Characterization of rat parotid protein kinase C

1. Protein kinase C (PK-C) from the rat parotid gland has been partially purified and characterized for the first time. During its purification, this enzyme exhibited the same chromatographic behavior as the rat brain enzyme. 2. Affinities for phosphatidylserine (3 micrograms/ml), ATP (8 microM) and calcium (8 microM) were determined kinetically and found to be similar for the enzymes from each tissue. 3. Experiments designed to detect agonist-stimulated translocation of PK-C activity during phosphatidylinositol turnover found no change in levels of soluble PK-C, suggesting that PK-C translocation may not be an obligatory correlate of its activation. The implications of this result are discussed.     

Expression and distribution of protein kinase C isozymes in brain tissue of spontaneous hypertensive rats.

Hypertension activates many endocrine, neuroendocrine and metabolic responses. How hypertension alters these functions remains unknown. Consequently the pathophysiology of hypertension related illnesses are incompletely understood. Protein kinase C (PKC) isoforms play an important role in cellular signal transduction and altered PKC activity has been reported in spontaneous hypertensive rats (SHR). In order to understand the role that PKC plays in hypertension, we hypothesized that PKC activity is significantly expressed in synaptosomal preparations from the brains of SHRs. In addition, the neuroanatomical distribution of this expression was mapped and compared to control animals. The brains were further studied for signs of neuropathology. Total PKC activity was significantly increased in synaptosomal samples isolated from the forebrain, midbrain, and hindbrain of SHR rats. Westem blot analysis identified PKC-alpha, -beta, -gamma, -delta, -epsilon and -zeta in all brain regions. Immunohistochemical analyses indicated that PKC-gamma was localized in cell bodies and processes in many autonomic cardiovascular control regions. These results suggest that PKC may be an important modulator of autonomic blood pressure control.     

Characterization of protein kinase C in rat and human prostates

The properties of protein kinase C (PKC) activity have been studied in cytosolic and membrane fractions from rat and human prostate. Ion exchange chromatography indicated the existence of different PKC isoforms, PKC from rat ventral prostate behaved as a classical Ca²⁺- and phospholipid-dependent enzyme and was activated by 1,2-diacylglycerol as well as by high concentrations of arachidonic acid. PKC activity in the cytosolic fraction was higher and presented different cofactor requirements than that in the membrane fraction. PKC from human benign hyperplastic prostate was also phospholipid dependent, activated by tumor-promotong phorbol esters, and appeared to belong to the group of PKC isozymes which lack Ca²⁺ sensitivity. Human prostatic PKC activity appeared to be of similar nature in both membrane and cytosolic fractions but the specific activity was higher in the particulate preparation which could be related to the stage of endogenous activation of the enzyme. These results extend previous observations in rat ventral prostate and present evidences on the human counterpart. Forthcoming experiments are needed to establish the exact nature of PKC isozymes and their physiological and pathophysiological role in this gland.     

Characterization of protein kinase C in Xenopus oocytes.

Protein kinase C (PKC) was partially purified from Xenopus laevis oocytes by ammonium sulfate fractionation followed by DEAE-cellulose and hydroxyapatite column chromatography. In the latter chromatography, two distinct PKC activities were identified. Both PKC fractions contained an 80 kDa protein which was recognized by three antisera raised against the conserved regions of mammalian PKC. However, specific antisera against alpha, beta I, beta II, and gamma-subspecies of rat PKC did not recognize the protein. Kinetic properties of the Xenopus PKCs were very similar to those of the rat alpha PKC, and only a subtle difference was found in the mode of activation by arachidonic acid. When oocytes were treated with the tumor promoter, phorbol 12-myristate 13-acetate, one of the Xenopus PKCs was found to disappear very rapidly, while the other remained unchanged up to 2 hr.     

Characterization of protein kinase C in early Xenopus embryogenesis

Recently, we presented evidence that protein kinase C (PKC) is involved in mediating the endogenous signals that induced competent Xenopus ectoderm to differentiate to neural tissue. We report here that PKC is already strongly activated in neural-induced ectoderm from midgastrula embryos and that this activation runs parallel with an increase in the level of inositol phosphates. We further identify several proteins that are phosphorylated, both in natural neural-induced ectoderm and in TPA-treated ectoderm, suggesting that they are phosphorylated through the PKC route. We found no major changes in PKC activity among different pregastrula stages, including the unfertilized egg. However, PKC isolated from animal, ectodermal cells is highly sensitive to Ca2+ and can be activated by low concentrations, (6-25 microM) of arachidonic acid, while PKC isolated from vegetal, endodermal cells is more insensitive to Ca2+ and cannot be activated by arachidonic acid. These results suggest that different PKC isozymes are present in animal and vegetal cells.     

Tissue distribution and developmental expression of protein kinase C isozymes

Protein kinase C is a ubiquitous enzyme found in a variety of mammalian tissues and is especially highly enriched in brain and lymphoid organs. Based on biochemical and immunological analyses, we have identified three types of protein kinase C isozyme (designated types I-III) from rat brain. Monospecific antibodies against each of the protein kinase C isozymes were prepared for the determination of tissue distribution, subcellular localization, and developmental changes of these enzymes. The various protein kinase C isozymes were found to be distinctively distributed in different tissues: the type I enzyme in brain; the type II enzyme in brain, pituitary and pineal glands, spleen, thymus, retina, lung, and intestine; and the type III enzyme in brain, pineal gland, retina, and spleen. The rat brain enzymes were differentially distributed in different subcellular fractions. The type I enzyme appeared to be most lipophilic and was recovered mostly in the particulate fractions (80-90%) regardless of the EGTA- or Ca2+-containing buffer used in the homogenization. Significant amounts (30-40%) of the type II and III enzymes were recovered in the cytosolic fraction with EGTA-containing buffer. The expressions of different protein kinase C isozymes appear to be differently controlled during development. In rat brain, both type II and III enzymes were found to increase progressively from 3 days before birth up to 2-3 weeks of age and remained constant thereafter. However, the expression of the type I enzyme displayed a different developmental pattern; it was very low within 1 week, and an abrupt increase was observed between 2 and 3 weeks of age. In thymus, the type II enzyme was found to be maximal shortly after birth; whereas the same kinase in spleen was very low within 2 weeks of age, and a significant increase was observed between 2 and 3 weeks. These results demonstrate that protein kinase C isozymes are distinctively distributed in different tissues and subcellular locales and that their expressions are controlled differently during development.     

Characterization of protein kinase C and its isoforms in human T lymphocytes

Protein kinase C (PKC) regulates numerous T cell functions and is present in abundance in normal human T cells and certain T cell lines. Although crude Triton X-100 soluble material obtained from T cell pellets contains minimal PKC activity, DEAE chromatography revealed that 12 to 37% of cellular PKC was membrane associated, probably due to removal of an inhibitor through column chromatography. As in other tissues, PKC from lymphoid tissue was phospholipid and Ca2+ dependent and diolein reduced the Ca2+ requirements for enzyme activity. Hydroxylapatite chromatography revealed that T cells possess two major peaks of PKC activity. Although, the enzyme in these peaks had similar m.w. and identical iso-electric mobility, the proteins differed with respect to their autophosphorylation sites and immunoreactivity toward an isoform specific antibody. Furthermore, differences in their activities in the presence of phospholipid, diolein, and limiting amounts of Ca2+ imply that these isoforms may be differentially activated. We discuss optimal conditions for activation of PKC and its isoforms for study of T lymphocyte cellular function.     

Expression of protein kinase C isoforms in cancerous breast tissue and adjacent normal breast tissue

The role of protein kinase C (PKC) in the carcinogenesis of human breast tissue has been studied at the molecular level for more than two decades. In this study, we employed Western blotting to determine the presence of PKC isoforms in cancerous and normal breast tissues. The results indicate significant expression of a conventional PKC (PKCα) and two atypical PKCs (PKC ζ and λ/ι) in both breast tumors and adjacent normal breast tissue. For the α,ζ and λ/ι isoforms, the expression of individual isoforms was higher in the breast tumors than in the adjacent normal breast tissue. Although the correlation coefficient was low, significant linear correlation was found among the activities of the isoforms. The data suggest a potential new direction in cancer chemotherapy, namely the blockage of the signal transduction pathway of specific PKC isoforms.     

Identification of type III protein kinase C in bovine aortic tissue

We identified a subtype of protein kinase C in bovine aortic tissue. In Western blots, both the soluble and the particulate fractions from the aorta reacted only with MC-3a. In the case of hydroxylapatite column chromatography, a single activity peak of protein kinase C from the soluble and the particulate fractions was obtained with about 140 mM of potassium phosphate, a finding similar to that with the Type III protein kinase C from rabbit brain. The sandwich-type enzyme immunoassay for protein kinase C, with which the contents of each protein kinase C isozyme can be determined in the crude extracts, revealed that the Type III bovine aortic protein kinase C included 25.9 ng/mg protein. These results strongly suggest that it is the Type III protein kinase C which is mainly expressed in aortic tissue. Kinetic parameters of the Type III protein kinase C of the soluble and the particulate fractions, with respect to the Km for ATP, were 33 and 15 microM and the Km values for myosin light chain from chicken gizzard were 6.3 and 4.6 microM, respectively.     

Activity of novel protein kinase C and distribution of protein kinase C theta in subcellular fractions of normal and Duchenne muscular dystrophic muscle

Phospholipid-dependent, Ca(2+)-independent isoenzymes termed novel protein kinase C or nPKC, include PKC delta, epsilon, eta, theta and mu. Status and role of nPKC and PKC theta in Duchenne muscular dystrophic (DMD) condition is unknown. In the present study, we have shown that most of the nPKC isoforms are translocated to the membrane fraction of DMD tissue specimen. It is well established that translocation plays a key role in signal transduction by individual PKC isoforms. In our experiment, the increased association of nPKC isoform PKC theta to membrane was further confirmed by Western blot. Increased expression of PKC theta mRNA was identified by dot blot analysis. The above results suggest that, the alterations in nPKC location and increased expression of PKC theta observed is a result of modification of PKC-mediated signal transduction and cell function.     

Alteration in protein kinase C activity and subcellular distribution in sickle erythrocytes

In agreement with previous data, membrane protein phosphorylation was found to be altered in intact sickle cells (SS) relative to intact normal erythrocytes (AA). Similar changes were observed in their isolated membranes. The involvement of protein kinase C (PKC) in this process was investigated. The membrane PKC content in SS cells, measured by [3H]phorbol ester binding, was about 6-times higher than in AA cells. In addition, the activity of the enzyme, measured by histone phosphorylation was also found to be increased in SS cell membranes but decreased in their cytosol compared to the activity in AA cell membranes and cytosol. The increase in membrane PKC activity was observed mostly in the light fraction of SS cells, fractionated by density gradient, whereas the decrease in cytosolic activity was only observed in the dense fraction. PKC activity, measured in cells from the blood of reticulocyte-rich patients, exhibited an increase in both membranes and cytosol, thus explaining some of the effects observed in the SS cell light fraction, which is enriched in reticulocytes. The increase in PKC activity in the membranes of SS cells is partly explained by their young age but the loss of PKC activity in their cytosol, particularly in that of the dense fraction, seems to be specific to SS erythrocytes. The relative decrease in membrane PKC activity between the dense and the light fractions of SS cells might be related to oxidative inactivation of the enzyme.     

Characterization and partial purification of rat submandibular gland protein kinase C

Membrane-bound protein kinase C of rat submandibular gland was characterized and the cytosolic kinase C of the tissue was partially purified. The membrane-bound kinase could be activated by Triton X-100 but not EGTA in the presence of both Ca2+ and phosphatidylserine (PS). The Km values for Ca2+ and PS were 150 microM and 5 micrograms, respectively. Addition of 10(-6) M diacylglycerol resulted in an increased affinity of the kinase for Ca2+ (Km = 10 microM). Phorbol 12,13-dibutyrate activated the kinase in the absence of exogenous Ca2+ and PS, suggesting that adequate amounts of each activator are present in the membrane itself. Polymyxin B inhibited the stimulated kinase C activity in a concentration-dependent manner. This inhibition could be overcome by addition of PS. The cytosolic kinase was partially purified 133-fold by chromatography on columns of DEAE-Sephacel and S-300 Sephacryl. The total kinase activity increased with respect to the kinase activity measured in the starting material with column chromatography, suggesting that an inhibitor is present in the cytosolic fraction of the tissue.     

Subcellular distribution of protein kinase C in rat adrenal glomerulosa cells

With the aid of a synthetic nonapeptide which is a selective substrate for protein kinase C the activity of this enzyme was determined in the crude cytosolic and particulate fractions of rat adrenal glomerulosa cells. When the cells were sonicated in the presence of Ca2+ chelators 65 per cent of their total protein kinase C activity was found in the cytosolic extract. The treatment of cells with angiotensin II under conditions where the maximal stimulation of inositol-lipid hydrolysis was observed did not cause a statistically significant change in the apparent subcellular distribution of protein kinase C. However, when the cytosolic extract was prepared in the presence of Ca2+ the protein kinase C activity was recovered nearly exclusively from the particulate fraction.     

Strain-specific postnatal changes in the activity and tissue levels of protein kinase C

The specific activity of protein kinase C from adult mouse lung and spleen was higher than in the corresponding tissues from neonatal mice. BALB/cBy mice had higher lung and spleen protein kinase C activities at both ages than did A/J mice, and the extent of this strain difference increased with age. These activity differences reflected the tissue levels of the 80 kD form of protein kinase C, as determined by quantitative immunoblotting. These genetic and ontogenetic differences provide an interesting model with which to study the regulation of protein kinase C gene expression.     

Targeted deletion of protein kinase C lambda reveals a distribution of functions between the two atypical protein kinase C isoforms

Protein kinase C lambda (PKClambda) is an atypical member of the PKC family of serine/threonine kinases with high similarity to the other atypical family member, PKCzeta. This similarity has made it difficult to determine specific roles for the individual atypical isoforms. Both PKClambda and PKCzeta have been implicated in the signal transduction, initiated by mediators of innate immunity, that culminates in the activation of MAPKs and NF-kappaB. In addition, work from invertebrates shows that atypical PKC molecules play a role in embryo development and cell polarity. To determine the unique functions of PKClambda, mice deficient for PKClambda were generated by gene targeting. The ablation of PKClambda results in abnormalities early in gestation with lethality occurring by embryonic day 9. The role of PKClambda in cytokine-mediated cellular activation was studied by making mouse chimeras from PKClambda-deficient embryonic stem cells and C57BL/6 or Rag2-deficient blastocysts. Cell lines derived from these chimeric animals were then used to dissect the role of PKClambda in cytokine responses. Although the mutant cells exhibited alterations in actin stress fibers and focal adhesions, no other phenotypic differences were noted. Contrary to experiments using dominant interfering forms of PKClambda, mutant cells responded normally to TNF, serum, epidermal growth factor, IL-1, and LPS. In addition, no abnormalities were found in T cell development or T cell activation. These data establish that, in vertebrates, the two disparate functions of atypical PKC molecules have been segregated such that PKCzeta mediates signal transduction of the innate immune system and PKClambda is essential for early embryogenesis.     

Characterization of protein kinase C theta activation loop autophosphorylation and the kinase domain catalytic mechanism

Protein kinase C theta (PKCtheta), a member of the Ca(2+)-independent novel subfamily of PKCs, is required for T-cell receptor (TCR) signaling and IL2 production. PKCtheta-deficient mice have impaired Th2 responses in a murine ova-induced asthma model, while Th1 responses are normal. As an essential component of the TCR signaling complex, PKCtheta is a unique T-cell therapeutic target in the specific treatment of T-cell-mediated diseases. We report here the PKCtheta autophosphorylation characteristics and elucidation of the catalytic mechanism of the PKCtheta kinase domain using steady-state kinetics. Key phosphorylated residues of the active PKCtheta kinase domain expressed in Escherichia coli were characterized, and mutational analysis of the kinase domain was performed to establish the autophosphorylation and kinase activity relationships. Initial velocity, product inhibition, and dead-end inhibition studies provided assignments of the kinetic mechanism of PCKtheta(362)(-)(706) as ordered, wherein ATP binds kinase first and ADP is released last. Effects of solvent viscosity and ATPgammaS on PKCtheta catalysis demonstrated product release is partially rate limiting. Our studies provide important mechanistic insights into kinase activity and phosphorylation-mediated regulation of the novel PKC isoform, PKCtheta. These results should aid the design and discovery of PKCtheta antagonists as therapeutics for modulating T-cell-mediated immune and respiratory diseases.     

Characterization of endogenous substrates for novel-type protein kinase C as well as conventional-type protein kinase C in primary cultured mouse epidermal cells

In primary cultured mouse epidermal cells, phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), induced changes in the phosphorylation levels of 10 proteins, termed KP-1 to 10, in two-dimensional PAGE. Seven of these proteins were phosphorylated and three were dephosphorylated. Similar changes were induced by other PKC activators, but not by inactive phorbol ester. Among these substrate proteins, phosphorylation of three proteins, i.e. KP-1 (pI 4.7/23,000 M_r), KP-2 (pI 4.7/20,700 M_r) and KP-10 (pI 4.7/25,000 M_r was markedly enhanced by PMA and inhibited by a potent PKC inhibitor staurosporine. In vitro phosphorylation studies and phosphoamino acid analysis, using these proteins as substrate and PKC preparations obtained from epidermal cell lysate, revealed that KP-1 and -2 were directly phosphorylated by Ca²⁺-, phospholipid-dependent protein kinase (conventional-type PKC; cPKC), but not by Ca²⁺-independent, phospholipid-dependent protein kinase (novel-type PKC; nPKC). On the other hand, KP-10 was mainly phosphorylated by nPKC in intact epidermal cells. These results indicate that cPKC and nPKC in epidermal cells have different substrate specificity for endogenous proteins and may induce different signal transduction.     

Evidence for protein kinase C regulation of ovarian theca-interstitial cell androgen biosynthesis

The hypothesis that protein kinase C may be an important regulator of ovarian theca-interstitial cell steroidogenesis was tested by using phorbol-12-myristate-13-acetate (PMA) and phorbol-12, 13-dibutyrate (PDB) to directly stimulate protein kinase C activity. Collagenase-dispersed cells (4 x 10(5) viable cells/dish) form ovaries of hypophysectomized immature rats were cultured in serum-free medium in the presence and absence of 0-100 ng/ml of luteinizing hormone (LH), PMA (0-100 nM), and/or PDB (0-100 nM). Treatment with 100 ng/ml LH stimulated androsterone production 100-fold at Day 4 of culture. The presence of 100 nM PMA or PDB had no effect on basal androsterone production; however, treatment with increasing concentrations of PMA or PDB (0-100 nM) caused a dose-related inhibition (maximum 70%) of LH-stimulated androsterone synthesis (ID50 = 1.8 nM and 2.4 nM, respectively). PMA and PDB did not significantly alter DNA, protein, or cell viability, indicating that their inhibitory effects were not due to changes in cell number or viability. Cells treated with LH and 100 nM 4 alpha-phorbol didecanoate (4 alpha-PDD; a phorbol ester that does not activate protein kinase C) failed to show significant decreases in androsterone production. Time-course studies revealed that when PMA treatment was delayed until Day 2 or 4 of culture, dramatic inhibitory effects on LH-stimulated androsterone production were still observed. These results suggest that the biological activity of protein kinase C is retained after the cells have expressed their differentiated state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activity and distribution of protein kinase C in liver during the acute-phase response

Activity and subcellular distribution of protein kinase C were estimated in liver cytosol and membrane fractions of rats carrying a turpentine-induced inflammation. Protein kinase C activity increases significantly 8 h after treatment in the membrane fraction, with concurrent reduction in the cytosol; 10 h after treatment the membrane-associated activity returns to normal, without concomitant recovery of that detected in the cytosol. The specific binding of phorbol dibutyrate to the liver membrane fraction increases but overall the effect is less evident and delayed in time. The changes are associated to alterations in the phosphorylation pattern of some liver proteins. Liver protein kinase C activity and intracellular distribution seem to be affected by a treatment which is known to induce an acute-phase response in the liver cells.