Phosphorylation by calcium calmodulin-dependent protein kinase II and protein kinase C modulates the activity of nitric oxide synthase.

Nitric oxide synthase purified from rat brain, which is Ca2+ and calmodulin dependent, was phosphorylated by calcium calmodulin-dependent protein kinase II as well as protein kinase C. Phosphorylation by calcium calmodulin-dependent protein kinase II resulted in a marked decrease in enzyme activity (33% of control) without changing the co-factor requirements, whereas a moderate increase in enzyme activity (140% of control) was observed after phosphorylation by protein kinase C. These findings indicate that brain nitric oxide synthase activity may be regulated not only by Ca2+/calmodulin and several co-factors, but also by phosphorylation.     

A sensitive method for detection of calmodulin-dependent protein kinase II activity in sodium dodecyl sulfate-polyacrylamide gel

A procedure for detecting protein kinase activities of the alpha and beta subunits of calmodulin-dependent protein kinase II separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. After electrophoresis, the gel was immersed in 6 M guanidine HCl for 1 h and then in a buffer containing 0.04% Tween 40 for 16 h at 4 degrees C for renaturation of the resolved polypeptides. The renatured polypeptides in the gel were incubated with [gamma-32P]ATP for phosphorylation of either the substrate included in the polyacrylamide gel or the kinase itself. After removal of the unreacted [gamma-32P]ATP, the protein kinase activities were visualized by autoradiography. Two radioactive protein bands of Mr 50,000 and 60,000, which corresponded to the alpha and beta subunits, were detected only when the phosphorylation was carried out in the presence of Ca2+ and calmodulin. Approximately 0.05 micrograms of the enzyme could be detected on a gel containing no protein substrate. When microtubule-associated protein 2 was included in the gel, the sensitivity of the detection of calmodulin-dependent protein kinase II in the gel was more than one order of magnitude higher than that in the gel containing no protein substrate.     

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.     

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

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

Calmodulin-dependent protein kinases purified from rat brain and rabbit liver

A calmodulin-dependent protein kinase was purified from rat brain by the same protocol used previously for a rabbit liver calmodulin-dependent glycogen synthase kinase. The rat brain kinase readily phosphorylated rabbit skeletal muscle glycogen synthase at sites 1b and 2, the same sites phosphorylated by rabbit liver calmodulin-dependent kinase. The two kinases have other similarities: substrate specificity, potent inhibition by sodium fluoride, and nearly equal Ka's (10-20 nM) for calmodulin. Also, both enzymes have similar Stokes radii, 70 A (rabbit liver) and 75 A (rat brain), but quite different sedimentation coefficients, 10.6 S and 17.4 S, respectively. Consequently, the calculated molecular weights are also different: 560,000 for the brain enzyme and 300,000 for the liver enzyme. The major subunit of the rat brain kinase appears to be a single 51-kDa peptide, not a doublet pattern of 51- and 53-kDa subunits that is characteristic of the rabbit liver enzyme. Our findings are consistent with the hypothesis that the rat brain and rabbit liver enzymes belong to a class of closely related calmodulin-dependent protein kinases, possibly isozymes. This class of enzymes may be responsible for regulating several of the known calcium-dependent physiological functions.     

Characterization of a soluble Mr-30,000 catalytic fragment of the neuronal calmodulin-dependent protein kinase II

Chymotryptic digestion of postsynaptic densities releases a soluble, catalytically active fragment of the alpha (Mr 50,000) subunit of the neuronal cytoskeletal calmodulin-dependent protein kinase II. The purified soluble form of the kinase likewise yields the fragment. Denaturation of the enzyme results in more extensive proteolytic degradation. 125I-Iodopeptide maps of the isolated catalytic portions of both forms of the enzyme are similar and are contained within the map of the isolated alpha subunit. Catalytic fragments of both forms of the enzyme comigrate on two-dimensional SDS-PAGE/isoelectric focusing with pI 6.7-7.2. The fragment phosphorylates microtubule-associated protein (MAP-2) but is not activated by Ca+2/calmodulin nor is it inhibited by trifluoperazine. Km values for MAP-2 and ATP are indistinguishable from those of the holoenzyme, while the Vmax is similar to that of the holoenzyme activated with Ca+2/calmodulin. Overlays of Western blots of fragment with 125I-calmodulin shows a loss of calmodulin binding. Both the number of phosphorylation sites and the ability to autophosphorylate are markedly reduced in the catalytic fragment. Evaluation of the hydrodynamic parameters of the purified fragment yielded Mr value of 25,600 with a frictional ratio (f/f0) of 1.12; the Mr value determined by SDS-PAGE was 30,000. Thus, the catalytic fragment appears to represent an activated form of the kinase with a monomeric, globular structure unlike the native enzyme which exhibits oligomerization and cytoskeletal association. These results are consistent with a tertiary structure for the calmodulin-dependent protein kinase that contains distinct domains responsible for catalytic activity, regulation by calmodulin, cytoskeletal association and the multimeric organization of enzyme subunits.     

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

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

Site-specific phosphorylation of the purified receptor for calcium-channel blockers by cAMP- and cGMP-dependent protein kinases, protein kinase C, calmodulin-dependent protein kinase II and casein kinase II

Five protein kinases were used to study the phosphorylation pattern of the purified skeletal muscle receptor for calcium-channel blockers (CaCB). cAMP kinase, cGMP kinase, protein kinase C, calmodulin kinase II and casein kinase II phosphorylated the 165-kDa and the 55-kDa proteins of the purified CaCB receptor. The 130/28-kDa and the 32-kDa protein of the receptor are not phosphorylated by these protein kinases. Among these protein kinases only cAMP kinase phosphorylated the 165-kDa subunit with 2-3-fold higher initial rate than the 55-kDa subunit. Casein kinase II phosphorylated the 165-kDa and the 55-kDa protein of the receptor with comparable rates. cGMP kinase, protein kinase C and calmodulin kinase II phosphorylated preferentially the 55-kDa protein. The 55-kDa protein is phosphorylated 50 times faster by cGMP kinase and protein kinase C than by calmodulin kinase II or casein kinase II and about 10 times faster by these enzymes than by cAMP kinase. Two-dimensional peptide maps of the 165-kDa subunit yielded a total of 11 phosphopeptides. Four or five peptides are phosphorylated specifically by cAMP kinase, cGMP kinase, casein kinase II and protein kinase C, whereas the other peptides are modified by several kinases. The same kinases phosphorylate 11 peptides in the 55-kDa subunit. Again, some of these peptides are modified specifically by each kinase. These results suggest that the 165-kDa and the 55-kDa subunit contain specific phosphorylation sites for cAMP kinase, cGMP kinase, casein kinase II and protein kinase C. Phosphorylation of these sites may be relevant for the in vivo function of the CaCB receptor.     

Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain

A calcium and calmodulin-dependent protein kinase has been purified from rat brain. It was monitored during the purification by its ability to phosphorylate the synaptic vesicle-associated protein, synapsin I. A 300-fold purification was sufficient to produce kinase that is 90-95% pure as determined by scans of stained sodium dodecyl sulfate-polyacrylamide gels and has a specific activity of 2.9 mumol of 32P transferred per min/mg of protein. Thus, the kinase is a relatively abundant brain enzyme, perhaps comprising as much as 0.3% of the total brain protein. The Stokes radius (95 A) and sedimentation coefficient (16.4 S) of the kinase indicate a holoenzyme molecular weight of approximately 650,000. The holoenzyme is composed of three subunits as judged by their co-migration with kinase activity during the purification steps and co-precipitation with kinase activity by a specific anti-kinase monoclonal antibody. The three subunits have molecular weights of 50,000, 58,000, and 60,000, and have been termed alpha, beta', and beta, respectively. The alpha- and beta-subunits are distinct peptides, however, beta' may have been generated from beta by proteolysis. All three of these subunits bind calmodulin in the presence of calcium and are autophosphorylated under conditions in which the kinase is active. The subunits are present in a ratio of about 3 alpha-subunits to 1 beta/beta'-subunit. We therefore postulate that the 650,000-Da holoenzyme consists of approximately 9 alpha-subunits and 3 beta/beta'-subunits. The abundance of this calmodulin-dependent protein kinase indicates that its activation is likely to be an important biochemical response to increases in calcium ion concentration in neuronal tissue.     

Calmodulin-dependent multifunctional protein kinase. Evidence for isoenzyme forms in mammalian tissues

Calcium/calmodulin-dependent multifunctional protein kinases, extensively purified from rat brain (with apparent molecular mass 640 kDa), rabbit liver (300 kDa) and rabbit skeletal muscle (700 kDa), were analysed for their structural, immunological, and enzymatic properties. The immunological cross-reactivity with affinity-purified polyclonal antibodies to the 50-kDa catalytic subunit of the brain calmodulin-dependent protein kinase confirmed the presence of common antigenic determinants in all subunits of the protein kinases. One-dimensional phosphopeptide patterns, obtained by digestion of the autophosphorylated protein kinases with S. aureus V8 protease, and two-dimensional fingerprints of the 125I-labelled proteins digested with a combination of trypsin and chymotrypsin, revealed a close similarity between the two subunits (51 kDa and 53 kDa) of the liver enzyme. Similar identity was observed between the 56-kDa and/or 58-kDa polypeptides of the skeletal muscle calmodulin-dependent protein kinase. The data suggest that the subunits of the liver and muscle protein kinases may be derived by partial proteolysis or by autophosphorylation. The peptide patterns for the 50-kDa and 60-kDa subunits of the brain enzyme confirmed that the two catalytic subunits represented distinct protein products. The comparison of the phosphopeptide maps and the two-dimensional peptide fingerprints, indicated considerable structural homology among the 50-kDa and 60-kDa subunits of the brain calmodulin-dependent protein kinase and the liver and muscle polypeptides. However, a significant number of unique peptides in the liver 51-kDa subunit, skeletal muscle 56-kDa, and the brain 50-kDa and 60-kDa polypeptides were observed and suggest the existence of isoenzyme forms. All calmodulin-dependent protein kinases rapidly phosphorylated synapsin I with a stoichiometry of 3-5 mol phosphate/mol protein. The two-dimensional separation of phosphopeptides obtained by tryptic/chymotryptic digestion of 32P-labelled synapsin I indicated that the same peptides were phosphorylated by all the calmodulin-dependent protein kinases. Such data represent the first structural and immunological comparison of the liver calmodulin-dependent protein kinase with the enzymes isolated from brain and skeletal muscle. The findings indicate the presence of a family of highly conserved calmodulin-dependent multifunctional protein kinases, with similar structural, immunological and enzymatic properties. The individual catalytic subunits appear to represent the expression of distinct protein products or isoenzymes which are selectively expressed in mammalian tissues.     

Ca2+/calmodulin-dependent protein kinase II. Isozymic forms from rat forebrain and cerebellum

Ca2+/calmodulin-dependent protein kinase II, an abundant brain protein proposed to mediate a number of Ca2+-regulated processes in neuronal tissue, is composed of autophosphorylatable subunits of Mr 50,000 and 60,000/58,000. A recent study (McGuinness, T. L., Lai, Y., Greengard, P., Woodgett, J.R., and Cohen, P. (1983) FEBS Lett. 163, 329-334) suggested that this kinase exists as isozymes which vary in the relative ratio of these subunits in different tissues or species. Other studies (Walaas, S. I., Nairn, A. C., and Greengard, P. (1983) J. Neurosci. 3, 291-301, 302-311) provided evidence which suggested that the ratio of these phosphopeptides might vary in different brain regions. In the present investigation, we have tested this possibility by comparing Ca2+/calmodulin-dependent protein kinase II purified from rat forebrain and cerebellum. The two kinases had similar purification characteristics, subunit compositions, physical properties, and substrate specificities. Gel filtration and sucrose density gradient centrifugation provided an estimated molecular weight of 550,000 for the forebrain kinase and 615,000 for the cerebellar kinase. The kinases from the two regions clearly differed in the relative proportions of the Mr 50,000 and 60,000/58,000 subunits. Three independent methods indicated that the forebrain kinase contained the Mr 50,000/(60,000/58,000) subunits in approximately a 3:1 ratio, while the cerebellar kinase contained the Mr 50,000/(60,000/58,000) subunits in approximately a 1:4 ratio. The forebrain kinase subunits were shown to be identical to the corresponding subunits of the cerebellar kinase by several criteria. The data are consistent with the existence in various brain regions of isozymic forms of Ca2+/calmodulin-dependent protein kinase II which differ in their relative subunit ratios.     

Purification and characterization of a brain-specific multifunctional calmodulin-dependent protein kinase from rat cerebellum.

A brain-specific multifunctional calmodulin-dependent protein kinase, calmodulin-dependent protein kinase IV, which exhibited characteristic properties quite different from those of calmodulin-dependent protein kinase II, was purified approximately 230-fold from rat cerebellum. The purified preparation gave two protein bands with molecular weights of 63,000 (alpha) and 66,000 (beta) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both of which showed protein kinase activity as examined by the activity gel method. The molecular weight of the enzyme was estimated as about 67,000 from sedimentation coefficient (3.2 S) and Stokes radius (50 A), indicating a monomeric structure of the enzyme. The enzyme phosphorylated smooth muscle myosin light chain, synapsin I, microtubule-associated protein 2, tau protein, myelin basic protein, histone H1, and tyrosine hydroxylase in a Ca2+/calmodulin dependent manner, suggesting that the enzyme is a multifunctional calmodulin-dependent protein kinase capable of phosphorylating a large number of substrates. A synthetic peptide, Lys-Ser-Asp-Gly-Gly-Val-Lys-Lys-Arg-Lys-Ser-Ser-Ser-Ser, was found to be a specific substrate for this kinase and, using this peptide as substrate, the distribution of the enzyme activity in various rat tissues was examined. The activity was found in cerebral cortex, brain stem, and cerebellum, most abundantly in cerebellum, but other tissues tested, including liver, spleen, kidney, lung, heart, skeletal muscle, and adrenal gland showed very little activity.     

A calmodulin-dependent protein kinase in Rous sarcoma virus-transformed rat cells and normal liver

A calmodulin-dependent protein kinase has been purified extensively from a Rous sarcoma virus-transformed rat cell line (RR1022) and from normal rat liver. The calmodulin-dependent protein kinase activity was manifested by in vitro phosphorylation of a single Mr 57 000 endogenous phosphoprotein (pp57) present in both the virally transformed cells and normal rat liver. The calmodulin-dependent protein kinase from transformed cells fractionated with the viral src gene product, pp60v-src, through a 650-fold purification of the oncogene product. However, purification of the calmodulin-dependent protein kinase from normal liver demonstrated that the calmodulin-dependent kinase was distinct from pp60v-src. Phosphorylation of pp57 by the kinase purified from the transformed cell line required Ca2+ and calmodulin, was inhibited by EDTA and was unaffected by cAMP or the heat- and acid-stable protein inhibitor of cAMP-dependent protein kinase. Troponin C did not substitute for calmodulin. A virtually identical calmodulin-dependent protein kinase activity was purified from rat liver by affinity chromatography on calmodulin-Sepharose. Phosphorylation of pp57 by the affinity-purified liver protein kinase was also observed, and required Ca2+ and calmodulin. EGTA and trifluoroperazine inhibited pp57 phosphorylation. The calmodulin-dependent protein kinase reported here did not phosphorylate substrates of known calmodulin-dependent protein kinases in vitro (myosin light chain, phosphorylase b, glycogen synthase, microtubule-associated proteins, tubulin, alpha-casein). Because none of these proteins served as substrates in vitro and pp57 was the only endogenous substrate found, the properties of this enzyme appear to be different from any previously described calmodulin-dependent protein kinase.     

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.     

Specific postsynaptic density proteins bind tubulin and calmodulin-dependent protein kinase type II

Cytoskeletal interactions which contribute to the assembly of the postsynaptic density (PSD) were investigated. PSDs bound 125I-tubulin specifically with an apparent Km of 2 X 10(-7) M and a Bmax of about 1 nmol/mg of protein. 125I-Tubulin blots revealed that a group of polypeptides between Mr 135,000 and 147,000 (P-140) was a major tubulin-binding PSD component. The P-140 polypeptides were highly enriched in the PSD fraction of purified synaptosomes and could not be detected in crude brain cytoplasm preparations. These polypeptides were subject to phosphorylation by endogenous calmodulin-dependent protein kinase type II, with a concomitant reduction in 125I-tubulin binding. The tubulin-binding polypeptides could also associate with the radiolabeled alpha- and beta-subunits of the calmodulin-dependent protein kinase. These observations are consistent with a role for the P-140 polypeptides in organizing the molecular structure of the PSD. The data also suggest that this structure may be modified by Ca2+-sensitive phosphorylation, thus permitting neuronal activity to modulate the cytoskeletal interactions of the PSD.     

High-level expression using baculovirus, purification, and characterization of a monomeric form of type II calmodulin-dependent protein kinase.

The type II calmodulin-dependent protein kinase is an oligomer existing in multiple isozymic forms. To facilitate investigations of the regulatory mechanisms of this complex enzyme, we have constructed a truncated, calmodulin-dependent monomer of the alpha subunit. The N-terminal enzyme fragment (alpha 315) was expressed at high levels in a baculovirus/insect cell expression system. The recombinant protein was purified chromatographically using DEAE-cellulose, calmodulin-Sepharose, and AffiGel blue, yielding 4 mg of kinase from 1.5 x 10(8) cells in 4 h. Characterization of the truncated kinase indicated that it is a monomer and that interactions of alpha 315 with calmodulin and substrates are indistinguishable from those observed for purified holoenzyme from rat brain. These results indicate that the baculovirus/insect cell expression system is well suited for producing alpha 315, a structurally simplified model of the type II calmodulin-dependent protein kinase.     

Conformation-sensitive modification of the type II calmodulin-dependent protein kinase by phenylglyoxal

Chemical modification by phenylglyoxal was used to investigate relationships between the structure, function, and regulation of the type II calmodulin-dependent protein kinase. Modification of the protein kinase by phenylglyoxal resulted in specific labeling of one distinct site, most likely an important arginine residue, with concomitant inactivation of the enzyme. Labeling and inactivation of the protein kinase was prevented by Mg2+-ADP which suggests that modification occurred at, or in close proximity to, its nucleotide-binding pocket. Half-maximal protection by Mg2+-ADP was enhanced by calmodulin which decreased the K0.5 for ADP from 540 to 61 microM. This response of the enzyme to calmodulin indicates that the modulator protein increases the affinity of the protein kinase for nucleotides. Inactivation of the enzyme by phenylglyoxal was dependent on the presence of Mg2+ or Ca2+/calmodulin, and further enhanced by the simultaneous addition of these effectors to the reaction. The Mg2+ effect is indicative of binding of this divalent metal ion to the protein kinase even in the absence of calmodulin and nucleotides. The stimulation of the modification reaction by calmodulin indicates an increase in the reactivity or accessibility of the modified residue in response to calmodulin-regulated conformational changes on the enzyme. The calmodulin-induced changes observed in this study may play important roles in the molecular mechanisms of activation of the type II calmodulin-dependent protein kinase.     

Further comparison of calmodulin-dependent protein kinase II from brain and calmodulin-dependent glycogen synthase kinase from skeletal muscle

Calmodulin-dependent protein kinase II was purified from rabbit brain and its properties were compared with those of calmodulin-dependent protein kinase II from rat brain and calmodulin-dependent glycogen synthase kinase from rabbit skeletal muscle. Rabbit brain calmodulin-dependent protein kinase II was clearly distinguished from rabbit skeletal muscle glycogen synthase kinase with respect to size, behavior on autophosphorylation, immunological cross-reactivity and peptide mapping, but was indistinguishable from rat brain calmodulin-dependent protein kinase II in all respects examined. Thus, differences between calmodulin-dependent protein kinase II and glycogen synthase kinase appear not to reflect a species difference but to reflect a tissue difference.     

Endogenous dephosphorylation of synaptosomal calmodulin-dependent protein kinase type II

Calmodulin-dependent protein kinase Type II autophosphorylation in synaptosomes is localized to the cytoskeleton (synaptic junction), while a potent dephosphorylating activity is present in the lipid bilayer. The dephosphorylating activity is operative in intact synaptosomes and in a reconstitution system comprised of the cytoskeletal and Triton X-100 - soluble fractions. Dephosphorylation is inhibited by EDTA and pyrophosphate, but not by EGTA or NaF. The present characterization of endogenous synaptosomal dephosphorylating activity completes the regulatory cycle operating on this enzyme in which phosphorylation of calmodulin-dependent protein kinase type II inhibits its response to Ca+2 and calmodulin.     

Purification and characterization of myosin light-chain kinase from the rat pancreas.

We have partially purified a protein kinase from rat pancreas that phosphorylates two light-chain subunits of pancreatic myosin, a doublet with components of 18 and 20 kDa. This protein kinase was purified approx. 1000-fold by sequential (NH4)2SO4 fractionation, gel filtration, ion-exchange and affinity chromatography on calmodulin-Sepharose 4B. The resultant enzyme preparation is free of cyclic AMP-dependent protein kinase, protein kinase C and calmodulin-dependent type I or II kinase activities. The purified protein kinase is completely dependent on Ca2+ and calmodulin, and phosphorylates a 20 kDa light-chain subunit of intact gizzard myosin, suggesting that it belongs to a class of enzymes known as myosin light-chain kinase (MLCK). The apparent Km values of the putative pancreatic MLCK for ATP (73 microM), gizzard myosin light chains (18 microM) and calmodulin (2 nM) are similar to those reported for MLCKs isolated from smooth muscle, platelet and other sources. The enzyme is half-maximally activated at a free Ca2+ concentration of 2.5 microM. A single component of the affinity-purified kinase reacts with antibodies to turkey gizzard MLCK. The apparent molecular mass of this component is 138 kDa. Immunoprecipitation of a pancreatic homogenate with these antibodies decreases calmodulin-dependent kinase activity for pancreatic myosin by over 85%. The immunoprecipitate contains a single electrophoretic band of 138 kDa. Tryptic phosphopeptide analyses of pancreatic myosin, phosphorylated by either gizzard or pancreatic MLCK, are identical. Thus the enzyme that we have purified from rat pancreas is a MLCK, as judged by (1) absolute dependence on Ca2+ and calmodulin, (2) high affinity for calmodulin, (3) narrow substrate specificity for the light-chain subunit of myosin, and (4) reactivity with antibodies to turkey gizzard MLCK. These studies establish the existence of a pancreatic MLCK which may be responsible for regulating myosin phosphorylation and enzyme secretion in situ.