Multiple ubiquitination of vertebrate calmodulin by reticulocyte lysate and inhibition of calmodulin conjugation by phosphorylase kinase

Mammalian calmodulin containing trimethyllysine 115 can be covalently coupled to ubiquitin in a Ca2+-dependent manner in the presence of ATP/Mg2+ by reticulocyte lysate. This conjugation reaction can be quantitated in a novel test employing fluphenazine-Sepharose. It is shown that at least 3 ubiquitin molecules can be coupled to calmodulin indicating that more than one lysine residue is involved in the ubiquitination reaction. In addition only the free form of calmodulin can be ubiquitinated. Neither calmodulin bound to phosphorylase kinase as an integral subunit (delta-subunit) nor that bound as a peripheral subunit (delta'-subunit) is ubiquitinated. A total binding of equimolar calmodulin to phosphorylase kinase occurs since the affinity of binding of calmodulin to phosphorylase kinase as integral (KCaMm unknown) or peripheral subunit (KCaMm ca. 30-50nM) is several order of magnitude higher than the corresponding affinity of calmodulin for the ubiquitin-conjugating enzyme (KCaMm ca. 8 microM). We conclude that the "protective" effect of phosphorylase kinase towards calmodulin conjugation is due to a changed conformation of bound calmodulin and/or inacessibility of the ubiquitination sites (e.g. at subunit-subunit interface). Thus Ca2+-dependent ubiquitination only of free calmodulin may provide an efficient scavanging mechanism (with subsequent breakdown) for all free calmodulin in excess of that amount which can be bound by the calmodulin-binding proteins in the cell.     

Synthetic peptides based on the calmodulin-binding domain of myosin light chain kinase inhibit activation of other calmodulin-dependent enzymes

Nanomolar concentrations of synthetic peptides corresponding to the calmodulin-binding domain of skeletal muscle myosin light chain kinase were found to inhibit calmodulin activation of seven well-characterized calmodulin-dependent enzymes: brain 61 kDa cyclic nucleotide phosphodiesterase, brain adenylate cyclase, Bordetella pertussis adenylate cyclase, red blood cell membrane Ca++-pump ATPase, brain calmodulin-dependent protein phosphatase (calcineurin), skeletal muscle phosphorylase b kinase, and brain multifunctional Ca++ (calmodulin)-dependent protein kinase. Inhibition could be entirely overcome by the addition of excess calmodulin. Thus, the myosin light chain kinase peptides used in this study may be useful antagonists for studying calmodulin-dependent enzymes and processes.     

High and intermediate affinity calmodulin binding domains of the alpha and beta subunits of phosphorylase kinase and their potential role in phosphorylation-dependent activation of the holoenzyme.

Phosphorylase kinase is a calcium-regulated multimeric enzyme of composition (alpha beta gamma delta)4, which contains calmodulin as the integral delta subunit and also is activated further by addition of extrinsic calmodulin. Previous studies by Dasgupta, M., Honeycutt, T., and Blumenthal, D.K. ((1989) J. Biol. Chem. 264, 17156-17163) have identified gamma 302-326 and gamma 342-366 as two calmodulin binding regions. Using peptides that were synthesized based on alpha and beta primary structure and that were predicted to contain the basic amphiphilic alpha-helix motif thought important for calmodulin binding, four additional potential calmodulin binding domains have now been identified: one of high affinity, beta 770-794; two of intermediate affinity, beta 5-28 and beta 920-946; and one with marginally low affinity, alpha 1070-1093. Peptide beta 770-794 was of higher calmodulin affinity than either gamma 302-326 or gamma 342-366; it was of higher affinity than the model synthetic peptide IV defined by O'Neil, K.T., and DeGrado, W.F. ((1990) Trends Biochem. Sci. 15, 59-64); and it is currently the most potent calmodulin-binding peptide so far described. Correlated with their affinity for calmodulin, all six phosphorylase kinase-derived peptides and several other established calmodulin-binding peptides inhibited phosphorylase kinase previously activated by cAMP-dependent phosphorylation, reducing its activity to the level of the nonactivated enzyme. However, these peptides did not inhibit (and some peptides slightly activated) the nonphosphorylated enzyme. Even in the presence of these peptides both activated and nonactivated enzyme remained fully Ca(2+)-dependent. The beta 770-794 peptide has at least a 5-fold greater calmodulin binding affinity than the holo-phosphorylase kinase. This, and its higher affinity for calmodulin than either of the sites on the gamma subunit, raises the possibility that in the native enzyme it may be involved in binding the intrinsic delta subunit. Further, inhibition of activated but not nonactivated enzyme by calmodulin-binding peptides would suggest that the phosphorylation-dependent activation of phosphorylase kinase may be mediated by changes in the binding interactions of the intrinsic calmodulin delta subunit.     

Expression of a cDNA for the catalytic subunit of skeletal-muscle phosphorylase kinase in transfected 3T3 cells.

Phosphorylase kinase is a multimeric enzyme of composition (alpha, beta, gamma, delta)4 whose catalytic activity resides in the gamma-subunit. As an approach to understand further its regulation, a cDNA for the gamma-subunit of phosphorylase kinase (gamma PhK) has been cloned into a mammalian expression vector behind the mouse metallothionein-1 promoter. NIH 3T3 cells were co-transfected with this construct (pEV gamma PhK) and pSV2neo, G418-resistant clones were selected, and several were found to have stably incorporated the gamma-subunit cDNA into their genomic DNA. Phosphorylase kinase activity was clearly present in extracts from cultures of pEV gamma PhK-transformed cells and increased several-fold after 24 h of incubation with Zn2+, whereas it was undetectable in the parent 3T3 cells. A significant, but variable, proportion (15-70%) of the activity was Ca2+-dependent. We conclude that the phosphorylase kinase activity expressed by the cells transformed with pEV gamma PhK is due to free gamma-subunit and gamma-subunit associated with cellular calmodulin, which replaces the delta-subunit normally associated with the gamma-subunit in the holoenzyme.     

Cardiac sarcoplasmic-reticulum calmodulin-binding proteins. Modulation of calmodulin binding to phospholamban by phosphorylation.

The gel-overlay technique with 125I-labelled calmodulin allowed the detection of several calmodulin-binding proteins of Mr 280 000, 150 000, 97 000, 56 000, 35 000 and 24 000 in canine cardiac sarcoplasmic reticulum. Only two calmodulin-binding proteins could be identified unambiguously. Among them, the 97 000-Mr protein that undergoes phosphorylation in the presence of Ca2+ and calmodulin, is likely to be glycogen phosphorylase. In contrast, the (Ca2+ + Mg2+)-activated ATPase did not appear to bind calmodulin under our experimental conditions. The second known calmodulin target is dephosphophospholamban, which migrates with an apparent Mr of 24 000. The dimeric as well as the monomeric form of phospholamban was found to bind calmodulin. Phospholamban shifts the apparent Kd of erythrocyte (Ca2+ + Mg2+)-activated ATPase for calmodulin, suggesting thus a tight binding of calmodulin to the proteolipid. Interestingly enough, phospholamban phosphorylation by either the catalytic subunit of cyclic AMP-dependent protein kinase or the Ca2+/calmodulin-dependent phospholamban kinase was found to inhibit calmodulin binding.     

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.     

Differential recognition of calmodulin-enzyme complexes by a conformation-specific anti-calmodulin monoclonal antibody

An anti-calmodulin monoclonal antibody having an absolute requirement for Ca2+ has been produced from mice immunized with a mixture of calmodulin and calmodulin-binding proteins. Radioimmune assays were developed for the determination of its specificity. the epitope for this antibody resides on the COOH-terminal half of the mammalian protein. Plant calmodulin or troponin C had little reactivity. The apparent affinity of the antibody for calmodulin was increased approximately 60-fold in the presence of heart calmodulin-dependent phosphodiesterase. The presence of heart phosphodiesterase in the radioimmune assay greatly enhanced the sensitivity for calmodulin. The intrinsic calmodulin subunit of phosphorylase kinase and calmodulin which was bound to brain phosphodiesterases was also recognized with high affinity by the antibody. The antibody reacted poorly with calmodulin which was bound to heart or brain calcineurin, skeletal muscle myosin light chain kinase, or other calmodulin-binding proteins. In direct binding experiments, most of the calmodulin-binding proteins studied were unreactive with the antibody. This selectivity allowed purification of heart and two brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes on immobilized antibody affinity columns. Phosphodiesterase activity was adsorbed directly from crude samples and specifically eluted with EGTA. Isozyme separation was accomplished using a previously described anti-heart phosphodiesterase monoclonal antibody affinity support. The brain isozymes differed not only in reactivity with the anti-phosphodiesterase antibody, but also in apparent subunit molecular weight, and relative specificity for cAMP and cGMP as substrates. The calmodulin activation constants for the brain enzymes were 10-20-fold greater than for the heart enzyme. The data suggest that the binding of ligands to Ca2+/calmodulin induce conformation changes in calmodulin which alter reactivity with the anti-calmodulin monoclonal antibody. The differential antibody reactivity toward calmodulin-enzyme complexes indicates that target proteins either induce very different conformations in calmodulin and/or interact with different geometries relative to the antibody binding site. The anti-calmodulin monoclonal antibody should be useful for the purification of other calmodulin-dependent phosphodiesterases as well as isozymes of phosphorylase kinase.     

Phosphorylation-dependent subcellular translocation of a Ca2+/calmodulin-dependent protein kinase produces an autonomous enzyme in Aplysia neurons

We have shown previously that the subcellular distribution of a major calmodulin-binding protein is altered under conditions causing increased synthesis of cAMP in Aplysia neurons (Saitoh, T., and J. H. Schwartz, 1983, Proc. Natl. Acad. Sci. USA, 80:6708-6712). We now provide evidence that this Mr 55,000 protein is a subunit of a Ca2+/calmodulin-dependent kinase: (a) both the Mr 55,000 calmodulin-binding protein and kinase activity are loosely attached to the membrane-cytoskeletal complex; (b) both kinase activity and the Mr 55,000 protein are translocated from the membrane-cytoskeleton complex to the cytoplasm under conditions that cause the change in the subcellular distribution of the Mr 55,000 calmodulin-binding protein; and (c) calmodulin-binding activity of the Mr 55,000 protein and the ability to carry out the Ca2+/calmodulin-dependent phosphorylation of synapsin I are purified in parallel. The subcellular localization of the Ca2+/calmodulin-dependent protein kinase appears to be under control of two second messengers: Ca2+ and cAMP. We find that the Mr 55,000 subunit is phosphorylated when the extracted membrane-cytoskeleton complex is incubated with Ca2+, calmodulin, and ATP, with the concomitant release of this phosphorylated peptide from the complex. Previously, we had found that, when translocation occurs in extracts in the presence of cAMP and ATP (but in the absence of Ca2+), there was no detectable phosphorylation of the Mr 55,000 subunit itself. The subcellular distribution of the subunit thus appears to be influenced by (a) cAMP-dependent phosphorylation, which, we infer, modifies some as yet unidentified structural component, causing the release of the enzyme; and (b) Ca2+/calmodulin-dependent phosphorylation of the Mr 55,000 subunit. These studies also suggest that phosphorylation has an important regulatory consequence: during the Ca2+/calmodulin-dependent translocation of the Mr 55,000 subunit, the kinase appears to be activated, becoming independent of added Ca2+/calmodulin.     

Identification and primary structure of calmodulin binding domains in the phosphorylase kinase holoenzyme

Fast twitch skeletal muscle phosphorylase kinase was isolated and incubated with a radioactive, bifunctional, photoactivable, and cleavable cross-linker conjugated to calmodulin. Incubation of the holoenzyme only resulted in the labeling of the alpha-subunit in the presence of Ca2+. After cleavage with CNBr (and subdigestion with Asp-N protease), a sequence was identified (residues 1069-1087) in the alpha-subunit which had the predominant basic character and the propensity to form an amphiphilic helix like other calmodulin binding domains. If cross-linked calmodulin was incubated with the isolated subunits of phosphorylase kinase, radioactivity was recovered in seven CNBr peptides: three came from the alpha-subunits, one of them corresponding to the sequence labeled in the holoenzyme. Three came from the beta-subunit, and one came from the gamma-subunit. The latter contained the two adjacent calmodulin binding domains recently identified in the gamma-subunit (Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1988) J. Biol. Chem. 264, 17156-17163).     

Characterization of a calmodulin-dependent protein phosphatase from human platelets

A calmodulin-dependent protein phosphatase has been identified in human platelets by its cross-reactivity with an antibody developed against a bovine brain calmodulin-dependent protein phosphatase and by its calmodulin-stimulated dephosphorylation of 32P-labeled substrates. The platelet enzyme was partially purified to separate it from calmodulin and calmodulin-independent phosphatases. The partially purified enzyme was stimulated by calmodulin, requiring 15 nM calmodulin for half-maximal activation. Calmodulin increased the Vmax of the phosphatase, with no significant effect on its Km. The enzyme was stimulated irreversibly and made calmodulin-independent by limited proteolysis. The optimal pH for the phosphatase was 7.5. After partial purification, phosphatase activity was significantly increased in the presence of Mn2+ and Ca2+ over that observed in the presence of Ca2+ alone. The enzyme effectively dephosphorylated casein, histone, protamine, and platelet actin. The holophosphatase was estimated to have a molecular weight of 76,900 as determined by sedimentation on sucrose gradients. Immunoblotting techniques using an antibody against the brain phosphatase suggests that the enzyme consists of 2 subunits of 60,000 and 16,500 daltons; the 60,000-dalton subunit co-migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a 60,000-dalton calmodulin-binding protein in the platelet suggesting that it is the calmodulin-binding subunit of the enzyme. The identification of a calmodulin-dependent protein phosphatase in human platelets suggests a role for Ca2+-dependent dephosphorylation in platelet activation.     

Mapping of calmodulin-binding domain of Ca2+/calmodulin-dependent protein kinase II from rat brain

Recent molecular cloning experiments have identified a 25 amino-acid region as the calmodulin-binding domain of the alpha-subunit of rat brain Ca2+/calmodulin-dependent multifunctional protein kinase II (CaM-K II). Synthetic peptides, derived from the deduced amino-acid sequence encompassing this region, were examined for their ability to bind calmodulin in a calcium dependent manner and to inhibit the Ca2+/calmodulin-dependent autophosphorylation of CaM-K II. Comparison of these structure-function relationships highlighted a region of 5 amino-acids, which was essential for calmodulin interaction and inhibition of kinase activity. This region demonstrated some homology with other calmodulin-binding peptides, and may represent a key site of interaction of the kinase with calmodulin. These analyses provide additional insight into the molecular mechanism underlying the Ca2+ regulation of CaM-K II.     

A monoclonal antibody showing cross-reactivity toward three calmodulin-dependent enzymes

The spleen cells of a Balb/c mouse immunized with purified bovine calmodulin-dependent cyclic nucleotide phosphodiesterase were fused with nonsecreting mouse myeloma cells (P3-X63-Ag8-653). Antibody producing hybridomas were screened by the enzyme-linked immunosorbent assay using purified phosphodiesterase as the antigen. One monoclonal cell line, CR-B1, was found to produce antibodies which showed positive enzyme-linked immunosorbent assay reactions with bovine brain calcineurin and rabbit muscle phosphorylase kinase in addition to phosphodiesterase. The antibody was purified and characterized. It was shown to immunoprecipitate the calmodulin (CaM)-dependent phosphodiesterase and phosphorylase kinase activities but not those of CaM itself, CaM-independent phosphodiesterase and the catalytic unit of cAMP-dependent protein kinase. The immunoprecipitation of phosphodiesterase could be inhibited by calcineurin and phosphorylase kinase. These results suggest that the antibody interacts at a common site on these calmodulin-dependent proteins. The antigenic determinant in phosphodiesterase does not appear to reside in the calmodulin-binding domain of the enzyme since the antibody and phosphodiesterase interaction is not inhibited by calmodulin, and the calmodulin activation of phosphodiesterase is not affected by CR-B1 antibody. It is therefore suggested that the structural similarity among the three calmodulin-dependent proteins extends beyond the calmodulin-binding domains.     

Studies on the regulatory domain of Ca2+/calmodulin-dependent protein kinase II by expression of mutated cDNAs in Escherichia coli.

The cDNAs encoding the alpha and beta subunits of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) were ligated into the bacterial expression vector pET and expressed in Escherichia coli. The bacterially expressed alpha and beta subunits exhibited Ca2+/calmodulin-dependent activity and were easily purified to apparent homogeneity from cell extracts. To determine the minimum size required for catalytic activity and the properties of the calmodulin-binding domain, mutated CaM kinase II cDNAs were expressed in E. coli and the enzymatic property of expressed proteins was examined. The replacement of Thr-286 of the alpha subunit with the negatively charged amino acid Asp or that of Arg-283 with the neutral amino acid Gly induced the partially Ca2+ independent activity. The mutant enzymes alpha-I(delta 283-478) and alpha-II(delta 359-478), which truncated the C-terminal region of the alpha subunit, exhibited CaM kinase II activity and the activities of alpha-I(delta 283-478) and alpha-II(delta 359-478) were completely independent of and partially dependent on Ca2+ and calmodulin, respectively. However, the truncated protein alpha(delta 250-478), which was only 33 amino acids shorter than the alpha-I(delta 283-478) protein had no enzymatic activity, indicating that alpha-I(delta 283-478) was close to the minimum size of the active form. The mutant enzyme alpha(delta 291-315), which lacked the calmodulin-binding domain exhibited Ca2+ independent activity. The molecular mass was, however, smaller than that expected from the amino acid sequence. The mutant enzyme alpha(delta 304-315), which lacked the C-terminal half of the calmodulin-binding domain of the alpha subunit, however, exhibited Ca(2+)-independent activity without a reduction in molecular size, indicating that residues 304-315 of the alpha subunit constituted the core calmodulin-binding domain.     

Characterization of the calmodulin-binding and catalytic domains in skeletal muscle myosin light chain kinase

Limited proteolysis has been utilized to study the structural organization of rabbit skeletal muscle myosin light chain kinase. The enzyme (Mr approximately 89,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) consists of an amino-terminal, protease-susceptible region of unidentified function and a carboxyl-terminal, protease-resistant region of Mr approximately 40,000 containing the catalytic and calmodulin-binding domains. Partial digestion with trypsin produced an intermediate 56,000-dalton fragment and a stable 38,000-dalton fragment, both of which were catalytically active and calmodulin-dependent. Chymotryptic digestion yielded three catalytically active fragments of about 37,000, 36,000, and 35,000 daltons. The Mr = 37,000 fragment was calmodulin-dependent with an apparent affinity equivalent to that of the native enzyme (approximately 1 nM). The 36,000-dalton fragment was also calmodulin-dependent but had a approximately 200-fold lower apparent affinity. The Mr = 35,000 fragment was calmodulin-independent. These three chymotryptic fragments, had identical amino termini. Nineteen residues were missing from the carboxyl terminus of the calmodulin-independent chymotryptic fragment whereas only 8 or 9 carboxyl-terminal residues were missing from the calmodulin-dependent tryptic fragments. These results suggest that the 11-residue sequence (IAVSAANRFKK) in the carboxyl-terminal region of myosin light chain kinase contributes directly to the binding of calmodulin. This conclusion is in accord with data (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, K., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3187-3191) that the carboxyl-terminal, 27-residue CNBr peptide of the native enzyme shows Ca2+-dependent, high affinity binding to calmodulin and that similar calmodulin-binding activity, although detectable in unfractionated CNBr digests of calmodulin-dependent enzyme forms, is much reduced in a CNBr digest of the calmodulin-independent, Mr = 35,000 chymotryptic fragment.     

The quaternary structure of phosphorylase kinase as influenced by low concentrations of urea. Evidence suggesting a structural role for calmodulin.

Skeletal-muscle phosphorylase kinase is a hexadecameric oligomer composed of equivalent amounts of four different subunits, (alpha beta gamma delta)4. The delta-subunit, which is calmodulin, functions as an integral subunit of the oligomer, and the gamma-subunit is catalytic. To learn more about intersubunit contacts within the hexadecamer and about the roles of individual subunits, we induced partial dissociation of the holoenzyme with low concentrations of urea. In the absence of Ca2+ the quaternary structure of phosphorylase kinase is very sensitive to urea over a narrow concentration range. Gel-filtration chromatography in the presence of progressively increasing concentrations of urea indicates that between 1.15 M- and 1.35 M-urea the delta-subunit dissociates, allowing extensive formation of complexes larger than the native enzyme that contain equivalent amounts of alpha-, beta- and gamma-subunits. As the urea concentration is increased to 2 M and 3 M, nearly all of the enzyme aggregates to the heavy species devoid of delta-subunit. Addition of Ca2+, which is known to block dissociation of the delta-subunit [Shenolikar, Cohen, Cohen, Nairn & Perry (1979) Eur. J. Biochem. 100, 329-337], also blocks aggregation of the enzyme induced by the low concentrations of urea. These results suggest that in native phosphorylase kinase the delta-subunit, in addition to activating the catalytic subunit and conferring upon it Ca2(+)-sensitivity, may also serve a structural role in preventing aggregation of the alpha-, beta- and gamma-subunits, thus limiting to four the number of alpha beta gamma delta protomers that associate under standard conditions. In gel-filtration chromatography with urea a protein peak containing equivalent amounts of alpha- and gamma-subunits is also observed, as is a peak containing only beta-subunits. Increasing concentrations of urea have a biphasic effect on the activity of the holoenzyme, being stimulatory up to 1 M and then inhibitory. The concentration-dependence of urea in the inhibitory phase parallels its ability to induce dissociation of the delta-subunit.     

Calmodulin-binding protein (55K + 17K) of sea urchin eggs has a Ca2+- and calmodulin-dependent phosphoprotein phosphatase activity

A calmodulin-binding protein from sea urchin eggs consisting of two subunits (55 and 17K-daltons) was identified as a Ca2+-dependent phosphoprotein phosphatase similar to calcineurin in mammalian brain and to phosphatase 2B in skeletal muscle. Peptide mappings showed that the 55K subunit was different from 61K subunit of calcineurin, whereas the 17K subunit was similar to 19K subunit of calcineurin but different from calmodulin. The 55K + 17K protein of sea urchin eggs dephosphorylated 32P-inhibitor-1 in a Ca2+- and calmodulin-dependent manner. Vmax and Km for inhibitor-1 in the presence of Ca2+ and calmodulin were 2,100 pmol Pi/min/mg and 2.7 microM. Ca2+-dependent phosphatase activity for inhibitor-1 was detected in homogenates of both unfertilized and fertilized eggs, but was not detected in isolated cortices and mitotic apparatus.     

Phosphorylation and characterization of bovine heart calmodulin-dependent phosphodiesterase.

Calmodulin-dependent phosphodiesterase was purified to apparent homogeneity from the total calmodulin-binding fraction of bovine heart in a single step by immunoaffinity chromatography. The isolated enzyme had significantly higher affinity for calmodulin than the bovine brain 60-kDa phosphodiesterase isozyme. The cAMP-dependent protein kinase was found to catalyze the phosphorylation of the purified cardiac calmodulin-dependent phosphodiesterase with the incorporation of 1 mol of phosphate/mol of subunit. The phosphodiesterase phosphorylation rate was increased severalfold by histidine without affecting phosphate incorporation into the enzyme. Phosphorylation of phosphodiesterase lowered its affinity for calmodulin and Ca2+. At constant saturating concentrations of calmodulin (650 nM), the phosphorylated calmodulin-dependent phosphodiesterase required a higher concentration of Ca2+ (20 microM) than the nonphosphorylated phosphodiesterase (0.8 microM) for 50% activity. Phosphorylation could be reversed by the calmodulin-dependent phosphatase (calcineurin), and dephosphorylation was accompanied by an increase in the affinity of phosphodiesterase for calmodulin.     

Phosphorylation-regulated calmodulin binding to a prominent cellular substrate for protein kinase C

We have evaluated the possibility that a major, abundant cellular substrate for protein kinase C might be a calmodulin-binding protein. We have recently labeled this protein, which migrates on sodium dodecyl sulfate-gel electrophoresis with an apparent Mr of 60,000 from chicken and 80,000-87,000 from bovine cells and tissues, the myristoylated alanine-rich C kinase substrate (MARCKS). The MARCKS proteins from both species could be cross-linked to 125I-calmodulin in a Ca2+-dependent manner. Phosphorylation of either protein by protein kinase C prevented 125I-calmodulin binding and cross-linking, suggesting that the calmodulin-binding domain might be located at or near the sites of protein kinase C phosphorylation. Both bovine and chicken MARCKS proteins contain an identical 25-amino acid domain that contains all 4 of the serine residues phosphorylated by protein kinase C in vitro. In addition, this domain is similar in sequence and structure to previously described calmodulin-binding domains. A synthetic peptide corresponding to this domain inhibited calmodulin binding to the MARCKS protein and also could be cross-linked to 125I-calmodulin in a calcium-dependent manner. In addition, protein kinase C-dependent phosphorylation of the synthetic peptide inhibited its binding and cross-linking to 125I-calmodulin. The peptide bound to fluorescently labeled 5-dimethylaminonaphthalene-1-sulfonyl-calmodulin with a dissociation constant of 2.8 nM, and inhibited the calmodulin-dependent activation of cyclic nucleotide phosphodiesterase with an IC50 of 4.8 nM. Thus, the peptide mimics the calmodulin-binding properties of the MARCKS protein and probably represents its calmodulin-binding domain. Phosphorylation of these abundant, high affinity calmodulin-binding proteins by protein kinase C in intact cells could cause displacement of bound calmodulin, perhaps leading to activation of Ca2+-calmodulin-dependent processes.     

Development and characterization of fluorescently-labeled myosin light chain kinase calmodulin-binding domain peptides

Calmodulin-dependent protein kinases such as myosin light chain kinase (MLCK), calmodulin kinase II, and phosphorylase kinase contain specific sequences responsible for binding calmodulin. These regions are known as calmodulin-binding domains and in many cases are contained within sequences that are short enough to be synthesized by solidphase techniques. The ability to chemically-synthesize target enzyme calmodulin-binding domains has permitted the use of a variety of biophysical techniques to study the interactions between calmodulin and calmodulin-binding domain peptides. The work reviewed here describes the development and characterization of peptides based on the sequence, of the calmodulin-binding domain of skeletal muscle myosin light chain kinase which were labeled with the fluorescent reagent, acrylodan. Data are presented demonstrating the use of fluorescently-labeled peptides to study various aspects of calmodulin-peptide interactions including binding affinity, stoichiometry, specificity, changes in peptide conformation, and thermal stability of the peptide-calmodulin complex. These data indicate the peptides exhibit many of the salient features seen with calmodulin-target enzyme interactions. The fluorescently-labeled peptides should thus serve as useful models for studying calmodulin-target enzyme interactions at the molecular level.     

Isolation of the native form of chicken gizzard myosin light-chain kinase.

A simple and rapid procedure for the purification of the native form of chicken gizzard myosin light-chain kinase (Mr 136000) is described which eliminates problems of proteolysis previously encountered. During this procedure, a calmodulin-binding protein of Mr 141000, which previously co-purified with the myosin light-chain kinase, is removed and shown to be a distinct protein on the basis of lack of kinase activity, different chymotryptic peptide maps, lack of cross-reactivity with a monoclonal antibody to turkey gizzard myosin light-chain kinase, and lack of phosphorylation by the purified catalytic subunit of cyclic AMP-dependent protein kinase. This Mr-141000 calmodulin-binding protein is identified as caldesmon on the basis of Ca2+-dependent interaction with calmodulin, subunit Mr, Ca2+-independent interaction with skeletal-muscle F-actin, Ca2+-dependent competition between calmodulin and F-actin for caldesmon, and tissue content.