. The first CaM-Sepharose column was run in the presence of Ca2+; the second was run in the presence of chelator in excess of Ca2+. P-57 was adsorbed by CaM-Sepharose only in the absence of bound Ca2+ and was eluted from the second column by buffers containing Ca2+. Sodium dodecyl sulfate (SDS)-polyacrylamide gels of the purified protein showed only one band at Mr 57 000. The major form of the protein on Bio-Gel A-1.5m and native polyacrylamide gradient gel electrophoresis ran with an apparent Stokes radius of 41 A. Photoaffinity labeling of P-57 with azido[125I]calmodulin yielded one cross-linked product on SDS gels with an Mr of 70 000. This interaction occurred only when excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was present and was inhibited by the presence of Ca2+ in excess of chelator. It appears that P-57 has novel binding properties for calmodulin distinct from all other calmodulin binding proteins described thus far.

Animals↗

PubMed1987

Affinity selection of chemically modified proteins: role of lysyl residues in the binding of calmodulin to calcineurin.

In affinity selection, calcineurin selects from a population of randomly modified calmodulins those species with which it prefers to interact. The method shows that acetylation of lysines affects calmodulin so as to interfere with its ability to interact with calcineurin. Monoacetylation of any lysine of calmodulin reduces its affinity for calcineurin by 5-10-fold. Multiple acetylations amplify the loss of affinity; none of the modifications are imcompatible with activity. The lack of selectivity of calcineurin against any particular modified lysine indicates that the loss of affinity reflects changes induced by the removal of the charged groups and suggests an important role for electrostatic interactions in the cooperative structural transitions which calmodulin undergoes upon binding its target proteins or calcium. In the presence of calcineurin, a large and specific decrease in the rate of acetylation of Lys-75 and -148 of calmodulin is observed. The reactivity of the same residues is greatly increased in the presence of calcium alone [Giedroc, D. P., Sinha, S. K., Brew, K., & Puett, D. (1985) J. Biol. Chem. 260, 13406-13413]. Lys-75, located in the central helix, and the C-terminal Lys-148 [Babu, Y. S., Sacks, J. S., Greenhouse, T. J., Bugg, C. E., Means, A. R., & Cook, W. J. (1985) Nature (London) 315, 37-40] may act as sensors of the calmodulin allosteric transitions. Their reactivity changes in opposite directions in response to calcium-induced or calcineurin-induced structural changes. The reactivity of other residues such as Lys-21, decreased in the presence of calcineurin but not calcium, is also affected by a conformational change which is induced specifically by calcineurin.

Acetylation↗

PubMed1990

Specific association of calmodulin-dependent protein kinase and related substrates with the junctional sarcoplasmic reticulum of skeletal muscle.

A systematic study of protein kinase activity and phosphorylation of membrane proteins by ATP was carried out with vesicular fragments of longitudinal tubules (light SR) and junctional terminal cisternae (JTC) derived from skeletal muscle sarcoplasmic reticulum (SR). Following incubation of JTC with ATP, a 170,000-Da glycoprotein, a 97,500-Da protein (glycogen phosphorylase), and a 55,000-60,000-Da doublet (containing calmodulin-dependent protein kinase subunit) underwent phosphorylation. Addition of calmodulin in the presence of Ca2+ (with no added protein kinase) produced a 10-fold increase of phosphorylation involving numerous JTC proteins, including the large (approximately 450,000 Da) ryanodine receptor protein. Calmodulin-dependent phosphorylation of the ryanodine receptor protein was unambiguously demonstrated by Western blot analysis. The specificity of these findings was demonstrated by much lower levels of calmodulin-dependent phosphorylation in light SR as compared to JTC, and by much lower cyclic AMP dependent kinase activity in both JTC and light SR. These observations indicate that the purified JTC contain membrane-bound calmodulin-dependent protein kinase that undergoes autophosphorylation and catalyzes phosphorylation of various membrane proteins. Protein dephosphorylation was very slow in the absence of added phosphatases, but was accelerated by the addition of phosphatase 1 and 2A (catalytic subunit) in the absence of Ca2+, and calcineurin in the presence of Ca2+. Therefore, in the muscle fiber, dephosphorylation of SR proteins relies on cytoplasmic phosphatases. No significant effect of protein phosphorylation was detected on the Ca2(+)-induced Ca2+ release exhibited by isolated JTC vesicles. However, the selective and prominent association of calmodulin-dependent protein kinase and related substrates with junctional membranes, its Ca2+ sensitivity, and its close proximity to the ryanodine and dihydropyridine receptor Ca2+ channels suggest that this phosphorylation system is involved in regulation of functions linked to these structures.

Adenosine Triphosphate↗

PubMed1998

The role of calmodulin-binding sites in the regulation of the Drosophila TRPL cation channel expressed in Xenopus laevis oocytes by ca2+, inositol 1,4,5-trisphosphate and GTP-binding proteins.

The roles of calmodulin-binding sites in the regulation by Ca2+, inositol 1,4,5-trisphosphate (InsP3) and GTP-binding regulatory proteins (G-proteins) of the Drosophila melanogaster TRPL (transient-receptor-potential-like) non-specific Ca2+ channel were investigated. Wild-type TRPL protein and two mutant forms, TRPL (W713G) and TRPL (W814G), in which a key tryptophan residue in each of the two putative calmodulin-binding sites (Sites 1 and 2, respectively) was replaced by glycine, were expressed heterologously in Xenopus laevis oocytes. Immunofluorescence studies indicated that the expressed TRPL, TRPL (W713G) and TRPL (W814G) proteins are located at the plasma membrane. TRPL oocytes (oocytes injected with trpl cRNA) and TRPL (W814G) oocytes [oocytes injected with trpl (W814G) cRNA] exhibited substantially greater rates of basal (constitutive) Ca2+ inflow (measured using fluo-3 and the Ca2+ add-back protocol) than mock-injected oocytes (mock oocytes). In TRPL (W713G) oocytes, this difference was abolished. In TRPL and TRPL (W814G) [oocytes injected with trpl (W713G) cRNA], but not in TRPL (W713G) oocytes, basal Ca2+ inflow was inhibited by W13, an inhibitor of calmodulin action. Calmodulin (3 muM intracellular) inhibited basal Ca2+ inflow in TRPL but not in TRPL (W713G) or TRPL (W814G) oocytes. Staurosporin, an inhibitor of protein kinase C (PKC), inhibited, while PMA (an activator of PKC) stimulated, basal Ca2+ inflow in TRPL oocytes. In oocytes incubated in the presence of PMA (to suppress Ca2+ inflow through endogenous receptor-activated Ca2+ channels), the InsP3-induced stimulation of Ca2+ inflow through TRPL channels was more clearly evident than in oocytes incubated in the absence of PMA. InsP3 caused a significant stimulation of Mn2+ inflow in TRPL but not in mock oocytes. Rates of InsP3-stimulated Ca2+ inflow through the TRPL, TRPL (W713G) and TRPL (W814G) channels were similar. The ability of GTPgammaS to stimulate Ca2+ inflow through TRPL channels was inhibited by 50% in TRPL (W713G) oocytes but was unaffected in TRPL (W814G) oocytes. It is concluded that, in the environment of the Xenopus oocyte, the Drosophila TRPL channel is activated by (a) interaction with Ca2+/calmodulin at calmodulin-binding Site 1; (b) PKC; (c) InsP3 in a process that does not involve Ca2+ and calmodulin; and (d) a trimeric G-protein(s) through both a Ca2+/calmodulin-dependent and a Ca2+/calmodulin-independent mechanism.

Animals↗

PubMed1991

Possible role for calmodulin and the Ca2+/calmodulin-dependent protein kinase II in postsynaptic neurotransmission.

The theory presented here is based on results from in vitro experiments and deals with three proteins in the postsynaptic density/membrane-namely, calmodulin, the Ca2+/calmodulin-dependent protein kinase, and the voltage-dependent Ca2+ channel. It is visualized that, in vivo in the polarized state of the membrane, calmodulin is bound to the kinase; upon depolarization of the membrane and the intrusion of Ca2+, Ca2(+)-bound calmodulin activates the autophosphorylation of the kinase. Calmodulin is visualized as having less affinity for the phosphorylated form of the kinase and is translocated to the voltage-dependent Ca2+ channel. There, with its bound Ca2+, it acts as a Ca2+ sensor, to close off the Ca2+ channel of the depolarized membrane. At the same time, it is thought that the configuration of the kinase is altered by its phosphorylated states; by interacting with Na+ and K+ channels, it alters the electrical properties of the membrane to regain the polarized state. Calmodulin is moved to the unphosphorylated kinase to complete the cycle, allowing the voltage-dependent Ca2+ channel to be receptive to Ca2+ flux upon the next cycle of depolarization. Thus, the theory tries to explain (i) why calmodulin and the kinase reside at the postsynaptic density/membrane site, and (ii) what function autophosphorylation of the kinase may play.

Calcium Channels↗

PubMed1996

The active species of plasma membrane Ca2+-ATPase are a dimer and a monomer-calmodulin complex.

The purified plasma membrane Ca2+-ATPase is fully activated through the enzyme concentration-dependent self-association at physiologically relevant Ca2+ concentrations (Kosk-Kosicka, D., and Bzdega, T. (1988) J. Biol. Chem. 263, 18184-18189; Kosk-Kosicka, D., Bzdega, T., and Wawrzynow, A. (1989) J. Biol. Chem. 264, 19495-19499). We have previously shown that the Ca2+-ATPase activity of the oligomeric enzyme is independent of calmodulin, in contrast to another active enzyme species, a presumable monomer, that is activated by calmodulin binding. Presently, we have succeeded in determining the molecular mass of the two active enzyme species by equilibrium ultracentrifugation. For the calmodulin-dependent species, the molecular mass is 170 +/- 30 kDa, which is consistent with predominantly monomeric Ca2+-ATPase with bound calmodulin. The molecular mass of calmodulin-independent oligomers is 260 +/- 34 kDa, indicating that they are dimers. Results of experiments performed under different calcium and potassium concentrations and in the presence of dextran that causes molecular crowding verify a strict Ca2+ requirement of the dimerization process. We conclude that the active species of the Ca2+-ATPase are a monomer-calmodulin complex and a dimer.

Calcium↗

PubMed2000

Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity.

Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like domain with three EF-hands. The neural visinin-like Ca(2+)-binding domain at the C-terminal end of the CaM-binding domain makes CCaMK unique among all the known calmodulin-dependent kinases. Biological functions of the plant visinin-like proteins or visinin-like domains in plant proteins are not well known. Using EF-hand deletions in the visinin-like domain, we found that the visinin-like domain regulated Ca(2+)-stimulated autophosphorylation of CCaMK. To investigate the effects of Ca(2+)-stimulated autophosphorylation on the interaction with calmodulin, the equilibrium binding constants of CCaMK were measured by fluorescence emission anisotropy using dansylated calmodulin. Binding was 8-fold tighter after Ca(2+)-stimulated autophosphorylation. This shift in affinity did not occur in CCaMK deletion mutants lacking Ca(2+)-stimulated autophosphorylation. A variable calmodulin affinity regulated by Ca(2+)-stimulated autophosphorylation mediated through the visinin-like domain is a new regulatory mechanism for CCaMK activation and calmodulin-dependent protein kinases. Our experiments demonstrate the existence of two functional molecular switches in a protein kinase regulating the kinase activity, namely a visinin-like domain acting as a Ca(2+)-triggered switch and a CaM-binding domain acting as an autophosphorylation-triggered molecular switch.

Adenosine Triphosphate↗

PubMed1985

Intracellular regulators of neuronal sprouting: calmodulin-binding proteins of nerve growth cones.

The focus of this study is a quantitative biochemical analysis of the calcium-dependent interactions of calmodulin with a nerve growth cone preparation from fetal rat brain (Pfenninger, K. H., L. Ellis, M. P. Johnson, L. B. Freidman, and S. Somlo, 1983, Cell 35:573-584). The presence of calmodulin as an integral component of this preparation is demonstrated, and quantitative binding studies are presented. The binding of 125I-calmodulin to nerve growth cone material is shown to be highly specific, calcium dependent, and saturable at nanomolar calmodulin concentrations. Additionally, the growth cones' binding components appear to be membrane proteins. The individual molecular mass species of growth cone proteins displaying calcium-dependent calmodulin binding are also detailed and presented in comparison with those of synaptosomes. This analysis reveals differences between the calmodulin binding proteins of the growth cone preparation and the synaptosome fraction, suggesting the presence in growth cones of a specialized set of components which may be involved in regulatory mechanisms controlling neuritic sprouting.

Animals↗

PubMed1989

Analysis of the calcium-modulated proteins, S100 and calmodulin, and their target proteins during C6 glioma cell differentiation.

We have analyzed the levels, subcellular distribution, and target proteins of two calcium-modulated proteins, S100 and calmodulin, in differentiated and undifferentiated rat C6 glioma cells. Undifferentiated and differentiated C6 cells express primarily the S100 beta polypeptide, and the S100 beta levels are four-fold higher in differentiated compared to undifferentiated cells. Double fluorescent labeling studies of undifferentiated cells demonstrated that S100 beta staining localized to a small region of the perinuclear cytoplasm and colocalized with the microtubule organizing center and Golgi apparatus. Analysis of differentiated C6 cells demonstrated that S100 beta distribution and S100 beta-binding protein profile changed significantly upon differentiation. In addition, the brain-specific isozyme of one S100-binding protein, fructose-1,6-bisphosphate aldolase C, can be detected in differentiated but not undifferentiated C6 cells. While changes in the subcellular distribution of calmodulin were not observed during differentiation, calmodulin levels and calmodulin-binding protein profiles did change. Altogether these data suggest that S100 beta and calmodulin regulate different processes in glial cells and that the regulation of the expression, subcellular distribution, and target proteins of S100 beta and calmodulin during differentiation is a complex process which involves multiple mechanisms.

Animals↗

PubMed1982

The cytoskeletal system of nucleated erythrocytes. II. presence of a high molecular weight calmodulin-binding protein.

Calmodulin was detected in dogfish erythrocyte lysates by means of phosphodiesterase activation. Anucleate dogfish erythrocyte cytoskeletons bound calmodulin. Binding of calmodulin was calcium-dependent, concentration-dependent, and saturable. Cytoskeletons consisted of a marginal band of microtubules containing primarily tubulin, and trans-marginal band material containing actin and spectrinlike proteins. Dogfish erythrocyte ghosts and cytoskeletons were found to contain a calcium-dependent calmodulin-binding protein, CBP, by two independent techniques: (a) 125I-calmodulin binding to cytoskeletal proteins separated by SDS PAGE, and (b) in situ azidocalmodulin binding in whole anucleate ghosts and cytoskeletons. CBP, with an apparent molecular weight of 245,000, co-migrated with the upper band of human and dogfish erythrocyte spectrin. CBP was present in anucleate ghosts devoid of marginal bands and absent from isolated marginal bands. CBP therefore appears to be localized in the trans-marginal band material and not in the marginal band. Similarities between CBP and high molecular weight calmodulin-binding proteins from mammalian species are discussed.

Animals↗

PubMed2001

Calmodulin levels are dynamically regulated in living vascular smooth muscle cells.

The total unbound calmodulin (i.e., not bound to target proteins) level in living smooth muscle cells from the ferret portal vein was monitored with a low-affinity, calmodulin-binding peptide tagged with an environmentally sensitive fluorophore. GS17C, a previously characterized peptide, from the calmodulin-binding domain of caldesmon was tagged with iodoacetyl nitrobenz-2-oxa-1,3-diazole (NBD) or, as a negative control, with iodoacetylfluorescein isothiocyanate. Increases in NBD-GS17C fluorescence were detected by using confocal microscopy when chemically loaded cells were stimulated with solutions of elevated [K(+)] or the calcium ionophore 4-bromoA-23187 to elicit increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) quantified by fura 2. Increases in peptide fluorescence were detected in response to a phorbol ester in the absence of changes in [Ca(2+)](i). These changes were blocked by the addition of the calmodulin antagonist calmidazolium. These results suggest that the total unbound intracellular calmodulin levels may be sufficient to regulate the activity of caldesmon and, furthermore, that phosphorylation of protein kinase C substrates may increase the level of available calmodulin in living smooth muscle cells.

4-Chloro-7-nitrobenzofurazan↗

PubMed1996

Inhibition of agonist-mediated calcium entry by calmodulin antagonists and by the Ca2+/calmodulin kinase II inhibitor KN-62. Studies with thyroid FRTL-5 cells.

Calmodulin and calmodulin-dependent mechanisms are probably important in regulating thyroid cell function. However, calmodulin antagonists may directly modify calcium fluxes in cells. In the present investigation the effects of several calmodulin inhibitors and of KN-62, a specific calcium/calmodulin kinase II inhibitor, on the ATP- and thapsigargin-evoked changes in intracellular free calcium ([Ca2+]i) were investigated in Fura-2 loaded thyroid FRTL-5 cells. All of the inhibitors tested attenuated agonist-evoked calcium entry. The inhibitor calmidazolium per se potently released sequestered calcium followed by enhanced calcium entry. Pretreatment of the cells with calmidazolium inhibited both the thapsigargin-and the ATP-evoked calcium entry. Our results show that calmodulin antagonists are potent inhibitors of calcium entry in thyroid cells, possibly by directly inhibiting the calcium entry pathway. This inhibition may explain, in part, the results obtained with calmodulin inhibitors in previous studies.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1999

Serine/threonine phosphorylation of calmodulin modulates its interaction with the binding domains of target enzymes.

The interaction of serine/threonine-phosphorylated calmodulin with synthetic peptides corresponding to the calmodulin-binding domains of six enzymes has been studied by fluorescence spectroscopy. For five peptides, the dissociation constant of the calmodulin-peptide complex (K(d)) increased when calmodulin was phosphorylated. An increase of more than one order of magnitude was observed with peptides derived from smooth-muscle myosin light-chain kinase and cAMP phosphodiesterase. In contrast, only a slight increase in K(d) was noted with two peptides derived from the plasma membrane Ca(2+)-ATPase and for the peptide derived from nitric oxide synthase. No significant change in affinity was detected with the peptide derived from calcineurin. In contrast, a decrease in the dissociation constant was observed with the peptide derived from the Ca(2+)-calmodulin dependent kinase II. Phosphorylation also affected the peptide-calmodulin binding stoichiometry: a decrease from two to one binding sites was observed with the peptides derived from myosin light-chain kinase and phosphodiesterase.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1992

The major calmodulin-binding protein in rabbit parietal cells is Ca2+/calmodulin-dependent protein kinase II.

Parietal cell secretion can be stimulated by both histaminergic and cholinergic agonists. We have recently found that inhibition of calmodulin-dependent protein kinase II (CaMK II) activity can abolish cholinergic but not histaminergic stimulation of parietal cell secretion (Am. J. Physiol. 262:G118-122). We have investigated the presence of calmodulin-binding proteins and CaMK II in isolated rabbit parietal cells. Calmodulin-binding proteins with apparent molecular masses of 50, 60, 85, 100, and 240 kDa were observed. The major calmodulin-binding species was a 50 kDa band which was enriched in 50,000 g. microsomal membranes. The 50 kDa calmodulin binding comigrated with immunoreactivity for CaMK II. Partial purification of the microsomal CaMK II demonstrated a 250 kDa oligomer. The results demonstrate that CaMK II is the major calmodulin-binding protein in parietal cells and is associated primarily with light microsomal membranes.

Animals↗

PubMed1991

A calmodulin-binding peptide of caldesmon.

Caldesmon is a major actin-binding protein identified in smooth muscle and many non-muscle cells. It also interacts with calmodulin and a number of other acidic proteins. We have shown previously that the polypeptide stretch from Val629 to Ser666 near the C terminus contains a calmodulin binding site (Wang, C.-L. A., Wang, L.-W. C., Xu, S., Lu, R. C., Saavedra-Alanis, V., and Bryan, J. (1991) J. Biol. Chem. 266, 9166-9172). On the other hand, Bartegi et al. (Bartegi, A., Fattoum, A., Derancourt, J., and Kassab, R. (1990) J. Biol. Chem. 265, 15231-15238) reported a cyanogen bromide fragment beginning at Trp659 which is also capable of binding both calmodulin and actin. A comparison of the overlapping sequence between these two peptides suggests that this calmodulin binding site is localized in a 7-residue segment, 659Trp-Glu-Lys-Gly-Asn-Val-Phe665. We have chemically synthesized an 18-residue peptide (GS17C, from Gly651 to Ser667 with an added cysteine at the C terminus) that contains this segment. This peptide was purified by high performance liquid chromatography and labeled with fluorescent probes at the terminal cysteine residue. We found that GS17C indeed binds calmodulin in a Ca(2+)-dependent manner (Kd = 8 x 10(-7) M) and appears to compete with caldesmon. Interestingly, this synthetic peptide also co-sediments with F-actin, binding to actin being displaceable by calmodulin, as in the case of the native caldesmon. But GS17C does not have any effect on the actomyosin ATPase activity, indicating that this peptide segment does not contain the inhibitory region.

Actins↗

PubMed1988

Calcium/calmodulin-dependent protein kinase II. Characterization of distinct calmodulin binding and inhibitory domains.

Regulatory domains of the multifunctional Ca2+/calmodulin-dependent protein kinase II were investigated utilizing synthetic peptides. These peptides were derived from the sequence between positions 281 and 319 as translated from the cDNA sequence of the rat brain 50-kDa subunit (Lin, C. R., Kapiloff, M. S., Durgerian, S., Tatemoto, K., Russo, A. F., Hanson, P., Schulman, H., and Rosenfeld, M. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5962-5966), which contain the putative calmodulin-binding region as well as potential autophosphorylation sites. Peptide 290 to 309 was found to be a potent calmodulin antagonist with an IC50 of 52 nM for inhibition of Ca2+/calmodulin-dependent protein kinase II. Neither truncation from the amino terminus (peptide 296-309) nor extension in the carboxyl-terminal direction (peptide 294-319) markedly affected calmodulin binding, whereas shortening the peptide from the carboxyl terminus (peptide 290-302) or from both ends (peptide 295-304) resulted in the elimination of this activity. Peptide 281-290 did not bind calmodulin, but was a good substrate for the enzyme, being phosphorylated at Thr-286. Several of the peptides inhibited the kinase in a partially competitive, substrate-directed manner, but were not themselves phosphorylated. These studies identify domains within Ca2+/calmodulin-dependent protein kinase II which may be involved in 1) inhibition of the kinase in the absence of calmodulin, 2) binding of calmodulin, and 3) the resulting activation. Additionally, it is suggested that phosphorylation of residues flanking these domains may be responsible for the known regulatory effects of autophosphorylation on the properties of the kinase.

Amino Acid Sequence↗

PubMed1988

Caldesmon-calmodulin interaction. Study by the method of protein intrinsic tryptophan fluorescence.

Addition of calmodulin to caldesmon causes a concentration-dependent shortwave shift and an increase of fluorescence intensity of caldesmon tryptophan residues. The existence of a protein complex is confirmed by the increase of the caldesmon sedimentation coefficient s0(20,w) from 3.0 S to 4.5 S in the presence of calmodulin. These findings allow application of the method of protein intrinsic tryptophan fluorescence for quantitative study of unmodified caldesmon and calmodulin in solution. The affinity of the caldesmon-calmodulin interaction (Kass = 1.8 x 10(6) M-1, in 0.1 M-KCl at 25 degrees C) and Ca2+ requirement (half-maximum binding at 0.8 microM-Ca2+) determined by means of the fluorescence technique are in agreement with previously reported values, thus confirming the validity of the method. The same approach was further used to provide information about the nature of interactions stabilizing the caldesmon-calmodulin complex. Association of the proteins and dissociation of the complex were studied in different physicochemical conditions, including variation of pH, temperature and ionic strength and the addition of quenchers, denaturants and anticalmodulin drugs. The results obtained suggest that caldesmon tryptophan residues, together with charged groups, are involved in calmodulin binding. Hydrophobic, electrostatic and hydrogen interactions contribute to the stability of the protein complex, making it insensitive to variations of physicochemical conditions within physiological limits.

Animals↗