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.     

Requirement of protein association with membranes for phosphorylation by protein kinase C.

To clarify the requirement of the association of substrate proteins with phospholipid membranes for phosphorylation by protein kinase C (PKC), we studied the relationship between membrane association of PKC-substrate proteins and their phosphorylation by PKC. In the presence of phosphatidylserine, 12-O-tetradecanoylphorbol-13-acetate induced PKC autophosphorylation in either the presence or the absence of Ca2+, and this phosphorylation was not inhibited by increasing salt concentration (up to 200 mM NaCl). Thus, Ca2+ and ionic strength did not markedly affect the enzymatic activity of PKC. Annexin I required Ca2+ for both its association with phospholipid membranes and phosphorylation by PKC, whereas histone and monomyristilated lysozyme (C14:0-lysozyme) did not. This result indicates that the membrane association of substrates closely correlates with their phosphorylation by PKC. Similar correlation was also observed in the effects of ionic strength on the membrane association of the substrates and their phosphorylation by PKC; increased ionic strength (200 mM NaCl) remarkably inhibited both the membrane association and the phosphorylation of histone and annexin I by PKC but C14:0-lysozyme was not markedly affected. These results suggest that the membrane association of PKC-substrate proteins is a prerequisite for their phosphorylation by PKC. This concept further conforms to the mechanisms of PKC inhibitors; some types of PKC inhibitors are mediated all or in part through inhibition of the substrate-membrane interaction.     

Misregulation of membrane trafficking processes in human nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) remodels the expression and function of genes and proteins that are critical for drug disposition. This study sought to determine whether disruption of membrane protein trafficking pathways in human NASH contributes to altered localization of multidrug resistance‐associated protein 2 (MRP2). A comprehensive immunoblot analysis assessed the phosphorylation, membrane translocation, and expression of transporter membrane insertion regulators, including several protein kinases (PK), radixin, MARCKS, and Rab11. Radixin exhibited a decreased phosphorylation and total expression, whereas Rab11 had an increased membrane localization. PKCδ, PKCα, and PKA had increased membrane activation, whereas PKCε had a decreased phosphorylation and membrane expression. Radixin dephosphorylation may activate MRP2 membrane retrieval in NASH; however, the activation of Rab11/PKCδ and PKA/PKCα suggest an activation of membrane insertion pathways as well. Overall these data suggest an altered regulation of protein trafficking in human NASH, although other processes may be involved in the regulation of MRP2 localization.     

Phorbol ester-induced actin assembly in neutrophils: role of protein kinase C

The shape changes and membrane ruffling that accompany neutrophil activation are dependent on the assembly and reorganization of the actin cytoskeleton, the molecular basis of which remains to be clarified. A role of protein kinase C (PKC) has been postulated because neutrophil activation, with the attendant shape and membrane ruffling changes, can be initiated by phorbol esters, known activators of PKC. It has become apparent, however, that multiple isoforms of PKC with differing substrate specificities exist. To reassess the role of PKC in cytoskeletal reorganization, we compared the effects of diacylglycerol analogs and of PKC antagonists on kinase activity and on actin assembly in human neutrophils. Ruffling of the plasma membrane was assessed by scanning EM, and spatial redistribution of filamentous (F)-actin was assessed by scanning confocal microscopy. Staining with NBD-phallacidin and incorporation of actin into the Triton X-100-insoluble ("cytoskeletal") fraction were used to quantify the formation of (F)-actin. [32P]ATP was used to detect protein phosphorylation in electroporated cells. Exposure of neutrophils to 4 beta-PMA (an activator of PKC) induced protein phosphorylation, membrane ruffling, and assembly and reorganization of the actin cytoskeleton, whereas the 4a-isomer, which is inactive towards PKC, failed to produce any of these changes. Moreover, 1,2-dioctanoylglycerol, mezerein, and 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol, which are nonphorbol activators of PKC, also promoted actin assembly. Although these effects were consistent with a role of PKC, the following observations suggested that stimulation of conventional isoforms of the kinase were not directly responsible for actin assembly: (a) Okadaic acid, an inhibitor of phosphatases 1 and 2A, potentiated PMA-induced protein phosphorylation, but not actin assembly; and (b) PMA-induced actin assembly and membrane ruffling were not prevented by the conventional PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, staurosporine, calphostin C, or sphingosine at concentrations that precluded PMA-induced protein phosphorylation and superoxide production. On the other hand, PMA-induced actin assembly was inhibited by long-chain fatty acid coenzyme A esters, known inhibitors of nuclear PKC (nPKC). We conclude that PMA-induced actin assembly is unlikely to be mediated by the conventional isoforms of PKC, but may be mediated by novel isoforms of the kinase such as nPKC.     

Deficient protein kinase C activity in turnip, a Drosophila learning mutant

The Drosophila mutant turnip was initially isolated based on poor learning performance (Quinn, W.G., Sziber, P.P., and Booker, R. (1979) Nature 277, 212-214). Here we show that turnip is dramatically reduced in protein kinase C (PKC) activity. In addition, turnip flies are deficient in phosphorylation of a 76-kDa head membrane protein (hereafter pp76) which is a major substrate for protein kinase C in homogenates of wild-type flies. Reduced PKC activity, defective pp76 phosphorylation, and most of turnip's learning deficiency co-map genetically to a region on the X-chromosome, 18A5-18D1-2, spanned by the deletion Df(1)JA27. Apparently turnip+ is not a structural gene for PKC because Drosophila PKC genes map elsewhere in the genome. Our results suggest that turnip gene product is required for activation of PKC and that PKC plays a role in associative learning in Drosophila.     

Suppression of protein kinase Cepsilon mediates 17beta-estradiol-induced neuroprotection in an immortalized hippocampal cell line

Although estrogens are neuroprotective in a variety of neuroprotection models, the precise underlying mechanisms are currently not well understood. Here, we examined the role of protein kinase C (PKC) in mediating estrogen-induced neuroprotection in the HT-22 immortalized hippocampal cell line. The neuroprotection model utilized calcein fluorescence to quantitate cell viability following glutamate insults. 17beta-Estradiol (betaE2) protected HT-22 cells when treatment was initiated before or after the glutamate insult. The inhibition of PKC by bis-indolylmaleimide mimicked and enhanced betaE2-induced neuroprotection. In contrast, the inhibition of specific PKC isozymes (alpha and beta) by Go6976, inhibition of 1-phosphatidylinositol 3 kinase by wortmannin, or inhibition of protein kinase A by H-89, did not alter cell viability, suggesting a specific involvement of PKC in an isozyme-dependent manner. We further examined whether estrogen interacts with PKC in a PKC isozyme-specific manner. Protein levels and activity of PKC isozymes (alpha, delta, epsilon, and zeta) were assessed by western blot analysis and radiolabeled phosphorylation assays respectively. Among the isozymes tested, betaE2 altered only PKCepsilon; it reduced the activity and membrane translocation of PKCepsilon in a manner that correlated with its protection against glutamate toxicity. Furthermore, betaE2 reversed the increased activity of membrane PKCepsilon induced by glutamate. These data suggest that the neuroprotective effects of estrogens are mediated in part by inhibition of PKCepsilon activity and membrane translocation.     

Kinetics of phosphorylation of Na+/K(+)-ATPase by protein kinase C

The kinetics of phosphorylation of an integral membrane enzyme, Na+/K(+)-ATPase, by calcium- and phospholipid-dependent protein kinase C (PKC) were characterized in vitro. The phosphorylation by PKC occurred on the catalytic alpha-subunit of Na+/K(+)-ATPase in preparations of purified enzyme from dog kidney and duck salt-gland and in preparations of duck salt-gland microsomes. The phosphorylation required calcium (Ka approximately 1.0 microM) and was stimulated by tumor-promoting phorbol ester (12-O-tetradecanoylphorbol 13-acetate) in the presence of a low concentration of calcium (0.1 microM). PKC phosphorylation of Na+/K(+)-ATPase was rapid and plateaued within 30 min. The apparent Km of PKC for Na+/K(+)-ATPase as a substrate was 0.5 microM for dog kidney enzyme and 0.3 microM for duck salt-gland enzyme. Apparent substrate inhibition of PKC activity was observed at concentrations of purified salt-gland Na+/K(+)-ATPase greater than 1.0 microM. Phosphorylation of purified kidney and salt-gland Na+/K+ ATPases occurred at both serine and threonine residues. The 32P-phosphopeptide pattern on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis after hydroxylamine cleavage of pure 32P-phosphorylated alpha subunit was the same for the two sources of enzyme, which suggests that the phosphorylation sites are similar. The results indicate that Na+/K(+)-ATPase may serve as a substrate for PKC phosphorylation in intact cells and that the Na+/K(+)-ATPase could be a useful in vitro model substrate for PKC interaction with integral membrane proteins.     

Selectivity of protein kinase inhibitors in human intact platelets

The specificity of commonly used protein kinase inhibitors has been evaluated in the intact human platelet. Protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA) were activated selectively by treating platelets with phorbol dibutyrate (PDBu) or prostacyclin (PGl2). PKC activity was quantitated by measuring PDBu-specific phosphorylation of a 47,000 molecular weight protein, and PKA activity monitored by measuring prostacyclin-dependent phosphorylation of a 22,000 molecular weight protein. Staurosporine and 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7) were found to be non-specific inhibitors in the intact platelet, consistent with their effects on the isolated enzymes. Tamoxifen inhibited PKC activity (IC50 = 80 microM) but increased PKA-dependent protein phosphorylation. These results support the use of human platelets for measuring the specificity of protein kinase inhibitors and indicate that tamoxifen might have value for experimental purposes as a relatively selective PKC inhibitor.     

AKAP79 selectively enhances protein kinase C regulation of GluR1 at a Ca2+-calmodulin-dependent protein kinase II/protein kinase C site

Enhancement of AMPA receptor activity in response to synaptic plasticity inducing stimuli may arise, in part, through phosphorylation of the GluR1 AMPA receptor subunit at Ser-831. This site is a substrate for both Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC). However, neuronal protein levels of CaMKII may exceed those of PKC by an order of magnitude. Thus, it is unclear how PKC could effectively regulate this common target site. The multivalent neuronal scaffold A-kinase-anchoring protein 79 (AKAP79) is known to bind PKC and is linked to GluR1 by synapse-associated protein 97 (SAP97). Here, biochemical studies demonstrate that AKAP79 localizes PKC activity near the receptor, thus accelerating Ser-831 phosphorylation. Complementary electrophysiological studies indicate that AKAP79 selectively shifts the dose-dependence for PKC modulation of GluR1 receptor currents approximately 20-fold, such that low concentrations of PKC are as effective as much higher CaMKII concentrations. By boosting PKC activity near a target substrate, AKAP79 provides a mechanism to overcome limitations in kinase abundance thereby ensuring faithful signal propagation and efficient modification of AMPA receptor-mediated responses.     

Modulation of erythrocyte membrane mechanical function by protein 4.1 phosphorylation

Erythrocyte membrane mechanical function is regulated by the spectrin-based membrane skeleton composed of alpha- and beta-spectrin, actin, protein 4.1R (4.1R), and adducin. Post-translational modifications of these proteins have been suggested to modulate membrane mechanical function. Indeed, beta-spectrin phosphorylation by casein kinase I has been shown to decrease membrane mechanical stability. However, the effects of the phosphorylation of skeletal proteins by protein kinase C (PKC), a serine/threonine kinase, have not been elucidated. In the present study, we explored the functional consequences of the phosphorylation of 4.1R and adducin by PKC. We identified Ser-312 in 4.1R as the PKC phosphorylation site. Using antibodies raised against phosphopeptides of 4.1R and adducin, we documented significant differences in the time course of phosphorylation of adducin and 4.1R by PKC. Although adducin was phosphorylated rapidly by the activation of membrane-bound atypical PKC by phorbol 12-myristate 13-acetate stimulation, there was a significant delay in the phosphorylation of 4.1R because of delayed recruitment of conventional PKC from cytosol to the membrane. This differential time course in the phosphorylation of 4.1R and adducin in conjunction with membrane mechanical stability measurements enabled us to document that, although phosphorylation of adducin by PKC has little effect on membrane mechanical stability, additional phosphorylation of 4.1R results in a marked decrease in membrane mechanical stability. We further showed that the phosphorylation of 4.1R by PKC results in its decreased ability to form a ternary complex with spectrin and actin as well as dissociation of glycophorin C from the membrane skeleton. These findings have enabled us to define a regulatory role for 4.1R phosphorylation in dynamic regulation of red cell membrane properties.     

Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous protein kinase C

Single IK(Ca) channels of human erythrocytes were studied with the patch-clamp technique to define their modulation by endogenous protein kinase C (PKC). The perfusion of the cytoplasmic side of freshly excised patches with the PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited channel activity. This effect was blocked by PKC(19-31), a peptide inhibitor specific for PKC. Similar results were obtained by perfusing the membrane patches with the structurally unrelated PKC activator 1-oleoyl-2-acetylglycerol (OAG). Blocking of this effect was induced by perfusion with PKC(19-31) or chelerythrine. Channel activity was not inhibited by the PMA analog 4alpha-phorbol 12,13-didecanoate (4alphaPDD), which has no effect on PKC. Activation of endogenous cAMP-dependent protein kinase (PKA), which is known to up-modulate IK(Ca) channels, restored channel activity previously inhibited by OAG. The application of OAG induced a reversible reduction of channel activity previously up-modulated by the activation of PKA, indicating that the effects of the two kinases are commutative, and antagonistic. Kinetic analysis showed that down-regulation by PKC mainly changes the opening frequency without significantly affecting mean channel open time and conductance. These results provide evidence that an endogenous PKC down-modulates the activity of native IK(Ca) channels of human erythrocytes. Our results show that PKA and PKC signal transduction pathways integrate their effects, determining the open probability of the IK(Ca) channels.     

Growth-dependent subcellular redistribution of protein kinase C in cultured porcine aortic endothelial cells

We have previously observed major differences in the phosphorylation of membrane proteins in sparse, proliferating versus confluent, quiescent pig aortic endothelial cells (EC) (Kazlauskas and DiCorleto, 1987). In the present study we examined whether EC growth state can influence the activity of a specific phosphorylating enzyme, protein kinase C (PKC) in cytosolic and membrane fractions of pig aortic EC. Levels of PKC were measured using two methods: 1) Ca2+ and phospholipid-dependent phosphorylation of exogenous histones using gamma-labeled [32P]ATP, and 2) [3H]phorbol-12,13-dibutyrate (PDBu) binding activity. The total amount of PKC activity in the quiescent versus proliferating cells was similar but the percentage of PKC activity in the membrane fraction correlated with the proliferative index of the cells: confluent, quiescent cultures exhibited a majority of PKC activity in the cytosolic fraction (67%), whereas sparse, proliferating cultures contained principally membrane-bound PKC (70%). We also examined the role of PKC in the mitogenic response of pig aortic EC to fetal calf serum. Following serum stimulation of sparse, serum-deprived pig aortic EC, PKC activity redistributed from the cytosolic to the membrane fraction in a rapid process that correlated with subsequent DNA synthesis. A potent activator of PKC, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced a minimal mitogenic response in pig aortic EC when added alone but acted synergistically with low concentrations of fetal calf serum to greatly stimulate DNA synthesis. Furthermore, pig aortic EC treated with TPA for 24 h to down-regulate PKC exhibited only 25% of the serum-stimulated mitogenic activity of control cultures. These results suggest a role for PKC activation and translocation in the proliferation of pig aortic EC.     

Protein kinase C and calmodulin effects on the plasma membrane Ca2+-ATPase from excitable and nonexcitable cells

We have purified Ca2+-ATPase from synaptosomal membranes (SM)1 from ratcerebellum by calmodulin affinity chromatography. The enzyme was identifiedas plasma membrane Ca2+-ATPase by its interaction with calmodulin andmonoclonal antibodies produced against red blood cell (RBC) Ca2+-ATPase, andby thapsigargin insensitivity. The purpose of the study was to establishwhether two regulators of the RBC Ca2+-ATPase, calmodulin and protein kinaseC (PKC), affect the Ca2+-ATPase isolated from excitable cells and whethertheir effects are comparable to those on the RBC Ca2+-ATPase. We found thatcalmodulin and PKC activated both enzymes. There were significantquantitative differences in the phosphorylation and activation of the SMversus RBC Ca2+-ATPase. The steady-state Ca2+-ATPase activity of SMCa2+-ATPase was approximately 3 fold lower and significantly less stimulatedby calmodulin. The initial rate of PKC catalyzed phosphorylation (in thepresence of 12-myristate 13-acetate phorbol) was approximately two timesslower for SM enzyme. While phosphorylation of RBC Ca2+-ATPase approachedmaximum level at around 5 min, comparable level of phosphorylation of SMCa2+-ATPase was observed only after 30 min. The PKC-catalyzedphosphorylation resulted in a statistically significant increase inCa2+-ATPase activity of up to 20-40%, higher in the SM Ca2+-ATPase.The differences may be associated with diversities in Ca2+-ATPase functionin erythrocytes and neuronal cells and different isoforms composition.     

Platelet-activating factor-stimulated protein tyrosine phosphorylation and eicosanoid synthesis in rat Kupffer cells. Evidence for calcium-dependent and protein kinase C-dependent and -independent pathways.

The lipid mediator platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, AGEPC) has been shown to elicit several important biochemical signaling responses in mammalian cells, including polyphosphoinositide hydrolysis, arachidonic acid release/eicosanoid production, and protein tyrosine phosphorylation. In the present study, the roles of Ca2+ and protein kinase C (PKC), two signaling components of the phospholipase C pathway, in AGEPC-stimulated eicosanoid production and protein tyrosine phosphorylation, were investigated in cultured rat Kupffer cells. AGEPC at nanomolar concentrations induced an increase in intracellular calcium concentration ([Ca2+]i), stimulated membrane PKC activity, and resulted in protein tyrosine phosphorylation. The maximal increase in [Ca2+]i and membrane PKC activity in response to AGEPC were observed within 30-50 s, whereas the AGEPC-induced protein tyrosine phosphorylation reached maximal levels within 2-5 min. [Ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) but not 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an inhibitor of calcium release from intracellular compartments, nearly abolished the AGEPC-induced increase in [Ca2+]i suggesting involvement of extracellular calcium influx in this event. Both EGTA and TMB-8 abolished or inhibited AGEPC-stimulated protein tyrosine phosphorylation and eicosanoid formation, respectively. The calcium ionophore A23187 alone stimulated eicosanoid production and protein tyrosine phosphorylation with an identical pattern to that of AGEPC. Phorbol myristate acetate (PMA), an activator of PKC, which did not affect [Ca2+]i, mimicked the actions of AGEPC, stimulating eicosanoid production and promoting tyrosine phosphorylation of a set of proteins similar to those phosphorylated following AGEPC stimulation. AGEPC-enhanced tyrosine phosphorylation of some of the protein substrates and eicosanoid production were inhibited in cells "down-regulated" for PKC. Furthermore, both PMA- and AGEPC-stimulated eicosanoid production and protein tyrosine phosphorylation were attenuated or abolished by at least one of the PKC inhibitors, staurosporine, and calphostin C. Taken together, these results are consistent with the conclusions that: (a) AGEPC stimulates the phospholipase-mediated arachidonic acid release/eicosanoid synthesis cascade and protein tyrosine phosphorylation through extracellular Ca(2+)-dependent and PKC-dependent and -independent mechanism(s) and (b) the Ca(2+)-PKC interaction determines the efficacy of the AGEPC-stimulated cellular events.     

Ciprofibrate stimulates protein kinase C-dependent phosphorylation of an 85 kDa protein in rat Fao hepatic derived cells

The effect of ciprofibrate on early events of signal transduction was previously studied in Fao cells. Protein kinase C (PKC) assays performed on permeabilized cells showed a more than two-fold increase in PKC activity in cells treated for 24 h with 500 microM ciprofibrate. To show the subsequent effect of this increase on protein phosphorylation, the in vitro phosphorylation on particulate fractions obtained from Fao cells was studied. Among several modifications, the phosphorylation of protein(s) with an apparent molecular mass of 85 kDa was investigated. This modification appeared in the first 24 h of treatment with 500 microM ciprofibrate. It was shown to occur on Ser/Thr residue(s). It was calcium but not calmodulin-dependent. The phosphorylation level of this/these protein(s) was reduced with kinase inhibitors and especially with 300 nM GF-109203X, a specific inhibitor of PKC. All these results suggest that the phosphorylation of the 85 kDa protein(s) is due to a PKC or to another Ser/Thr kinase activated via a PKC pathway. A possible biochemical candidate for 85 kDa protein seems to be the beta isoform of phosphatidylinositol 3-kinase regulatory subunit.     

Direct activation of gastric H,K-ATPase by N-terminal protein kinase C phosphorylation. Comparison of the acute regulation mechanisms of H,K-ATPase and Na,K-ATPase

In this study we compared the protein kinase dependent regulation of gastric H,K-ATPase and Na,K-ATPase. The protein kinase A/protein kinase C (PKA/PKC) phosphorylation profile of H,K-ATPase was very similar to the one found in the Na,K-ATPase. PKC phosphorylation was taking place in the N-terminal part of the alpha-subunit with a stoichiometry of approximately 0.6 mol Pi/mole alpha-subunit. PKA phosphorylation was in the C-terminal part and required detergent, as is also found for the Na,K-ATPase. The stoichiometry of PKA-induced phosphorylation was approximately 0.7 mol Pi/mole alpha-subunit. Controlled proteolysis of the N-terminus abolished PKC phosphorylation of native H,K-ATPase. However, after detergent treatment additional C-terminal PKC sites became exposed located at the beginning of the M5M6 hairpin and at the cytoplasmic L89 loop close to the inner face of the plasma membrane. N-terminal PKC phosphorylation of native H,K-ATPase alpha-subunit was found to stimulate the maximal enzyme activity by 40-80% at saturating ATP, depending on pH. Thus, a direct modulation of enzyme activity by PKC phosphorylation could be demonstrated that may be additional to the well-known regulation of acid secretion by recruitment of H,K-ATPase to the apical membranes of the parietal cells. Moreover, a distinct difference in the regulation of H,K-ATPase and Na,K-ATPase is the apparent absence of any small regulatory proteins associated with the H,K-ATPase.     

Altered levels and protein kinase C-mediated phosphorylation of substrates in normal and transformed mouse lung epithelial cells.

Protein phosphorylation and protein kinase C (PKC) levels were analyzed in intact cultures of spontaneously transformed, chemically transformed, and untransformed mouse pulmonary epithelial cell lines. It was found that although the transformed cell lines contained about 80% less protein kinase C, measured as total enzyme activity or binding of [3H]phorbol ester, phosphorylation events after phorbol ester treatment could still be easily detected. A commonly described Mr 80-kDa protein kinase C substrate (p80, 80 K, MARKS) was identified using 2D-PAGE, following phosphorylation in intact cells, and found to have reduced availability for phosphorylation in the transformed cell lines C4SE9, C1SA5 and NULB5 in comparison to the untransformed C4E10 and C1C10. Available levels of p80 were further analyzed in heat-denatured extracts from all cell lines using partially purified bovine brain PKC and correlated well with changes seen in intact cells. It was also noted that all transformed cell lines contained large amounts of a family of phosphoproteins of Mr 55-65 kDa, that could not be detected in the untransformed cell lines and whose phosphorylation state was increased by protein kinase C activation. This protein was found to be located in the nucleus. Hence, spontaneously and chemically transformed mouse pulmonary epithelial cells exhibit reduced levels of PKC, along with an altered pattern of PKC-mediated phosphorylation.     

Differential stimulation of protein kinase C activity by phorbol ester or calcium/phosphatidylserine in vitro and in intact synaptosomes.

Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) was compared with calcium/phosphatidylserine (Ca/PS). The substrate specificity of PKC was more limited with PS/PMA. Substrates could be divided into three overlapping groups according to their relative level of phosphorylation: C1, relatively preferred substrates with Ca/PS, included dephosphin, histone, and peptide GS1-10. C2, relatively preferred with PS/PMA, included myelin basic protein and MARCKS. C3, substrates independent of activators. PS/PMA altered the Vmax of PKC for substrate, and decreased the Km for Mg2+. Differential substrate phosphorylation by PS/PMA also occurred for PKC isozymes resolved by hydroxylapatite chromatography and was most dramatic for PKC-alpha, which could no longer phosphorylate histone or GS1-12. Differential activities of PKC were also observed in synaptosol and in intact synaptosomes where PMA stimulated phosphorylation of MARCKS, but not dephosphin. It was further shown that dephosphin was indeed a substrate of PKC in the intact synaptosomes by use of a repolarization-dependent dephosphin phosphorylation assay. The differential PKC activities could also be distinguished by inhibitors. H-7 was equipotent, palmitoylcarnitine did not inhibit in vitro C2 phosphorylation, but inhibited dephosphin in intact synaptosomes, and sphingosine did not inhibit C1 substrates and was without effect on dephosphin in intact synaptosomes. Therefore PS/PMA alters or limits the substrate specificity of PKC, leading to a differential substrate phosphorylation in vitro and in intact synaptosomes and differential inhibitor sensitivity. The pattern of protein phosphorylation observed after PKC activation in intact cells will therefore be dependent upon the activator.     

Effect of feeding protein deficient diet on phospholipid turnover and protein kinase C mediated protein phosphorylation in rat brain

Feeding of protein deficient diet is known to alter the transmembrane signalling in brain of rat by reducing total protein kinase C (PKC) activity. Phospholipid metabolism regulates the activation of PKC through generation of second messengers and the extent of PKC activation accordingly influences the magnitude of phosphorylation of its endogenous substrate proteins. Thus it was speculated that ingestion of protein deficient diet may modify the turnover rate of membrane phospholipids and magnitude of phosphorylation of endogenous substrate proteins of PKC. The experiments were conducted on rats fed on three different types of laboratory prepared diets viz. casein (20% casein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threonine) for 28 days. The metabolism of phosphoinositides (PIs) and phosphatidyl choline (PC) was studied by equilibrium labeling with [3H] myo inositol and [14C methyl] choline chloride respectively. The phosphorylation of endogenous substrate proteins of PKC was studied by using 32P-gamma-ATP followed by SDS-PAGE and autoradiography. The results suggest that in deficient group, there is an increased incorporation of [3H] myo inositol in PIs and inositol phosphate pool in comparison to the casein group. The phosphatidyl inositol (PI) turnover reduced, although there was a marginal increase in the phosphatidyl inositol monophosphate (PIP) and phosphatidyl inositol bis phosphate (PIP2). Supplementation of diet showed a reversal of the pattern towards control to a considerable extent. In the deficient group, PC metabolism showed an increased incorporation of [14C methyl] choline in choline phospholipids but decreased incorporation in phosphoryl choline in comparison with the casein group. The increase in total PC contents was significant but marginal in residue contents. The turnover rate of PC increased only marginally and that of residue declined. Supplementation of diet reduced the total contents of PC and residue, but the turnover rate of PC and residue remained still higher. Phosphorylation of endogenous proteins showed four different proteins of 78, 46, 33 and 16 kDa to be the substrates of PKC in casein group. In deficient group, phosphorylation of these proteins increased markedly while supplementation of diet had a reversing effect rendering the values to be intermediate between casein and the supplemented group. The changes in phospholipid metabolism and in phosphorylation of endogenous substrate proteins of PKC suggest that dietary protein deficiency causes alterations in transmembrane signalling mechanism in rat brain. These effects are partially reversed by improving the quality of proteins in the diet.     

Phospholipid metabolism and protein kinase C mediated protein phosphorylation in dietary protein deficiency in rat lung

Nutritional deprivation of proteins decreases the protein kinase C (PKC) activity in rat lung. The activity of (PKC) is influenced by lipid metabolism. Changes in PKC activity may influence phosphorylation of its substrate proteins in the tissues. Therefore, alterations in phospholipid metabolism and PKC mediated protein phosphorylation in dietary protein deficiency in rat lung were envisaged. The study was conducted on rats fed on three different types of diet viz., casein (20% protein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threoning). Feeding of protein deficient diet caused reduction in incorporation of [3H] myo-inositol in the total phosphoinositides in lungs and an increase in total inositol phosphate pool. There was a significant reduction in the contents and turnover rate of phosphatidyl inositol and phosphatidyl inositol monophosphate. Supplementation of diet with L-lysine and DL-threonine had a reversing effect on total pool of phosphoinositides and, the metabolism of phosphatidyl inositol bisphosphate and phosphatidyl inositol. In phosphatidyl choline metabolism, the dietary protein deficiency led to a decrease in incorporation of [14C-methyl] choline-chloride in total phospholipids. In contrast, its incorporation increased in phosphatidyl choline pool. The contents of phosphatidyl choline and residue, incorporation of [14C-methyl] choline-chloride in them and their turnover rate also increased. Supplementation of diet had a reversal effect on most of these parameters. Phosphorylation of proteins of 84, 47, 35 and 16 kDa was identified to be mediated by PKC. In dietary protein deficiency, phosphorylation of all these proteins, except that of 47 kDa, increased. Supplementation of diet reversed the pattern except that of 84 kDa. The findings suggest that changes in phospholipid metabolism in dietary protein deficiency may effect the activity of PKC thereby influencing the phosphorylation of its substrate proteins and hence associated functions that may lead to pathophysiology of lung.