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

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

Studies on protein kinase C tightly-bound to rat liver plasma membrane and its protease-activated form

1. Rat liver plasma membrane contained two types of protein kinase C which could be extracted by Ca2(+)-chelator and detergent, respectively. The activities of these two enzymes were nearly equivalent. 2. The detergent-extracted protein kinase C, tightly-bound to membrane, was separated into two subtypes by hydroxyapatite column chromatography. Based on the elution profile and the Ca2+/phospholipid requirement, the major and the minor components were identified as type III and type II protein kinase C, respectively. 3. The detergent-extracted protein kinase C was converted to an active fragment with Mr 45,000 by limited proteolysis with trypsin. Incubation under physiological level of ionic strength increased the stability of this active enzyme and protected it from further inactivation by trypsin. 4. Phosphorylation of H1 histone by the protease-activated kinase was stimulated 1.5-2-fold by phosphatidylserine. However, this enzyme phosphorylated multiple proteins in rat liver subcellular fractions in Ca2(+)- and phospholipid-independent manner. 5. These results suggest that the protein kinase C (mainly type III enzyme) tightly-bound to rat liver plasma membrane may have important role through protein phosphorylation by the native or the protease-activated kinase.     

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

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

Further studies on the ionic strength-dependent proteolytic activation of protein kinase C in rat liver plasma membrane by endogenous trypsin-like protease

cAMP and Ca2(+)-independent histone kinase was generated from rat liver plasma membrane in an ionic strength-dependent manner by the action of an endogenous trypsin-like protease (Hashimoto, E. et al. (1986) FEBS Lett. 200, 63-66). In addition to the effect of ionic strength, this proteolytic activation of protein kinase proceeded faster at alkaline pH. In an attempt to identify the activated kinase as the protease-activated form of protein kinase C (protein kinase M), the active enzyme released from plasma membrane was highly purified and characterized. Various properties including Mg2+ requirement in histone phosphorylation, substrate specificity, effects of protein kinase activators, and inhibitors and comparison of catalytic properties by peptide map analysis were compatible with those of protein kinase M reported earlier. Immunoblot analyses also supported the idea that the protein kinase subjected to proteolytic activation was protein kinase C. The subtype of protein kinase C detected in this study was identified as type III enzyme encoding alpha-type sequence from the elution profile from hydroxyapatite column. These results suggest that type III protein kinase C bound to rat liver plasma membrane has an ability to be activated by endogenous trypsin-like protease dependently on the alteration of ionic strength and pH around the plasma membrane.     

Protease-activated protein kinase in rat liver plasma membrane

Upon limited proteolysis with trypsin, a cAMP and Ca2+-independent protein kinase was produced from rat liver plasma membrane. This enzyme showed a multifunctional capacity and phosphorylated calf thymus histone and rat liver ribosomal proteins. The molecular weight was estimated to be 5.0 X 10(4). When plasma membrane was treated with a buffer containing Triton X-100, a proenzyme with a molecular weight of 8.4 X 10(4) was extracted. By tryptic digestion, the proenzyme was converted to an active protein kinase which was similar to the enzyme obtained by the direct digestion of membrane. However, this proenzyme phosphorylated H1 histone in the presence of Ca2+ and phospholipid without proteolytic digestion. These results indicate the existence of a protease-activated protein kinase in rat liver plasma membrane and the proenzyme seems to be same as protein kinase C.     

Mechanism of proteolytic activation of rat liver protein kinase C generating Ca2(+)-phospholipid-independent form with apparent molecular mass of 80,000

1. New Ca2(+)-phospholipid-independent form of protein kinase C was produced by limited proteolysis with trypsin. 2. The molecular mass of this active enzyme was slightly smaller than that of original protein kinase C. 3. The active enzyme cross-reacted with antibody against the pseudosubstrate region on amino-terminal end of protein kinase C. 4. The active enzyme was inhibited by the peptide inhibitor derived from the pseudosubstrate region. 5. These results suggest that the limited proteolysis at or near the pseudosubstrate region made protein kinase C active without Ca2+ and phospholipid.     

Purification and properties of protein kinase C from rabbit reticulocyte lysates

We have previously reported that addition of Ca2+ and phospholipid (PL) inhibits translation in hemin-containing reticulocyte lysates through activation of a eukaryotic protein synthesis initiation factor (eIF-2) kinase. The possibility that this activation was mediated by a Ca2+-PL-dependent protein kinase (protein kinase C, PKC) appeared unlikely by the observation that it was prevented or reversed by NADPH-generating systems. Nevertheless, reticulocyte lysates contain a potent PKC activity and we deemed it desirable to isolate this enzyme to answer unequivocally the question whether it does or does not activate eIF-2 alpha kinase. We have purified reticulocyte PKC to near homogeneity with Mr 95,500 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme absolutely depended upon both Ca2+ and phosphatidylserine for activity on histone H1 or the beta-subunit of initiation factor eIF-2 and underwent autophosphorylation in a Ca2+- and PL-dependent manner. Mild treatment with trypsin yielded an Mr 82,000 polypeptide that still required Ca2+ and PL for activity. This Mr agrees with that reported for other PKCs, suggesting that these enzymes may undergo limited degradation during isolation. Further proteolytic treatment converted the reticulocyte enzyme into a Ca2+- and PL-dependent form, as is known for PKCs from other sources. The highly purified PKC had no effect on translation in hemin-supplemented reticulocyte lysates.     

Phosphorylation of myosin light chain by a protease-activated kinase from rabbit skeletal muscle

A protease-activated protein kinase that phosphorylates the P light chain of myosin in the absence of Ca2+ and calmodulin has been isolated from rabbit skeletal muscle. The enzyme has properties similar to protease-activated kinase I from rabbit reticulocytes [S. M. Tahara and J. A. Traugh (1981) J. Biol. Chem. 256, 11588-11564], which has been shown to phosphorylate the P light chain of myosin [P. T. Tuazon, J. T. Stull, and J. A. Traugh (1982) Biochem. Biophys. Res. Commun. 108, 910-917]. The protease-activated kinase from skeletal muscle has been partially purified by chromatography on DEAE-cellulose, phosphocellulose and hydroxyapatite. The enzyme phosphorylates histone as well as the P light chain of myosin following activation by proteolysis. Stoichiometric phosphorylation of myosin light chain was observed with the protease-activated kinase and myosin light chain kinase. The sites phosphorylated by the protease-activated kinase and myosin light chain kinase were examined by two-dimensional peptide mapping following chymotryptic digestion. The phosphopeptides observed with the protease-activated kinase were different from those obtained with the Ca2+-dependent myosin light chain kinase, indicating that the two enzymes phosphorylated different sites on the P light chain of skeletal muscle myosin. When actomyosin from skeletal muscle was examined as substrate, the P light chain was phosphorylated following activation of the protease-activated kinase by limited proteolysis.     

Ionic strength-dependent proteolytic activation of protein kinase C by trypsin-like protease

Protein kinase C, reversibly bound to rat liver plasma membrane through Ca2+, was activated by endogenous trypsin-like protease in an ionic strength-dependent manner. In an attempt to understand the reaction mechanism, the EGTA-extracted protein kinase C and the trypsin-like protease (Tanaka, K. et al. (1986) J. Biol. Chem. 261, 2610-2615) were separately purified from plasma membrane. In the reaction system using these purified enzymes, increasing the ionic strength with NaCl (140-210 mM) effectively enhanced the proteolytic activation of the protein kinase C in the presence of Ca2+ and phospholipid. These results suggest that ionic strength is an important factor for the proteolytic activation of membrane-bound rat liver protein kinase C.     

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

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

Modulation of Ca2+-activated, phospholipid-dependent protein kinase in platelets treated with a tumor-promoting phorbol ester

Incubation of human platelets with 12-0-tetradecanoylphorbol-13-acetate (TPA) caused a rapid decrease in soluble Ca2+, phospholipid-dependent protein kinase activity (protein kinase C) and an increase in protein kinase C associated with the particulate fraction. TPA also induced an increased activity of a Ca2+, phospholipid-independent protein kinase activity in both the soluble and the particulate fractions of platelets. This latter kinase eluted from DEAE cellulose columns at a higher salt concentration than protein kinase C, and was shown by Sephadex G-100 chromatography to have a MW of approx. 50,000 compared with an MW of 80,000 for protein kinase C. The data suggest that TPA treatment of platelets causes irreversible activation of protein kinase C by proteolysis of the enzyme to a form active in the absence of Ca2+ and phospholipid.     

Autophosphorylation of rat brain Ca2+-activated and phospholipid-dependent protein kinase

Ca2+-activated and phospholipid-dependent protein kinase (protein kinase C) isolated from rat brain cytosol undergoes autophosphorylation in the presence of Mg2+, ATP, Ca2+, phosphatidylserine, and diolein. Approximately 2-2.5 mol of phosphate were incorporated per mol of the kinase. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, the phosphorylated kinase showed a single protein band of Mr = 82,000 compared to the Mr = 80,000 of the nonphosphorylated enzyme. Analysis of the 32P-labeled tryptic peptides derived from the autophosphorylated kinase by peptide mapping revealed that multiple sites were phosphorylated. Both serine and threonine residues were found to be labeled with 32P. Limited proteolysis of the autophosphorylated kinase with trypsin resulted in the conversion of the kinase into a phospholipid- and Ca2+-independent form. Two major 32P-labeled fragments, Mr = 48,000 and 38,000, were formed as a result of proteolysis, suggesting that the catalytic domain and possibly the Ca2+- and phospholipid-binding region were both phosphorylated. Protein kinase C autophosphorylation has a Km for ATP (1.5 microM) about 10-fold lower than that for phosphorylation of exogenous substrates. The kinetically preferred autophosphorylation appears to be an intramolecular reaction. The autophosphorylated protein kinase C, unlike the protease-degraded enzyme, still depends on Ca2+ and phospholipid for maximal activity. However, the autophosphorylated form of the kinase has a lower Ka for Ca2+ and a higher affinity for the binding of [3H]phorbol-12, 13-dibutyrate. These findings suggest that autophosphorylation of protein kinase C may be important in the regulation of the enzymic activity subsequent to signal transduction.     

Autophosphorylation of the type II calmodulin-dependent protein kinase is essential for formation of a proteolytic fragment with catalytic activity. Implications for long-term synaptic potentiation

Autophosphorylation plays an essential role in proteolytic activation of the type II calmodulin-dependent protein kinase (CaM kinase II). Limited proteolysis of CaM kinase II by trypsin, alpha-chymotrypsin, and Ca2+-stimulated neutral protease (calpain) yielded a catalytically active kinase fragment only when the holoenzyme was autophosphorylated prior to proteolysis. Slightly larger, inactive fragments were obtained from nonphosphorylated CaM kinase II, regardless of whether Ca2+/calmodulin or Mg2+/ATP were present or absent. The active fragment exhibited Ca2+/calmodulin-dependent kinase activity with kinetic parameters identical with those of the activated holoenzyme. The key autophosphorylation site of CaM kinase II was absent from the active fragment which indicates that proteolysis can effectively uncouple the activation state and Ca2+/calmodulin independence of the kinase from the action of phosphoprotein phosphatases. Because autophosphorylation exerts such a tight control over this irreversible process, proteolytic activation of CaM kinase II by intracellular proteases offers an attractive mechanism for prolonging the effects of Ca2+ at the synapse.     

Conversion of protein kinase C from a Ca2+-dependent to an independent form of phorbol ester-binding protein by digestion with trypsin

Tryptic fragments of protein kinase C containing the kinase (45 KDa) and phorbol ester-binding activity (38 KDa) were separated by Mono O column chromatography. The purified phorbol ester-binding fragment exhibits a higher affinity for phosphatidylserine than the native enzyme but comparable Kd for [3H]phorbol 12,13-dibutyrate as the native enzyme. This proteolytic fragment binds phorbol ester equally efficient either in the presence or absence of Ca2+ and the addition of the kinase fragment did not restore the Ca2+-requirement for the binding. These results indicate that protein kinase C is composed of two functionally distinct units which can be expressed independently after limited proteolysis with trypsin.     

Protein kinase C from chicken gizzard: characterization and detection of an inhibitor and endogenous substrates

Protein kinase C was purified from the cytosolic fraction of chicken gizzard by Ca2+ -dependent hydrophobic interaction chromatography, anion-exchange chromatography, and hydrophobic chromatography. The molecular weight was estimated as 61,500 by gel filtration and 80,000 by denaturing gel electrophoresis, indicating that the native enzyme is a monomer. Using the mixed micellar assay, with histone III-S as the substrate, protein kinase C required Ca2+, phospholipid, and diacylglycerol for activity, with half-maximal activation at approximately 5 x 10(-7) M Ca2+ in the presence of L-alpha-phosphatidyl-L-serine and 1,2-diolein. No activation by Ca2+ was observed in the absence of diacylglycerol. Protein kinase C requires free Mg2+, in addition to the MgATP2- substrate, for activity. The Km for ATP was determined to be 20 microM. Activity was sensitive to ionic strength, with half-maximal inhibition at 70 mM NaCl. Using the liposomal assay, phosphorylation of platelet P47 protein and smooth muscle vinculin was more strongly dependent on Ca2+ and lipids than was histone phosphorylation. Partial digestion of protein kinase C with trypsin yielded a constitutively active fragment. A heat-stable inhibitor and three major endogenous protein substrates of protein kinase C were also detected in chicken gizzard smooth muscle.     

Does protein kinase C require Ca2+ for activation?

Ca2+ requirement for protein kinase C activation is a matter of controversy. In this report we have examined Ca2+ dependency of the reaction in different assay systems and shown that the enzyme response to Ca2+, as well as diacylglycerol, depends upon phospholipid species, protein substrate and lipid conformation (micelles or sonicates). These results emphasize that the enzyme characteristics as defined in reconstituted membrane systems may not have a physiological relevance.     

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

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

Differential down-regulation of protein kinase C isozymes

Types I, II, and III protein kinase C have been shown to be products of, respectively, gamma, beta, and alpha genes of this enzyme family (Huang, F. L., Yoshida, Y., Nakabayashi, H., Knopf, J. L., Young, W. S., III, and Huang, K.-P. (1987) Biochem. Biophys. Res. Commun. 149, 946-952). Incubation of the highly purified rat brain protein kinase C isozymes with trypsin (kinase/trypsin (w/w) = 100) under identical conditions results in a preferential degradation of types I and II enzymes, whereas the type III enzyme was relatively resistant to tryptic proteolysis. Degradation of the type III enzyme by trypsin could be facilitated with the addition of Ca2+, phosphatidylserine, and dioleoylglycerol; none of these components alone was effective. Limited proteolysis of the three protein kinase C isozymes generated distinctive fragments for each isozyme, indicating that each isozyme has different trypsin-sensitive sites. Tryptic digestion of the type III protein kinase C was used as a model to determine the effects of various modulators on protein kinase C degradation. While Ca2+ and phosphatidylserine together were sufficient to convert the type III protein kinase C from a trypsin-insensitive to a -sensitive form, addition of dioleoylglycerol greatly reduced the Ca2+ requirement for such a conversion. Among the various phospholipids tested, in the presence of either dioleoylglycerol or phorbol ester, phosphatidylserine, cardiolipin, and phosphatidic acid were the most effective, and phosphatidylcholine and phosphatidylethanolamine were the least effective in supporting the digestion of type III protein kinase. Other acidic phospholipids, such as lysophosphatidylserine and phosphatidylinositol, were also effective in supporting the degradation in the presence of phorbol ester but not in the presence of dioleoylglycerol. The relevance of these proteolytic reactions to physiological responses was assessed with phorbol ester on rat basophilic leukemia RBL-2H3 cells, which contained both types II and III protein kinase C. Immunoblot analysis with the isozyme-specific antibodies revealed that phorbol ester induced a faster degradation of type II than that of type III isozyme in these cells. The results demonstrate that the various protein kinase C isozymes have different susceptibilities to proteolysis in vitro, when tested with trypsin, as well as to endogenous proteases in intact cells.     

Protein kinase C as a possible receptor protein of tumor-promoting phorbol esters

A tumor-promoting phorbol ester, [3H]phorbol-12,13-dibutyrate, may bind to a homogeneous preparation of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) in the simultaneous presence of Ca2+ and phospholipid. This tumor promoter does not bind simply to phospholipid nor to the enzyme per se irrespective of the presence and absence of Ca2+. All four components mentioned above appear to be bound together, and the quaternary complex thus produced is enzymatically fully active for protein phosphorylation. Phosphatidylserine is most effective. Various other phorbol derivatives which are active in tumor promotion compete with [3H]phorbol-12,13-dibutyrate for the binding, and an apparent dissociation binding constant of the tumor promoter is 8 nM. This value is identical with the activation constant for protein kinase C and remarkably similar to the dissociation binding constant that is described for intact cell surface receptors. The binding of the phorbol ester is prevented specifically by the addition of diacylglycerol, which serves as activator of protein kinase C under physiological conditions. Scatchard analysis suggests that one molecule of the tumor promoter may bind to every molecule of protein kinase C in the presence of Ca2+ and excess phospholipid. It is suggestive that protein kinase C is a phorbol ester-receptive protein, and the results presented seem to provide clues for clarifying the mechanism of tumor promotion.     

Proteolytic cleavage and nuclear translocation of fibrocystin is regulated by intracellular Ca2+ and activation of protein kinase C

Fibrocystin, a type I membrane protein of unknown function, is the protein affected in the autosomal recessive form of polycystic kidney disease. Here we show that fibrocystin undergoes regulated proteolysis. Several proteolytic cleavages occur within the predicted ectodomain, whereas at least one cleavage occurs within the cytoplasmic portion. The latter generates a C-terminal intracellular fragment that harbors the nuclear localization signal KRKVSRLAVTGERTATPAPKIPRIT and translocates to the nucleus. Proteolytic cleavage of fibrocystin occurs constitutively in long term cultures of polarized inner medullary collecting duct cells (mIMCD-3). Activation of protein kinase C and release of intracellular Ca2+ are required for proteolysis under these conditions. In short term cultures of human embryonic kidney 293 cells (HEK-293), proteolytic cleavage of fibrocystin can be elicited by stimulation of intracellular Ca2+ release or activation of protein kinase C. These results identify a novel Ca2+-dependent pathway that signals from fibrocystin located in the cell membrane to the nucleus.