The effects of calcium site occupancy and reagent length on reactivity of calmodulin lysyl residues with heterobifunctional aryl azides. Mapping interaction domains with specific calmodulin photoprobe derivatives.

The relationship of structural and functional moieties on calmodulin is important in all venues of cell activity. In this study, we investigate the effect of lysine modification on calmodulin function. Azidosalicylate reagents containing different "linker arm" lengths, between the photoactive terminus and an amine-reactive N-hydroxysuccinimidyl ester moiety were used to modify calmodulin lysines at three different positions in a calcium-dependent manner. The short cross-linker, (ASNE-2 (where ASNE represents azidosalicylate N-hydroxysuccinimidyl ester), modifies Lys-75, whereas the longer reagent, ASNE-6, modifies lysines 21, 75, and 94. The modification of these different lysines is shown to be calcium-dependent. At 1-100 microM levels of calcium, only Lys-94 is modified, suggesting that modification of this residue is directed by both the binding of calcium to calcium-binding loops III and IV and the hydrophobic pocket exposed between these two loops as a result of calcium binding. At higher calcium concentrations (> 200 microM), where sites I and II become filled, modification of Lys-21 or Lys-75 also was observed. All the modified calmodulins were able to stimulate 3',5'-cyclic-nucleotide phosphodiesterase fully although the Kact for the Lys-75 and Lys-21 derivatives increased 10- and 50-fold, respectively. None of the modifications affected the activation of erythrocyte plasma membrane Ca(2+)-ATPase. Only the ASNE-6 Lys-75 derivative showed efficient (40%) photocross-linking to the Ca(2+)-ATPase. The ASNE-2 Lys-75 derivative as well as the ASNE-6 Lys-21 and Lys-94 derivatives did not show efficient calcium-dependent photocross-linking to this enzyme.     

Topographical mapping of calmodulin-target enzyme interaction domains

Calmodulin derivatives, specifically biotinylated in domains I and III, were synthesized to address the structures of calmodulin necessary for binding to its target enzymes in active conformations. By binding avidin to these biotinylated calmodulins, the role of specific sequences of the calmodulin molecule in target enzyme interactions could then be evaluated. The role of domain I in these interactions was assessed by biotinylation of Cys-27 of wheat germ calmodulin with N-ethylmaleimidobiotin. This modification did not affect the ability of this calmodulin to activate 3'-5'-cyclic nucleotide phosphodiesterase (PDE) or human erythrocyte Ca2+-Mg2+ ATPase. The addition of avidin to form a stable calmodulin-avidin complex also did not affect activation. Bovine testes calmodulin was biotinylated on Lys-94 by calcium-dependent reaction with N-hydroxysuccinimido ester-biotin at pH 6.0. This derivative was used to probe the Ca+2 binding region of domain III. The incorporation of biotin at Lys-94 of bovine calmodulin did not affect calmodulin activation of PDE. However, compared to unmodified calmodulin, a 4-fold higher concentration of this derivative was required to fully activate the ATPase. The addition of excess avidin to this derivative abolished all activation for both PDE and the ATPase. Sites of modification were determined by sequence analysis of labeled peptides.     

Azidocalmodulin derivatives. Activation of, and binding to, three target proteins: aorta myosin light-chain kinase, erythrocyte (Mg2+ + Ca2+)-dependent ATPase and cardiac sarcoplasmic-reticulum kinase.

Different azidocalmodulin derivatives were synthesized by modification of either one carboxylic acid group or one or several arginine residues and their binding and activation capacity investigated in three target enzyme systems. The systems studied were smooth-muscle myosin light-chain kinase, cardiac sarcoplasmic-reticulum kinase and erythrocyte (Mg2+ + Ca2+)-dependent ATPase. The results indicated that the activation ability of each calmodulin derivative was different depending on the system studied. Binding studies carried out by the displacement of 125I-calmodulin indicated that the monosubstitutions did not greatly alter the apparent Kd of calmodulin for the enzymes but that the modification of four arginine residues caused a 4-8-fold increase in the apparent Kd in all systems. These results have shown that azidocalmodulin derivatives may have different degrees of usefulness in the study of calmodulin target proteins in different systems, with the behaviour of the derivatives not predictable on the basis of the nature (soluble or membrane-bound) or the type (ATPase or kinase) of enzyme system to be investigated. However, the monosubstituted calmodulin and, in particular, the carboxylic acid-group-modified derivative (where the modification was statistically dispersed over the protein chain) are good candidates for photolabelling calmodulin-binding proteins.     

The control by Ca2+ of the polyphosphoinositide phosphodiesterase and the Ca2+-pump ATPase in human erythrocytes

1. Both the Ca(2+)-pump ATPase and the polyphosphoinositide phosphodiesterase of the erythrocyte membrane can, when assayed under appropriate conditions, be activated by Ca(2+) in the micromolar range. We have therefore compared the mechanisms and affinities for Ca(2+) activation of the two enzymes in human erythrocyte membranes, to see whether the polyphosphoinositide phosphodiesterase would be active in normal healthy erythrocytes. 2. At physiological ionic strength and in the presence of calmodulin, the Ca(2+)-pump ATPase was activated by Ca(2+) in a highly co-operative manner, with half-maximal activation occurring at about 0.3mum-Ca(2+). At an optimal Ca(2+) concentration, calmodulin stimulated the Ca(2+)-sensitive ATPase activity about 10-fold. 3. Ca(2+) activated the polyphosphoinositide phosphodiesterase in a non-co-operative manner. The Ca(2+) requirements for breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were identical, which supports our previous conclusion that Ca(2+) activates a single polyphosphoinositide phosphodiesterase that degrades both lipids with equal facility. Added calmodulin did not affect the activity of the polyphosphoinositide phosphodiesterase. 4. At low ionic strength in the absence of Mg(2+), half-maximal activation of the phosphodiesterase was at about 3mum-Ca(2+). The presence of 1mm-Mg(2+) shifted the Ca(2+) activation curve to the right, as did elevation of the ionic strength. When the Ca(2+)-pump ATPase and the polyphosphoinositide phosphodiesterase were assayed in the same incubations and under conditions of intracellular ionic strength and Mg(2+) concentration, the ATPase was fully activated at 3mum-Ca(2+), whereas no polyphosphoinositide phosphodiesterase activity was detected below 100mum-Ca(2+). 5. The Ca(2+)-pump ATPase of the erythrocyte membrane normally maintains the Ca(2+) concentration of healthy erythrocytes below approx. 0.1mum. It therefore seems unlikely that the polyphosphoinositide phosphodiesterase of the erythrocyte membrane ever expresses its activity in a healthy erythrocyte.     

Allosteric regulation of Ca2+-calmodulin-dependent phosphodiesterase activity from the bovine brain

The Ca2+-calmodulin-dependent interaction of phosphodiesterase with phenyl-Sepharose was demonstrated. BSA caused incomplete competitive inhibition of phosphodiesterase activation by calmodulin. The 17-fold increase of the constant for phosphodiesterase activation by calmodulin was accompanied by an insignificant rise in the maximum rate of cAMP hydrolysis; in this case the value of the inhibition constant amounted to Ki approximately 6 microM. In the absence of calmodulin saturating concentrations of BSA reduced the enzyme activity nearly 3-4-fold. The effect of BSA on phosphodiesterase was incompetitive with respect to cAMP (Ki approximately 1.4 microM). Both phenomena are characteristic of incompetitive binding of BSA to the enzyme with respect to cAMP and calmodulin. Gel filtration data reflect the changes in the enzyme molecular weight during its interaction with BSA. All the above reactions of the enzyme are reversible.     

Anomeric O-acylation of Kdo using alkyl and aryl isocyanates

To develop a convenient method for the preparation of an alpha-Kdo derivative carrying a functional spacer at the reducing end, we examined anomeric O-acylation using Kdo and halogenated alkyl/aryl isocyanates as nucleophile and electrophiles, respectively. Reaction of a Kdo derivative with 2-chloroethyl isocyanate in the presence of DMAP gave an alpha-spiro product (82%) and an alpha-Kdo derivative of a dimeric isocyanate adduct (10%). Similar reaction with 4-(chloromethyl)phenyl isocyanate gave only the corresponding alpha-spiro product (81%). The NMR data show that the pyranose rings of both the alkyl and aryl spiro products adopt the 5C2 conformation. Thus, we accomplished alpha-selective anomeric O-acylation by coupling the Kdo derivative with alkyl and aryl isocyanates.     

Specific acylation of calmodulin. Synthesis and adduct formation with a fluorenyl-based spin label

The spin-labeling reagent, N4-(9'-fluorenylmethyloxycarbonyl)-4-amino-1-oxyl-4-succinimidyloxyca rbonyl- 2,2,6,6-tetramethylpiperidine, and the same enriched in 14C at the 4-formyl group, were synthesized as new acylating compounds for protein amino groups that can preserve charge. Porcine testicular calmodulin was modified with this reagent at pH 7.8 in the presence of Ca2+ under conditions that yielded a fairly homogeneous derivative as judged by electrophoretic analysis and tryptic digestion patterns. The tryptic peptides were separated by gel filtration and reverse-phase high-performance liquid chromatography, and the resulting, highly purified 14C-labeled peptides were hydrolyzed and their amino acid compositions determined. The results indicate that at least 87% of the modifications occur at lysyl residues 75 and 148, and the former appears to be the most reactive. This bilabeled calmodulin adduct does not activate a bovine brain cyclic nucleotide phosphodiesterase preparation. The fluorenylmethyloxycarbonyl portion of this inactive calmodulin derivative can, however, be removed by conditions that do not diminish native calmodulin activity in the phosphodiesterase assay. The resulting calmodulin adduct is active in the enzymic assay, although with diminished potency compared to calmodulin. The specificity of the reaction of this acylating reagent with calmodulin may be due to recognition of the tricyclic fluorene ring by the phenothiazine-binding sites since it was found that trifluoperazine inhibited the labeling reaction. Also, calmodulin was far more reactive to this reagent than were several other proteins. This is the first report of a specific, characterized lysine modification on calmodulin, and it is possible that other phenothiazine-binding proteins may also exhibit similar selectivity for acylation. Electron paramagnetic resonance spectra of the calmodulin adducts suggest a high degree of spin immobilization in both the Ca2+-free and Ca2+-saturated states.     

A model for the regulation of the calmodulin-dependent enzymes erythrocyte Ca2+-transport ATPase and brain phosphodiesterase by activators and inhibitors.

Acidic phospholipids, unsaturated fatty acids and limited proteolysis mimic the activating effect of calmodulin on erythrocyte Ca2+-transport ATPase and on brain cyclic nucleotide phosphodiesterase, as has been reported previously in several studies. Three different antagonists of calmodulin-induced activation of these enzymes were tested for their inhibitory potency on the stimulation produced by the other activators. Trifluoperazine and penfluridol were found to antagonize all the above mentioned types of activation of Ca2+-transport ATPase in the same concentration range. Both inhibitors also can reverse the activation of phosphodiesterase by oleic acid, phosphatidylserine and calmodulin at similar concentrations. However, in contrast with erythrocyte Ca2+-transport ATPase, activation of phosphodiesterase by limited tryptic digestion cannot be antagonized by penfluridol and trifluoperazine. Calmidazolium, formerly referred to as compound R 24571, was found to be a relatively specific inhibitor of calmodulin-induced activation of phosphodiesterase and Ca2+-transport ATPase, since antagonism of the other activators required much higher concentrations of the drug. The results suggest that the investigated drugs exert their inhibitory effect on calmodulin-regulated enzymes not solely via their binding to calmodulin but may also interfere directly with the calmodulin effector enzyme. In addition, a general mechanism of activation and inhibition of calmodulin-dependent enzymes is derived from our results.     

Replacement of Lys-75 of calmodulin affects its interaction with smooth muscle caldesmon

The interaction of smooth muscle caldesmon with synthetic calmodulin (SynCam) and its five mutants with replacement of Lys-75 was analyzed by means of intrinsic Trp fluorescence, zero-length crosslinking and by caldesmon-induced inhibition of actomyosin ATPase activity. SynCam and its double mutant with replacement K75P and simultaneous insertion of KGK between residues 80 and 81 have a comparably low affinity to caldesmon and the probability of crosslinking of this mutant to caldesmon was the lowest among all mutants analyzed. SynCam and its double mutant (K75P+KGK) induced nearly complete reversion of caldesmon inhibition of actomyosin ATPase activity with half-maximal reversion achieved at about 1 microM. Two mutants, K75A and K75V, with partially stabilized less positive central domain have higher affinity to caldesmon. These mutants induce 80-85% reversion of caldesmon inhibition of actomyosin ATPase and the half-maximal reversion was achieved at about 0.3-0.4 microM. Two last mutants, K75P and K75E, with distorted central domain have high affinity to caldesmon and the probability of crosslinking of K75P to caldesmon was the highest among calmodulin mutants tested. These mutants induced complete reversion of caldesmon inhibition with half-maximal effect observed at 0.3-0.4 microM. We suggest that the length, flexibility and charge of the central domain affect binding of calmodulin mutants and their ability to reverse caldesmon-induced inhibition of actomyosin ATPase activity.     

Effects of carbamoylation with alkyl isocyanates on the assay of proteins by dye binding

The effect of carbamoylation with alkyl isocyanate was used both to monitor the stability of the isocyanates and to study the influence of charge modification on protein assay. Carbamoylation of poly (L-lysine) with methyl isocyanate, ethyl isocyanate and 2-chloroethyl isocyanate was observed to decrease binding of methyl orange. The data emphasized the lability of alkyl isocyanates and indicated the importance of preparing aqueous solutions at low temperatures for studies on protein carbamoylation. After carbamoylation of several proteins, there was decreased metachromasia on binding to Coomassie Blue G. Poly (L-lysine) and H1 histone showed anomalous behavior in that with low concentrations of Coomassie Blue G the metachromasia was increased by carbamoylation, but at high concentrations of the dye the metachromasia was decreased by carbamoylation. In contrast to some reports in the literature, the data indicated that there is not always a simple relationship between the positive charge on a protein and the interaction with anionic dyes.     

Activation of a cyclic nucleotide phosphodiesterase and of a protein kinase by chemically modified calmodulin

1. Several calmodulin derivatives prepared by chemical modification of lysine residues were tested using bovine heart cyclic nucleotide phosphodiesterase and wheat germ calmodulin-dependent protein kinase. 2. The effect of chemical modification on the activation capacity of calmodulin for the two studied enzymes was different. 3. This was particularly noticeable in the case of alkylated derivatives which exhibited a higher affinity than native calmodulin towards phosphodiesterase but a lower affinity towards protein kinase. 4. The efficiency of these derivatives (maximal activation) was higher than that of native calmodulin in relation with the protein kinase.     

A calcium-sensitive fluorescent analog of calmodulin based on a novel calmodulin-binding fluorophore

Structure-activity studies of tetramethinemerocyanine fluorophores enabled the synthesis of novel dyes which showed spectral changes during reversible, calcium-dependent association with calmodulin. These spectral changes were greatly enhanced in dyes with a quaternary nitrogen and specifically placed hydrophobic chains. One such dye was covalently attached to calmodulin, producing a calmodulin analog with calcium-sensitive fluorescence. The analog, MeroCaM, showed a calcium-induced 3.4-fold increase in excitation ratio (608/532 nm excitation, 623 nm emission), which was fully reversed by lowering free calcium levels. MeroCaM's excitation ratio showed a half-maximal change at 300-400 nM calcium, below calcium concentrations reported to produce half-maximal saturation of calcium-calmodulin binding. However, the calcium dependence of MeroCaM's phosphodiesterase activation paralleled that of calmodulin. MeroCaM's fluorescence changes therefore appear to reflect primarily calcium binding to high affinity sites. MeroCaM's maximal phosphodiesterase activation was 30-40% that of calmodulin. In myosin light chain kinase activation, MeroCaM and calmodulin displayed indistinguishable maximal activation levels and concentration dependence of activation. Changes in MeroCaM's calcium affinity induced by magnesium, phosphodiesterase, and melittin were similar to those reported for calmodulin. Experiments with melittin revealed that target protein interaction could alter the fluorescence changes produced by calcium binding. MeroCaM showed promising brightness and photostability when imaged in individual living fibroblasts. The long excitation and emission wavelengths of MeroCaM, and the strong dependence of its excitation ratio on calcium concentrations, suit it well for use as a probe of calmodulin-dependent calcium signaling in living cells, as well as for experiments in vitro.     

自旋标记钙调蛋白与酸枣仁皂甙A相互作用研究

中草药有效成份酸枣仁皂甙A是钙调蛋白CaM的一种天然非竞争性拮抗剂。利用氮氧自由基马来酰亚胺衍生物标记CaM研究了二者的相互作用。结果表明,每分子CaM至少可以结合二个分子的酸枣仁皂钙A,二者的作用影响CaM上Lys残基主要是75,94)的环境,推测酸枣仁皂钙A是通过疏水作用结合到CaM分子两端的疏水沟区。通过比较三氟拉嗪TFP与酸枣仁皂甙A的结构特点,抑制性质与结合位点,提出了CaM调节环核苷酸磷酸二酯酶PDE的模式。     

Response of three enzymes to oleic acid, trypsin, and calmodulin chemically modified with a reactive phenothiazine

Calmodulin was covalently modified with 10-(1-propionyloxysuccinimide)-2-trifluoromethylphenothiazine++ + to stoichiometries between 0 and 2 mol/mol in the presence of Ca2+. The modified calmodulins, oleic acid, and trypsin were assayed for their ability to activate pea plant NAD kinase, bovine brain 3',5'-cAMP phosphodiesterase, and human erythrocyte Ca2+-ATPase. All modified calmodulins activated both phosphodiesterase and Ca2+-ATPase; at the highest concentration assayed, calmodulin modified with 2 mol of reagent/mol activated phosphodiesterase and Ca2+-ATPase to 53% and 100%, respectively, of the activation obtained with unmodified calmodulin. However, higher concentrations of the modified calmodulins were required to observe the same activation; at least 900-fold and 100-fold higher concentrations were required for the two enzymes, respectively. NAD kinase was not activated by any calmodulin labeled to a stoichiometry greater than 1 mol/mol even when a concentration equal to 17,000 times the apparent dissociation constant of calmodulin for NAD kinase was assayed. Therefore, the modified protein (and not some fraction resistant to labeling) is active toward the mammalian enzymes but inactive toward plant NAD kinase. The different response of the three enzymes to the chemical modification suggests that the enzymes may utilize different binding domains on calmodulin. NAD kinase also was not activated by other known activators of the two mammalian enzymes, namely lipids and limited proteolysis. In parallel experiments using the same agents on each enzyme, NAD kinase was the only enzyme of the three that was not activated by oleic acid and several other lipids or by limited trypsin digestion. These results show that NAD kinase possesses several attributes which would not be predicted by current models of the mechanism of activation of enzymes by calmodulin.     

Interaction of a kinesin-like protein with calmodulin isoforms from Arabidopsis.

In Arabidopsis and other plants there are multiple calmodulin isoforms. However, the role of these isoforms in regulating the activity of target proteins is obscure. Here, we analyzed the interaction between a kinesin-like calmodulin-binding motor protein (Reddy, A. S. N., Safadi, F., Narasimhulu, S. B., Golovkin, M., and Hu, X. (1996) J. Biol. Chem. 271, 7052-7060) and three calmodulin isoforms (calmodulin-2, -4, and -6) from Arabidopsis using different approaches. Gel mobility and fluorescence shift assays revealed that the motor binds to all calmodulin isoforms in a calcium-dependent manner. Furthermore, all calmodulin isoforms were able to activate bovine calcium/calmodulin-dependent phosphodiesterase. However, the concentration of calmodulin-2 required for half-maximal activation of phosphodiesterase is 2- and 6-fold lower compared with calmodulin-4 and -6, respectively. The dissociation constants of the motor to calmodulin-2, -4, and -6 are 12.8, 27.0, and 27.8 nM, respectively, indicating that calmodulin-2 has 2-fold higher affinity for the motor than calmodulin-4 and -6. Similar results were obtained using another assay that involves the binding of (35)S-labeled calmodulin isoforms to the motor. The binding saturation curves of the motor with calmodulin isoforms have confirmed that calmodulin-2 has 2-fold higher affinity to the motor. However, the affinity of calmodulin-4 and -6 isoforms for the motor was about the same. Based on these studies, we conclude that all calmodulin isoforms bind to the motor protein but with different affinities.     

Calmodulin fragments can not activate target enzymes

Under conditions where nM level of calmodulin was able to show full activation of myosin light chain kinase and cyclic-nucleotide phosphodiesterase, the fragments of calmodulin at concentrations as high as 20 microM failed to activate these enzymes in the presence of Ca2+. The fragments tested were Ala1-Lys75 (F12), Ala1-Arg74 (F12'), Lys75-Lys148 (F34'), Met76-Lys148 (F34'), Asp78-Lys148 (F34), Ala1-Arg106 (F123), and His107-Lys148 (F4). Purification of the proteolytic fragments through HPLC was necessary to remove contaminant calmodulin. Among the fragments, that corresponding to the C-terminal half domain inhibited myosin light chain kinase activity with the inhibition constant of 13 microM. The integrated structure of calmodulin consisting of N-terminal half domain, C-terminal half domain, and the linker peptide was indispensable for the enzyme activation. We discuss the functions of the two structural domains (N-domain and C-domain) in the activation of various enzymes.     

Differential reactivities of lysines in calmodulin complexed to phosphatase

Calmodulin and calmodulin complexed with calcineurin phosphatase were trace labeled with [3H]acetic anhydride and the incorporation of [3H]acetate into each epsilon-amino lysine of calmodulin was measured. The relative reactivities of calmodulin lysines were higher in the presence of Ca2+ than in the presence of EGTA, and the order was: Lys-75 greater than Lys-94 greater than Lys-148 greater than or equal to Lys-77 greater than Lys-13 greater than or equal to Lys-21 greater than Lys-30. The changes in relative reactivity implied a change in conformation. When calmodulin was complexed with the phosphatase, Lys-21, Lys-77, and Lys-148 were most protected, implying that these residues are at or near the interaction sites or are conformationally perturbed by the interaction. Lys-30 and Lys-75 were slightly protected, lysine 13 showed no change, while lysine 94 significantly increased in reactivity. Comparison with results obtained from myosin light chain kinase using a similar technique (Jackson, A. E., Carraway, K. L., III, Puett, D., and Brew, K. (1986) J. Biol. Chem. 261, 12226-12232) reveals that calmodulin may interact with each of the two enzymes similarly at or near Lys-21, Lys-75, and Lys-148; one difference with phosphatase is that complex formation also involved Lys-77. These findings suggest that calmodulin interacts differently with its target enzymes.     

A continuous fluorescence assay for cyclic nucleotide phosphodiesterase hydrolysis of cyclic GMP

The fluorescent 2'-methylanthraniloyl derivative of cyclic GMP undergoes a 45% decrease in fluorescence when it is cleaved by brain phosphodiesterase in the presence of calmodulin. This fluorescence decrease is dependent upon calcium, calmodulin, and phosphodiesterase, and correlates well (r = 0.996) with the disappearance of substrate as monitored by high-performance liquid chromatography. The Kd values determined by this fluorescence method and HPLC suggest that cyclic GMP and its fluorescent derivative exhibit similar kinetic parameters in their hydrolysis.     

Regulation of Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II

The 63-kDa subunit, but not the 60-kDa subunit, of brain calmodulin-dependent cyclic nucleotide phosphodiesterase was phosphorylated in vitro by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II. When calmodulin was bound to the phosphodiesterase, 1.33 +/- 0.20 mol of phosphate was incorporated per mol of the 63-kDa subunit within 5 min with no significant effect on enzyme activity. Phosphorylation in the presence of low concentrations of calmodulin resulted in a phosphorylation stoichiometry of 2.11 +/- 0.21 and increased about 6-fold the concentration of calmodulin necessary for half-maximal activation of the phosphodiesterase. Peptide mapping analyses of complete tryptic digests of the 63-kDa subunit revealed two major (P1, P4) and two minor (P2, P3) 32P-peptides. Calmodulin-binding to the phosphodiesterase almost completely inhibited phosphorylation of P1 and P2 with reduced phosphorylation rates of P3 and P4, suggesting the affinity change of the enzyme for calmodulin may be caused by phosphorylation of P1 and/or P2. When Ca2+/calmodulin-dependent protein kinase II was added without prior autophosphorylation, there was no phosphorylation of the 63-kDa phosphodiesterase subunit or of the kinase itself in the presence of a low concentration of calmodulin, and with excess calmodulin the phosphodiesterase subunit was phosphorylated only at P3 and P4. Thus the 63-kDa subunit of phosphodiesterase has a regulatory phosphorylation site(s) that is phosphorylated by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II and blocked by Ca2+/calmodulin binding to the subunit.     

Differentiation of the drug-binding sites of calmodulin

Calmodulin contains several binding sites for hydrophobic compounds. The apparent specificity of various 'calmodulin antagonists' for these sites was investigated. The Ki values for the inhibition of calmodulin-activated cyclic-nucleotide phosphodiesterase and myosin light-chain kinase was determined. In addition, the Kd values of the same compounds for binding to calmodulin were measured. The compounds could be separated into four groups. Group I and II compounds inhibited competitively the activation of the phosphodiesterase and myosin light-chain kinase by calmodulin. Group I compounds inhibited the activation of the phosphodiesterase and myosin light-chain kinase at identical concentrations. In contrast, group II compounds inhibited the activation of the phosphodiesterase at 5-10-fold lower concentrations than that of myosin light-chain kinase. Group III compounds inhibited the activation of these enzymes by an uncompetitive mechanism. Group IV compounds inhibited the activation of the phosphodiesterase with Ki values above 10 microM and did not affect the activation of myosin light-chain kinase. Binding of [3H]bepridil to calmodulin under equilibrium conditions yielded one high-affinity site (apparent Kd 0.4 microM) and four low affinity sites (apparent Kd 44 microM). Group I compounds interfered with the binding of bepridil to the high and low-affinity sites in a competitive manner. Group II compounds interfered in a non-competitive manner with the high-affinity site and apparently competed only with one of the low-affinity sites. Group III compounds did not compete with any of the bepridil-binding sites. Nimodipine, a group III compound, bound to one site on calmodulin with a Kd value of 1.1 microM. Other dihydropyridines competed with [3H]nimodipine for this site. The group I and II compounds, trifluoperazine and prenylamine, did not affect the binding of [3H]nimodipine. These data show that 'calmodulin antagonists' can be differentiated into at least three distinct groups. Kinetic and binding data suggest that the three groups bind to at least three different sites on calmodulin. Selective occupation of these sites may inhibit specifically the activation of distinct enzymes.