Ligand-induced conformational changes in cytosolic protein kinase C

The changes in intrinsic spectral properties of protein kinase C were monitored upon association with its divalent cation and lipid activators in a model membrane system. The enzyme demonstrated changes in both its intrinsic fluorescence and far ultraviolet circular dichroism spectra upon association with lipid vesicles in the absence of calcium. The acidic phospholipid, phosphatidylserine, significantly quenched the intrinsic tryptophan fluorescence and was also the most potent lipid support for the phosphorylating activity of the enzyme. The enzyme was fully activated by a number of Ca2(+)-lipid combinations which correlated with maximal fluorescence quenching (40-50%) of available tryptophan residues in hydrophobic domains. The circular dichroism structure of the associated active-protein Ca2(+)-lipid complexes suggested different active enzyme secondary structures. However, the Ca2(+)-dependent changes in fluorescence and circular dichroism spectra were observed only after the enzyme associated with the lipid vesicles. These data suggest that protein kinase C has the properties of a complex multidomain protein and provides an additional perspective into the mechanism of protein kinase C activation.     

Differential sensitivity of protein kinase C isozymes to phospholipid-induced inactivation

Interactions of types I, II, and III protein kinase C (PKC) with phospholipids were investigated by following the changes in protein kinase activity and phorbol ester binding. The acidic phospholipids such as phosphatidylserine (PS), phosphatidic acid, phosphatidyl-glycerol, and cardiolipin, which are activators of PKC in the assay of protein phosphorylation, could differentially inactivate PKC I, II, and III during preincubation in the absence of divalent cation. The phospholipid-induced inactivation of PKC was concentration and time dependent and only affected the kinase activity without influencing phorbol ester binding. PKC I was the most susceptible to the phospholipid-induced inactivation, and PKC III was the least. The IC50 values of PS for PKC I, II, and III were 5, 45, and greater than 120 microM, respectively. Addition of divalent cation such as Ca2+ or Mg2+ suppressed the phospholipid-induced inactivation of PKC. In the absence of divalent cation, PKC I, II, and III all formed complexes with PS vesicles, although to a slightly different degree, as analyzed by molecule sieve chromatography. [3H]Phorbol 12,13-dibutyrate binding for PKC I, II, and III was recovered after chromatography; however, the kinase activities of all these enzymes were greatly reduced. In the presence of Ca2+, all three PKCs formed complexes with PS vesicles, and both the kinase and phorbol ester-binding activities of PKC II and III were recovered following chromatography. Under the same conditions, the phorbol ester-binding activity of PKC I was also recovered, but the kinase activity was not. The phospholipid-induced inactivation of PKC apparently results from a direct interaction of phospholipid with the catalytic domain of PKC; this interaction can be suppressed by divalent cations. In the presence of divalent cations, PS interacted preferentially with the regulatory domain of PKC and resulted in the activation of the kinase.     

Biochemical characterization of rat brain protein kinase C isozymes

Biochemical characteristics of three rat brain protein kinase C isozymes, types I, II, and III, were compared with respect to their protein kinase and phorbol ester-binding activities. All three isozymes appeared to be alike in their phorbol ester-binding activities as evidenced by their similar Kd for phorbol 12,13-dibutyrate and requirements for Ca2+ and phospholipids. However, differences with respect to the effector-mediated stimulation of protein kinase activity were detectable among these isozymes. The type I enzyme could be stimulated by cardiolipin to a greater extent than those of the type II and III enzymes. In the presence of cardiolipin, the concentrations of dioleoylglycerol or phorbol 12,13-dibutyrate required for half-maximal activation (A1/2) of the type I enzyme were nearly an order of magnitude lower than those for the type II and III enzymes. In the presence of phosphatidylserine, differences in the A1/2 of dioleoylglycerol and phorbol 12,13-dibutyrate for the three isozymes of protein kinase C were less significant than those measured in the presence of cardiolipin. Nevertheless, the A1/2 of these two activators for the type I enzyme were lower than those for the type II and III enzymes. At high levels of phosphatidylserine (greater than 15 mol %), binding of phorbol 12,13-dibutyrate to the type I enzyme evoked a corresponding stimulation of the kinase activity, whereas binding of this phorbol ester to the type II and III enzymes produced a lesser degree of kinase stimulation. For all three isozymes, the concentrations of phosphatidylserine required for half-maximum [3H]phorbol 12,13-dibutyrate binding were almost an order of magnitude less than those for kinase stimulation. Consequently, neither isozyme exhibited a significant kinase activity at lower levels of phosphatidylserine (less than 5 mol %) and phorbol 12,13-dibutyrate (50 nM), a condition sufficient to promote near maximal phorbol ester binding. In addition to their different responses to the various activators, the three protein kinase C isozymes also have different Km values for protein substrates. The type I enzyme appeared to have lower Km values for histone IIIS, myelin basic protein, poly(lysine, serine) (3:1) polymer, and protamine than those for the type II and III enzymes. These results documented that the three protein kinase C isozymes were distinguishable in their biochemical properties. In particular, the type I enzyme, which is a brain-specific isozyme, is distinct from the type II and III enzymes, both have a widespread distribution among different tissues.     

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.     

Differential inhibition of protein kinase C subtypes

Catalytic properties of protein kinase C isoforms purified from rat brain and bovine adrenocortical tissues were examined. The results showed that known inhibitors of PKC activity such as gossypol and H-7 were active on all the three isolated enzyme isoforms with similar IC50 values. However, whereas the type III brain isozyme activity was not affected by a preincubation with phosphatidylserine (PS), the same treatment resulted in a virtually complete loss of the type I and II isoform activities within 4 min at 30 degrees. This kinase inactivation caused by PS preincubation was prevented in the presence of ATP-Mg2+ or its competitive inhibitor H-7. These findings indicate that the type III isoform can clearly be distinguished from the other members of the PKC family by this specific property. This approach was used to confirm the characterization of the single form of PKC detected in bovine adrenocortical tissue as a type III isotype. This specific behavior toward phosphatidylserine suggests that the molecular organization of the phospholipid sensitive, regulatory domain of the PKC isoform III with regard to its catalytic site and thus its mechanism of activation may differ from that of other PKC isotypes.     

Identification of two cytosolic diacylglycerol kinase isoforms in rat brain, and in NIH-3T3 and ras-transformed fibroblasts.

Two major species of diacylglycerol kinase (type I and type II) were separated from brain cytosol and from NIH-3T3 or ras-transformed 3T3 cells by heparin-agarose chromatography. Multiple species of diacylglycerol kinase were also detected by non-denaturing isoelectric focusing. The two peaks of activity were of similar size, both co-eluted at approximately 95 kDa from a Superose f.p.l.c. column. Type II enzyme (pI 8.0) was more active when substrate was presented in a deoxycholate/phosphatidylserine undefined environment, as opposed to an octyl glucoside/phosphatidylserine micellar environment. Type II activity was also enhanced by the presence of phosphatidylcholine as cofactor. Type I enzyme (pI 4.0) was more active in the presence of either phosphatidylserine or phosphatidylinositol. Type I and II enzymes had different ATP affinities. Both enzymes showed a preference for diacylglycerol substrates with saturated acyl chains of 10-12 carbon atoms. The cytosolic enzyme activity was able to bind to diacylglycerol-enriched membranes in NIH-3T3 fibroblasts, and this translocation was unaffected in ras-transformed 3T3 cells. These results demonstrate the presence of multiple diacylglycerol kinases in brain cytosol and NIH-3T3 and ras-transformed 3T3 cells. The enzymes differ in cofactor, ATP and substrate requirements. These results can explain some of the contradictions between previous studies of cytosolic diacylglycerol kinase activity, and suggest the presence of a family of such kinases that are differentially regulated by phospholipid cofactors.     

High cooperativity, specificity, and multiplicity in the protein kinase C-lipid interaction

The number of phosphatidylserine molecules involved in activating protein kinase C was determined in a mixed micelle system where one monomer of protein kinase C binds to one detergent:lipid micelle of fixed composition. Unusually high cooperativity, specificity, and multiplicity in the protein kinase C-phospholipid interaction are demonstrated by examining the lipid dependence of enzymatic activity. The rates of autophosphorylation and substrate (histone) phosphorylation are specifically regulated by the phosphatidylserine content of the micelles. Hill coefficients of 8-11 were calculated for phosphatidylserine-dependent stimulation of enzyme activity, with a maximum occurring in micelles containing greater than or equal to 12 phosphatidylserine molecules. The high specificity that exists is illustrated by the fact that phosphatidylethanolamine and phosphatidylglycerol, but not phosphatidylcholine or phosphatidic acid, can replace only some of the phosphatidylserine molecules. We propose that Ca2+ and acidic phospholipids cause the protein to undergo a conformation change revealing multiple phosphatidylserine binding sites and resulting in the highly cooperative and specific interaction of protein kinase C with phosphatidylserine. Consistent with this, the proteolytic sensitivity of protein kinase C increases approximately 10-fold in the presence of phosphatidylserine and Ca2+ compared to Ca2+ alone. The high degree of cooperativity and specificity may provide a sensitive method for the physiological regulation of protein kinase C by phospholipid.     

Phosphorylation of aortic plasma membranes by protein kinase C

A calcium-sensitive, phospholipid-dependent protein kinase (protein kinase C) and its three isozymes were purified from rat heart cytosolic fractions utilizing a rapid purification method. The purified protein kinase C enzyme showed a single polypeptide band of 80 KDa on SDS-polyacrylamide gel electrophoresis, and was totally dependent on the presence of Ca²⁺ and phospholipid for activity. Diacylglycerol was also found to stimulate enzymatic activity. Autophosphorylation of the purified PKC showed an 80 KDa polypeptide. The identity of the purified protein was also verified with monoclonal antibodies specific for PKC. Further fractionation of the purified PKC on a hydroxylapatite column yielded three distinct peaks of enzyme activity, corresponding to type I, II and III based on similar chromatographic behaviour as the rat brain enzyme. All three forms were entirely Ca^2– and phosphatidylserine dependent. Type II was found to be the most abundant. Type I was found to be highly unstable. PKC activity studies demonstrate that types II and III isozymic forms are different with respect to their sensitivity to Ca²⁺.Abbreviations PKC Protein Kinase C - SDS Sodium Dodecyl Sulfate - PAGE Polyacrylamide Gel Electrophoresis - K_m Michaelis constant - NBT Nitro-Blue Tetrazolium - BCIP 5-Bromo-4-Chloro-3-Indolyl Phosphate     

Interaction of protein kinase C isozymes with phosphatidylinositol 4,5-bisphosphate.

Interaction of protein kinase C (PKC) isozymes with phosphatidylinositol 4,5-bisphosphate (PIP2) was investigated by monitoring the changes in the intrinsic fluorescence of the enzyme, the kinase activity, and phorbol ester binding. Incubation of PKC I, II, and III with PIP2 resulted in different rates of quenching of PKC fluorescence and different degrees of inactivation of these enzymes. Other inositol-containing phospholipids such as phosphatidylinositol and phosphatidylinositol 4-phosphate also caused differential rates of quenching of the intrinsic fluorescence of these enzymes. These latter two phospholipids were, however, less potent in the inactivation of PKCs than PIP2. The IC50 of PIP2 were 2, 4, and 11 microM for PKC I, II, and III, respectively. Inactivation of PKCs by PIP2 cannot be reversed by extensive dilution of PIP2 with Nonidet P-40 nor by digestion of PIP2 with phospholipase C. Interaction of PIP2 with the various PKC isozymes was greatly facilitated in the presence of Mg2+ or Ca2+ as evidenced by the accelerated quenching of the PKC fluorescence, however, these divalent metal ions protected PKC from the PIP2-induced inactivation. Binding of PIP2 to PKC in the absence of divalent metal ion also caused a reduction of [3H]phorbol 12,13-dibutyrate binding as a result of reducing the affinity of the enzyme for phorbol ester. Based on gel filtration chromatography, it was estimated that one molecule of PKC interacted with one PIP2 micelle with an aggregation number of 80-90. The PIP2-bound PKC could further interact with phosphatidylserine in the presence of Ca2+ to form a larger complex. Binding of PKC to both PIP2 and phosphatidylserine in the presence of Ca2+ was also evident by changes in the intrinsic fluorescence of PKC. As the interaction of PKC with PIP2, but not with phosphatidylserine, could be enhanced by millimolar concentrations of Mg2+, we propose that PIP2 may be a component of the membrane anchor for PKC under basal physiological conditions when [Ca2+]i is low and Mg2+ is plentiful. Under the in vitro assay conditions, PIP2 could stimulate PKC activity to a level approximately 10-20% of that by diacylglycerol. The stimulatory effect of PIP2 on PKC apparently is not due to binding to the same site recognized by diacylglycerol or phorbol ester, because PIP2 cannot effectively compete with phorbol 12,13-dibutyrate in the binding assay.     

Effect of phospholipid unsaturation on protein kinase C activation.

To examine the hypothesis that physical features of the membrane contribute to protein kinase C activation, phosphatidylcholine/phosphatidylserine/diolein (70:25:5) vesicles of defined acyl chain composition were tested for their ability to activate the enzyme. Maximal activation was found to correlate with the mole percent unsaturation in the system. Unsaturation could be provided by either the phosphatidylserine or the phosphatidylcholine component. Vesicles containing 5 mol% diolein but lacking any unsaturation in the phospholipid did not support activity, indicating that acidic head groups alone are not sufficient for activity. The saturated lipid vesicles could be rendered effective but only at very high (25 mol%) concentrations of diolein. The degree of acyl chain unsaturation and the positioning of the double bond had little effect on the activity, suggesting that the effect of the unsaturation is due to some physical property of the lipid rather than to a specific lipid-protein interaction. Addition of cholesterol to both saturated and unsaturated systems indicated that fluidity, as assessed by fluorescence anisotropy, did not correlate with activity. These results suggest that a physical property of the membrane other than fluidity is important for the activation of protein kinase C. A model for protein kinase C activation involving phase separation and/or head group spacing is discussed.     

Enzyme systems involved in phosphorylation and dephosphorylation of two endogenous phosphoproteins in neuronal membranes.

Adenosine 3′:5′-monophosphate-dependent protein kinase and phosphoprotein phosphatases were solubilized by Triton X-100, from a particulate fraction of bovine cerebral cortex enriched in synaptic membranes, and partially purified. The properties of these partially purified enzymes were studied using two substrates, Protein I and Protein II, prepared from the synaptic membrane fraction, as well as the substrates protamine and histone. The results suggest that the phosphorylation of Protein I and Protein II, as well as protamine and histone, are catalyzed by a single species of cAMP-deperident protein kinase. Thus, a single peak of protein kinase activity was observed, upon DEAE-cellulose hromatography of the Triton X-100 extract of the synaptic membrane preparation, which catalyzed the phosphorylation of all four substrate proteins. Moreover, the activity of this partially purified protein kinase toward the various substrate proteins was altered in a parallel fashion, either when the protein kinase preparation was subjected to heat inactivation or pH inactivation, or when the enzyme was assayed in the presence of various concentrations of cyclic nucleotides or of a protein kinase modulator. The individual protein substrates acted as competitive inhibitors with respect to one another. Upon sucrose density gradient centrifugation, the protein kinase activity toward the various substrates sedimented as a single peak. Finally, the relative specific activities toward the various substrates did not change significantly during a 2000-fold purification of the enzyme. In contrast to these observations with protein kinase, two peaks of protein phosphatase activity, with markedly different specificities toward Protein I and Protein II, were found upon DEAE-cellulose and Bio-Gel P-200 column chromatography of the Triton X-100 extract of the synaptic membrane fractions. One peak catalyzed the dephosphorylation of Phosphoprotein I but not of Phosphoprotein II, whereas the other peak catalyzed the dephosphorylation of Phosphoprotein II but not of Phosphoprotein I. The dephosphorylation of Phosphoprotein I by Phosphoprotein I phosphatase was not affected by adenosine 3':5'-monophosphate, whereas the dephosphorylation of Phosphoprotein II by Phosphoprotein II phosphatase required the presence of this nucleotide. Moreover, the two phosphatases differed from one another with respect to Stokes' radius as well as sedimentation coefficient.     

The protein-tyrosine kinase substrate, calpactin I heavy chain (p36), is part of the primer recognition protein complex that interacts with DNA polymerase alpha

Primer recognition proteins (PRP) stimulate the activity of DNA polymerase alpha on DNA substrates with long single-stranded template containing few primers. Purified PRP from HeLa cells and human placenta are composed of two subunits of 36,000 (PRP 1) and 41,000 (PRP 2) daltons. By amino acid sequence homology, we have identified PRP 2 as the glycolytic enzyme 3-phosphoglycerate kinase. Here we present data that establishes PRP 1 to be the protein-tyrosine kinase substrate, calpactin I heavy chain. Amino acid sequence analysis of six tryptic peptides of PRP 1 followed by homology search in a protein sequence data base revealed 100% identity of all six peptides with the deduced amino acid sequence of human calpactin I heavy chain. The activities of PRP and calpactin I coelute on gel filtration columns, and a high correlation of PRP and calpactin I activities was seen at different stages of purification. A rabbit polyclonal anti-chicken calpactin I antibody was shown to cross-react with PRP 1 polypeptide at various stages of PRP purification, and the homogeneous preparation of PRP exhibits 3-phosphoglycerate kinase (PRP 2) and calpactin I (PRP 1) activities. PRP activity is neutralized by a mouse monoclonal anti-calpactin II antibody although having no effect on the polymerase alpha activity itself. Calpactin II has a 50% amino acid sequence homology with calpactin I. However, PRP 1 is not calpactin II as shown by lack of cross-reaction to a monoclonal anti-calpactin II antibody on Western blots. Calpactin I and 3-phosphoglycerate kinase, purified independently, cannot be efficiently reconstituted into the PRP complex, indicating that their association in the PRP complex involves specific protein-protein interactions that remain to be elucidated. The biochemical and immunological data presented here revealing the identity of PRP 1 as calpactin I provide evidence for one physiological role of calpactin I in the cell.     

Retinoic acid as a modulator of the activity of protein kinase Calpha

All-trans-retinoic acid (atRA) is a derivative of vitamin A and possesses antitumor activity. We demonstrate that atRA is able to modulate the activity of protein kinase C alpha (PKCalpha), which is related to tumor development. In vitro, it was found that atRA activated PKCalpha in the presence of Ca(2+) and in the absence of phosphatidylserine, although such activity is considerably inhibited in mutations affecting residues D246 and D248 and also residue N189, all of which are known to be essential for the interaction with Ca(2+) and phosphatidylserine in the C2 domain. It was concluded that atRA substitutes phosphatidylserine although with lower specific activities. However, atRA had a biphasic effect on PKCalpha activity in the presence of activating phospholipids, such as phosphatidylserine and phosphatidylinositol 4,5-bisphosphate, yielding activation at low concentrations but inactivation at higher ones. This second inhibitory characteristic was not shown with K209 and K211 mutations (residues located in the Lys-rich cluster in the C2 domain) in PKCalpha. This interesting effect revealed the importance of phospholipid binding at this site to ensure maximum activity for the wild-type PKCalpha. The C1 domain was not related with the atRA effect on PKCalpha. It was concluded that whereas atRA may activate PKCalpha through the Ca(2+)-phosphatidylserine-binding site of the C2 domain, it may also inhibit the activity of this enzyme when displacing the phospholipid from the Lys-rich cluster also located in the C2 domain.     

Isolation and characterization of the calcium- and phospholipid-dependent protein kinase (protein kinase C) subtypes from bovine heart

Protein kinase C was isolated from bovine heart by chromatography on DEAE-Sephacel, phenyl-Sepharose, poly(L-lysine) agarose, and hydroxylapatite. Estimates based upon enzyme recovery indicate 10-20 nmol/min of protein kinase C activity per gram of bovine ventricular myocardium. Hydroxylapatite column chromatography resolved the preparation into two peaks of calcium- and phospholipid-dependent protein kinase activity. By Western blot analysis, peaks 1 and 2 contained subtypes II (beta 2) and III (alpha), respectively. No cross-reactivity was observed, indicating that separation was complete. Type III, the major subtype detected, was subsequently purified to apparent homogeneity by chromatography on phosphatidylserine (PS) acrylamide. Type II activity could not be recovered following phosphatidylserine affinity chromatography. Phospho amino acid analysis showed that type III autophosphorylated at serine residues, whereas type II autophosphorylated at both serine and threonine residues. Among the various phospholipids tested for activity, PS was the most effective. Both subtypes were activated by 1-stearoyl-2-arachidonylglycerol (SAG) in the presence of phosphatidylserine and calcium. Activation of both subtypes occurred at calcium concentrations of less than 1 microM. In addition to several similarities, these two subtypes showed differences in activation and kinetic properties: type II was activated by cardiolipin, 1,2-and 1,3-dioleoylglycerol, and both cis- and trans-unsaturated fatty acids. Type III was activated to a lesser degree by cardiolipin and showed no response to 1,3-dioleoylglycerol. Type III was activated to a greater extent by 1,2-diacylglycerols and by cis-unsaturated fatty acids. In the presence of PS and SAG, type II exhibited substantial activity in the presence of 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) without added calcium. Activation of types II and III by unsaturated fatty acids was independent of phospholipid and showed a lower apparent calcium affinity than that observed for activation by phosphatidylserine. These results show that cardiac protein kinase C subtypes II and III were functionally distinguishable and may play unique roles in the regulation of cardiac function.     

Differential activation of protein kinase C isozymes by short chain phosphatidylserines and phosphatidylcholines

To investigate the importance of the physical state of phospholipids for activation of protein kinase C, we have used short chain phospholipids, which, depending on their concentration, can exist as either monomers or micelles. We previously reported that short chain phosphatidylcholines (PC) can activate protein kinase C at concentrations that correlate with the critical micelle concentration of the activating lipid (Walker, J. M., and Sando, J. J. (1988) J. Biol. Chem. 263, 4537-4540). We have now expanded this work to short chain phosphatidylserine (PS) systems in order to examine the role of Ca2(+)-phospholipid interactions in the activation process. Short chain PS were synthesized from corresponding PC and purified by reverse-phase high pressure liquid chromatography. Use of the short chain system has revealed significant differences in the activation of type II and type III protein kinase C isozymes. The type II isozyme required Ca2+ in the presence of long chain PS vesicles; in the presence of the short chain phospholipid micelles (PC or PS), most of the activity was Ca2+ independent. Addition of diacylglycerol caused a small increase in type II activity in all phospholipid systems. In contrast, type III protein kinase C was Ca(+)-dependent in all of the lipid systems. The concentration of Ca2+ required to activate type III protein kinase C was independent of the phospholipid type despite large differences in the ability of these lipids to bind Ca2+. This isozyme required diacylglycerol only in the PC micelle system or with vesicles composed of long chain saturated PS. The presence of short chain PS micelles or long chain PS with unsaturated fatty acyl chains rendered this Ca2(+)-dependent protein kinase C virtually diacylglycerol independent. These results are consistent with a model in which type II protein kinase C requires Ca2+ primarily for membrane association, a requirement which is bypassed with the micelle system, whereas type III protein kinase C has an additional Ca2+ requirement for activity that does not involve Ca2(+)-phospholipid interactions.     

Calphobindins (placental annexins) inhibit protein kinase C.

Calphobindins (CPBs, placental annexins) are intracellular Ca(2+)- and phospholipid-dependent proteins like protein kinase C [EC 2.7.1.37]. We investigated the inhibitory effects of calphobindins on the protein kinase C activity in vitro. CPB I inhibited the protein kinase C activity for both histone phosphorylation and lipocortin phosphorylation, but CPB II and CPB III inhibited only the protein kinase C activity for histone phosphorylation. In the case of histone phosphorylation, all CPBs inhibited the protein kinase C activity in a concentration-dependent manner, and the IC50 (concentration required for 50% inhibition) value of CPB I was 70 nM. The inhibition of protein kinase C by CPB I was Ca(2+)-dependent, and did not disappear upon increasing the concentration of phosphatidyl-serine. Kinetic analysis by double-reciprocal plots indicated that CPB I interacted not only with phosphatidylserine but also with protein kinase C. Although CPB I partially interacts with phospholipid, it is conceivable that the inhibitory action of CPB I on protein kinase C results from direct interaction of CPB I with protein kinase C. Since CPBs are mainly present under the plasma membrane, it is presumed that CPB I is an endogenous inhibitor of protein kinase C, and according to intracellular circumstances, CPB II and CPB III may also be endogenous inhibitors.     

Reversible exposure of the pseudosubstrate domain of protein kinase C by phosphatidylserine and diacylglycerol.

The lipid activators of protein kinase C, phosphatidylserine and diacylglycerol, induce a reversible conformational change that exposes the auto-inhibitory pseudosubstrate domain of the enzyme. The pseudosubstrate domain of beta-II protein kinase C is cleaved after the first residue, arginine 19, by the endoproteinase Arg-C only when the kinase is bound to the activating lipid phosphatidylserine. Exposure of this residue is markedly enhanced by diacylglycerol. In contrast, the pseudosubstrate domain is not cleaved in the absence of lipids, when protein kinase C is bound to non-activating acidic lipids, when the kinase has autophosphorylated on the amino terminus, or after dilution of the activating lipids. This work reveals specificity in the interaction of protein kinase C with phosphatidylserine since only this phospholipid causes the specific conformational change detected in the regulatory domain of the enzyme, and demonstrates that allosteric regulators expose the intramolecular auto-inhibitory domain of a kinase.     

Phospholipid dependence and liposome reconstitution of purified hyaluronan synthase

Previous radiation inactivation and enzyme characterization studies demonstrated that the Streptococcus equisimilis hyaluronan synthase (seHAS) is phospholipid-dependent and that cardiolipin (CL) is the best phospholipid for enzyme activation. Here we investigated the ability of seHAS, purified in the absence of added lipid, to be activated by synthetic phosphatidic acid (PA), phosphatidylserine, or CL lipids containing fatty acyl chains of different length or different numbers of double bonds. The most effective lipid was tetraoleoyl CL (TO-CL), whereas tetramyristoyl CL (TM-CL) was ineffective. None of the phosphatidylserine species tested gave significant activation. PAs containing C10 to C18 saturated acyl chains were not effective activators, and neither were oleoyl lyso PA, dilinoleoyl PA, or PA containing one oleoyl chain and either a palmitoyl or stearoyl chain. In contrast, dioleoyl PA stimulated seHAS approximately 10-fold, to approximately 20% of the activity observed with TO-CL. The tested acidic lipids such as PA and CL activated the enzyme most efficiently if they contained only oleic acid. Mixing experiments showed that the enzyme interacts preferentially with TO-CL in the presence of TM-CL. Similarly, seHAS incorporated into phosphotidylcholine-based liposomes showed increasing activity with increasing TO-CL, but not TM-CL, content. Inactivation of membrane-bound seHAS by solubilization with Nonidet P-40 was prevented by TO-CL, but not TM-CL. The pH dependence of seHAS in the presence of synthetic or naturally occurring CLs showed the same pattern of lipid preference between pH 6 and 10.5. Unexpectedly, HAS showed lipid-independent activity at pH 11.5. The results suggest that Class I HAS enzymes are lipid-dependent and that assembly of active seHAS-lipid complexes has high specificity for the phospholipid head group and the nature of the fatty acyl chains.     

Purification, characterization and substrate specificity of rabbit lung phospholipid transfer proteins.

Three phospholipid transfer proteins, namely proteins I, II and III, were purified from the rabbit lung cytosolic fraction. The molecular masses of phospholipid transfer proteins I, II and III are 32 kilodaltons (kDa), 22 kDa and 32 kDa, respectively; their isoelectric point values are 6.5, 7.0 and 6.8, respectively. Phospholipid transfer proteins I and III transferred phosphatidylcholine (PC) and phosphatidylinositol (PI) from donor unilamellar liposomes to acceptor multilamellar liposomes; protein II transferred PC but not PI. All the three phospholipid transfer proteins transferred phosphatidylethanolamine poorly and showed no tendency to transfer triolein. The transfer of [14C]PC from unilamellar liposomes to multilamellar liposomes facilitated by each protein was affected differently by the presence of acidic phospholipids in the PC unilamellar liposomes. In an equal molar ratio of acidic phospholipid and PC, phosphatidylglycerol (PG) reduced the activities of proteins I and III by 70% (P = 0.0004 and 0.0032, respectively) whereas PI and phosphatidylserine (PS) had an insignificant effect. In contrast, the protein II activity was stimulated 2-3-times more by either PG (P = 0.0024), PI (P = 0.0006) or PS (P = 0.0038). In addition, protein II transferred dioleoylPC (DOPC) about 2-times more effectively than dipalmitoylPC (DPPC) (P = 0.0002), whereas proteins I and III transferred DPPC 20-40% more effectively than DOPC but this was statistically insignificant. The markedly different substrate specificities of the three lung phospholipid transfer proteins suggest that these proteins may play an important role in sorting intracellular membrane phospholipids, possibly including lung surfactant phospholipids.     

Differential regulation of calcium-dependent and calcium-independent cyclic nucleotide phosphodiesterases from heart by palmitoylcarnitine and palmitoyl coenzyme A

Regulation of Ca2+-dependent (peak I) and Ca2+-independent (peak II) phosphodiesterases from the heart by various fatty acyl esters and phospholipids were studied. DL-Palmitoylcarnitine stimulated the basal activity (in the absence of Ca2+) of peak I enzyme, while non-competitively inhibiting peak II enzyme with respect to cyclic AMP. It had no effect on other species of Ca2+-independent phosphodiesterases, including cyclic AMP- and cyclic GMP-specific enzymes from the lung, and cyclic CMP enzyme from the liver Palmitoyl-CoA and phosphatidylserine also stimulated the basal activity of peak I enzyme, but they were without effect on peak II enzyme. In comparison, DL-palmitoylcarnitine inhibited Ca2+-dependent activity of cardiac myosin light chain kinase, whereas phosphatidylserine was without effect. It is conceivable that differential regulation of phosphodiesterases by these lipids could profoundly alter the levels or effects, or both, of cyclic nucleotides and Ca2+ in the myocardium.