Histones H3 and H4 inhibit protein kinase C specifically

The lysine-rich histone H1 is a preferred substrate for the Ca2+-phospholipid-dependent protein kinase (protein kinase C). Histones H3 and H4 are poor substrates but potent inhibitors of the enzyme. The inhibitory effect of H3 and H4 seems to result mainly from a decreased sensitivity of protein kinase C to stimulation by phosphatidylserine (PS). These observations suggest that site-specific phosphorylation of one histone type can be regulated by other histones.     

Protein kinase C effectors bind to multidrug ABC transporters and inhibit their activity

P-Glycoprotein and homologous multidrug transporters contain a phosphorylatable linker sequence that was proposed to control drug efflux on the basis that it was indeed phosphorylated in vitro and in vivo, and that inhibitors of protein kinase C (PKC) inhibited both P-glycoprotein phosphorylation and activity. However, site-directed mutagenesis of all phosphorylatable residues did not alter the drug resistance. The present work shows that PKC effectors are able to bind directly to multidrug transporters, from either cancer cells (mouse P-glycoprotein), yeast (Saccharomyces cerevisiae Pdr5p), or protozoan parasite (Leishmania tropica ltmdr1), and to inhibit their energy-dependent drug-efflux activity. The binding of staurosporine and derivatives such as CGP 41251 is prevented by preincubation with ATP, suggesting at least partial interaction at the ATP-binding site. In contrast, more hydrophobic compounds such as calphostin C and CGP 42700 bind outside the ATP-binding site and strongly interfere with drug interaction. A direct correlation is obtained between the efficiencies of PKC effectors to inhibit energy-dependent interaction of rhodamine 6G with yeast Pdr5p, to promote intracellular drug accumulation in various multidrug resistant cells, and to chemosensitize growth of resistant cells. The noncompetitive inhibition by PKC effectors of rhodamine 6G interaction with Pdr5p suggests that the binding might interfere with signal transduction between nucleotide hydrolysis and drug interaction. The overall results indicate that the multidrug transporters from different species display common features for interaction with PKC inhibitors. The hydrophobic derivative of staurosporine, CGP 42700, constitutes a potentially powerful modulator of P-glycoprotein-mediated multidrug resistance.     

Monoclonal antibodies specific for type 3 protein kinase C recognize distinct domains of protein kinase C and inhibit in vitro functional activity

Monoclonal antibodies (mAbs) which distinguish Type 3 protein kinase C (PKC) from Types 1 and 2 have been obtained from mice immunized with purified Type 3 PKC from rabbit brain cytosol. Most of these mAbs (seven out of eight) selectively recognize Type 3 versus Types 1 and 2 PKC in both enzyme-linked immunosorbent and immunoblot assays. Trypsin treatment of Type 3 PKC reduced the immunoreactivity with 82-kDa PKC and generated immunoreactive fragments of 45 and 35 kDa. The mAbs can be divided into two classes based on their ability to recognize the 45-kDa catalytic fragment (5/8) or the 35 kDa regulatory domain fragment (3/8). Each of the mAbs inhibits phosphorylation of histone or lipocortin by PKC, although the extent of the inhibition varied. Only those mAbs that recognize the 35-kDa regulatory domain inhibited phorbol ester binding. The inhibition of both kinase and binding activities by this group of mAbs was sensitive to the concentration of phospholipid used in the assay. This functional inhibition suggests that these mAbs may be useful for defining the phospholipid binding domain(s) of Type 3 PKC. The mAbs recognized 82-kDa PKC in a variety of cell types; the presence of smaller molecular weight fragments was not consistently found. Distinct immunofluorescence staining patterns were observed with mAbs directed toward different epitopes, suggesting that there may be heterogeneity in the subcellular localization of PKC. The type specificity of these mAbs will make them valuable tools for studying activation and regulation of Type 3 PKC in cell culture model systems.     

Antagonists of protein kinase C inhibit rat retinal glutamate transport activity in situ

Neuronal and glial high‐affinity transporters regulate extracellular glutamate concentration, thereby terminating synaptic transmission and preventing neuronal excitotoxicity. Glutamate transporter activity has been shown to be modulated by protein kinase C (PKC) in cell culture. This is the first study to demonstrate such modulation in situ, by following the fate of the non‐metabolisable glutamate transporter substrate, d ‐aspartate. In the rat retina, pan‐isoform PKC inhibition with chelerythrine suppressed glutamate uptake by GLAST (glutamate/aspartate transporter), the dominant excitatory amino acid transporter localized to the glial Müller cells. This effect was mimicked by rottlerin but not by Gö6976, suggesting the involvement of the PKCδ isoform, but not PKCα, β or γ. Western blotting and immunohistochemical labeling revealed that the suppression of glutamate transport was not due to a change in transporter expression. Inhibition of PKCδ selectively suppressed GLAST but not neuronal glutamate transporter activity. These data suggest that the targeting of specific glutamate transporters with isoform‐specific modulators of PKC activity may have significant implications for the understanding of neurodegenerative conditions arising from compromised glutamate homeostasis, e.g. glaucoma and amyotrophic lateral sclerosis.     

Alkyl-linked diglycerides inhibit protein kinase C activation by diacylglycerols

Alkylacylglycerols are synthesized when choline-phospholipids are degraded by a phospholipase C. This class of compounds has been shown to have biological activities; however, the mechanism of action is unknown. A series of alkyl-linked diglycerides were synthesized and tested for activity in an in vitro assay for protein kinase C. When protein kinase C activity was stimulated with the synthetic diacylglyceride analog 1-oleoyl-2-acetyl-sn-glycerol, the addition of alkyl glycerides caused a concentration-dependent inhibition of protein kinase C activity. Comparison of the protein kinase C inhibition by this series of 1-O-alkyl-2-acyl analogs revealed that both saturated and unsaturated long-chain groups in position 1 were effective and that dietherglycerols with short-chain moieties in position 2 were also effective. It is concluded from these studies that the biological activity of alkyl-linked glycerides may be expressed through protein kinase C inhibition.     

Detection of protein kinase activity specifically activated at metaphase-anaphase transition

We have previously reported that Ser13 and Ser34 on glial fibrillary acidic protein (GFAP) in the cleavage furrow of glioma cells are phosphorylated during late mitotic phase (Matsuoka, Y., K. Nishizawa, T. Yano, M. Shibata, S. Ando, T. Takahashi, and M. Inagaki. 1992, EMBO (Eur. Mol. Biol. Organ.) J. 11:2895-2902). This observation implies a possibility that there is a protein kinase specifically activated at metaphase-anaphase transition. To further analyze the cell cycle- dependent GFAP phosphorylation, we prepared monoclonal antibodies KT13 and KT34 which recognize the phosphorylation of GFAP at Ser13 and Ser34, respectively. Immunocytochemical studies with KT13 and KT34 revealed that the GFAP phosphorylation in the cleavage furrow during late mitotic phase occurred not only in glioma cells but also in human SW-13 and mouse Ltk- cells in which GFAP was ectopically expressed, thus the phosphorylation can be monitored in a wide range of cell types. Furthermore, we detected kinase activity which phosphorylates GFAP at Ser13 and Ser34 in the lysates of late mitotic cells but not in those of interphase cells or early mitotic cells. These results suggest that there exists a protein kinase which is specifically activated at the transition of metaphase to anaphase not only in GFAP-expressing cells but also in cells without GFAP.     

Polycyclic aromatic hydrocarbon o-quinones inhibit the activity of the catalytic fragment of protein kinase C

Polycyclic aromatic hydrocarbons (PAHs) require metabolic activation to exert their carcinogenic effects. PAH trans-dihydrodiol proximate carcinogens are oxidized by aldo-keto reductases (AKRs) to their corresponding reactive and redox-active o-quinones which may have the properties of initiators and promoters. To determine whether these o-quinones target protein kinase C (PKC), their effects on human recombinant PKCalpha and PKCdelta and the catalytic fragment of rat brain PKC were determined. Naphthalene-1,2-dione (NP-1,2-dione), benzo[a]pyrene-7,8-dione (BP-7,8-dione), and 7,12-dimethylbenz[a]anthracene-3,4-dione (DMBA-3,4-dione) potently inhibited (IC(50) values 3-5 microM) the basal and stimulated activity of the holoenzymes PKCalpha and PKCdelta in a dose-dependent manner. Inhibition of PKC by BP-7,8-dione was observed irrespective of whether PKCalpha activity was stimulated with phorbol 12-myristate 13-acetate (PMA), phosphatidylserine (PS), or Ca(2+) or whether PKCdelta was stimulated with phorbol 12-myristate 13-acetate (PMA) or phosphatidylserine (PS), suggesting that the inhibition was not cofactor-specific. All three quinones inhibited the catalytic fragment of PKC in vitro, yielding identical IC(50) values (3-5 microM), indicating that they interact with the catalytic domain of PKC rather than the cofactor/activator sites. In contrast, no effect on either the holoenzyme or the catalytic fragment was observed with the corresponding PAH trans-dihydrodiols, indicating that inhibition was o-quinone-specific. Irreversible inhibition of the catalytic fragment of PKC was observed since activity could not be restored by dialysis, suggesting that arylation of the fragment had occurred. NP-1,2-dione and BP-7,8-dione also suppressed PKC activity in human breast cancer MCF-7 cell lysates which express PKCalpha, -beta, -delta, -epsilon, -iota, and -lambda isozymes. These data suggest that PAH o-quinones, generated by AKRs, may affect cellular signaling through suppression of the activity of PKC isoforms.     

N-alpha-Tosyl-L-lysine chloromethyl ketone and N-alpha-tosyl-L-phenylalanine chloromethyl ketone inhibit protein kinase C

TLCK (N-alpha-tosyl-L-lysine chloromethyl ketone) inhibits protein kinase C whether or not the enzyme is under the regulation of Ca2+ and phospholipid. TLCK (IC50 = 1 mM) is a much more potent inhibitor of protein kinase C than TPCK (N-alpha-tosyl-L-phenylalanine chloromethyl ketone) (IC50 = 8 mM), suggesting that the lysyl moiety of TLCK may be specifically recognized by the active site of protein kinase C. These results extend the evidence that the active site of protein kinase C recognizes basic amino acids, and suggest that the active sites of protein kinase C and the cAMP-dependent protein kinase, which is also inhibited by TLCK and TPCK, are structurally related.     

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.     

Products of the porcine group C rotavirus NSP3 gene bind specifically to double-stranded RNA and inhibit activation of the interferon-induced protein kinase PKR.

The porcine group C rotavirus (Cowden strain) NSP3 protein (the group C equivalent of the group A gene 7 product, formerly called NS34) shares homology with known double-stranded RNA-binding proteins, such as the interferon-induced, double-stranded RNA-dependent protein kinase PKR. A clone of NSP3, expressed both in vitro and in COS-1 cells, led to the synthesis of minor amounts of a product with an M(r) of 45,000 (the expected full-length M(r) of NSP3) and major amounts of products with M(r)s of 38,000 and 8,000. Restriction enzyme digestion analysis prior to expression in vitro and amino-terminal sequence analysis suggest that the products with M(r)s of 38,000 and 8,000 are cleavage products of the protein with an M(r) of 45,000. The full-length protein and the product with an M(r) of 8,000, both of which contain the motif present in double-stranded RNA-binding proteins, bound specifically to double-stranded RNA. The products with M(r)s of 45,000 and 8,000 were also detected in Cowden strain-infected MA104 cells. NSP3 products expressed in COS-1 cells were capable of inhibiting activation of the double-stranded RNA-dependent protein kinase similar to other double-stranded RNA-binding proteins, and NSP3 products expressed in HeLa cells were capable of rescuing the replication of an interferon-sensitive deletion mutant of vaccinia virus.     

Control of parathyroid hormone-degrading activity in the opossum kidney cell: possible involvement of protein kinase C

To clarify the possible role of protein kinase C in the control of parathyroid hormone (PTH)-degrading activity (PTHDA) in a PTH-responsive opossum kidney (OK) cell line, we investigated the effects of protein kinase C activators, 12-O-tetradecanoyl phorbol 13-acetate (TPA), 1-oleoyl-2-acetyl-glycerol (OAG), and 4 beta-phorbol 12, 13-didecanoate (4 beta-PDD). TPA, OAG, and 4 beta-PDD enhanced PTHDA in a dose-dependent fashion (10-50 ng/ml, 10-100 microgram/ml, and 10-50 nM, respectively), whereas 4 alpha-PDD, a non-activator of protein kinase C, did not affect it. HPLC analysis of TPA-treated samples revealed increase of all immunoreactive PTH fragments produced by OK cells. These findings suggested that activation of protein kinase C in OK cells would augment PTHDA in the cells.     

Vitamin E inhibits protein kinase C activity

Vitamin E (dl-alpha-tocopherol) has been found to inhibit in vitro brain protein kinase c with a half inhibitory concentration of 450 microM. The known plasma concentrations of vitamin E are one order of magnitude lower than the protein kinase c half-inhibitory concentration but it is also known that, at the membrane level where the active protein kinase c is located, the lipophilic vitamin E is more concentrated (Burton, G.W., Joyce, A. and Ingold, K.U. and Locke, S. (1983) Arch. Biochem. Biophys. 221, 281-290). It appears that vitamin E, in addition to its antioxidant function, may play a role in regulating the activity of protein kinase c.     

Ontogeny of pineal protein kinase C activity

The developmental appearance of protein kinase C (PKC) activity in the rat pineal gland was investigated. Enzyme activity could be detected before birth in both cytosol and membrane fractions. A small peak in activity was seen between -2 and 4 days of age, coinciding with a temporary redistribution of activity to the membrane fraction (4% increasing to 17%). After 10 days of age both cytosol and membrane activity increased progressively to reach adult levels by 30 days. Inhibition of daily adrenergic stimulation to the gland by decentralizing or removing the superior cervical ganglia or exposing rats to constant light for 14 days did not reduce PKC activity. These results indicate that PKC activity is located in pinealocytes rather than in the presynaptic adrenergic terminals and that adrenergic stimulation is not necessary to maintain the high level of PKC activity in the pineal.     

Ethanol-induced mitogen activated protein kinase activity mediated through protein kinase C.

The aim of this study was to determine the pathway(s) by which ethanol activates mitogen-activated protein kinase (MAPK) signaling and to determine the role of Ca2+ in the signaling process. MAPK signaling was determined by assessing MAPK activity, measuring phosphorylated extracellular signaling-regulated kinase (pp 44 ERK-1 and pp 42 ERK-2) expression and ERK activity by measuring ERK-2-dependent phosphorylation of a synthetic peptide as a MAPK substrate in rat vascular smooth muscle cells. Ethanol activated extracellular signal-regulated kinase expression (ERK 1 and 2) could be observed when vascular smooth muscle cells (VSMCs) were stimulated for 5 min or less, but was inhibited when cells are treated for 10 min or more with 1-16 mM of ethanol. Maximum ethanol-induced MAPK activity was observed within 5 min with 4 or 8 mM. Ethanol stimulated MAPK activity was blocked by the protein kinase C (PKC) inhibitor (GF109203X) and epidermal growth factor (EGF) receptor antagonist (PD153035) by 41 +/- 24 and 34 +/- 12.3%, respectively. The calcium channel blocker, diltiazem and the chelating agent, BAPTA, reduced the activation of MAPK activity by ethanol, significantly. The data demonstrate that ethanol-stimulated MAPK expression is mediated partially through both the EGF-receptor and PKC intermediates and that activation through the PKC intermediate is calcium-dependent.     

GTP-binding proteins as possible targets for protein kinase C action

The alpha-subunits of two guanine nucleotide binding proteins Gi and transducin, as well as the beta-subunit of transducin, serve as substrates for phosphorylation by the Ca2+- and phospholipid-dependent protein kinase C (PKC). Phosphorylation of the alpha-subunit of transducin is strictly dependent on its conformation and it is only the inactive form that is subjected to phosphorylation by PKC. This review will focus on the proposition that G proteins may serve as cellular targets for modulatory actions of PKC.     

Activation of protein kinase C with cardiolipin-containing liposomes in relation to membrane-protein interaction

Many cytoplasmic proteins, including Ca2+- and phospholipid-dependent protein kinase (protein kinase C) of polymorphonuclear leukocytes (PMNs) associate in Ca2+-dependent manner with phospholipid liposomes containing cardiolipin (CL), as in the case of phosphatidylserine (PS)-containing liposomes. A crude protein kinase C fraction was purified by association of the enzyme with CL-containing liposomes (flotation method). The partially purified protein kinase C from rat brain or guinea pig PMN was activated by the CL-containing liposomes in the presence of dioleoylglycerol (DG) and Ca2+. This activation was analogous to that of PS. The half maximum activity was obtained with 20 microM CL in the presence of 1 microM Ca2+ and 5 microM DG. Many of the cytoplasmic proteins which associate with CL-containing liposomes were preferentially phosphorylated by membrane-associated protein kinase C in the presence of DG and Ca2+. These results suggest that the association of cytoplasmic protein kinase C with the membrane has an important role in regulation of protein kinase C activity in relation to the association of other cytoplasmic proteins to the membrane.     

Regulation of protein kinase C activity by lipids

Protein kinase C is activated by the simultaneous presence of phospholipid, a diglyceride, and Ca2+. Under physiological conditions the activity of the enzyme is regulated by the availability of diglycerides, which are the products of phosphoinositide hydrolysis. The phospholipid-kinase interactions appear not to be of a highly specific nature. Phosphatidylserine (PS) is presumed to be the endogenous lipid that interacts with the kinase, but other acidic lipids can substitute. On the other hand, the kinase-diglyceride interactions are highly specific in nature, as would be expected of a physiological regulator. These interactions are stereo-specific and stoichiometric with respect to diglyceride. The specificity is directed toward the glycerol backbone and hydrophilic oxygen moieties of the diglyceride. The removal of one or more of the oxygen atoms or the addition of a single methyl group to the glycerol backbone virtually abolishes the activity of a putative diglyceride activator. The extreme specificity of the kinase toward the diglycerides, however, must be contrasted with the abilities of structurally diverse tumor promotors and irritants to activate the kinase. Specific small-molecule antagonists of protein kinase C have yet to be developed. The small-molecule antagonists that have been developed so far have been relatively nonspecific cationic lipids that appear to function by interfering with the interaction between the acidic phospholipids and Ca2+.