Membrane-associated Protein Kinase C Research Articles

Sex-specific effects of 17β-estradiol and dihydrotestosterone (DHT) on growth plate chondrocytes are dependent on both ERα and ERβ and require palmitoylation to translocate the receptors to the plasma membrane

Rat costochondral cartilage growth plate chondrocytes exhibit cell sex-specific responses to 17β-estradiol (E2), testosterone, and dihydrotestosterone (DHT). Mechanistically, E2 and DHT stimulate proliferation and extracellular matrix synthesis in chondrocytes from female and male rats, respectively, by signaling through protein kinase C (PKC) and phospholipase C (PLC). Estrogen receptors (ERα; ERβ) and androgen receptors (ARs) are present in both male and female cells, but it is not known whether they interact to elicit sex-specific signaling. We used specific agonists and antagonists of these receptors to examine the relative contributions of ERs and ARs in membrane-mediated E2 signaling in female chondrocytes and DHT signaling in male chondrocytes. PKC activity in female chondrocytes was stimulated by agonists of ERα and ERβ and required intact caveolae; PKC activity was inhibited by the E2 enantiomer and by an inhibitor of ERβ. Western blots of cell lysates co-immunoprecipitated for ERα suggested the formation of a complex containing both ERα and ERß with E2 treatment. DHT and DHT agonists activated PKC in male cells, while AR inhibition blocked the stimulatory effect of DHT on PKC. Inhibition of ERα and ERβ also blocked PKC activation by DHT. Western blots of whole-cell lysates, plasma membranes, and caveolae indicated the translocation of AR to the plasma membrane and specifically to caveolae with DHT treatment. These results suggest that E2 and DHT promote chondrocyte differentiation via the ability of ARs and ERs to form a complex. The results also indicate that intact caveolae and palmitoylation of the membrane receptor(s) or membrane receptor complex containing ERα and ERβ is required for E2 and DHT membrane-associated PKC activity in costochondral cartilage cells.

Ethanol Activation of Protein Kinase A Regulates GABAAα1 Receptor Function and Trafficking in Cultured Cerebral Cortical Neurons

Ethanol exposure produces alterations in GABAergic signaling that are associated with dependence and withdrawal. Previously, we demonstrated that ethanol-induced protein kinase C (PKC) γ signaling selectively contributes to changes in GABAA α1 synaptic receptor activity and surface expression. Here, we demonstrate that protein kinase A (PKA) exerts opposing effects on GABAA receptor adaptations during brief ethanol exposure. Cerebral cortical neurons from day 0-1 rat pups were tested after 18 days in culture. Receptor trafficking was assessed by Western blot analysis, and functional changes were measured using whole-cell patch-clamp recordings of evoked and miniature inhibitory postsynaptic current (mIPSC) responses. One-hour ethanol exposure increased membrane-associated PKC and PKA, but steady-state GABAA α1 subunit levels were maintained. Activation of PKA by Sp-adenosine 3',5'-cyclic monophosphothioate triethylamine alone increased GABAA α1 subunit surface expression and zolpidem potentiation of GABA responses, whereas coexposure of ethanol with the PKA inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine decreased α1 subunit expression and zolpidem responses. Exposure to the PKC inhibitor calphostin-C with ethanol mimicked the effect of direct PKA activation. The effects of PKA modulation on mIPSC decay τ were consistent with its effects on GABA currents evoked in the presence of zolpidem. Overall, the results suggest that PKA acts in opposition to PKC on α1-containing GABAA receptors, mediating the GABAergic effects of ethanol exposure, and may provide an important target for the treatment of alcohol dependence/withdrawal.

Neurobehavioral teratogenicity of perfluorinated alkyls in an avian model

Perfluorinated alkyls are widely-used agents that accumulate in ecosystems and organisms because of their slow rate of degradation. There is increasing concern that these agents may be developmental neurotoxicants and the present study was designed to develop an avian model for the neurobehavioral teratogenicity of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). Fertilized chicken eggs were injected with 5 or 10mg/kg of either compound on incubation day 0. On the day of hatching, imprinting behavior was impaired by both compounds. We then explored underlying mechanisms involving the targeting of protein kinase C (PKC) isoforms (α, β, γ) in the intermedial part of the hyperstriatum ventrale, the region most closely associated with imprinting. With PFOA exposure, cytosolic PKC concentrations were significantly elevated for all three isoforms; despite the overall increase in PKC expression, membrane-associated PKC was unaffected, indicating a defect in PKC translocation. In contrast, PFOS exposure evoked a significant decrease in cytosolic PKC, primarily for the β and γ isoforms, but again without a corresponding change in membrane-associated enzyme; this likely partial, compensatory increases in translocation to offset the net PKC deficiency. Our studies indicate that perfluorinated alkyls are indeed developmental neurotoxicants that affect posthatch cognitive performance but that the underlying synaptic mechanisms may differ substantially among the various members of this class of compounds, setting the stage for disparate outcomes later in life.

Synthesis of phosphatidylserine by base exchange in Triton‐insoluble floating fractions from rat cerebellum

Phosphatidylserine (PS), which is synthesized in mammalian tissues by the exchange between free serine and the nitrogen bases present in membrane glycerophospholipids, is strictly required for protein kinase C (PKC) activity. PKC, as other molecules involved in signal transduction, is present in lipid rafts, considered as a platform for molecular signaling. Membrane microdomains enriched in components of rafts can be isolated on the basis of their insolubility in Triton X-100 at 4 degrees C and their low density in sucrose density gradient. This study demonstrates the existence of serine base exchange enzyme (SBEE) in Triton-insoluble floating fractions containing associated PKC. Using two fractions of detergent-resistant membranes from rat cerebellum, we observed a correlation between the level of SBEE activity and that of membrane-associated PKC. This suggests that SBEE, synthesizing PS in the binding area for PKC, participates to signal transduction. The capability of SBEE to utilize not only serine but also ethanolamine, as free exchanging base, suggests a mechanism for modulating in loco PS concentration.

Requirement of Aldose Reductase for the Hyperglycemic Activation of Protein Kinase C and Formation of Diacylglycerol in Vascular Smooth Muscle Cells

Activation of protein kinase C (PKC) has been linked to the development of secondary diabetes complications. However, the underlying molecular mechanisms remain unclear. We examined the contribution of aldose reductase, which catalyzes the first, and the rate-limiting, step of the polyol pathway of glucose metabolism, to PKC activation in vascular smooth muscle cells (VSMCs) isolated from rat aorta and exposed to high glucose in culture. Exposure of VSMCs to high glucose (25 mmol/l), but not iso-osmotic mannitol, led to an increase in total membrane-associated PKC activity, which was prevented by the aldose reductase inhibitors tolrestat or sorbinil or by the ablation of aldose reductase by small interfering RNA (siRNA). The VSMCs were found to express low levels of sorbitol dehydrogenase, and treatment with the sorbitol dehydrogenase inhibitor CP-166572 did not prevent high-glucose-induced PKC activation. Stimulation with high glucose caused membrane translocation of conventional (alpha, beta1, beta2, and gamma) and novel (delta and epsilon) isoforms of PKC. Inhibition of aldose reductase prevented membrane translocation of PKC-beta2 and -delta and delayed the activation of PKC-beta1 and -epsilon, whereas membrane translocation of PKC-alpha and -gamma was not affected. Treatment with tolrestat prevented phosphorylation of PKC-beta2 and -delta. High glucose increased the formation of diacylglycerol (DAG) and enhanced phosphorylation of phospholipase C-gamma1 (PLC-gamma1). Inhibition of aldose reductase prevented high glucose-induced DAG formation and phosphorylation of PLC-gamma1 and PLC-beta2 and -delta. Inhibition of phospholipid hydrolysis by D609, but not by the synthetic alkyl-1-lysophospholipid 1-O-octadecyl-2-O-methyl-rac-glycerophosphocholine, or edelfosine, prevented DAG formation. Treatment with sorbinil decreased the levels of reactive oxygen species in high-glucose-stimulated VSMCs. Hence, inhibition of aldose reductase, independent of sorbitol dehydrogenase, appears to be effective in diminishing oxidative stress and hyperglycemic changes in signaling events upstream to the activation of multiple PKC isoforms and PLC-gamma1 and may represent a useful approach for preventing the development of secondary vascular complications of diabetes.

Identification of myristoylated alanine-rich C kinase substrate (marcks) in astrocytes

We have characterized membrane-associated substrates of Ca2+-dependent kinases in primary rat astrocytes by in vitro phosphorylation, 2-dimensional gel electrophoresis and autoradiography. The most prominent among these were three acidic, protein kinase C (PKC) substrates. These are important because they likely transduce cytokine and other neuro-immune modulatory signals mediated by PKC. We now show that one of these phosphoproteins is myristoylated alanine-rich PKC kinase substrate (MARCKS) or phosphomyristin C. The identity was corroborated by one- and 2- dimensional immunoblotting with an MARCKS-specific polyclonal antibody. Exposing primary astrocytes to phorbol 12-myristate 13-acetate stimulated phosphorylation of this protein. The level of MARCKS appeared inversely proportional to the proliferative potential of astrocytes because it was lower in spontaneously transformed as compared to passaged or confluent cells. These data are consistent with previous reports and indicate that one of three major acidic membrane-associated PKC substrates in astrocytes is MARCKS. Thus, MARCKS is likely near-proximal transducer of PKC-mediated signals in astrocytes.

Lithium and valproic acid treatments reduce PKC activation and receptor-G protein coupling in platelets of bipolar manic patients

Dysregulated protein kinase C (PKC) distribution and activation, and abnormal receptor-G protein coupling, have been implicated in the pathophysiology of bipolar affective disorder (BD). The therapeutic effectiveness of lithium has also been correlated with its ability to reduce PKC activation and G protein-mediated signaling. We examine the cellular distribution and activation of PKC and receptor-G protein coupling in blood platelets from normal controls, patients with BD mania or schizophrenia during treatment-free state, and after lithium or valproic acid administration. PKC activity was measured under basal and 50 nM phorbol 12-myristate, 13-acetate (PMA), 1 μM serotonin or 0.5 U/ml thrombin-stimulated conditions. The coupling of G proteins to serotonin or thrombin receptors were assessed by serotonin or thrombin-mediated [ 35S]GTPγS binding to membrane Gα proteins. The results demonstrate that membrane-associated PKC activity and stimulus-induced PKC translocation are increased in BD manic, whereas stimulus-elicited PKC translocation is attenuated in schizophrenic patients. Lithium and valproic acid treatments attenuated the stimulus-induced PKC translocations to a similar degree and decreased PKC activity in both cytosolic and membranous fractions after two weeks of drug administration. An increase in 5-HT or thrombin stimulated [ 35S]GTPγS binding to Gα proteins was detected in BD manic but not in schizophrenic patients although basal [ 35S]GTPγS binding was not different across the diagnostic groups. Lithium and valproic acid treatments similarly reduced receptor-G protein coupling with comparable time courses. Thus, increased membrane-associated PKC, cytosol to membrane PKC translocation and receptor-G protein coupling in platelets of BD manic patients were alleviated by lithium or valproic acid treatments.

Dose-dependent effects of acute exercise on PKC levels in rat heart: is PKC the heart's prophylactic?

Epidemiological studies have demonstrated that chronic exercise is cardioprotective, and recent evidence from our laboratory suggests a key role for protein kinase C (PKC)-dependent pathways, at least in part, as a cellular basis for this response. However, the dose-response relationship linking exercise volume and the time course of isoform-specific PKC activation are poorly understood. The purpose of this investigation was to determine the effects of acute exercise of varying durations on PKC subcellular distribution and phosphorylation in the rat left ventricle. Adult (5 months) male Fischer-344 more rats were subjected to a single bout (OB) or 7 days (SB) of treadmill running (n = 6/group; 23 m min-1, 20 min), and compared with sedentary controls (SED; n = 8). Hearts were isolated immediately after [early window (EW); n = 3/group] or 24 h after the last exercise bout [late window (LW); n = 3/group] in OB and SD, respectively. Total PKC and subcellular distribution for the alpha, delta, epsilon, betaI, and betaII isoforms, as well as phosphorylated (phospho-) PKC epsilon (pSer729), PKC alpha (pSer657) and PKCdelta (pThr507) levels were assessed by western blotting. Protein kinase C epsilon and PKC alpha mRNA levels were assessed by real time polymerase chain reaction. Following OB, PKCbetaI protein levels were reduced, while total phospho-PKC epsilon (pSer729), PKC alpha (pSer657) and PKC delta (pThr507) levels were increased during EW (P < 0.05). Interestingly, total PKC delta (31%) and membrane-associated PKC alpha (24%) levels decreased from EW to LW (P < 0.05). In contrast, SB yielded chronic increases in total PKC epsilon (80.5%) levels and PKC delta (20.0%) levels (P < 0.03), with reversal of effects on phospho-PKC epsilon (Ser729), phospho-PKC alpha (Ser657) and phospho-PKC delta (Thr507) levels observed with OB. Reductions in total phospho-PKC alpha (Ser657) persisted at SB (26.1%; P < 0.02). Interestingly, mRNA levels for PKC epsilon were significantly increased following SB while PKC alpha mRNA levels were reduced, respectively. These data suggest that divergent patterns of PKC activation occur following OB and SB at both the transcriptional and translational levels. That similar patterns of PKC translocation are observed in experimental models of ischaemic preconditioning and genetic PKC manipulation provide evidence for a dose-dependent cardioprotective phenotype induced by physical activity.

Differential effects of NMDA and group I mGluR antagonists on both nociception and spinal cord protein kinase C translocation in the formalin test and a model of neuropathic pain in rats

Coincident with nociception, both noxious chemical stimulation of the hind paw and chronic constriction injury (CCI) of the sciatic nerve produce an increase in protein kinase C (PKC) translocation in the spinal cord of rats. Noxious stimulus-induced PKC translocation likely depends on glutamate activity at either N-methyl- d-aspartate (NMDA) receptors or group I metabotropic glutamate receptors (mGluR1/5) in the spinal cord dorsal horn. This study compares nociceptive responses to, and the alterations in membrane-associated PKC, induced by noxious chemical stimulation of the hindpaw and CCI of the sciatic nerve, as well as their modulation by both NMDA and mGluR1/5 receptor antagonists. Three groups of rats were given a single intrathecal (i.t.) injection of either vehicle, dizocilpine maleate (MK-801, 60 nmol), an NMDA receptor antagonist, or ( S)-4-carboxyphenylglycine ( S)-4CPG, (150 nmol), an mGluR1/5 antagonist, 10 min prior to a 50 μl of 2.5% formalin injection into the ventral surface of one hind paw. Another three groups of rats were given twice daily injections of either vehicle, MK-801 (30 nmol) or ( S)-4CPG (90 nmol) i.t. for 5 days starting 30 min before CCI or sham injury of the sciatic nerve. Nociceptive responses were assessed for a 60 min period after the formalin injection in the first three groups, and tests of mechanical and cold allodynia were performed on days 4, 8, 12 and 16 after CCI for the latter three groups. Furthermore, changes in the levels of membrane-associated PKC, as assayed by quantitative autoradiography of the specific binding of [ 3H]-phorbol 12,13-dibutyrate ([ 3H]-PDBu) in the dorsal horn of the lumbar spinal cord sections, were assessed in formalin-injected rats (at 5, 25 and 60 min) and in neuropathic rats 5 days after CCI, treated (as above) with vehicle, MK-801 or ( S)-4CPG. The results indicate that i.t. treatment with MK-801 significantly reduced nociceptive scores in the formalin test and also produced a significant suppression of formalin-induced increases in [ 3H]-PDBu binding in laminae I–II, III–VI and X of the lumbar spinal cord. In contrast, i.t. treatment with ( S)-4CPG failed to significantly affect either nociceptive behaviours in the formalin test or formalin-induced increases in [ 3H]-PDBu binding in laminae I–II and III–VI of the lumbar spinal cord. On the other hand, i.t. treatment with either MK-801 or ( S)-4CPG produced a significant reduction in mechanical and cold hypersensitivity, as well as [ 3H]-PDBu binding in laminae I–II and III–VI of the lumbar spinal cord, after CCI. These results suggest that while NMDA, but not mGluR1/5, receptors are involved in translocation of PKC and nociception in a model of persistent acute pain, both types of receptors influence the translocation of PKC in dorsal horn and mechanical and cold allodynia in a model of chronic neuropathic pain.

Advanced glycosylation end products stimulate collagen mRNA synthesis in mesangial cells mediated by protein kinase C and transforming growth factor–beta

Advanced glycosylation end products (AGE) seem to be implicated in the pathogenesis of diabetic nephropathy. The present study has examined the effects of AGE on protein kinase C (PKC) activity and transforming growth factor–β1 (TGF-β1) in relation to collagen gene regulation in cultured human mesangial cells (HMCs). Quiescent HMCs were exposed to serum-free media containing bovine serum albumin (BSA), AGE-modified BSA (AGE-BSA), or glycated BSA in which AGE formation was prevented by the use of aminoguanidine (BSA-AM). AGE-BSA (200 μg/mL) induced a peak membrane-associated PKC activity, particularly PKC-a, at 4 hours. AGE-BSA stimulated α1(I) and α1(IV) collagen mRNA expression after 24-hour incubation with HMCs, which remained elevated until hour 60. HMCs incubated with AGE-BSA induced a significant inhibition of cell proliferation compared with cells incubated with BSA. AGE-BSA stimulated TGF-β mRNA and protein expression in HMCs. The TGF-β secreted by HMCs was shown by CCL-64 mink lung cell assay to be bioactive. In contrast, BSA-AM did not affect either collagen or TGF-β mRNA or protein expression in HMCs. The stimulatory effects of AGE-BSA on collagen gene regulation in HMCs could be negated by the pretreament of HMCs with GF 109203X for 30 minutes or with phorbol myristate acetate for 24 hours before AGE-BSA administration. Neutralizing antibody to TGF-β inhibited increased collagen mRNA expression by HMCs exposed to AGE-BSA. These results suggest that AGE-BSA stimulates collagen mRNA expression by activating PKC and the transcriptional upregulation of TGF-β1 in HMCs. Thus, PKC and TGF-β may function as key signaling intermediaries in the AGE–up-regulated collagen gene expression pathway in HMCs. (J Lab Clin Med 2001;138:59-68)

Protein kinase C activation: isozyme-specific effects on metabolism and cardiovascular complications in diabetes.

Protein kinase C (PKC) is a family of multifunctional isoenzymes, activated by diacylglycerols (DAGs), which play a central role in signal transduction and intracellular crosstalk by phosphorylating at serine/threonine residues an array of substrates, including cell-surface receptors, enzymes, contractile proteins, transcription factors and other kinases. Individual isozymes vary in their pattern of tissue and subcellular distribution, function and Ca2+/phospholipid cofactor requirements, and in diabetes there is widespread activation of the DAG-PKC pathway in metabolic, cardiovascular and renal tissues. In liver, muscle and adipose tissue, PKC isozymes have been implicated both as mediators and inhibitors of insulin action. Activation of DAG-sensitive PKC isoforms, such as PKC-theta and PKC-epsilon, down-regulates insulin receptor signalling and could be an important biochemical mechanism linking dysregulated lipid metabolism and insulin resistance in muscle. On the other hand, atypical PKC isozymes, such as PKC-zeta and PKC-lambda, have been identified as downstream targets of PI-3-kinase involved in insulin-stimulated glucose uptake, especially in adipocytes. Glucose-induced de novo synthesis of (palmitate-rich) DAG and sustained isozyme-selective PKC activation (especially but not exclusively PKC-beta) has been strongly implicated in the pathogenesis of diabetic microangiopathy and macroangiopathy through a host of undesirable effects on endothelial function, VSM contractility and growth, angiogenesis, gene transcription (in part by MAP-kinase activation) and vascular permeability. Interventions that increase DAG metabolism (e. g. vitamin E) and/or inhibit PKC isozymes (e. g. the beta-selective inhibitor LY333531) ameliorate the biochemical and functional consequences of DAG-PKC activation in experimental diabetes, for example improving retinal blood flow and albuminuria in parallel with reductions in membrane-associated PKC isozyme activities. Thus, a greater understanding of the functional diversity and pathophysiological regulation of PKC isozymes is likely to have important clinical and therapeutic benefits.

Increased platelet aggregation in diabetic patients with microangiopathy despite good glycemic control

The pathogenesis of diabetic micro- and macroangiopathy cannot be fully explained by hyperglycemia alone. To clarify diabetic complications mediated by increased platelet activity, we have studied platelet aggregation and its second messenger molecules such as protein kinase C (PKC), RhoA, and phosphatidylinositol 3-kinase (PI3- kinase), in six diabetic patients with diabetic retinopathy and other diabetic complications in spite of good glycemic control. Their HbA 1c levels throughout the observation period had been less than 6% with diet treatment alone, despite which diabetic retinopathy developed to the pre-proliferative stage during 2-8 years observation. Low-dose thrombin (< 0.5 U/ml)-stimulated platelet aggregation in the diabetic patients was enormously elevated compared with healthy control subjects. PKC, RhoA and PI3-kinase activities in the cytosol- and membrane-associated fractions were examined in the platelets from the two patients (Cases 2 and 4). Platelet membrane-associated RhoA and PI3-kinase activity in Case 2 were increased before the stimulation. Platelet RhoA and PI 3-kinase activities in Case 4 were increased after the stimulation with low-dose thrombin (0.01 U/ml). Membrane-associated immunoreactive PKC f , but not PKC g in Cases 2 and 4 was elevated. Although platelet hyperactivity in these four patients was observed, PKC and RhoA in mononuclear leukocytes from these patients were not different from healthy subjects. Membrane-associated PKC f and RhoA immunoreactivities also increased in the other three cases. These results suggest that hyperreactivity of PKC f may lead to increased RhoA and PI3-kinase activities and platelet hyperfunction in diabetic patients with good glycemic control, and that raised platelet PKC f may be implicated in the pathogenesis of diabetic complications.

ATP-Sensitive potassium channels modulate glucose transport in cultured human skeletal muscle cells.

Several lines of evidence suggest that ATP-sensitive potassium (KATP) channels are involved in glucose uptake by insulin target tissues. The aim of the present study was to prove directly the effect of KATP channel activity on glucose transport into cultured human skeletal muscle cells. We used potassium channel openers PCO-400 and nicorandil alone or in combination with channel blockers glibenclamide and gliclazide to examine their effects on insulin- or high glucose concentration-induced glucose uptake using 2-deoxy-D-3H-glucose or 3-O-methyl-D-3H-glucose as tracer, respectively. PCO-400 inhibited the basal (non-stimulated) uptake of 2-DG or 3-OMG at the glucose concentration of 5 mM. PCO-400 and nicorandil dose-dependently inhibited insulin-stimulated glucose uptake, and their inhibitory effects were reversed by glibenclamide or gliclazide. In addition, PCO-400 inhibited high glucose concentration-facilitated glucose transport in the absence of insulin, and this effect was also antagonized by both sulfonylurea drugs. Regarding the mechanism by which KATP channels modulate glucose transport, we focused on protein kinase C (PKC), because PKC has been supposed to participate in both insulin- and high glucose concentration-stimulated glucose transport. PMA (phorbol 12-myristate 13-acetate) dose-dependently reversed the PCO-400-induced suppression of insulin-stimulated glucose uptake. On the other hand, PCO-400 at the concentration that inhibited glucose uptake caused no alteration of membrane-associated PKC activity in the presence of insulin or PMA. From these results we conclude that KATP channels modulate the basal and insulin-or high glucose level-stimulated glucose transport in skeletal muscle through a mechanism independent of PKC.

Intracellular pH on translocation of protein kinase C isozymes in rat pinealocytes.

In rat pinealocytes, cytoplasmic alkalization causes protein kinase C (PKC) translocation, but the isozyme involved is not known. In this study, we investigated the effect of cytoplasmic alkalization on membrane-associated PKCalpha, delta, epsilon, and zeta, four isozymes present in the rat pineal gland. Treatment with NH(4)Cl, which had no effect on PKCzeta, caused a sustained increase in membrane-associated PKCalpha, delta, and epsilon that lasted for at least 60 min. The effect of NH(4)Cl on PKCalpha, delta, and epsilon was reduced by sodium propionate, an agent that counteracts the effect of NH(4)Cl on intracellular pH. Both sodium propionate and 5-(N,N-hexamethylene)amiloride (HMA), two treatments that abolished the effect of norepinephrine on cytoplasmic alkalization, also reduced norepinephrine-mediated increases in membrane-associated PKCalpha, delta, and epsilon. In contrast, these two treatments did not have an effect on the increase in membrane-associated PKC isozymes caused by 4beta-phorbol 12-myristate 13-acetate (PMA), an active phorbol ester, even though HMA was effective in abolishing PMA-mediated increases in intracellular pH. These results, apart from demonstrating that cytoplasmic alkalization by itself can cause translocation of PKCalpha, delta, and epsilon in rat pinealocytes, also indicate that the norepinephrine-stimulated cytoplasmic alkalization plays an important role in transducing signals from the adrenergic receptor to selective PKC isozymes. However, PKC translocation stimulated directly by PMA does not appear to be sensitive to changes in intracellular pH.

Lithium regulates PKC-mediated intracellular cross-talk and gene expression in the CNS in vivo.

It has become increasingly appreciated that the long-term treatment of complex neuropsychiatric disorders like bipolar disorder (BD) involves the strategic regulation of signaling pathways and gene expression in critical neuronal circuits. Accumulating evidence from our laboratories and others has identified the family of protein kinase C (PKC) isozymes as a shared target in the brain for the long-term action of both lithium and valproate (VPA) in the treatment of BD. In rats chronically treated with lithium at therapeutic levels, there is a reduction in the levels of frontal cortical and hippocampal membrane-associated PKC alpha and PKC epsilon. Using in vivO microdialysis, we have investigated the effects of chronic lithium on the intracellular cross-talk between PKC and the cyclic AMP (cAMP) generating system in vivo. We have found that activation of PKC produces an increase in dialysate cAMP levels in both prefrontal cortex and hippocampus, effects which are attenuated by chronic lithium administration. Lithium also regulates the activity of another major signaling pathway the c-Jun N-terminal kinase pathway--in a PKC-dependent manner. Both Li and VPA, at therapeutically relevant concentrations, increase the DNA binding of activator protein 1 (AP-1) family of transcription factors in cultured cells in vitro, and in rat brain ex vivo. Furthermore, both agents increase the expression of an AP-1 driven reporter gene, as well as the expression of several endogenous genes known to be regulated by AP-1. Together, these results suggest that the PKC signaling pathway and PKC-mediated gene expression may be important mediators of lithium's long-term therapeutic effects in a disorder as complex as BD.

Protein Kinase C-regulated Dynamitin-Macrophage-enriched Myristoylated Alanine-Rice C Kinase Substrate Interaction Is Involved in Macrophage Cell Spreading

Macrophage spreading requires the microtubule cytoskeleton and protein kinase C (PKC). The mechanism of involvement of the microtubules and PKC in this event is not fully understood. Dynamitin is a subunit of dynactin, which is important for linking the microtubule-dependent motor protein dynein to vesicle membranes. We report that dynamitin is a Ca(2+)/calmodulin-binding protein and that dynamitin binds directly to macrophage-enriched myristoylated alanine-rice C kinase substrate (MacMARCKS), a membrane-associated PKC substrate involved in macrophage spreading and integrin activation. Dynamitin was found to copurify with MacMARCKS both during MacMARCKS purification with conventional chromatography and during the immunoabsorption of MacMARCKS using anti-MacMARCKS antibody. Vice versa, MacMARCKS was also found to cosediment with the 20 S dynactin complex. We determined that the effector domain of MacMARCKS is required to interact with the N-terminal domain of dynamitin. MacMARCKS and dynamitin also partially colocalized at peripheral regions of macrophages and in the cell-cell border of 293 epithelial cells. Treatment with phorbol esters abolished this colocalization. Disrupting the interaction with a short peptide derived from the MacMARCKS-binding domain of dynamitin caused macrophages to spread and flatten. These data suggest that the dynamitin-MacMARCKS interaction is involved in cell spreading. Furthermore, the regulation of this interaction by PKC and Ca(2+)/calmodulin provides a possible regulatory mechanism for cell adhesion and spreading.

Macrophage-enriched Myristoylated Alanine-rich C Kinase Substrate and Its Phosphorylation Is Required for the Phorbol Ester-stimulated Diffusion of β2 Integrin Molecules

An early event of beta(2) integrin activation is the increased diffusion rate of this molecule on the cell surface, thereby providing integrin molecules with a better chance to meet the ligands. The activation of protein kinase C (PKC) stimulates integrin diffusion by releasing the cytoskeletal constraint on integrin molecules. We report here that macrophage-enriched myristoylated alanine-rich C kinase substrate (MacMARCKS), a membrane-associated PKC substrate involved in integrin activation, is required for this PKC-stimulated diffusion of integrin molecules. Using the single-particle tracking technique, we observed that the activation of PKC stimulated an 11-fold increase in the diffusion rate of beta(2) integrins in wild type J774 macrophage cells but not in those expressing mutant MacMARCKS. Further evidence is provided from a MacMARCKS-deficient cell line in which phorbol esters failed to stimulate the diffusion of integrin. Transfection of wild type MacMARCKS into these cells restored the rapid diffusion rate of the beta(2) integrins. The phosphorylation of MacMARCKS is important because transfection of a nonphosphorylatable MacMARCKS mutant or the addition of staurosporine eliminates the rapid diffusion rate of integrin. Furthermore, adding cytochalasin D bypasses the MacMARCKS deficiency and stimulates beta(2) integrin diffusion, suggesting that MacMARCKS's involvement in integrin activation is prior or at the site of cytoskeleton. Therefore, we conclude that MacMARCKS is required for releasing the cytoskeletal constraint on integrin molecules during PKC-mediated integrin activation.

1,25(OH)(2)-vitamin D(3) affects the subcellular distribution of protein kinase C isoenzymes in rat duodenum: influence of aging.

We have previously shown that the steroid hormone 1, 25-dihydroxy-vitamin D(3) [1,25(OH)(2)D(3)] stimulates total cell protein kinase C (PKC) activity in rat duodenum, an effect that is severely impaired in old animals. We further examined the role of 1, 25(OH)(2)D(3) on PKC as it relates to aging by measuring hormone-induced changes in subcellular localization of PKC activity and isoenzymes in duodenal mucosae from young (three-month-old) and aged (24-month-old) rats. Short treatment of duodenum with 1, 25(OH)(2)D(3) (0.1 nM, 1 min) increased membrane-associated PKC activity, whereas it decreased the activity in the cytosol of young rats but was without significant effect in aged animals. Furthermore, the ability to translocate was present in young animals after a short treatment with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA; 100 nM) or dioctanoyl-glycerol (50 microM), whereas the ability was absent in aged rats, suggesting that PKC function was impaired with aging independent of agonist stimulation. The expression of specific PKC isoenzymes and changes in their subcellular distribution after short exposure of the duodenum to the hormone were determined. Western blot analysis of total homogenates using antibodies to various PKC isoforms allowed detection of PKC alpha, beta, and delta. The expression of the straight theta and the zeta isoforms was in addition demonstrated by reverse transcription-polymerase chain reaction. The pattern of isoenzymes present in the duodenum was unaffected by aging. In young rats, 1, 25(OH)(2)D(3) translocates PKC alpha, beta, and delta to the membrane and nucleus; however, no translocation of PKC isoforms was observed in 24-month-old animals in response to the hormone. In summary, in rat duodenum, 1,25(OH)(2)D(3) modulation of PKC activity and isoenzyme subcellular distribution are impaired with aging and may explain age-induced alterations in the intestinal processes under the control of the hormone.

Abnormalities in protein kinase C signaling and the pathophysiology of bipolar disorder.

Protein kinase C (PKC) is a group of calcium and phospholipid-dependent enzymes, which plays a pivotal role in cell signaling systems. Recently accumulated evidence indicates that alterations in PKC activity play a significant role in the pathophysiology of bipolar disorder. A number of laboratories investigated the effect of mood stabilizers on the regulation of PKC activity in bipolar patients, in animals, and in cultured cells. Following chronic lithium treatment, PKC activation was significantly reduced in rat brains, as measured by the translocation of cytoplasmic PKC to the membrane compartment, or by quantitative binding of the PKC ligand, PDBu. The effect of the therapeutic concentration of lithium in attenuating PKC-dependent intracellular parameters was also demonstrated in cultured cells. More importantly, alterations in platelet PKC was shown in bipolar patients during the manic state of the illness. In comparison to patients with major depressive disorder, schizophrenia, or healthy controls, PKC activity was significantly increased in manic patients, suggesting that changes in PKC may be an illness-specific marker. Interestingly, enhanced PKC activity during mania was suppressed following mood-stabilizer treatment as manic symptoms improved. In parallel to the findings in platelets, postmortem studies demonstrate that membrane-associated PKC and stimulation-induced translocation of cytosolic enzyme to the membrane were also increased in frontal cortex of bipolar patients. Other studies suggest alterations in other signal transduction mechanisms in bipolar disorder. These include alterations in G protein activation, phosphatidylinositol (PI) signaling, cyclic AMP formation, and intracellular calcium homeostasis. The alterations of PKC activity in bipolar disorder may be related to changes in these other intracellular signaling mechanisms. Alternatively, the changes of PKC activity may be the core pathology of the illness. More studies are required to further characterize the association of PKC changes with bipolar disorder, using a proper neuronal model.

Conformation of the C1 phorbol-ester-binding domain participates in the activating conformational change of protein kinase C

The fluorescent phorbol ester 12-N-methylanthraniloylphorbol 13-acetate [sapintoxin D (SAPD)] was used as both the activator and the probe for the activating conformational change of the C1 domain of recombinant protein kinase C (PKC)alpha. Fluorescence emission spectra and steady-state anisotropy measurements of SAPD in fully active membrane-associated PKC show that there is a relatively hydrophobic environment and restricted motional freedom characterizing the phorbol-ester-binding site. SAPD also interacts with the membrane lipids so that it was necessary to resort to time-resolved anisotropy measurements to resolve the signals corresponding to PKC-bound SAPD from that associated with buffer and lipid. In the presence of membrane lipids (unilamellar vesicles of phosphatidylcholine and phosphatidylserine, 4:1 molar ratio) and Ca(2+), at a concentration sufficient to activate the enzyme fully, a long correlation time characteristic of highly restricted motion was observed for PKC-associated SAPD. The fraction of SAPD molecules displaying this restricted motion, in comparison with the total SAPD including that in lipids and in buffer, increased with increasing concentrations of Ca(2+) and paralleled the appearance of enzyme activity, whereas the rotational correlation time remained constant. This could be rationalized as an increase in the number of active PKC conformers in the total population of PKC molecules. It therefore seems that there is a distinct conformation of the C1 activator-binding domain associated with the active form of PKC. The addition of SAPD and dioleoyl-sn-glycerol together produced an activity higher than that achievable by either activator alone both at concentrations that alone induced maximal activity for the respective activator; this higher activity was associated with a further restriction in SAPD motion. Increasing the cholesterol concentration, the phosphatidylethanolamine concentration, the sn-2 unsaturation in phosphatidylcholine and the vesicle curvature each also elevated SAPD-induced PKC activity and again increased the PKC-associated SAPD rotational correlation time. In summary, the rotational correlation time of PKC-bound SAPD, extractable from a single time-resolved fluorescence anisotropy measurement, provides a novel probe for the involvement of interactions between the C1 domain and phorbol ester in the modulation of PKC activity.