Ca2+-dependent hydrophobic-interaction chromatography. Isolation of a novel Ca2+-binding protein and protein kinase C from bovine brain.

Several bovine brain proteins have been found to interact with a hydrophobic chromatography resin (phenyl-Sepharose CL-4B) in a Ca2+-dependent manner. These include calmodulin, the Ca2+/phospholipid-dependent protein kinase (protein kinase C) and a novel Ca2+-binding protein that has now been purified to electrophoretic homogeneity. This latter protein is acidic (pI 5.1) and, like calmodulin and some other high-affinity Ca2+-binding proteins, exhibits a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, with an apparent Mr of 22 000 in the absence of Ca2+ and Mr 21 000 in the presence of Ca2+. This novel calciprotein is distinct from known Ca2+-binding proteins on the basis of Mr under denaturing conditions, Cleveland peptide mapping and amino acid composition analysis. It may be a member of the calmodulin superfamily of Ca2+-binding proteins. This calciprotein does not activate two calmodulin-dependent enzymes, namely cyclic nucleotide phosphodiesterase and myosin light-chain kinase, nor does it have any effect on protein kinase C. It may be a Ca2+-dependent regulatory protein of an as-yet-undefined enzymic activity. The Ca2+/phospholipid-dependent protein kinase is also readily purified by Ca2+-dependent hydrophobic-interaction chromatography followed by ion-exchange chromatography, during which it is easily separated from calmodulin. A preparation of protein kinase C that lacks contaminating kinase or phosphatase activities is thereby obtained rapidly and simply. Such a preparation is ideal for the study of phosphorylation reactions catalysed in vitro by protein kinase C.     

A Protein Modulator Stimulates C Kinase-Dependent Phosphorylation of a 90K Substrate in Synaptic Membranes

We have identified and partially purified an acidic, heat-stable, noncalmodulin protein from bovine brain cytosol that stimulates Ca2+-dependent phosphorylation of an Mr 90K substrate in crude rat brain synaptic membranes. We show that this modulator of phosphorylation (MOP) enhances Ca2+- and phospholipid-dependent protein kinase (C kinase) phosphorylation of this 90K substrate. The 90K substrate is a higher Mr form of an 87K substrate that is a major C kinase substrate in rat brain. The Ca2+-dependent phosphorylation of both substrates is inhibited by the Ca2+-binding proteins S-100 and calmodulin. Both substrates yield phosphopeptide fragments of Mr 9K and 13K after limited proteolysis with V8 protease. Two-dimensional polyacrylamide gel electrophoresis reveals that they have similar acidic isoelectric points (pI 5.0). MOP enhances Ca2+-dependent phosphorylation of the 90K substrate whereas the phosphorylation of 87K is diminished. This reciprocal relationship suggests that the mobility of the 87K substrate in sodium dodecyl sulfate-polyacrylamide gels is decreased to 90K with increasing phosphorylation. MOP may be a novel protein modulator of C kinase-mediated phosphorylation in the nervous system.     

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.     

Protease-activated protein kinase C in rat liver

In regenerating rat liver, an elevated protein kinase activity was detected which phosphorylated ribosomal protein S6 and histones. The properties of this enzyme were closely similar with those of protease-activated protein kinase C with Mr 45,000. During the study of the mechanism of proteolytic activation, type III protein kinase C (encoding alpha-sequence) was shown to be subjected to limited proteolysis by trypsin-like protease and converted to protein kinase M in ionic strength- and pH-dependent manner. This reaction was stimulated in the presence of Ca2+ and phospholipid under slightly higher ionic strength condition than physiological level (greater than 140 mM NaCl) and alkaline pH (7.5-8.0). These results suggest that activation of Na+/H+ exchanger in plasma membrane may trigger this type of proteolytic activation of protein kinase C. In addition to protein kinase M, another type of protease-activated kinase with Mr 80,000 was detected when limited proteolysis of protein kinase C was performed on inactive form of this enzyme (in the absence of either Ca2+ or phospholipid or both activators) under lower ionic strength condition. The molecular mass of this active enzyme was slightly smaller (approximately 200) than that of native protein kinase C. However, it is not clear at this time whether this small fragment was released from amino-terminal or carboxy-terminal domain to make protein kinase C partially active in the absence of Ca2+ and phospholipid. Although it has been proposed that proteolytic degradation of protein kinase C is involved in down regulation of this enzyme, the physiological significance of these two types of protease-activated forms of protein kinases in liver has remained obscure.     

Calmodulin increases Ca-dependent inhibition of the Na,K-ATPase in human red blood cells.

Proteins in human red cell hemolysate were purified to determine which of them increase inhibition of the Na,K-ATPase in the presence of 2 microM free Ca. Samples purified 600,000-fold inhibited the Na,K-ATPase of human red cells in a Ca-dependent manner and stimulated the (Ca+Mg)-ATPase. These samples contained two proteins as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): calmodulin (18,000 Mr), which comprised most (greater than 90%) of the total protein, and an unidentified protein of approximately 13,000 Mr. Both proteins were a distinctive light yellow when stained with silver. Calmodulin from bovine testes also inhibited the Na,K-ATPase and stimulated the (Ca+Mg)-ATPase. This preparation also contained two proteins as analyzed by SDS-PAGE: calmodulin (95 to 99% of the total protein) and another protein of approximately 13,000 Mr (1 to 5% of the total protein). Both were light yellow when stained with silver. Since the amount of red cell protein was limited, the remainder of the study was carried out with the bovine testes preparation. Heating the testes preparation decreased, but did not abolish, inhibition of the Na,K-ATPase and reduced stimulation of the (Ca+Mg)-ATPase. When corrected for denatured calmodulin, both heated and unheated proteins increased inhibition of the Na,K-ATPase to the same extent. The Na,K-ATPase was inhibited at 2 microM free Ca in a dose-dependent manner over a range of 15 to 100 nM calmodulin. To establish if the inhibition was due to the calmodulin or the 13,000 Mr protein, both were electroeluted after SDS-PAGE. Electroeluted calmodulin stimulated the (Ca+Mg)-ATPase and increased Ca inhibition of the Na,K-ATPase. Electroeluted amounts of the smaller Mr protein slightly stimulated the (Ca+Mg)-ATPase, but had no effect on the Na,K-ATPase. This protein was digested with cyanogen bromide, partially sequenced, and thereby identified as a fragment of calmodulin. We conclude that intact calmodulin increases inhibition of the Na,K-ATPase at 2 microM free Ca. We suggest that calmodulin is part of a mechanism mediating the effects of physiological free Ca on the Na,K-ATPase.     

Differential activation of two protease-activated protein kinases from reticulocytes by a Ca2+-stimulated protease and identification of phosphorylated translational components

Two protein kinases have been partially purified from rabbit reticulocytes and shown to be activated by limited proteolysis with trypsin [S.M. Tahara and J.A. Traugh (1981) J. Biol. Chem. 256, 11558-11564; P.T. Tuazon, W.C. Merrick, and J.A. Traugh (1980) J. Biol. Chem. 255, 10954-10958]. Reticulocyte lysate was examined for protease activities which might be involved in activation of the protein kinases in vivo. Two neutral proteases, differentially activated by Fe2+ and Ca2+, were identified and partially purified. The Ca2+-stimulated protease specifically activated protease-activated kinase II; no effect was observed on protease-activated kinase I. The Fe2+-stimulated protease was not active on either protein kinase. The protease-activated kinases were examined using initiation factors (eIF) and 40-S ribosomal subunits as substrate. Protease-activated kinase I phosphorylated one subunit of eIF-3 (Mr 130000), eIF-4B and 40-S ribosomal protein S10. Protease-activated kinase II modified the beta subunit of eIF-2 (Mr 53000) and 40-S ribosomal protein S6. The substrate specificities are unique when compared with other cAMP-dependent and cAMP-independent protein kinases from reticulocytes.     

Functional domain structure of calcineurin A: mapping by limited proteolysis

Limited proteolysis of calcineurin, the Ca2+/calmodulin-stimulated protein phosphatase, with clostripain is sequential and defines four functional domains in calcineurin A (61 kDa). In the presence of calmodulin, an inhibitory domain located at the carboxyl terminus is rapidly degraded, yielding an Mr 57,000 fragment which retains the ability to bind calmodulin but whose p-nitrophenylphosphatase is fully active in the absence of Ca2+ and no longer stimulated by calmodulin. Subsequent cleavage(s), near the amino terminus, yield(s) an Mr 55,000 fragment which has lost more than 80% of the enzymatic activity. A third, slower, proteolytic cleavage in the carboxyl-terminal half of the protein converts the Mr 55,000 fragment to an Mr 42,000 polypeptide which contains the calcineurin B binding domain and an Mr 14,000 fragment which binds calmodulin in a Ca2+-dependent manner with high affinity. In the absence of calmodulin, clostripain rapidly severs both the calmodulin-binding and the inhibitory domains. The catalytic domain is preserved, and the activity of the proteolyzed 43-kDa enzyme is increased 10-fold in the absence of Ca2+ and 40-fold in its presence. The calcineurin B binding domain and calcineurin B appear unaffected by proteolysis both in the presence and in the absence of calmodulin. Thus, calcineurin A is organized into functionally distinct domains connected by proteolytically sensitive hinge regions. The catalytic, inhibitory, and calmodulin-binding domains are readily removed from the protease-resistant core, which contains the calcineurin B binding domain. Calmodulin stimulation of calcineurin is dependent on intact inhibitory and calmodulin-binding domains, but the degraded enzyme lacking these domains is still regulated by Ca2+.     

ATPase activity and Ca2+ transport by reconstituted tryptic fragments of the Ca2+ pump of the erythrocyte plasma membrane

The purified Ca2+ ATPase of the erythrocyte plasma membrane has been submitted to controlled trypsin proteolysis under conditions that favor either its (putative) E1 or E2 configurations. The former configuration has been forced by treating the enzyme with Ca2+-saturated calmodulin, the latter with vanadate and Mg2+. The E1 conformation leads to the accumulation of a polypeptide of Mr 85 KDa which still binds calmodulin, the E2 conformation to the accumulation of one of Mr 81 KDa which does not. Both fragments arise from the hydrolysis of a transient 90 KDa product which has Ca2+-calmodulin dependent ATPase activity, and which retains the ability to pump Ca2+ in reconstituted liposomes. Highly enriched preparations of the 85 and 81 KDa fragments have been obtained and reconstituted into liposomes. The former has limited ATPase and Ca2+ transport ability and is not stimulated by calmodulin. The latter has much higher ATPase and Ca2+ transport activity. It is proposed that the Ca2+ pumping ATPase of erythrocytes plasma membrane contains a 9 KDa domain which is essential for the interaction of the enzyme with calmodulin and for the full expression of the hydrolytic and transport activity. This putative 9 KDa sequence contains a 4 KDa "inhibitory" domain which limits the activity of the ATPase. In the presence of this 4 KDa sequence, i.e., when the enzyme is degraded to the 85 KDa product, calmodulin can still be bound, but no longer stimulates ATPase and Ca2+ transport.     

Protease-activated form of protein kinase C with Mr 80,000 generated from rat liver plasma membrane by trypsin-like protease

New type of protease-activated form of protein kinase C was generated from rat liver plasma membrane by action of endogenous trypsin-like protease. The molecular mass was estimated to be about 80,000 by immunoblot analysis which was slightly smaller (approximately 2,000) than that of native protein kinase C. The protein kinase activity was 2-times stimulated by Ca2+ and phospholipid and inhibited by the synthetic peptide derived from the pseudosubstrate region of protein kinase C. This type of activated kinase was produced in purified enzyme system in the absence of either Ca2+ or phospholipid or both. These results suggest that limited proteolysis generating the active form of Mr 80,000 may occur on the inactive form of protein kinase C.     

Characterization and partial purification of cardiac sarcoplasmic reticulum phospholamban kinase

Phospholamban, the cardiac sarcoplasmic reticulum proteolipid, is phosphorylated by cAMP-dependent protein kinase, by Ca2+/phospholipid-dependent protein kinase, and by an endogenous Ca2+/calmodulin-dependent protein kinase, the identity of which remains to be defined. The aim of this study was therefore to characterize the latter kinase, called phospholamban kinase. Phospholamban kinase was purified approximately 42-fold with a yield of 11%. The purified fraction exhibits a specific activity of 6.5 nmol of phosphate incorporated into exogenous phospholamban per minute per milligram of protein. Phospholamban kinase appears to be a high molecular weight enzyme and presents a broad substrate specificity, synapsin-1, glycogen synthase, and smooth muscle myosin regulatory light chain being the best substrates. Phospholamban kinase phosphorylates synapsin-1 on a Mr 30 000 peptide. The enzyme exhibits an optimum pH of 8.6, a Km for ATP of 9 microM, and a requirement for Mg2+ ions. These data suggest that phospholamban kinase might be an isoenzyme of the multifunctional Ca2+/calmodulin-dependent protein kinase. Consequently we have searched for Mr 50 000-60 000 phosphorylatable subunits among cardiac sarcoplasmic reticulum proteins. A Mr 56 000 protein was found to be phosphorylated in the presence of Ca2+/calmodulin. Such phosphorylation alters the electrophoretic migration velocity of the protein. In addition, this protein that binds calmodulin was always found to be present in fractions containing phospholamban kinase activity. This Mr 56 000 protein is therefore a good candidate for being a subunit of phospholamban kinase. However, the Mr 56 000 calmodulin-binding protein and the Mr 53 000 intrinsic glycoprotein which binds ATP are two distinct entities.     

Proteolytic activation of calcium-activated, phospholipid-dependent protein kinase by calcium-dependent neutral protease

A Ca2+-dependent protease I), which hydrolyzes casein at Ca2+ concentrations lower than the 10(-5) M range, is purified roughly 4000-fold from the soluble fraction of rat brain. This protease is able to activate Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) by limited proteolysis analogously to the previously known Ca2+-dependent analogously to the previously known Ca2+-dependent protease (Ca2+ protease II) which is active at the millimolar range of Ca2+ (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616). The protein kinase fragment thus produced shows a molecular weight of about 5.1 X 10(4), and is significantly smaller than native protein kinase C (Mr = 7.7 X 10(4). Although protein kinase C may be normally activated in a reversible manner by the simultaneous presence of phospholipid and diacylglycerol at Ca2+ concentrations less than 10(-6) M, this enzyme fragment is fully active without any lipid fractions and independent of Ca2+. The limited proteolysis of protein kinase C is markedly enhanced in the velocity by the addition of phospholipid and diacylglycerol, which are both required for the reversible activation of the enzyme. However, casein hydrolysis by this protease is not affected by phospholipid and diacylglycerol. Available evidence suggests that, at lower concentrations of this divalent cation, Ca2+ protease I reacts preferentially with the active form of protein kinase C which is associated with membrane, and converts it to the permanently active form. In contrast, the inactive form of protein kinase C, which is free of membrane phospholipid, does not appear to be very susceptible to the proteolytic attack. It remains unknown, however, whether this mechanism of irreversible activation of protein kinase C does operate in physiological processes. It is noted that Ca2+ protease II, which is active at higher concentrations of Ca2+, proteolytically activates protein kinase C irrespective of the presence and absence of phospholipid and diacylglycerol.     

A monoclonal antibody against brain calmodulin-dependent protein kinase type II detects putative conformational changes induced by Ca2+-calmodulin

A mouse monoclonal IgG1 antibody has been generated against the soluble form of the calmodulin-dependent protein kinase type II. This antibody recognizes both the soluble and cytoskeletal forms of the enzyme, requiring Ca2+ (EC50 = 20 microM) for the interaction. Other divalent cations such as Zn2+, Mn2+, Cd2+, Co2+, and Ni2+ will substitute for Ca2+, while Mg2+ and Ba2+ will not. The antibody reacts with both the alpha- and beta-subunits on Western blots in a similar Ca2+-dependent fashion but with a lower sensitivity. The affinity of the antibody for the kinase is 0.13 nM determined by displacement of 125I Bolton-Hunter-labeled kinase with unlabeled enzyme. A variety of other proteins including tubulin do not compete for antibody binding. The Mr 30,000 catalytic fragment obtained by proteolysis of either the soluble or the cytoskeletal form of the kinase fails to react with the antibody. Calmodulin and antibody reciprocally potentiate each other's interaction with the enzyme. This is illustrated both by direct binding studies and by a decrease of the Kmapp for calmodulin and an increase in the Vmax for the autophosphorylation reaction of the enzyme. The antibody thus appears to recognize and stabilize a conformation of the kinase which favors calmodulin binding although it does not itself activate the kinase in the absence of calmodulin. Since the Mr 30,000 catalytic fragment of the kinase is not immunoreactive, either the antibody combining site of the kinase must be present in the noncatalytic portion of the protein along with the calmodulin binding site or proteolysis interferes with the putative Ca2+-dependent conformational change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-calmodulin-dependent phosphorylation and passive transport of Ca2+ in the myocardial sarcolemma

Highly purified vesicles of rabbit myocardium sarcolemma with predominant inside-out orientation possess the Ca2+-calmodulin-dependent protein kinase activity. At optimal concentrations of calmodulin (0.5 microM) and Ca2+ (0.1 mM), the activity of protein kinase is 0.21 nmol 32P X min X mg of protein. The Km(app) value for ATP is 3.0 X 10(-6) M, V = 0.27 nmol 32P X mg of protein X min. Endogenous Ca2+-calmodulin-dependent protein kinase phosphorylates four protein substrates in sarcolemmal vesicles (Mr = 145, 22, 11.5, and 6-8 KD). Studies with passive efflux of Ca2+ from the SL vesicles showed that the Ca2+-calmodulin-dependent phosphorylation of protein components of sarcolemma inhibits this reaction.     

Calmodulin inhibits the epidermal growth factor receptor tyrosine kinase.

We demonstrate in this report that the epidermal growth factor (EGF) receptor from rat liver can be isolated by calmodulin affinity chromatography by binding in the presence of Ca2+ and elution with a Ca(2+)-chelating agent. The bulk of the EGF receptor is not eluted by a NaCl gradient in the presence of Ca2+. We ascertained the identity of the isolated receptor by immunoblot and immunoprecipitation using a polyclonal antibody against an EGF receptor from human origin. The purified receptor is autophosphorylated in tyrosine residues in an EGF-stimulated manner, and EGF-dependent phosphorylation of serine residues was also detected. Both the EGF and the transforming growth factor-alpha stimulate the tyrosine-directed protein kinase activity of the isolated receptor with similar affinities. Furthermore, we demonstrate that calmodulin inhibits the EGF-dependent tyrosine-directed protein kinase activity associated to the receptor in a concentration-dependent manner. This inhibition is partially Ca2+ dependent and is not displaced by increasing the concentration of EGF up to an EGF/calmodulin ratio of 10 (mol/mol). In addition, calmodulin was phosphorylated in an EGF-stimulated manner in the presence of a basic protein (histone) as cofactor and in the absence, but not in the presence, of Ca2+.     

Purification, characterization and substrate specificity of calmodulin-dependent myosin light-chain kinase from bovine brain.

A substrate-specific calmodulin-dependent myosin light-chain kinase (MLCK) was purified 45,000-fold to near homogeneity from bovine brain in 12% yield. Bovine brain MLCK phosphorylates a serine residue in the isolated turkey gizzard myosin light chain (MLC), with a specific activity of 1.8 mumol/min per mg of enzyme. The regulatory MLC present in intact gizzard myosin is also phosphorylated by the enzyme. The Mr-19,000 rabbit skeletal-muscle MLC is a substrate; however, the rate of its phosphorylation is at best 30% of that obtained with turkey gizzard MLC. Phosphorylation of all other protein substrates tested is less than 1% of that observed with gizzard MLC as substrate. SDS/polyacrylamide-gel electrophoresis of purified MLCK reveals the presence of a major protein band with an apparent Mr of 152000, which is capable of binding 125I-calmodulin in a Ca2+-dependent manner. Phosphorylation of MLCK by the catalytic subunit of cyclic-AMP-dependent protein kinase results in the incorporation of phosphate into the Mr-152,000 protein band and a marked decrease in the affinity of MLCK for calmodulin. The presence of Ca2+ and calmodulin inhibits the phosphorylation of the enzyme. Bovine brain MLCK appears similar to MLCKs isolated from platelets and various forms of muscle.     

Decoding calcium signals by multifunctional CaM kinase.

Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) is one of the three major protein kinases coordinating cellular responses to hormones and neurotransmitters. It mediates the action of Ca2+ on neurotransmitter synthesis and release, on carbohydrate metabolism and on the cytoskeleton. CaM kinase has structural/functional properties that facilitate its response to distinctive attributes of Ca2+ signals which often involve transient increases that span a narrow concentration range and increases that are pulsatile rather than persistent. The kinase responds to the narrow working range of Ca2+ signals by the use of calmodulin as the Ca2+ sensor. It is activated by the binding of calmodulin to an autoinhibitory domain that keeps the kinase inactive in the basal state. The transient nature of the signal is accommodated by autophosphorylation of this autoinhibitory domain which allows the kinase to remain partially active after calmodulin dissociates and thereby switches it to a Ca(2+)-independent species. The pulsatile nature of Ca2+ signals may also be decoded by CaM kinase. Autophosphorylation traps calmodulin on autophosphorylated subunits by greatly reducing its off-rate. At high frequency of stimulation, calmodulin would remain trapped during the brief interval between Ca2+ oscillations and each successive rise in Ca2+ would recruit more calmodulin. This may enable a stimulus frequency dependent activation of CaM kinase.     

Ganglioside-modulated protein phosphorylation. Partial purification and characterization of a ganglioside-stimulated protein kinase in brain

Gangliosides have profound modulatory effects on protein phosphorylation in brain. A protein kinase activated directly by gangliosides has been partially purified from the particulate fractions of guinea pig brain through extraction with nonionic detergent, ion-exchange chromatography, hydrophobic chromatography, and gel filtration. This novel ganglioside-stimulated protein kinase is distinct from cAMP-dependent, Ca2+/calmodulin-dependent, and Ca2+/phospholipid-dependent protein kinases. The partially purified kinase preparation could undergo ganglioside-stimulated autophosphorylation of a major phosphoprotein with Mr corresponding to 68,000. It also could phosphorylate exogenous substrates such as the synthetic peptide Leu-Arg-Arg-Ala Ser-Leu-Gly. The requirement of gangliosides for the activation of kinase activity is dose-dependent and specific. Among the various gangliosides tested, GT1b and GD1a were found to be the most potent activators, whereas GD1b and GM1 were slightly less effective. The activation process is rapid and does not require the presence of Ca2+, suggesting that the stimulatory effect of gangliosides is not mediated through limited proteolysis or Ca2+-glycolipid complexes. Although the exact physiological significance of the ganglioside-stimulated protein kinase is not known at present, it is possible that certain functions related to gangliosides in the nervous system are mediated through the activation of this novel enzyme.     

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.     

Myosin light chain kinase structure function analysis using bacterial expression

A 40-kDa fragment of chicken smooth muscle myosin light chain kinase was produced and partially purified from a bacterial expression system. This fragment exhibits calmodulin binding and substrate phosphorylation properties similar to those of the isolated chicken gizzard enzyme. A series of 3'-deletion mutants was prepared and used to produce proteins with the same NH2 terminus but with COOH termini varying over 180 amino acids. Results show that truncation of the enzyme at Ser-512 (based on the amino acid numbering system described for the partial cDNA clone by Guerriero, V., Jr., Russo, M. A., Olson, N. J., Putkey, J. A., and Means, A. R. (1986) Biochemistry 25, 8372-8381) does not alter calmodulin binding, calmodulin regulation, or enzymatic properties. Removal of an additional 5 residues from the COOH terminus completely inhibits calmodulin binding and results in an inactive kinase that can be fully activated by limited proteolysis. Site specific mutations within these 5 residues demonstrate that Gly-508 and Arg-509 are independently involved in calmodulin-dependent binding and activation of myosin light chain kinase. Truncation of the enzyme at residues within the protein kinase catalytic domain results in inactive protein that cannot be activated by proteolysis.     

Purification and characterization of a calcium-calmodulin-dependent phospholamban kinase from canine myocardium

A Ca2+-calmodulin-dependent protein kinase was purified to apparent homogeneity from the cytosolic fraction of canine myocardium, with phospholamban as substrate. Purification involved sequential chromatography on DEAE-cellulose, calmodulin-agarose, DEAE-Bio-Gel A, and phosphocellulose. This procedure resulted in a 987-fold purification with a 5.4% yield. The purified enzyme migrated as a single band on native polyacrylamide gels, and it exhibited an apparent molecular weight of 550,000 upon gel filtration. Gel electrophoresis under denaturing conditions revealed a single protein band with Mr 55,000. The purified kinase could be autophosphorylated in a Ca2+-calmodulin-dependent manner, and under optimal conditions, 6 mol of Pi was incorporated per mole of 55,000-dalton subunit. The activity of the enzyme was dependent on Ca2+, calmodulin, and ATP.Mg2+. Other ions which could partially substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations were Sr2+ greater than Mn2+ greater than Zn2+ greater than Fe2+. The substrate specificity of the purified Ca2+-calmodulin-dependent protein kinase for cardiac proteins was determined by using phospholamban, troponin I, sarcoplasmic reticulum membranes, myofibrils, highly enriched sarcolemma, and mitochondria. The protein kinase could only phosphorylate phospholamban and troponin I either in their purified forms or in sarcoplasmic reticulum membranes and myofibrils, respectively. Exogenous proteins which could also be phosphorylated by the purified protein kinase were skeletal muscle glycogen synthase greater than gizzard myosin light chain greater than brain myelin basic protein greater than casein. However, phospholamban appeared to be phosphorylated with a higher rate as well as affinity than glycogen synthase.(ABSTRACT TRUNCATED AT 250 WORDS)