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The Ca2+-phospholipid-regulated protein kinase has been purified to homogeneity from a 100,000 X g supernatant fluid of rat brain homogenate by a procedure that includes DEAE-cellulose chromatography and successive filtrations on Ultrogel AcA 34 in EGTA and in phosphatidylserine and Ca2+. A more rapid purification consisting of DEAE-cellulose chromatography, Ultrogel AcA 34 gel filtration chromatography, and DEAE-trisacryl chromatography, all in the presence of EGTA, was also developed. Although the enzyme obtained by the latter procedure is not homogeneous, it exhibits properties similar to those of the pure enzyme and is more stable. In addition, the DEAE-trisacryl step permitted resolution of a contaminating Ca2+-inhibitable protein kinase that can interfere with studies of the Ca2+-phospholipid-stimulated enzyme. The homogeneous enzyme, purified about 300-fold, was estimated to have a Mr of 84,000. Its activity was 20- to 30-fold higher in the presence of phospholipid and Ca2+ than in the presence of phospholipid and EGTA, EGTA, or Ca2+ alone. The specific activity of the activated kinase was 852 nmol of P incorporated into histone per min/mg at 20 degrees C. The pure enzyme underwent autophosphorylation in a Ca2+- and phospholipid-dependent manner. This reaction was inhibited in the presence of histones without affecting the kinetic properties of the enzyme. Under optimal assay conditions, the homogeneous enzyme was activated 10-20% by either 10 microM diolein or 100 nM phorbol 12-myristate 13-acetate. Activation of the purified enzyme by diolein or the phorbol ester was far greater (3- to 4-fold) when aggregated instead of freshly sonicated phospholipids were used, suggesting that these compounds affect the interaction of the enzyme with phospholipids and Ca2+. The purified enzyme catalyzed the phosphorylation of the 40S ribosomal subunit protein S6. The Km for S6 was approximately equal to 1 microM and it was estimated that 2 mol of phosphate were incorporated per mol of S6. The observation that protein S6 can be phosphorylated by the purified Ca2+-phospholipid-dependent protein kinase may link recent reports that phorbol ester tumor promoters activate the Ca2+-phospholipid-dependent protein kinase in vitro and stimulate phosphorylation of the ribosomal protein S6 in vivo.

Muscular contraction is triggered by the increase in free calcium concentration and modulated by cyclic nucleotide-dependent phosphorylation. Beside a direct trigger of sarcomeric muscle contraction through binding of troponin C, calcium ions trigger or modulate contractility through calcium-calmodulin-dependent myosin light chain kinases, and increase the rate of relaxation through the calmodulin-dependent phosphorylation of phospholamban, the activator of the cardiac sarcoplasmic reticulum calcium pump. In both cases, a concerted regulation by calcium and cyclic nucleotides is observed. Hyperactivation of the calcium pump is brought about by additional phosphorylation of phospholamban by cAMP-dependent protein kinase. Similarly myofibrillar myosin light chain kinases from smooth and skeletal muscles are substrates of the cAMP-dependent protein kinase. The calmodulin-dependent protein kinases are probably organized into supramolecular regulatory complexes.

We recently reported that phospholamban, the activator of the cardiac sarcoplasmic reticulum calcium pump, is phosphorylated by both cAMP-dependent protein kinase and a membrane-bound, Ca2+/calmodulin-dependent phospholamban kinase. Phospholamban kinase and glycogen phosphorylase b kinase share the same substrate specificity. They differ however in that phospholamban kinase exhibits an absolute requirement for exogenous calmodulin. In line with the latter observation, phospholamban kinase is shown in this report to be inhibited by fluphenazine. Lower concentrations of the drug induced an activation of the kinase, presumably by hydrophobic interaction with either membrane phospholipids or integral proteins. Also, phospholamban kinase was found to be totally insensitive to antibodies elicited against phosphorylase kinase. Since antipsychotic drugs fail to inhibit the delta-subunit-dependent activity of phosphorylase kinase, the above findings confirm that the two kinases are distinct molecular entities. After detergent solubilization of the sarcoplasmic reticulum, the phospholamban-ATPase complex remains a substrate for phospholamban kinase activity, which retains the ability to catalyze the phosphorylation of exogenous phosphorylase b. However, the Ca2+ dependence is entirely lost upon solubilization and no kinase activity is retained on calmodulin-Sepharose in the presence of Ca2+ ions. Phospholamban and phosphorylase kinase activities copurify with the pump-phospholamban complex upon fractionation of the solubilized proteins by density gradient ultracentrifugation, suggesting a tight interaction between the ATPase, its activator, and the phospholamban kinase. A tentative schematic representation of this supramolecular assembly is based upon the results described in this and preceding papers.

Polyphosphoinositides were quantified in metaphase II-arrested eggs of the amphibian Xenopus laevis and 8-10 min later in eggs activated by pricking. The content of phosphatidylinositol 4,5-biphosphate (PIP2) was remarkably high in metaphase II-arrested eggs with respect to that of phosphatidylinositol 4-phosphate (PIP). It was found to drop dramatically at activation. In contrast PIP content did not change significantly.

Protein kinase C was first described some eight years ago. Recent results indicate that this kinase may have a crucial role in signal transduction for substances involved in cellular differentiation and division. Protein kinase C is activated by attachment to plasma membranes, in the presence of calcium and diacylglycerol. The activator is produced in the membrane following the signal-induced breakdown of phosphoinositides. Tumor promoters, such as phorbol ester, can substitute for diacylglycerol. The recent findings that: tyrosine kinases might be involved in the phosphoinositide turnover and, phosphorylation of growth factor receptors by protein kinase C regulates some of their functions, indicate more and more clearly that this kinase is involved in the control of cell growth division and differentiation. Purification procedures, properties and mechanisms of regulation will be summarized and discussed.

Ions appear as very important second messenger since steep ionic gradients are established between subcellular compartments as well as between the outside and the inside of the cell and ions are very diffusible molecules. Ions have been shown to induce many biological events as exemplified by the triggering of skeletal muscle contraction by calcium ions. There is more and more evidence that the same ions might play as tuning systems for biological functions. Therefore their concentration must be precisely controlled. This paper summarizes evidences that phosphorylation of regulatory proteins modulates the cytoplasmic level of some ions.

It has been described that phosphorylation, and dephosphorylation, of specific proteins is associated with key events of the cell cycle and is likely to be due to activation of kinase(s). From our results, the presence of calcium-phospholipid-dependent protein kinase (PKC) was clearly demonstrated in both the cytosolic and particulate fractions of immature Xenopus laevis oocytes and in the cytosolic fraction of mature oocytes. However, it was less active in metaphase II- than in prophase I-arrested oocytes. The enzyme was partially purified by DEAE-cellulose and phenyl-Sepharose chromatography. It was activated in vitro by the tumor-promoting phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA) as already described for PKC from other tissues. On the other hand, a calcium-phospholipid-independent histone kinase activity 4-fold higher in metaphase II- than in prophase I-arrested oocytes was detected. The possible role of PKC and phospholipid-independent histone kinase in the maturation process is discussed.

A two-dimensional radioelectrophoretic method is described, by which parvalbumins from minute biopsy samples (approx. 50 mg) can be detected and quantitated by their 45Ca2+-binding properties. In the first dimension, parvalbumins are purified by sieving through a gradient polyacrylamide gel and collected at the bottom of the electrophoresis tubes. The second dimension is a disc electrophoresis in the presence of 45Ca2+. Parvalbumins can thus be identified and quantitated by three criteria: low molecular weight, acidic character and calcium-binding properties, since they are never exposed to denaturing conditions. Validity of the technique was demonstrated on carp myogen, and on extracts from rabbit psoas and heart muscles. Application of this method to the shrew fast beating myocardium shows that it does contain parvalbumin, in agreement with the proposed role of soluble relaxing factor (Pechère et al. (1977) FEBS Lett. 75, 111--1141. When applied to human muscle biopsies, radioelectrophoresis points to an uneven distribution of parvalbumin among different skeletal muscles. For the human limb muscles tested in this study, the parvalbumin content is similar to that of rabbit psoas muscle.

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a (Ca2+ + Mg2+)-ATPase activity of about 10 nmol (min . mg)-1 and an ATP-dependent Ca2+ uptake of about 10 nmol (min . mg)-1. When incubated in the presence of Mg[gamma-32P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [32P]phosphate-labeled Ca2+ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca2+ or Ca2+ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of 125I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol (Mr = 94 000, 87 000, 60 000 and 43 000). Some minor calmodulin-binding proteins were enriched in the membrane fractions (Mr = 69 000, 57 000, 39 000 and 37 000). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca2+ uptake is essentially unaltered, while the Ca2+ capacity is diminished as a consequence of a doubling in the rate of Ca2+ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca2+ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 microM calmodulin result in increased levels of vesicle phosphorylation.

L2C B lymphocytes have a constant high DNA synthesis due to their continuous proliferative state. The addition of polymyxin B (PmB), a rather selective inhibitor of protein kinase C, stopped (3H)thymidine incorporation with an IC50 of 10 microM when added 18 h before measuring DNA synthesis. Interestingly, PmB inhibition of DNA synthesis was suppressed when 4 nM 12-O-tetradecanoylphorbol-13-acetate was added along with PmB, indicating that PmB may act through inhibition of protein kinase C. In the node and spleen lymphocytes of normal guinea pigs, protein kinase C activity was entirely cytosolic and was eluted at 0.12 M NaCl when adsorbed on DEAE-cellulose. In L2C leukemic lymphocytes, total protein kinase C activity was of the same order of magnitude, but 20% of it was associated with the membrane fraction. The lipid-dependent activity, eluted at 0.12 M NaCl from cytosolic and membrane fractions, was suppressed by staurosporine with an IC50 of 10-40 nM and by polymyxin B with an IC50 of 2-6 microM. Phosphoinositide metabolism was studied in the transformed cells. Incorporation of 32Pi into polyphosphoinositides was considerable, whereas much more time was required for a tiny incorporation of inositol. We detected no release of radioactive inositol triphosphate. Taken together, these results suggest that protein kinase C function is indispensible for triggering L2C leukemic lymphocyte proliferation. The causes of this permanent activation merit further investigation.

The partial purification and characterization of (a) factor(s) from rat brain which inhibit(s) the activity of calcium and phospholipid-dependent protein kinase from the same tissue is described. This factor, present in 100 000 X g rat brain homogenate supernatant, is inactivated upon treatment by trypsin and pepsin and is therefore assumed to be a protein. It was partially purified by ion-exchange chromatography on DEAE-cellulose, ammonium sulfate precipitation and gel filtration. This inhibitor is not stable to heating at 70 degrees C for 10 min, however partial renaturation of the inhibitory activity can be observed after incubation of the denatured inhibitor for 24 h at 4 degrees C. It is precipitable by 10% trichloroacetic acid and by 2 M ammonium sulfate. It exhibits a Stokes radius of 20 A by gel exclusion chromatography, corresponding to a molecular mass of 20 kDa assuming a globular shape. Kinetic analysis of the inhibition of calcium-phospholipid-dependent histone kinase activity indicates that the inhibitor is competitive with respect to the protein substrate. No change was observed in the kinetic values of the kinase for ATP, Ca2+ and phospholipids.