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.     

A calcium-binding protein from rabbit lung cytosol identified as the product of growth-regulated gene (2A9) and its binding proteins

Using Ca(2+)-dependent affinity chromatography on a synthetic compound (W-77)-coupled Sepharose 4B column, we purified two different Ca(2+)-binding proteins from rabbit lung extracts. The molecular weights of these proteins were estimated to be 17 kDa (calmodulin) and 10 kDa, respectively. The partial amino acid sequence of the 10-kDa protein revealed that it has two EF-hand structures. In addition, the 10-kDa protein was highly homologous (91%) to the product of growth-regulated gene, 2A9 (calcyclin). The Ca(2+)-binding property of the 10-kDa protein was observed by a change in the uv difference spectrum. Equilibrium dialysis showed that 1 mol of the 10-kDa protein bound to 2.04 +/- 0.05 mol of Ca2+ in the presence of 10(-4) M Ca2+. However, the protein failed to activate calmodulin-dependent enzymes such as Ca2+/CaM kinase II, myosin light chain kinase, and phosphodiesterase. We found that a 50-kDa cytosolic protein of the rabbit lung, intestine, and spleen bound to the 10-kDa protein, in a Ca(2+)-dependent manner. The distribution of calcyclin and calcyclin binding proteins was unique and seems to differ from that of calmodulin and calmodulin-binding proteins. Thus, calcyclin probably plays a physiological role through its binding proteins for the Ca(2+)-dependent cellular response.     

Dephosphorylation of neuromodulin by calcineurin

Neuromodulin (p57, GAP-43, F1, B-50) is a major neural-specific, calmodulin binding protein found in brain, spinal cord, and retina that is associated with membranes. Phosphorylation of neuromodulin by protein kinase C causes a significant reduction in its affinity for calmodulin (Alexander, K. A., Cimler, B. M., Meirer, K. E., and Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). It has been proposed that neuromodulin may function to bind and concentrate calmodulin at specific sites within neurons and that activation of protein kinase C causes the release of free calmodulin at high concentrations near its target proteins. It was the goal of this study to determine whether bovine brain contains a phosphoprotein phosphatase that will utilize phosphoneuromodulin as a substrate. Phosphatase activity for phosphoneuromodulin was partially purified from a bovine brain extract using DEAE-Sephacel and Sephacryl S-200 gel filtration chromatography. The neuromodulin phosphatase activity was resolved into two peaks by Affi-Gel Blue chromatography. One of these phosphatases, which represented approximately 60% of the total neuromodulin phosphatase activity, was tentatively identified as calcineurin by its requirement for Ca2+ and calmodulin (CaM) and inhibition of its activity by chlorpromazine. Therefore, bovine brain calcineurin was purified to homogeneity and examined for its phosphatase activity against bovine phosphoneuromodulin. Calcineurin rapidly dephosphorylated phosphoneuromodulin in the presence of micromolar Ca2+ and 3 microM CaM. The apparent Km and Vmax for the dephosphorylation of neuromodulin, measured in the presence of micromolar Ca2+ and 2 microM CaM, were 2.5 microM and 70 nmol Pi/mg/min, respectively, compared to a Km and Vmax of 4 microM and 55 nmol Pi/mg/min, respectively, for myosin light chain under the same conditions. Dephosphorylation of neuromodulin by calcineurin was stimulated 50-fold by calmodulin in the presence of micromolar free Ca2+. Half-maximal stimulation was observed at a calmodulin concentration of 0.5 microM. We propose that phosphoneuromodulin may be a physiologically important substrate for calcineurin and that calcineurin and protein kinase C may regulate the levels of free calmodulin available in neurons.     

Interaction between lipids and bovine brain calmodulin: lysophosphatidylcholine-induced conformation change

We have monitored the interaction of several lipids with the bovine brain calmodulin(CaM) and analyzed the effect of lysophosphatidylcholine(lyso-PC, 2-50 micrograms/ml) on conformation of CaM and the interaction between CaM and CaM-binding protein(CaMBP), using a fluorescence signal of 1-(dimethylamino)naphthalene-5-sulfonate-labeled CaM(DNS-CaM). Lyso-PC(egg, 20 micrograms/ml), among various natural lipids including phosphatidylserine(PS), phosphatidylinositol(PI), phosphatidylethanolamine (PE) and their lyso forms, greatly and dose-dependently enhanced the intensity of DNS fluorescence of DNS-CaM in the presence (100 microM CaCl2) and absence (1 mM EGTA) of Ca2+. Apparent dissociation constants calculated from the fluorometric titrations of binding of lyso-PC to DNS-CaM were 0.6 and 3.7 micrograms/ml in the presence and absence of Ca2+, respectively. Lyso-PC remarkably prevented both trypsin-induced quenching of the fluorescence of DNS-CaM and tryptic digestion of native CaM in the absence of Ca2+. Enhancement of DNS fluorescence of DNS-CaM by CaMBP was observed only in the presence of Ca2+ and lyso-PC could further increase the fluorescence intensity of the complex. These all results suggest that lyso-PC can modulate the interaction between CaM and CaMBP as a result of its direct effect on conformation of CaM.     

Molecular and biochemical analysis of calmodulin interactions with the calmodulin-binding domain of plant glutamate decarboxylase.

We previously provided what to our knowledge is the first evidence that plant glutamate decarboxylase (GAD) is a calmodulin (CaM)-binding protein. Here, we studied the GAD CaM-binding domain in detail. A synthetic peptide of 26 amino acids corresponding to this domain forms a stable complex with Ca2+/CaM with a 1:1 stoichiometry, and amino acid substitutions suggest that tryptophan-485 has an indispensable role in CaM binding. Chemical cross-linking revealed specific CaM/GAD interactions even in the absence of Ca2+. However, increasing KCI concentrations or deletion of two carboxy-terminal lysines abolished these interactions but had a mild effect on CaM/GAD interactions in the presence of Ca2+. We conclude that in the presence of Ca(2+)-hydrophobic interactions involving tryptophan-485 and electrostatic interactions involving the carboxy-terminal lysines mediate CaM/GAD complex formation. By contrast, in the absence of Ca2+, CaM/GAD interactions are essentially electrostatic and involve the carboxy-terminal lysines. In addition, a tryptophan residue and carboxy-terminal lysines are present in the CaM-binding domain of an Arabidopsis GAD. Finally, we demonstrate that petunia GAD activity is stimulated in vitro by Ca2+/CaM. Our study provides a molecular basis for Ca(2+)-dependent CaM/GAD interactions and suggests the possible occurrence of Ca(2+)-independent CaM/GAD interactions.     

Binding of the Neuronal Protein B-50, but Not the Metabolite B-60, to Calmodulin

Protein kinase C phosphorylates the neurone-specific protein B-50 at a single Ser41 residue, which is also the point for a major proteolytic cleavage in vitro, and probably in vivo, that produces a B-50 phosphorylation-inhibiting N-terminal fragment and a large C-terminal metabolite B-60 (B-50(41-226]. The intact purified protein will bind to calmodulin in the absence of calcium, but the interaction has an absolute requirement for dephospho-B-50. In an attempt to unify two aspects of B-50 biochemistry, we have examined the interaction of B-50 binding to calmodulin and B-50 proteolysis. HPLC- and affinity-purified B-50 bound to calmodulin, but purified B-60 did not. To ensure that this effect was not due to the phosphorylation state of pure, isolated B-60, the metabolite was generated in vitro using a Triton extract of synaptosomal plasma membranes, which contains the as yet uncharacterized B-50 protease. B-60 derived from dephospho-B-50 also failed to bind calmodulin. The results demonstrate a direct connection between B-50 binding to calmodulin and B-50 proteolysis. The position of the proposed calmodulin-binding domain within intact B-50 is discussed in light of the failure of calmodulin to bind B-60.     

Glutamate Transport and Not Glutamate Receptor Binding Is Stimulated by Gangliosides in a Ca2+-Dependent Manner in Rat Brain Synaptic Plasma Membranes

Crude as well as purified synaptic plasma membrane (SPM) preparations were analyzed for the influence of the ganglioside galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylgluc osyl ceramide (GM1) on high-affinity binding of L-[3H]glutamate. Assayed in two different buffer systems, SPM consistently exhibited increased (40-50%) binding upon incubation with GM1 plus Ca2+, as compared to controls without GM1. Incorporation experiments with 3H-labeled GM1 proved trypsin-stable insertion of GM1 into SPM, with a maximum incorporation of four times the endogenous amount (35 nmol/mg of protein). The observed increase in glutamate binding was not due to a change in the affinity of the binding sites, but to a change in the number of binding sites, and it was absolutely dependent on the presence of Ca2+. A pharmacological profile of the GM1/Ca2+-stimulated glutamate binding is presented. The original classification of the stimulatory effect as an effect on glutamate receptor binding had to be revised to take into account the observed temperature sensitivity of the ganglioside effect, its sensitivity to high osmolarity and to ultrasonication, and the lack of binding stimulation after detergent treatment of membranes or after receptor solubilization. Vesicular space measured in both SPM preparations was found to be around 7 microliters/mg of protein, in ganglioside-treated as well as in control membranes. From the data, it is concluded that a special, Na+- and Cl- -independent form of glutamate transport into resealed membrane vesicles is stimulated by gangliosides in the presence of Ca2+.     

Ca(2+)-dependent and Ca(2+)-independent calmodulin binding sites in erythrocyte protein 4.1. Implications for regulation of protein 4.1 interactions with transmembrane proteins

In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH(2)-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca(2+) and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca(2+)/CaM to down-regulate 4. 1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca(2+) and CaM requires binding of CaM to both Ca(2+)-independent and Ca(2+)-dependent sites in protein 4.1.     

KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II

1-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipera zine (KN-62), a selective inhibitor of rat brain Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) was synthesized and its inhibitory properties in vitro and in vivo were investigated. KN-62 inhibited phosphorylation of exogenous substrate (chicken gizzard myosin 20-kDa light chain) by Ca2+/CaM kinase II with Ki value of 0.9 microM, but no significant effect up to 100 microM on activities of chicken gizzard myosin light chain kinase, rabbit brain protein kinase C, and bovine heart cAMP-dependent protein kinase type II. KN-62 also inhibited the Ca2+/calmodulin-dependent autophosphorylation of both alpha (50 kDa) and beta (60 kDa) subunits of Ca2+/CaM kinase II dose dependently in the presence or absence of exogenous substrate. Kinetic analysis indicated that this inhibitory effect of KN-62 was competitive with respect to calmodulin. However, KN-62 did not inhibit the activity of autophosphorylated Ca2+/CaM kinase II. Moreover, Ca2+/CaM kinase II bound to a KN-62-coupled Sepharose 4B column, but calmodulin did not. These results suggest that KN-62 affects the interaction between calmodulin and Ca2+/CaM kinase II following inhibition of this kinase activity by directly binding to the calmodulin binding site of the enzyme but does not affect the calmodulin-independent activity of already autophosphorylated (activated) enzyme. We examined the effect of KN-62 on cultured PC12 D pheochromocytoma cells. KN-62 suppressed the A23187 (0.5 microM)-induced autophosphorylation of the 53-kDa subunit of Ca2+/CaM kinase in PC12 D cells, which was immunoprecipitated with anti-rat forebrain Ca2+/CaM kinase II polypeptides antibodies coupled to Sepharose 4B, thereby suggesting that KN-62 could inhibit the Ca2+/CaM kinase II activity in vivo.     

Modulation of erythrocyte Ca2+-ATPase by selective calpain cleavage of the calmodulin-binding domain

The activity of the membrane-bound and the purified erythrocyte Ca2+-ATPase in the absence of calmodulin was stimulated by calpain digestion but could be further increased to maximal levels by calmodulin (CaM). Thus, CaM sensitivity was retained by the digested ATPase, at least at short times of incubation. In membranes digested at higher temperatures and in the purified ATPase digested at higher calpain/ATPase ratios, the ATPase became fully activated. The membrane-bound and the purified 138-kDa ATPase were converted by calpain to a fragment of approximately 124 kDa which still bound CaM and could be isolated on CaM columns when proteolysis occurred slowly but not when it occurred rapidly. Carboxypeptidase digestion of the purified enzyme and of its fragment of about 124 kDa has shown that calpain attacked the CaM-binding domain near the C terminus of the ATPase. This has also been supported by digestion of the purified enzyme and of its fragment of about 124 kDa. A first cut occurred in the middle of the domain producing a fragment of about 14 kDa and a (CaM-binding) fragment of about 124 kDa. A second cut closer to the N terminus of the domain also produced a fragment of about 124 kDa and accounted for the loss of CaM binding at prolonged times of incubation of the ATPase with calpain.     

Purification of Maize Cytosolic 70 kD Stress Protein: a Calmodulin-binding Protein in Plants

The maize cytosolic 70 kD stress protein (HSC70) has been purified by a two-step procedure employing affinity chromatography on ATP-agarose followed by DEAE52 ion-exchange chromatography. Using a biotinylated cauliflower calmodulin (CAM) gel-overlay technique in the presence of 1 mmol/L Ca2+ , the HSCT0 could bind to CAM. No band was shown on sodium dodecyl sulfate-polyacrylamide gel overlayed with biotinylated cauliflower CaM when 1 mmoL/L Ca2+ was replaced by 5 mmol/L EGTA. It indicated that the binding of HSC70 to CaM was dependent on Ca2+. The purified HSC70 inhibited the activity of CaM-dependent NADK and the degree of inhibition increased with augmentation of the HSC70, which appeared to be typically characteristic to CaM- binding protein.     

Identification of the Ca(2+)-dependent calmodulin-binding region of chromogranin A.

Chromogranin A (CGA), the most abundant protein in bovine adrenal chromaffin granules, is a high-capacity, low-affinity Ca(2+)-binding protein found in most neuroendocrine cells, and binds calmodulin (CaM) in a Ca(2+)-dependent manner. The binding of chromogranin A to calmodulin was determined by measuring the intrinsic tryptophan fluorescence of chromogranin A in the presence and absence of Ca2+. Binding was specifically Ca(2+)-dependent; neither Mg2+ nor Mn2+ could substitute for Ca2+. Chelation of Ca2+ by EGTA completely eliminated the chromogranin A-calmodulin interaction. CaM binding was demonstrated by a synthetic CGA peptide representing residues 40-65. When the CGA peptide and CaM were mixed in the presence of 15 mM CaCl2, the intrinsic tryptophan fluorescence emission underwent a substantial blue-shift, shifting from 350 to 330 nm. Like the intact CGA, the peptide-CaM binding was specifically Ca(2+)-dependent, and neither Mg2+ nor Mn2+ could induce the binding. Calmodulin bound both to CGA and to the synthetic CGA peptide with a stoichiometry of one to one. The dissociation constants (Kd) determined by fluorometric titration were 13 nM for the peptide-CaM binding and 17 nM for intact CGA-CaM binding. The Kd values are comparable to those (approximately 10(-9) M) of other CaM-binding proteins and peptides, demonstrating a tight binding of CaM by CGA. The CaM-binding CGA residues 40-65 are 100% conserved among all the sequenced CGAs in contrast to 50-60% conservation found in the entire sequence, implying essential roles of this region.     

Ca2+/calmodulin-dependent protein kinase II is an essential mediator in the coordinated regulation of electrocyte Ca2+-ATPase by calmodulin and protein kinase A

The aim of this study was to investigate (a) whether Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) participates in the regulation of plasma membrane Ca2+-ATPase and (b) its possible cross-talk with other kinase-mediated modulatory pathways of the pump. Using isolated innervated membranes of the electrocytes from Electrophorus electricus L., we found that stimulation of endogenous protein kinase A (PKA) strongly phosphorylated membrane-bound CaM kinase II with simultaneous substantial activation of the Ca2+ pump (approximately 2-fold). The addition of cAMP (5-50 pM), forskolin (10 nM), or cholera toxin (10 or 100 nM) stimulated both CaM kinase II phosphorylation and Ca2+-ATPase activity, whereas these activation processes were cancelled by an inhibitor of the PKA alpha-catalytic subunit. When CaM kinase II was blocked by its specific inhibitor KN-93, the Ca2+-ATPase activity decreased to the levels measured in the absence of calmodulin; the unusually high Ca2+ affinity dropped 2-fold; and the PKA-mediated stimulation of Ca2+-ATPase was no longer seen. Hydroxylamine-resistant phosphorylation of the Ca2+-ATPase strongly increased when the PKA pathway was activated, and this phosphorylation was suppressed by inhibition of CaM kinase II. We conclude that CaM kinase II is an intermediate in a complex regulatory network of the electrocyte Ca2+ pump, which also involves calmodulin and PKA.     

Phosphorylation of the Casein Kinase II Domain of B-50 (GAP-43) in Rat Cortical Growth Cones

Abstract: Growth-associated phosphoprotein B-50 is a neural protein kinase C (PKC) substrate enriched in nerve growth cones that has been implicated in growth cone plasticity. Here we investigated whether B-50 is a physiological substrate for casein kinase II (CKII) in purified rat cortical growth cone preparations. Using site-specific proteolysis and known modulators of PKC, in combination with immunoprecipitation, mass spectrometry, and phosphoamino acid analysis, we demonstrate that endogenous growth cone B-50 is phosphorylated at multiple sites, on both serine and threonine residues. Consistent with previous reports, stimulation of PKC activity increased the phosphorylation of only those proteolytic fragments containing Ser⁴¹. Under basal conditions, however, phosphorylation was predominantly associated with fragments not containing Ser⁴¹. Mass spectrometry of tryptic digests of B-50, which had been immunoprecipitated from untreated growth cones, revealed that in situ phosphorylation occurs within peptides B-50_181–198 and B-50_82–98. These peptides contain the major and minor in vitro CKII phosphosites, respectively. In addition, cyanogen bromide digestion of immunoprecipitated chick B-50 generated a 4-kDa C-terminal B-50 phosphopeptide, confirming that phosphorylation of the CKII domain occurs across evolutionary diverse species. We conclude that B-50 in growth cones is not only a substrate for PKC, but also for CKII.     

Evidence for multisite ADP-ribosylation of neuronal phosphoprotein B-50/GAP-43

The neuronal phosphoprotein B-50/GAP-43 is associated with neuronal growth and regeneration and is involved in the calcium/CaM and G_o signal transduction systems. In particular, B-50 interacts uniquely with CaM by binding in the absence of Ca²⁺. Previously identified as a major neuronal substrate for protein kinase C, which releases CaM via phosphorylation, B-50 has more recently been shown to be a substrate for endogenous ADP-ribosyltransferases. In the present study, we utilized amino acid modification with iodoacetamide and chemical stability to mercury and neutral hydroxylamine to demonstrate that the predominant site of ADP-ribosylation is Cys 3 and/or Cys 4. Chymotryptic peptide mapping further revealed a second, less labelled site of ribosylation in the C-terminal region. The results also demonstrate that, in contrast to PKC phosphorylation, ADP-ribosylation of B-50 does not mediate CaM binding. Since Cys 3 and Cys 4, by palmitoylation, are important for membrane anchoring, our findings suggest that ADP-ribosylation of B-50 may have a role in directing the intracellular localization of the protein. Hence, ribosylation of B-50 may mediate where B-50 interacts with signal transduction pathways.     

N-Terminal-Specific Anti-B-50 (GAP-43) Antibodies Inhibit Ca2+-Induced Noradrenaline Release, B-50 Phosphorylation and Dephosphorylation, and Calmodulin Binding

Abstract: B-50 (GAP-43) is a presynaptic protein kinase C (PKC) substrate implicated in the molecular mechanism of noradrenaline release. To evaluate the importance of the PKC phosphorylation site and calmodulin-binding domain of B-50 in the regulation of neurotransmitter release, we introduced two monoclonal antibodies to B-50 into streptolysin O-permeated synaptosomes isolated from rat cerebral cortex. NM2 antibodies directed to the N-terminal residues 39–43 of rat B-50 dose-dependently inhibited Ca²⁺-induced radiolabeled and endogenous noradrenaline release from permeated synaptosomes. NM6 C-terminal-directed (residues 132–213) anti-B-50 antibodies were without effect in the same dose range. NM2 inhibited PKC-mediated B-50 phosphorylation at Ser⁴¹ in synaptosomal plasma membranes and permeated synaptosomes, inhibited ³²P-B-50 dephosphorylation by endogenous synaptosomal phosphatases, and inhibited the binding of calmodulin to synaptosomal B-50 in the absence of Ca²⁺. Similar concentrations of NM6 did not affect B-50 phosphorylation or dephosphorylation or B-50/calmodulin binding. We conclude that the N-terminal residues 39–43 of the rat B-50 protein play an important role in the process of Ca²⁺-induced noradrenaline release, presumably by serving as a local calmodulin store that is regulated in a Ca²⁺- and phosphorylation-dependent fashion.     

Calmodulin X (Ca2+)4 is the active calmodulin-calcium species activating the calcium-, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum in the regulation of the calcium pump

Calcium-, calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum increases the rate of calcium transport. The complex dependence of calmodulin-dependent phosphoester formation on free calcium and total calmodulin concentrations can be satisfactorily explained by assuming that CaM X (Ca2+)4 is the sole calmodulin-calcium species which activates the calcium-, calmodulin-dependent, membrane-bound protein kinase. The apparent dissociation constant of the E X CaM X (Ca2+)4 complex determined from the calcium dependence of calmodulin-dependent phosphoester formation over a 100-fold range of total calmodulin concentrations (0.01-1 microM) was 0.9 nM; the respective apparent dissociation constant at 0.8 mM free calcium, 1 mM free magnesium with low calmodulin concentrations (0.1-50 nM) was 2.60 nM. These results are in good agreement with the apparent dissociation constant of 2.54 nM of high affinity calmodulin binding determined by 125I-labelled calmodulin binding to sarcoplasmic reticulum fractions at 1 mM free calcium, 1 mM free magnesium and total calmodulin concentration ranging from 0.1 to 150 nM, i.e. conditions where approximately 98% of the total calmodulin is present as CaM X (Ca2+)4. The apparent dissociation constant of the calcium-free calmodulin-enzyme complex (E X CaM) is at least 100-fold greater than the apparent dissociation constant of the E X CaM X (Ca2+)4 complex, as judged from non-saturation 125I-labelled calmodulin binding at total calmodulin concentrations of up to 150 nM, in the absence of calcium.     

Calmodulin-Dependent Protein Phosphorylation in Synaptic Junctions

Synaptic junctions (SJs) from rat forebrain were examined for Ca2+/calmodulin (CaM)-dependent kinase activity and compared to synaptic plasma membrane (SPM) and postsynaptic density (PSD) fractions. The kinase activity in synaptic fractions was examined for its capacity to phosphorylate endogenous proteins or exogenous synapsin I, in the presence or absence of Ca2+ plus CaM. When assayed for endogenous protein phosphorylation, SJs contained approximately 25-fold greater amounts of Ca2+/CAM-dependent kinase activity than SPMs, and fivefold more activity than PSDs. When kinase activities were measured by phosphorylation of exogenous synapsin I, SJs contained fourfold more activity than SPMs, and 10-fold more than PSDs. The phosphorylation of SJ proteins of 60- and 50-kilodalton (major PSD protein) polypeptides were greatly stimulated by Ca2+/CaM; levels of phosphorylation for these proteins were 23- and 17-fold greater than basal levels, respectively. Six additional proteins whose phosphorylation was stimulated 6-15-fold by Ca2+/CAM were identified in SJs. These proteins include synapsin I, and proteins of 240, 207, 170, 140, and 54 kilodaltons. The 54-kilodalton protein is a highly phosphorylated form of the major PSD protein and the 170-kilodalton component is a cell-surface glycoprotein of the postsynaptic membrane that binds concanavalin A. The CaM-dependent kinase in SJ fractions phosphorylated endogenous phosphoproteins at serine and/or threonine residues. Ca2+-dependent phosphorylation in SJ fractions was strictly dependent on exogenous CaM, even though SJs contained substantial amounts of endogenous CaM (15 micrograms CaM/mg SJ protein). Exogenous CaM, after being functionally incorporated into SJs, was rapidly removed by sequential washings. These observations suggest that the SJ-associated CaM involved in regulating Ca2+-dependent protein phosphorylation may be in dynamic equilibrium with the cytoplasm. These findings indicate that a brain CaM-dependent kinase(s) and substrate proteins are concentrated at SJs and that CaM-dependent protein phosphorylation may play an important role in mechanisms that underlie synaptic communication.     

Regulation of calmodulin content in synaptic plasma membranes by glucocorticoids

Synaptic plasma membranes (SPM) from the brain are known to have specific binding sites for several steroid hormones, but the mechanisms of membrane transduction of steroid signals is not understood. In this study, corticosterone was found to prevent temperature-dependent dissociation of endogenous calmodlin (CaM) from highly purified SPM from rat cerebral cortex. The steroid stabilizes Ca²⁺-dependent membrane binding of endogenous CaM (78% of total CaM), whereas Ca²⁺-independent binding of CaM (the other 22%) is not affected. The stabilization of membrane binding of endogenous CaM by corticosterone is concentration-dependent, with the maximal effect occurring at steroid concentration of 1 M. The EC₅₀ is estimated as 130 nM, which is almost identical to the K_d of specific binding of the steroid to SPM (120 nM) reported previously. The effect in stabilizing membrane binding of CaM is specific to corticosterone and other glucocorticoids (cortisol, dexamethasone and triamcinolone); gonadal steroids (17-estradiol, progesterone and testosterone) are ineffective. Furthermore, corticosterone administration in vivo (2 mg/kg, i.p.) produced a rapid increase of CaM content in SPM, occurring within 5 min after steroid injection and persisting for at least 20 min. Since CaM mediates a variety of biochemical processes in synaptic membranes, we hypothesize that the effect of glucocorticoids in promoting membrane binding of CaM may lead to a cascade of consequences in synaptic membrane function.Special issue dedicated to Dr. Sidney Ochs.