Poly (ADP-ribose) synthetase is phosphorylated by protein kinase C in vitro

Poly (ADP-ribose) synthetase from bovine thymus was phosphorylated effectively by protein kinase C in vitro. The phosphorylation was dependent on the activators of this kinase, Ca2+ and phospholipid. The apparent Km for the synthetase was about 8 microM, which was lower than that for histone H1. Though the synthetase was a weak substrate for Ca2+/calmodulin-dependent protein kinase II, other protein kinases, cyclic AMP-dependent and cofactor-independent protein kinases did not phosphorylate the synthetase. Phosphorylation of the synthetase by protein kinase C resulted in appreciable inhibition of the synthetase activity.     

Purification and characterization of two cyclic AMP-independent casein/glycogen synthase kinases from rat liver cytosol

Two cyclic AMP-independent protein kinases (ATP: protein phosphotransferase, EC 2.7.1.37) (casein kinase 1 and 2) have been purified from rat liver cytosol by a method involving chromatography on phosphocellulose and casein-Sepharose 4B. Both kinases were essentially free of endogeneous protein substrates and capable of phosphorylating casein, phosvitin and I-form glycogen synthase, but were inactive on histone IIA, protamine and phosphorylase b. They were neither stimulated by cyclic AMP, Ca2+ and calmodulin, nor inhibited by the cyclic AMP-dependent protein kinase inhibitor protein. The casein and glycogen synthase kinase activities of each enzyme decreased at the same rate when incubated at 50 degrees C. Casein kinase 1 and casein kinase 2 showed differences in molecular weight, sensitivity to KCl, Km for casein and phosvitin and Ka for Mg2+, whereas their Km values for ATP and I-form glycogen synthase were similar. The phosphorylation of glycogen synthase by these kinases correlated with a decrease in the +/- glucose 6-phosphate activity ratio (independence ratio). However, casein kinase 1 catalyzed the incorporation of about 3.6 mol of 32P/85000 dalton subunit, decreasing the independence ratio from 83 to about 15, whereas the phosphorylation achieved by casein kinase 2 was only about 1.9 mol of 32P/850000 dalton subunit, decreasing the independence ratio to about 23. The independence ratio decrease was prevented by the presence of casein but was unaffected by phosphorylase b. These data indicate that casein/glycogen synthase kinases 1 and 2 are different from cyclic AMP-dependent protein kinase and phosphorylase kinase.     

Independent Protein Kinases Associated with the Rat Cerebral Synaptic Junction: Comparison with Cyclic AMP-Dependent and Ca2+/Calmodulin-Dependent Protein Kinases in the Synaptic Junction

Independent protein kinases in the synaptic junction (SJ) isolated from rat cerebrum were characterized. SJ showed a protein kinase activity, phosphorylating intrinsic proteins, even in the absence of cyclic AMP or Ca2+ plus calmodulin (CaM) exogenously added. The activity was affected neither by Ca2+ concentrations in the physiological fluctuation range nor by the addition of specific ligands such as glutamate, aspartate, acetylcholine, and concanavalin A. The activity was not due to cyclic AMP-dependent protein kinase in SJ, since the activity was not inhibited by an inhibitor protein for cyclic AMP-dependent protein kinase, and since synapsin I was not specifically phosphorylated whereas cyclic AMP-dependent kinase appeared to phosphorylate selectively the protein in SJ. Phosphorylation of SJ proteins by the independent kinases was about one-third of that of the Ca2+/CaM-dependent protein kinase intrinsic to SJ. The apparent Km for ATP was estimated to be 700 microM. Proteins of 16K Mr and 117K Mr were specifically phosphorylated under the basic condition (in the absence of the substances known to activate specifically protein kinases), as well as six other proteins both under the basic conditions and in the presence of Ca2+ and CaM. The phosphorylation of 150K Mr, 60K Mr, 51K Mr, and 16K Mr SJ proteins was enhanced after prephosphorylation of SJ proteins by intrinsic kinase in the presence of Ca2+ and CaM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial purification and characterization of a new phosphoprotein kinase from cells infected with pseudorabies virus

Cytoplasmic fractions from normal baby hamster kidney fibroblasts and from fibroblasts infected with pseudorabies virus were fractionated by DEAE-cellulose chromatography and fractions assayed for protein kinase activity. In preparations from uninfected and infected cells protein kinase activities identified as casein kinase I and II, the two isoforms of the cyclic-AMP-dependent protein kinase, protein kinase C, and a presumed proteolytic fragment of protein kinase C were present in comparable amounts. However in infected cells a new protein kinase activity was detected, appearing about 4 h after infection and increasing during the following 6 h at least. This new protein kinase was purified 100-fold by high-performance gel-permeation and ion-exchange chromatography, and characterized. It has an apparent relative molecular mass of 68 000 on the basis of gel-permeation chromatography, and a sedimentation coefficient of 4.3 S. It catalysed the phosphorylation of serine residues of basic proteins in vitro, with protamine a better substrate than mixed histones; and used ATP (apparent Km = 60 microM), but not GTP, as phosphoryl donor. Molecules that can serve as effectors for other protein kinases (cyclic AMP, cyclic GMP, Ca2+ + calmodulin, Ca2+ + phospholipid, double-stranded RNA, and heparin) did not significantly alter the activity of this enzyme. A distinguishing characteristic of the protein kinase was a high KCl concentration optimum with the persistence of activity up to 800 mM KCl, at least.     

Inhibition by melittin of phospholipid-sensitive and calmodulin-sensitive Ca2+-dependent protein kinases.

Effects of melittin, an amphipathic polypeptide, on various species of protein kinases were investigated. It was found that melittin inhibited the newly identified phospholipid-sensitive Ca2+-dependent protein kinase (from heart, brain, spleen and neutrophils) and the cardiac myosin light-chain kinase, a calmodulin-sensitive Ca2+-dependent enzyme. In contrast, melittin had little or no effect on either the holoenzymes of the cardiac cyclic AMP-dependent and cyclic GMP-dependent protein kinases or the catalytic subunit of the former. Kinetic analysis indicated that melittin inhibited phospholipid-sensitive Ca2+-dependent protein kinase non-competitively with respect to ATP (Ki = 1.3 microM); although exhibiting complex kinetics, its inhibition of the enzyme was overcome by phosphatidylserine (a phospholipid cofactor), but not by protein substrate (histone H1) or Ca2+. On the other hand, melittin inhibited myosin light-chain kinase non-competitively with respect to ATP (Ki = 1.4 microM) or Ca2+ (Ki = 1.9 microM), and competitively with respect to calmodulin (Ki = 0.08 microM); although exhibiting complex kinetics, its inhibition of the enzyme was reversed by myosin light chains (substrate protein). The present findings indicate the presence of functionally important hydrophobic or hydrophilic loci on the Ca2+-dependent protein kinases, but not on the cyclic nucleotide-dependent class of protein kinase, with which melittin can interact. Moreover, the kinetic data suggest that melittin inhibited myosin light-chain kinase by interacting with a site on the enzyme the same as, or proximal to, the calmodulin-binding site, thus interfering with the formation of active enzyme-calmodulin-Ca2+ complex.     

Characterization of bovine aortic protein kinase C with histone and platelet protein P47 as substrates.

A Ca2+- and phospholipid-dependent protein kinase (protein kinase C) was partially purified from the media of bovine aortas by chromatography on DEAE-Sephacel and phenyl-Sepharose. Enzyme activity was characterized with both histone and a 47 kDa platelet protein (P47) as substrates, because the properties of protein kinase C can be modified by the choice of substrate. Both phosphatidylserine and Ca2+ were required for kinase activity. With P47 as substrate, protein kinase C had a Ka for Ca2+ of 5 microM. Addition of diolein to the enzyme assay caused a marked stimulation of activity, especially at low Ca2+ concentrations, but the Ka for Ca2+ was shifted only slightly, to 2.5 microM. With histone as substrate, the enzyme had a very high Ka (greater than 50 microM) for Ca2+, which was substantially decreased to 3 microM-Ca2+ by diolein. A Triton X-100 mixed-micelle preparation of lipids was also utilized to assay protein kinase C with histone as the substrate. Under these conditions kinase activity was almost totally dependent on the presence of diolein; again, diolein caused a large decrease in the Ka for Ca2+, from greater than 100 microM to 2.5 microM. The increased sensitivity of protein kinase C to Ca2+ with P47 rather than histone, and the ability of diacylglycerol to activate protein kinase C without shifting the Ka for Ca2+, when P47 is the substrate, illustrate that the mechanism of protein kinase C activation is influenced by the exogenous substrate used to assay the enzyme.     

N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide, a novel activator of protein kinase C

Naphthalenesulfonamide derivatives were used to study the mechanism of regulation of Ca2+-dependent smooth muscle myosin light chain phosphorylation catalyzed by Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) and myosin light chain kinase. Derivatives such as N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9), with a hydrophobic residue at the end of a hydrocarbon chain, stimulated Ca2+-activated, phospholipid-dependent myosin light chain phosphorylation in a Ca2+-dependent fashion. There was no significant effect of these compounds on Ca2+-calmodulin (CaM) dependent myosin light chain phosphorylation. On the other hand, derivatives with the guanidino or amino residue at the same position had an inhibitory effect on both Ca2+-phospholipid- and Ca2+-CaM-dependent myosin light chain phosphorylation. These observations suggest that activation of Ca2+-activated, phospholipid-dependent myosin light chain phosphorylation by naphthalenesulfonamide derivatives depends on the chemical structure at the end of hydrocarbon chain of each compound. SC-9 was similar to phosphatidylserine with regard to activation, and the apparent Km values for Ca2+ of the enzyme with this compound and phosphatidylserine were 40 microM and 80 microM, respectively. Kinetic analysis indicated that 12-O-tetradecanoylphorbol 13-acetate increased the affinity of the enzyme with SC-9 for calcium ion. However, kinetic constants revealed that the Km value of protein kinase C activated by SC-9 for substrate myosin light chain was 5.8 microM, that is, about 10 times lower than that of the enzyme with phosphatidylserine, and that the Vmax value with SC-9 was 0.13 nmol X min-1, that is, 3-fold smaller than that seen with phosphatidylserine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lysophosphatidylcholine metabolism and lipoprotein secretion by cultured rat hepatocytes deficient in choline.

A protein kinase activity was identified in pig brain that co-purified with microtubules through repeated cycles of temperature-dependent assembly and disassembly. The microtubule-associated protein kinase (MTAK) phosphorylated histone H1; this activity was not stimulated by cyclic nucleotides. Ca2+ plus calmodulin, phospholipids or polyamines. MTAK did not phosphorylate synthetic peptides which are substrates for cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase. Ca2+/calmodulin-dependent protein kinase II, protein kinase C or casein kinase II. MTAK activity was inhibited by trifluoperazine [IC50 (median inhibitory concn.) = 600 microM] in a Ca2+-independent fashion. Ca2+ alone was inhibitory [IC50 = 4 mM). MTAK was not inhibited by heparin, a potent inhibitor of casein kinase II, nor a synthetic peptide inhibitor of cyclic AMP-dependent protein kinase. MTAK demonstrated a broad pH maximum (7.5-8.5) and an apparent Km for ATP of 45 microM. Mg2+ was required for enzyme activity and could not be replaced by Mn2+. MTAK phosphorylated serine and threonine residues on histone H1. MTAK is a unique cofactor-independent protein kinase that binds to microtubule structures.     

In Vitro Phosphorylation of Bovine Adrenal Chromaffin Cell Tyrosine Hydroxylase by Endogenous Protein Kinases

Under phosphorylating conditions, addition of Ca2+ or cyclic AMP to the 100,000 g supernatant of purified bovine adrenal chromaffin cells increases both the incorporation of 32P into tyrosine hydroxylase and the activity of the enzyme. Combining maximally effective concentrations of each of these stimulating agents produces an additive increase in both the level of 32P incorporation into tyrosine hydroxylase and the degree of activation of the enzyme. The increased phosphorylation by Ca2+ is due to stimulation of endogenous Ca2+-dependent protein kinase activity and not inhibition of phosphoprotein phosphatases. When the chromaffin cell supernatant is subjected to diethylaminoethyl (DEAE) chromatography to remove calmodulin and phospholipids, tyrosine hydroxylase is no longer phosphorylated or activated by Ca2+; on the other hand, phosphorylation and activation of tyrosine hydroxylase by cyclic AMP are not affected. Subsequent replacement of either Ca2+ plus calmodulin or Ca2+ plus phosphatidylserine to the DEAE-fractionated cell supernatant restores the phosphorylation, but not activation of the enzyme. Reverse-phase HPLC peptide mapping of tryptic digests of tyrosine hydroxylase from the 100,000 g supernatant shows that the Ca2+-dependent phosphorylation occurs on three phosphopeptides, whereas the cyclic AMP-dependent phosphorylation occurs on one of these peptides. In the DEAE preparation, either cyclic AMP alone or Ca2+ in the presence of phosphatidylserine stimulates the phosphorylation of only a single phosphopeptide peak, the same peptide phosphorylated by cyclic AMP in the crude supernatant. In contrast, Ca2+ in the presence of calmodulin stimulates the phosphorylation of three peptides having reverse-phase HPLC retention times that are identical to peptides phosphorylated by Ca2+ addition to the crude unfractionated 100,000 g supernatant. Rechromatography of the peaks from each of the in vitro phosphorylations, either in combination with each other or in combination with each of the seven peaks generated from phosphorylation of tyrosine hydroxylase in situ, established that cyclic AMP, Ca2+/phosphatidylserine, and Ca2+/calmodulin all stimulate the phosphorylation of the same reverse-phase HPLC peptide: in situ peptide 6. Ca2+/calmodulin stimulates the phosphorylation of in situ peptides 3 and 5 as well. Thus, tyrosine hydroxylase can be phosphorylated in vitro by protein kinases endogenous to the chromaffin cell. Phosphorylation occurs on a maximum of three of the seven in situ phosphorylated sites, and all three of these sites can be phosphorylated by a Ca2+/calmodulin-dependent protein kinase.     

Protein kinase activity associated with pancreatic zymogen granules.

Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues.     

Adrenocortical cytochrome P-450 responsible for cholesterol side chain cleavage (P-450scc) is phosphorylated by the calcium-activated, phospholipid-sensitive protein kinase (protein kinase C)

Purified bovine adrenocortical cytochrome P-450scc (specific for cholesterol side chain cleavage in the inner mitochondrial membrane) was selectively phosphorylated in vitro by a Ca2+-activated, phospholipid-sensitive protein kinase (protein kinase C) preparation, whereas cyclic AMP dependent and two cyclic nucleotide independent kinases were ineffective. Cytochrome P-450scc incorporated a maximum of 4 mol of phosphate in the presence of protein kinase C within 15 min at 30 degrees C, with apparent Km and Vmax of 0.14 mumol and 0.76 pmol/min, respectively. Serine and threonine were the two target aminoacids phosphorylated in a ratio of about 1:1. In the presence of 1 microM Ca2+, a mixture of phosphatidylserine and diolein (or a potent tumor promoter phorbol ester) was required for optimal cytochrome P-450scc phosphorylation. In addition, purified inner mitochondrial membrane preparations from adrenocortical mitochondria were found to contain protein kinase C activity. These findings, together with the previous demonstration that activators of protein kinase C such as a potent phorbol ester activates steroidogenesis of intact adrenocortical cells, suggest that phosphorylation of P-450scc should be examined for its possible role in the regulation of adrenocortical functions.     

Purification and characterization of protein kinase C from rabbit iris smooth muscle. Myosin light-chain phosphorylation in vitro and in intact muscle.

Protein kinase C of rabbit iris smooth muscle was purified by the sequential use of three chromatographic steps, i.e. anion-exchange (DEAE-cellulose), gel filtration (Sephadex G-150) and substrate affinity (protamine-agarose), and its properties were investigated by using as substrate myosin light-chain protein (MLC) isolated from the same tissue. The enzyme appeared as a single band on SDS/polyacrylamide-gel electrophoresis, with a molecular mass of approx. 80 kDa. Histone H-1 and iris muscle MLC, but not rabbit skeletal-muscle MLC, were effective substrates for the enzyme, with apparent Km values of 3.0 and 16.6 microM respectively. The enzyme, with MLC as substrate, had the following characteristics. (a) Its activity was dependent on Ca2+ and phosphatidylserine (PS). In the presence of Ca2+ and PS, diolein and phorbol dibutyrate (PDBu) increased its activity by 61 and 65% respectively. Half-maximal activation of the enzyme (Ka) occurred at 10 microM free Ca2+, and in the presence of diolein and PDBu the apparent Ka for Ca2+ was decreased to 3 microM and 2 microM respectively. (b) Studies on the relative potency of various cofactors in activating the enzyme revealed that PS, phorbol myristate acetate and 1-stearoyl-2-arachidonylglycerol were the most potent of the phospholipids, phorbol esters and diacylglycerols respectively. (c) H-7, a protein kinase C inhibitor, inhibited MLC phosphorylation in a dose-dependent manner, with 50% inhibition at 10 microM. (d) Addition of carbamoylcholine (for 1 min) or PDBu (for 25 min) to iris sphincter muscle prelabelled with [32P]Pi specifically increased MLC phosphorylation, and only the stimulatory effect of the muscarinic agonist was blocked by atropine. The data provide additional support for a role for protein kinase C in the contractile response of the iris smooth muscle.     

Affinity purification and biochemical characterization of histolysin, the major cysteine proteinase of Entamoeba histolytica.

Insulin receptor was co-purified from human placenta together with insulin-stimulated kinase activity that phosphorylates the insulin receptor on serine residues. By using this 'in vitro' system, the mechanism of activation of the serine kinase by insulin was explored. Peptide 1150, histone, poly(Glu-Tyr), eliminating Mn2+ (Mg2+ only), treatment at 37 degrees C (1 h), N-ethylmaleimide, phosphate, beta-glycerol phosphate and anti-phosphotyrosine antibody all inhibited insulin-receptor tyrosine kinase activity and the ability of insulin to stimulate phosphorylation of the insulin receptor on serine. Additionally, direct stimulation of the receptor tyrosine kinase by vanadate increased serine phosphorylation of the insulin receptor. Insulin-stimulated tyrosine phosphorylation preceded insulin-stimulated serine phosphorylation of the insulin receptor. The activity of the insulin-sensitive receptor serine kinase was not augmented by cyclic AMP, cyclic GMP, Ca2+, Ca2+ + calmodulin, Ca2+ + phosphatidylserine + diolein or spermine, or inhibited appreciably by heparin. Additionally, the serine kinase phosphorylated casein or phosvitin poorly and was active with Mn2+. This indicates that it is distinct from Ca2+, Ca2+/phospholipid, Ca2+/calmodulin, cyclic AMP- and cyclic GMP-dependent protein kinases, casein kinases I and II and insulin-activated ribosomal S6 kinase. Taken together, these data indicate that a novel species of serine kinase catalyses the insulin-dependent phosphorylation of the insulin receptor and that activation of this receptor serine kinase by insulin requires an active insulin-receptor tyrosine kinase.     

Purification and characterization of myosin light-chain kinase from porcine myometrium and its phosphorylation and modulation by cyclic AMP-dependent protein kinase

Myosin light-chain kinase was purified from porcine myometrium to apparent homogeneity at about 262-fold with an Mr of 130 000 as determined by SDS-polyacrylamide gel electrophoresis and a sedimentation coefficient of 4.5 S. The approximate content of the soluble myosin light-chain kinase was estimated to be about 0.85 microM. The purified enzyme exhibited strict substrate specificity only for 20-kDa myosin light chain and Ka values of 0.6 nM and 0.3 microM for calmodulin and Ca2+, respectively. The enzyme was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase, which resulted in a decrease in the affinity for calmodulin of 4-7-fold without effect on the Vmax. The maximal amount of phosphate incorporated into the enzyme was 0.5-0.8 and 1.0-1.4 mol per mol of the enzyme in the presence and absence of Ca2+ and calmodulin, respectively. In the presence of a subsaturating concentration of calmodulin, the enzyme showed a lower sensitivity for Ca2+ by phosphorylation.     

Purification and characterization of a phosvitin kinase from the thyroid gland

1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands. Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 300 mM, phosphorylated only phosvitin and was not retained on phosphocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhibited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent Km of 100 micrograms/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent Km of 25 and 28 microM, respectively. It had an absolute requirement for Mg2+ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecular weight of 35000 suggesting a polymeric structure of the enzyme.     

Regulation of erythrocyte Ca2+ pump activity by protein kinase C

Using either inside-out vesicles (IOV) prepared from human erythrocytes or purified Ca2+-ATPase from the same source, the effects of protein kinase C (Ca2+/phospholipid-dependent enzyme) on Ca2+ transport and Ca2+-ATPase activity were measured. Incubation of IOV with protein kinase C in the presence, but not absence, of either 12-O-tetradecanoylphorbol-13-acetate or diolein led to a Ca2+-dependent stimulation of ATP-dependent calcium uptake. The effect was a 5-7-fold increase of Vmax without a significant change in the apparent Km for Ca2+. By comparison, the effect of calmodulin was a 14-fold stimulation of Vmax and a 4-fold reduction in apparent Km. The effect of protein kinase C and calmodulin on Ca2+ uptake were nearly additive. Stimulation of IOV Ca2+ transport by protein kinase C was entirely reversible by treatment of activated IOV with alkaline phosphatase. Incubation of purified Ca2+-ATPase with protein kinase C in the presence of 12-O-tetradecanoylphorbol-13-acetate or diolein led to a stimulation of Ca2+-dependent ATPase activity. These results indicate that protein kinase C stimulates the activity of the plasma membrane Ca2+ pump by a direct effect on the pump protein.     

Phosphorylation of a pancreatic zymogen granule membrane protein by endogenous calcium/phospholipid-dependent protein kinase

The occurrence of phospholipid-sensitive calcium-dependent protein kinase (referred to as C kinase) and its endogenous substrate proteins was examined in a membrane preparation from rat pancreatic zymogen granules. Using exogenous histone H1 as substrate, C kinase activity was found in the membrane fraction. The kinase was solubilized from membranes using Triton X-100 and partially purified using DEAE-cellulose chromatography. An endogenous membrane protein (Mr approximately equal to 18 000) was found to be specifically phosphorylated in the combined presence of Ca2+ and phosphatidylserine. Added diacylglycerol was effective in stimulating phosphorylation of exogenous histone by the partially purified C kinase, but had no effect upon phosphorylation of the endogenous 18 kDa protein by the membrane-associated C kinase. Phosphorylation of the 18 kDa protein was rapid (detectable within 30 s following exposure to Ca2+ and phosphatidylserine), and highly sensitive to Ca2+ (Ka = 4 microM in the presence of phosphatidylserine). These findings suggest a role for this Ca2+-dependent protein phosphorylation system in the regulation of pancreatic exocrine function.     

Guanosine cyclic monophosphate-dependent protein kinase from foetal calf heart. Purification, general properties and catalytic subunit.

Cyclic GMP-dependent protein kinase was purified from foetal calf hearts, and its general properties and subunit structure were studied. The enzyme was purified over 900-fold from the heart extract by pH 5.3-isoelectric precipitation, DEAE-cellulose chromatography, Sephadex G-200 filtration and hydroxyapatite treatment. The purified myocardial enzyme, free from cyclic AMP-dependent protein kinase contamination, exhibited an absolute requirement of stimulatory modulator (or crude modulator containing the stimulatory modulator component) for its cyclic GMP-stimulated activity. Inhibitory modulator (protein inhibitor) of cyclic AMP-dependent protein kinase could not stimulate nor inhibit the cyclic GMP target enzyme. The enzyme had Ka values of 0.013, 0.033 and 3.0 micronM for 8-bromo cyclic GMP, cyclic GMP and cyclic AMP respectively. The cyclic GMP-dependent enzyme required Mg2+ and Co2+ for its activity, with optimal concentrations of about 30 and 0.5 mM respectively. The pH optimum for the enzyme activity ranged from 6 to 9. Histones were generally effective substrate proteins. The enzyme exhibited a greater affinity for histones than did the cyclic AMP-dependent class of protein kinase. The holoenzyme (apparent mol.wt. 150 000) of the myocardial cyclic GMP-dependent protein kinase was dissociated into a cyclic GMP-independent catalytic subunit (apparent mol.wt. 60 000) by cyclic GMP and histone. The catalytic subunit required the stimulatory modulator for its activity, as in the case of the holoenzyme in the presence of cyclic GMP.     

Monoclonal antibodies against type II rat brain protein kinase C

Monoclonal antibodies (8/1, 10/10, and 25/3) against rat brain type II protein kinase C were used for the immunochemical characterization of this kinase. These antibodies immunoprecipitated the type II protein kinase C in a dose-dependent manner but did neither to the type I nor III isozyme. Immunoblot analysis of the tryptic fragments from protein kinase C revealed that all three antibodies recognized the 27-38-kDa fragments, the phospholipid/phorbol ester-binding domain, but not the 45-48-kDa fragments, the kinase catalytic domain. The immune complexes of the kinase and the antibodies retained 70-80% of the kinase activity which was dependent on Ca2+ and phosphatidylserine and further activated by diacylglycerol or tumor-promoting phorbol ester. With antibody 8/1, the kinetic parameters with respect to Km for ATP and histone and K alpha for phosphatidylserine and phorbol 12,13-dibutyrate were not significantly influenced. However, the antibody causes variable effects on the K alpha for Ca2+ under different assay conditions. When determined in the presence of phosphatidylserine, the K alpha for Ca2+ was reduced by an order of magnitude (37 +/- 8 to 2.0 +/- 1.8 microM); in the presence of phosphatidylserine and phorbol 12,13-dibutyrate, the K alpha for Ca2+ was not significantly altered; and in the presence of phosphatidylserine and dioleoylglycerol, the kinase became an apparently Ca2+-independent enzyme. The effects of antibody 8/1 on the kinetic parameters of the enzyme for phorbol ester binding were different from those for kinase activity. This antibody causes a 20-30% reduction in phorbol ester binding and a 2-fold increase (1.9 +/- 0.2 to 3.9 +/- 0.3 micrograms/ml) in the concentration of phosphatidylserine required for half-maximal binding, but is without significant influence on those parameters for Ca2+ and phorbol 12,13-dibutyrate. The differential effects of antibody 8/1 on kinase activity and phorbol ester binding with respect to the kinetic parameter of phosphatidylserine suggest that the roles of this phospholipid in supporting phorbol ester binding and kinase activation are different. In the presence of the antibody, the autophosphorylations of the phospholipid/phorbol ester-binding domain and the kinase domain were reduced; the reduction was more pronounced for the former than for the latter. These results suggest that the epitope for antibody 8/1 is localized within the phospholipid/phorbol ester-binding domain at the region adjacent to the kinase domain so that the autophosphorylations of both domains are affected.     

Kinetic characterization of the Ca2+-pumping ATPase of cardia sarcolemma in four states of activation

The Ca2+ dependence of the Ca2+-pumping ATPase of bovine cardiac sarcolemma was studied for four states of activation: (a) unactivated, (b) cAMP-dependent protein kinase (cAMP protein kinase C-subunit)-activated, (c) calmodulin (CAM)-activated, and (d) CAM plus cAMP protein kinase C-subunit-activated. Analysis of the Ca2+ dependence of active transport gave the following Vmax (nanomoles Ca2+/(mg x min], Km (nM) for Ca2+, and Hill coefficient values for the four states at pH 7.4, 37 degrees C: (a) 1.7 +/- 0.3, 1800 +/- 100, 1.6 +/- 0.1; (b) 3.1 +/- 0.5, 1100 +/- 100, 1.7 +/- 0.1; (c) 15.0 +/- 2.5, 64 +/- 1.4, 3.7 +/- 0.2; and (d) 36.0 +/- 6.5, 63 +/- 1.7, 3.7 +/- 0.1. CAM has the most dramatic effect, increasing the apparent Ca2+ affinity by a factor of 28, increasing the Hill coefficient 2.0 units to a value approaching 4 and increasing the Vmax by a factor of 9 or 12. The effective Ca2+ concentration (EC50) for the Ca2+-induced activation of the enzyme in the presence of 5 microM calmodulin is close to the Km for Ca2+ for the CAM-activated state (64 nM). Activation by cAMP protein kinase C-subunit had only minor effects on the Km and Hill coefficient, but increased the Vmax of both the unactivated and the CAM-activated forms of the pump by factor of 1.8 and 2.4, respectively. Analysis suggests that CAM activation is the result of direct binding of Ca2-CAM or high complexes, conferring higher Ca2+ affinity to the enzyme. Analysis suggests that regulatory phosphorylation (cAMP protein kinase C-subunit) increases the rates of processes subsequent to or distinct from Ca2+ binding. The CAM-activated form of the pump was further characterized. Unexpectedly, this form of the enzyme is stimulated a factor of 1.9 by ADP, with half-maximal stimulation between 0.4 and 0.7 mM. Analysis of the progress curves for uptake show that the CAM-activated enzyme is highly resistant to inhibition by transported Ca2+, with an IC50 of 32 mM. The implications of these findings for the pump mechanism and for its role in the regulation of cardiac contractility are discussed.