Activation of a calmodulin-dependent phosphatase by a Ca2+-dependent protease

A calmodulin-dependent protein phosphatase (calcineurin) was converted to an active, calmodulin-independent form by a Ca2+-dependent protease (calpain I). Proteolysis could be blocked by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, leupeptin, or N-ethylmaleimide, but other protease inhibitors such as phenylmethanesulfonyl fluoride, aprotinin, benzamidine, diisopropyl fluorophosphate, and trypsin inhibitor were ineffective. Phosphatase proteolyzed in the absence of calmodulin was insensitive to Ca2+ or Ca2+/calmodulin; the activity of the proteolyzed enzyme was greater than the Ca2+/calmodulin-stimulated activity of the unproteolyzed enzyme. Proteolysis of the phosphatase in the presence of calmodulin proceeded at a more rapid rate than in its absence, and the proteolyzed enzyme retained a small degree of sensitivity to Ca2+/calmodulin, being further stimulated some 15-20%. Proteolytic stimulation of phosphatase activity was accompanied by degradation of the 60-kilodalton (kDa) subunit; the 19-kDa subunit was not degraded. In the absence of calmodulin, the 60-kDa subunit was sequentially degraded to 58- and 45-kDa fragments; the 45-kDa fragment was incapable of binding 125I-calmodulin. In the presence of calmodulin, the 60-kDa subunit was proteolyzed to fragments of 58, 55 (2), and 48 kDa, all of which retained some ability to bind calmodulin. These data, coupled with our previous report that the human platelet calmodulin-binding proteins undergo Ca2+-dependent proteolysis upon platelet activation [Wallace, R. W., Tallant, E. A., & McManus, M. C. (1987) Biochemistry 26, 2766-2773], suggest that the Ca2+-dependent protease may have a role in the platelet as an irreversible activator of certain Ca2+/calmodulin-dependent reactions.     

Calmodulin and Ca2+-dependent phosphorylation and dephosphorylation of 63-kDa subunit-containing bovine brain calmodulin-stimulated cyclic nucleotide phosphodiesterase isozyme

Bovine brain contains calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes which are composed of two distinct subunits: Mr 60,000 and 63,000. The 60-kDa but not the 63-kDa subunit-containing isozyme can be phosphorylated by cAMP-dependent protein kinase resulting in decreased affinity of this subunit toward calmodulin (Sharma, R. K., and Wang, J. H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 2603-2607). In contrast, purified 63-kDa subunit-containing isozyme has been found to be phosphorylated by a preparation of bovine brain calmodulin-binding proteins in the presence of Ca2+ and calmodulin. The phosphorylation resulted in the maximal incorporation of 2 mol of phosphate/mol of the phosphodiesterase subunit with a 50% decrease in the enzyme affinity toward calmodulin. At a constant calmodulin concentration of 6 nM, the phosphorylated isozyme required a higher concentration of Ca2+ for activation than the nonphosphorylated phosphodiesterase. The Ca2+ concentrations at 50% activation by calmodulin of the nonphosphorylated and phosphorylated isozymes were 1.1 and 1.9 microM, respectively. Phosphorylation can be reversed by the calmodulin-dependent phosphatase, calcineurin, but not by phosphoprotein phosphatase 1. The results suggest that the Ca2+ sensitivities of brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes can be modulated by protein phosphorylation and dephosphorylation mechanisms in response to different second messengers.     

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.     

Ca(2+)/calmodulin-independent activation of calcineurin from Dictyostelium by unsaturated long chain fatty acids

This study describes a novel mode of activation for the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin. Using purified calcineurin from Dictyostelium discoideum we found a reversible, Ca(2+)/calmodulin-independent activation by the long chain unsaturated fatty acids arachidonic acid, linoleic acid, and oleic acid, which was of the same magnitude as activation by Ca(2+)/calmodulin. Half-maximal stimulation of calcineurin occurred at fatty acid concentrations of approximately 10 microM with either p-nitrophenyl phosphate or RII phosphopeptide as substrates. The methyl ester of arachidonic acid and the saturated fatty acids palmitic acid and arachidic acid did not activate calcineurin. The activation was shown to be independent of the regulatory subunit, calcineurin B. Activation by Ca(2+)/calmodulin and fatty acids was not additive. In binding assays with immobilized calmodulin, arachidonic acid inhibited binding of calcineurin to calmodulin. Therefore fatty acids appear to mimic Ca(2+)/calmodulin action by binding to the calmodulin-binding site.     

Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm

Preliminary data demonstrated that the inhibition of reactivated sperm motility by calcium was correlated with inhibited protein phosphorylation. The inhibition of phosphorylation by Ca2+ was found to be catalyzed by the calmodulin-dependent protein phosphatase (calcineurin). Sperm from dog, pig, and sea urchin contain both the Ca2+-binding B subunit of the enzyme (Mr 15,000) and the calmodulin-binding A subunit with an Mr of 63,000. The sperm A subunit is slightly higher in Mr than reported for other tissues. Inhibition of endogenous calmodulin-dependent protein phosphatase activity with a monospecific antibody revealed the presence of 14 phosphoprotein substrates in sperm for this enzyme. The enzyme was localized to both the flagellum and the postacrosomal region of the sperm head. The flagellar phosphatase activity was quantitatively extracted with 0.6 M KCl from isolated flagella from dog, pig, and sea urchin sperm. All salt-extractable phosphatase activity was inhibited with antibodies against the authentic enzyme. Preincubation of sperm models with the purified phosphatase stimulated curvolinear velocity and lateral head amplitude (important components of hyperactivated swimming patterns) and inhibited beat cross frequency suggesting a role for this enzyme in axonemal function. Our results suggest that calmodulin-dependent protein phosphatase plays a major role in the calcium-dependent regulation of flagellar motility.     

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.     

Purification and characterization of a Ca2+/calmodulin-dependent protein kinase from rat brain

A soluble Ca2+/calmodulin-dependent protein kinase has been purified from rat brain to near homogeneity by using casein as substrate. The enzyme was purified by using hydroxylapatite adsorption chromatography, phosphocellulose ion-exchange chromatography, Sepharose 6B gel filtration, affinity chromatography using calmodulin-Sepharose 4B, and ammonium sulfate precipitation. On sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels, the purified enzyme consists of three protein bands: a single polypeptide of 51 000 daltons and a doublet of 60 000 daltons. Measurements of the Stokes radius by gel filtration (81.3 +/- 3.7 A) and the sedimentation coefficient by sucrose density sedimentation (13.7 +/- 0.7 S) were used to calculate a native molecular mass of 460 000 +/- 29 000 daltons. The kinase autophosphorylated both the 51 000-dalton polypeptide and the 60 000-dalton doublet, resulting in a decreased mobility in NaDodSO4 gels. Comparison of the phosphopeptides produced by partial proteolysis of autophosphorylated enzyme reveals substantial similarities between subunits. These patterns, however, suggest that the 51 000-dalton subunit is not a proteolytic fragment of the 60 000-dalton doublet. Purified Ca2+/calmodulin-dependent casein kinase activity was dependent upon Ca2+, calmodulin, and ATP X Mg2+ or ATP X Mn2+ when measured under saturating casein concentrations. Co2+, Mn2+, and La3+ could substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations. In addition to casein, the purified enzyme displayed a broad substrate specificity which suggests that it may be a "general" protein kinase with the potential for mediating numerous processes in brain and possibly other tissues.     

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.     

Dephosphorylation of the small heat shock protein hsp25 by calcium/calmodulin-dependent (type 2B) protein phosphatase.

The dephosphorylation of the mouse small heat shock protein hsp25 within an extract obtained from Ehrlich ascites tumor cells is inhibited by the calcium chelator EGTA and at concentrations of microcystin-LR which are characteristic for inhibition of calcium/calmodulin-dependent (2B type) protein phosphatases. Furthermore, the dephosphorylation of hsp25 in the cell-free system derived from Ehrlich ascites tumor could be increased specifically by addition of the calcium/calmodulin-dependent (2B type) protein phosphatase calcineurin. Dephosphorylation of the heat shock protein hsp25 is also obtained in an in vitro system containing phosphorylated recombinant hsp25, 1 mM Ca2+, calmodulin, and calcineurin specifying hsp25 as the direct substrate for this enzyme. The expression of two isoforms of the catalytic subunit of the mouse calcium/calmodulin-dependent (2B type) protein phosphatases in Ehrlich ascites tumor cells is demonstrated by polymerase chain reaction using specific oligonucleotide primers to the catalytic and calmodulin-binding domain, respectively. Northern blot analysis using the amplified fragments as probes shows that the mRNA of one isoform of the mouse calcium/calmodulin-dependent protein phosphatase is of medium abundance in EAT cells. These data suggest a calcium/calmodulin-dependent dephosphorylation of the small stress protein in EAT cells also in vivo. Since it is known that heat shock increases the intracellular calcium level and that thermotolerance is influenced by calcium chelators, ionophores, and anti-calmodulin drugs, the changes in the degree of hsp25 phosphorylation induced by thermal stress resulting in an altered thermoresistance could be explained at least partially by the calcium/calmodulin-dependent dephosphorylation through protein phosphatases 2B.     

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.     

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.     

Activation of type II calcium/calmodulin-dependent protein kinase by Ca2+/calmodulin is inhibited by autophosphorylation of threonine within the calmodulin-binding domain

It is now well established that autophosphorylation of a threonine residue located next to each calmodulin-binding domain in the subunits of type II Ca2+/calmodulin-dependent protein kinase causes the kinase to remain active, although at a reduced rate, after Ca2+ is removed from the reaction. This autophosphorylated form of the kinase is still sensitive to Ca2+/calmodulin, which is required for a maximum catalytic rate. After removal of Ca2+, new sites are autophosphorylated by the partially active kinase. Autophosphorylation of these sites abolishes sensitivity of the kinase to Ca2+/calmodulin (Hashimoto, Y., Schworer, C. M., Colbran, R. J., and Soderling, T. R. (1987) J. Biol. Chem. 262, 8051-8055). We have identified two pairs of homologous residues, Thr305 and Ser314 in the alpha subunit and Thr306 and Ser315 in the beta subunit, that are autophosphorylated only after removal of Ca2+ from an autophosphorylation reaction. The sites were identified by direct sequencing of labeled tryptic phosphopeptides isolated by reverse-phase high pressure liquid chromatography. Thr305-306 is rapidly dephosphorylated by purified protein phosphatases 1 and 2A, whereas Ser314-315 is resistant to dephosphorylation. We have shown by selective dephosphorylation that the presence of phosphate on Thr305-306 blocks sensitivity of the kinase to Ca2+/calmodulin. In contrast, the presence of phosphate on Ser314-315 is associated with an increase in the Kact for Ca2+/calmodulin of only about 2-fold, producing a relatively small decrease in sensitivity to Ca2+/calmodulin.     

Human platelet calmodulin-binding proteins: identification and Ca2+-dependent proteolysis upon platelet activation

Calmodulin-binding proteins have been identified in human platelets by using Western blotting techniques and 125I-calmodulin. Ten distinct proteins of 245, 225, 175, 150, 90, 82 (2), 60, and 41 (2) kilodaltons (kDa) bound 125I-calmodulin in a Ca2+-dependent manner; the binding was blocked by ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA), trifluoperazine, and nonradiolabeled calmodulin. Proteins of 225 and 90 kDa were labeled by antisera against myosin light chain kinase; 60- and 82-kDa proteins were labeled by antisera against the calmodulin-dependent phosphatase and caldesmon, respectively. The remaining calmodulin-binding proteins have not been identified. Calmodulin-binding proteins were degraded upon addition of Ca2+ to a platelet homogenate; the degradation could be blocked by either EGTA, leupeptin, or N-ethylmaleimide which suggests that the degradation was due to a Ca2+-dependent protease. Activation of intact platelets by thrombin, adenosine 5'-diphosphate, and collagen under conditions which promote platelet aggregation (i.e., stirring with extracellular Ca2+) also resulted in limited proteolysis of calmodulin-binding proteins including those labeled with antisera against myosin light chain kinase and the calmodulin-dependent phosphatase. Activation by the Ca2+ ionophores A23187 and ionomycin also promoted degradation of the calmodulin-binding proteins in the presence of extracellular Ca2+; however, degradation in response to the ionophores did not require stirring of the platelet suspension to promote aggregation. Many Ca2+/calmodulin-regulated enzymes are irreversibly activated in vitro by limited proteolysis. Our data indicate that limited proteolysis of Ca2+/calmodulin-regulated enzymes also occurs in the intact platelet and suggest that the proteolysis is triggered by an influx of extracellular Ca2+ associated with platelet aggregation.     

Calmodulin-independent inhibition of platelet phospholipase A2 by calmodulin antagonists

We tested the effects of calmodulin, two types of calmodulin antagonists, and various phospholipids on the phospholipase A2 activities of intact platelets, platelet membranes, and partially purified enzyme preparations. Trifluoperazine, chlorpromazine (phenothiazines) and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide (W-7), at concentrations which antagonize the effects of calmodulin, significantly inhibited thrombin- and Ca2+ ionophore-induced production of arachidonic acid metabolites by suspensions of rabbit platelets and Ca2+-induced arachidonic acid release from phospholipids of membrane fractions, but not phospholipase A2 activity in purified enzyme preparations. The addition of acidic phospholipids, but not calmodulin, stimulated phospholipase A2 activity in purified enzyme preparations while decreasing its Km for Ca2+. The dose-response and kinetics of inhibition by calmodulin antagonists of acidic phospholipid-activated phospholipase A2 activity in purified preparations were similar to those of Ca2+-induced arachidonic acid release from membrane fractions. Calmodulin antagonists were also found to inhibit Ca2+ binding to acidic phospholipids in a similar dose-dependent manner. Our results suggest that the platelet phospholipase A2 is the key enzyme involved in arachidonic acid mobilization in platelets and is regulated by acidic phospholipids in a Ca2+-dependent manner and that calmodulin antagonists inhibit phospholipase A2 activity via an action on acidic phospholipids.     

Inactivation and Reactivation of the Multifunctional Calmodulin-Dependent Protein Kinase from Brain by Autophosphorylation and Dephosphorylation: Involvement of Protein Phosphatases from Brain

The multifunctional calmodulin-dependent protein kinase (calmodulin-kinase) from rat brain was autophosphorylated in a Ca2+- and calmodulin-dependent manner. The activity of the autophosphorylated enzyme was independent of Ca2+ and calmodulin. Calmodulin-kinase was dephosphorylated by protein phosphatase C from bovine brain, which is the catalytic subunits of protein phosphatases 1 and 2A. The holoenzyme of protein phosphatase 2A was also involved in the dephosphorylation of the enzyme. The autophosphorylated sites of calmodulin-kinase were universally dephosphorylated by protein phosphatase C. Calmodulin-kinase was inactivated and reactivated by autophosphorylation and dephosphorylation, respectively. Furthermore, the regulation of calmodulin-kinase by autophosphorylation and dephosphorylation was observed using calmodulin-kinase from canine heart. These results suggest that the activity of calmodulin-kinase is regulated by autophosphorylation and dephosphorylation, and that the regulation is the universal phenomenon for many other calmodulin-kinases in various tissues.     

Subunit A of calmodulin-dependent protein phosphatase requires Mn2+ for activity

Bovine brain calmodulin-dependent protein phosphatase comprises a catalytic subunit A (Mr 60,000) and a regulatory subunit B (Mr 19,000). The native enzyme was active with Ca²⁺ or Mn²⁺. Upon resolution into its subunits in 6 M urea and 15 mM EDTA, subunit A was active with Mn²⁺; Co²⁺ and Ni²⁺ partially substituted for Mn²⁺, but Ca²⁺, Mg²⁺ and Zn²⁺ were ineffective. The stimulating effect of Mn²⁺ was not easily reversed by EGTA. Like the native phosphatase, subunit A was markedly stimulated by calmodulin or by controlled trypsinization. Unlike the native enzyme, however, trypsinized subunit A still required Mn²⁺ for activity. These findings provide evidence that the catalytic subunit of phosphatase may be a metallo (possibly Mn²⁺) enzyme.     

Identification of Endogenous Calmodulin-Dependent Kinase and Calmodulin-Binding Proteins in Cold-Stable Microtubule Preparations from Rat Brain

Calmodulin-dependent kinase activity was investigated in cold-stable microtubule fractions. Calmodulin-dependent kinase activity was enriched approximately 20-fold over cytosol in cold-stable microtubule preparations. Calmodulin-dependent kinase activity in cold-stable microtubule preparations phosphorylated microtubule-associated protein-2, alpha- and beta-tubulin, an 80,000-dalton doublet, and several minor phosphoproteins. The endogenous calmodulin-dependent kinase in cold-stable microtubule fractions was identical to a previously purified calmodulin-dependent kinase from rat brain by several criteria including (1) subunit molecular weights, (2) subunit isoelectric points, (3) calmodulin-binding properties, (4) subunit autophosphorylation, (5) calmodulin-binding subunit composition on high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (6) isolation of kinase on calmodulin affinity resin, (7) kinetic parameters, (8) phosphoamino acid phosphorylation sites on beta-tubulin, and (9) phosphopeptide mapping. Endogenous cold-stable calmodulin-dependent kinase activity was isolated from the microtubule fraction by calmodulin affinity resin column chromatography and specifically eluted with EGTA. This kinase fraction contained the calmodulin-binding, autophosphorylating rho and sigma subunits of the previously purified kinase. The rho and sigma subunits of this kinase represented the major calmodulin-binding proteins in the cold-stable microtubule fractions as assessed by denaturing and non-denaturing procedures. These results indicate that calmodulin-dependent kinase is a major calmodulin-binding enzyme system in cold-stable microtubule fractions and may play an important role in mediating some of the effects of calcium on microtubule and cytoskeletal dynamics.     

Escherichia coli S-adenosylhomocysteine/5''-methylthioadenosine nucleosidase. Purification, substrate specificity and mechanism of action.

Ca2+-activated protein phosphatase activity was demonstrated in mouse pancreatic acinar cytosol with alpha-casein and skeletal-muscle phosphorylase kinase as substrates. This phosphatase activity preferentially dephosphorylated the alpha subunit of phosphorylase kinase. After DEAE-cellulose chromatography, the Ca2+-activated phosphatase activity became dependent on exogenous calmodulin for maximal activity. Half-maximal activation was achieved at 0.5 +/- 0.1 microM-Ca2+. Trifluoperazine completely inhibited Ca2+-activated phosphatase activity, with half-maximal inhibition occurring at 8.5 +/- 0.6 microM. Mn2+, but not Mg2+, at 1 mM concentration could substitute for Ca2+ in eliciting full enzyme activation. The apparent Mr of the phosphatase as determined by Sephadex G-150 chromatography was 93000 +/- 1000. Submitting active fractions obtained after Sephadex chromatography to calmodulin affinity chromatography resulted in the resolution of a major protein of Mr 55500 +/- 300. In conclusion, Ca2+-activated protein phosphatase activity has been identified in exocrine pancreas and has several features in common with Ca2+-activated calmodulin-dependent protein phosphatases previously isolated from brain and skeletal muscle. It is possible that this Ca2+-activated phosphatase may utilize as substrates certain acinar-cell phosphoproteins previously shown to undergo dephosphorylation in response to Ca2+-mediated secretagogues.     

Regulation of human platelet membrane Ca2+ transport by cAMP- and calmodulin-dependent phosphorylation

We have examined the effects of added cAMP-dependent protein kinase and endogenous calmodulin-dependent kinase on Ca2+ transport in purified internal membranes from human platelets. Both Ca2+ uptake and Ca2+-ATPase activity were maximally stimulated about 2-fold by addition of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase inhibitor reduced both Ca2+ uptake and Ca2+-ATPase activities at concentrations which also inhibited cAMP-dependent protein phosphorylation. In addition, concerted stimulation of Ca2+-ATPase by exogenous calmodulin and added catalytic subunit of cAMP-dependent protein kinase was observed. A 22-kDa protein was phosphorylated by both cAMP-dependent and calmodulin-dependent kinases at the same rate as stimulation of the Ca2+-ATPase. Cyclic AMP-dependent phosphorylation of the 22-kDa polypeptide was inhibited by the protein kinase inhibitor and calmodulin-dependent phosphorylation was inhibited by chlorpromazine and EGTA. These results are consistent with the hypothesis that one mode of control of Ca2+ homeostasis in platelets may be similar to the phospholamban system in cardiac muscle.     

Phorbol myristate acetate stimulates formation of phosphatidyl inositol 4-phosphate and phosphatidyl inositol 4,5-bisphosphate in human platelets

There are conflicting data in the literature as to whether or not the Ca2+ activation of phospholipase A2 is mediated by the calcium binding protein calmodulin. In the present study the membrane-bound phospholipase A2 enzymes in rat and human platelets were shown to be absolutely Ca2+ dependent but were not stimulated by the addition of calmodulin. A partially purified phospholipase A2 from rat platelet membrane, which contained little endogenous calmodulin, also was not stimulated by calmodulin addition. Both isolated and membrane-bound phospholipase A2 were inhibited by the non-specific calmodulin antagonist trifluoperazine but the inhibition was not overcome by adding calmodulin. There was thus no evidence from these studies that phospholipase A2 is calmodulin regulated.