Activation Of Protein Kinase Cα Research Articles

Protein kinase Cα activation switches YAP1 from TEAD-mediated signaling to p73-mediated signaling.

Yes‐associated protein 1 (YAP1) interacts with TEAD transcription factor in the nucleus and upregulates TEAD‐target genes. YAP1 is phosphorylated by large tumor suppressor (LATS) kinases, the core kinases of the Hippo pathway, at 5 serine residues and is sequestered and degraded in the cytoplasm. In human cancers with the dysfunction of the Hippo pathway, YAP1 becomes hyperactive and confers malignant properties to cancer cells. We have observed that cold shock induces protein kinase C (PKC)‐mediated phosphorylation of YAP1. PKC phosphorylates YAP1 at 3 serine residues among LATS‐mediate phosphorylation sites. Importantly, PKC activation recruits YAP1 to the cytoplasm even in LATS‐depleted cancer cells and reduces the cooperation with TEAD. PKC activation induces promyelocytic leukemia protein‐mediated SUMOylation of YAP1. SUMOylated YAP1 remains in the nucleus, binds to p73, and promotes p73‐target gene transcription. Bryostatin, a natural anti‐neoplastic reagent that activates PKC, induces YAP1/p73‐mediated apoptosis in cancer cells. Bryostatin reverses malignant transformation caused by the depletion of LATS kinases. Therefore, bryostatin and other reagents that activate PKC are expected to control cancers with the dysfunction of the Hippo pathway.

IGFBP-1 hyperphosphorylation in response to nutrient deprivation is mediated by activation of protein kinase Cα (PKCα)

Fetal growth restriction (FGR) is associated with decreased nutrient availability and reduced insulin-line growth factor (IGF)-I bioavailability via increased IGF binding protein (IGFBP)-1 phosphorylation. While protein kinase C (PKC) is implicated in IGFBP-1 hyperphosphorylation in nutrient deprivation, the mechanisms remain unclear. We hypothesised that the interaction of PKCα with protein kinase CK2β and activation of PKCα under leucine deprivation (L0) mediate fetal hepatic IGFBP-1 hyperphosphorylation. Parallel Reaction Monitoring Mass Spectrometry (PRM-MS) followed by PKCα knockdown demonstrated the PKCα isoform interacts with IGFBP-1 and CK2β under L0. Pharmacological PKCα activation with phorbol 12-myristate 13-acetate (PMA) increased whereas inhibition with bisindolylmaleimide II (Bis II) decreased IGFBP-1 phosphorylation (Ser101/119/169, Ser98 + 101 and Ser169 + 174), respectively. Furthermore, PMA mimicked L0-induced PKCα translocation and IGFBP-1 expression. PKCα expression was increased in baboon fetal liver in FGR, providing biological relevance in vivo. In summary, we report a novel nutrient-sensitive mechanism for PKCα in mediating IGFBP-1 hyperphosphorylation in FGR.

G-protein-coupled receptor 40 agonist GW9508 potentiates glucose-stimulated insulin secretion through activation of protein kinase Cα and ε in INS-1 cells.

ObjectiveThe mechanism by which G-protein-coupled receptor 40 (GPR40) signaling amplifies glucose-stimulated insulin secretion through activation of protein kinase C (PKC) is unknown. We examined whether a GPR40 agonist, GW9508, could stimulate conventional and novel isoforms of PKC at two glucose concentrations (3 mM and 20 mM) in INS-1D cells.MethodsUsing epifluorescence microscopy, we monitored relative changes in the cytosolic fluorescence intensity of Fura2 as a marker of change in intracellular Ca2+ ([Ca2+]i) and relative increases in green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate (MARCKS-GFP) as a marker of PKC activation in response to GW9508 at 3 mM and 20 mM glucose. To assess the activation of the two PKC isoforms, relative increases in membrane fluorescence intensity of PKCα-GFP and PKCε-GFP were measured by total internal reflection fluorescence microscopy. Specific inhibitors of each PKC isotype were constructed and synthesized as peptide fusions with the third α-helix of the homeodomain of Antennapedia.ResultsAt 3 mM glucose, GW9508 induced sustained MARCKS-GFP translocation to the cytosol, irrespective of changes in [Ca2+]i. At 20 mM glucose, GW9508 induced sustained MARCKS-GFP translocation but also transient translocation that followed sharp increases in [Ca2+]i. Although PKCα translocation was rarely observed, PKCε translocation to the plasma membrane was sustained by GW9508 at 3 mM glucose. At 20 mM glucose, GW9508 induced transient translocation of PKCα and sustained translocation as well as transient translocation of PKCε. While the inhibitors (75 μM) of each PKC isotype reduced GW9508-potentiated, glucose-stimulated insulin secretion in INS-1D cells, the PKCε inhibitor had a more potent effect.ConclusionGW9508 activated PKCε but not PKCα at a substimulatory concentration of glucose. Both PKC isotypes were activated at a stimulatory concentration of glucose and contributed to glucose-stimulated insulin secretion in insulin-producing cells.

Curcumin Inhibits Protein Kinase Cα Activity by Binding to Its C1 Domain.

Curcumin is a polyphenolic nutraceutical that acts on multiple biological targets, including protein kinase C (PKC). PKC is a family of serine/threonine kinases central to intracellular signal transduction. We have recently shown that curcumin selectively inhibits PKCα, but not PKCε, in CHO-K1 cells [Pany, S. (2016) Biochemistry 55, 2135-2143]. To understand which domain(s) of PKCα is responsible for curcumin binding and inhibitory activity, we made several domain-swapped mutants in which the C1 (combination of C1A and C1B) and C2 domains are swapped between PKCα and PKCε. Phorbol ester-induced membrane translocation studies using confocal microscopy and immunoblotting revealed that curcumin inhibited phorbol ester-induced membrane translocation of PKCε mutants, in which the εC1 domain was replaced with αC1, but not the PKCα mutant in which αC1 was replaced with the εC1 domain, suggesting that αC1 is a determinant for curcumin's inhibitory effect. In addition, curcumin inhibited membrane translocation of PKCε mutants, in which the εC1A and εC1B domains were replaced with the αC1A and αC1B domains, respectively, indicating the role of both αC1A and αC1B domains in curcumin's inhibitory effects. Phorbol 13-acetate inhibited the binding of curcumin to αC1A and αC1B with IC50 values of 6.27 and 4.47 μM, respectively. Molecular docking and molecular dynamics studies also supported the higher affinity of curcumin for αC1B than for αC1A. The C2 domain-swapped mutants were inactive in phorbol ester-induced membrane translocation. These results indicate that curcumin binds to the C1 domain of PKCα and highlight the importance of this domain in achieving PKC isoform selectivity.

Dynamic Response of the C2 Domain of Protein Kinase Cα to Ca2+ Binding

Ca2+-dependent conserved-region 2 (C2) domains target their host signaling proteins to anionic membranes. The Ca2+-binding event is a prerequisite for membrane association. Here, we investigate multiscale metal-ion-dependent dynamics of the C2 domain of protein kinase Cα (C2α) using NMR spectroscopy. Interactions with metal ions attenuate microsecond-timescale motions of the loop regions, indicating that preorganization of the metal-binding loops occurs before membrane insertion. Binding of a full complement of Ca2+ ions has a profound effect on the millisecond-timescale dynamics of the N- and C-terminal regions of C2α. We propose that Ca2+ binding allosterically destabilizes the terminal regions of C2α and thereby facilitates the conformational rearrangement necessary for full membrane insertion and activation of protein kinase Cα.

Activation of protein kinase Cα increases phosphorylation of the UT-A1 urea transporter at serine 494 in the inner medullary collecting duct.

Hypertonicity increases urea transport, as well as the phosphorylation and membrane accumulation of UT-A1, the transporter responsible for urea permeability in the inner medullary collect duct (IMCD). Hypertonicity stimulates urea transport through PKC-mediated phosphorylation. To determine whether PKC phosphorylates UT-A1, eight potential PKC phosphorylation sites were individually replaced with alanine and subsequently transfected into LLC-PK1 cells. Of the single mutants, only ablation of the S494 site dampened induction of total UT-A1 phosphorylation by the PKC activator phorbol dibutyrate (PDBu). This result was confirmed using a newly generated antibody that specifically detected phosphorylation of UT-A1 at S494. Hypertonicity increased UT-A1 phosphorylation at S494. In contrast, activators of cAMP pathways (PKA and Epac) did not increase UT-A1 phosphorylation at S494. Activation of both PKC and PKA pathways increased plasma membrane accumulation of UT-A1, although activation of PKC alone did not do so. However, ablating the PKC site S494 decreased UT-A1 abundance in the plasma membrane. This suggests that the cAMP pathway promotes UT-A1 trafficking to the apical membrane where the PKC pathway can phosphorylate the transporter, resulting in increased UT-A1 retention at the apical membrane. In summary, activation of PKC increases the phosphorylation of UT-A1 at a specific residue, S494. Although there is no cross talk with the cAMP-signaling pathway, phosphorylation of S494 through PKC may enhance vasopressin-stimulated urea permeability by retaining UT-A1 in the plasma membrane.

Trans-regulation of Syndecan Functions by Hetero-oligomerization

Syndecans, a family of transmembrane heparansulfate proteoglycans, are known to interact through their transmembrane domains to form non-covalently linked homodimers, a process essential for their individual functions. Because all syndecan transmembrane domains are highly conserved and thus might mediate interactions between different members of the syndecan family, we investigated syndecan interactions in detail. All recombinant syndecan-2 and -4 protein variants containing the transmembrane domain formed not only sodium dodecyl sulfate (SDS)-resistant homodimers but also SDS-resistant heterodimers. Biochemical and structural data revealed that recombinant syndecan-2 and -4 formed intermolecular interactions in vitro, and the GXXXG motif in transmembrane domain mediated this interaction. When exogenously expressed in rat embryonic fibroblasts, syndecan-2 interacted with syndecan-4 and vice versa. Furthermore, bimolecular fluorescence complementation-based assay demonstrated specific hetero-molecular interactions between syndecan-2 and -4, supporting hetero-oligomer formation of syndecans in vivo. Interestingly, hetero-oligomerization significantly reduced syndecan-4-mediated cellular processes such as protein kinase Cα activation and protein kinase Cα-mediated cell adhesion as well as syndecan-2-mediated tumorigenic activities in colon cancer cells such as migration and anchorage-independent growth. Taken together, these data provide evidence that hetero-oligomerization produces distinct syndecan functions and offer insights into the underlying signaling mechanisms of syndecans.

Cardiomyocyte-specific Loss of Diacylglycerol Acyltransferase 1 (DGAT1) Reproduces the Abnormalities in Lipids Found in Severe Heart Failure

Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final step in triglyceride synthesis, the conversion of diacylglycerol (DAG) to triglyceride. Dgat1(-/-) mice exhibit a number of beneficial metabolic effects including reduced obesity and improved insulin sensitivity and no known cardiac dysfunction. In contrast, failing human hearts have severely reduced DGAT1 expression associated with accumulation of DAGs and ceramides. To test whether DGAT1 loss alone affects heart function, we created cardiomyocyte-specific DGAT1 knock-out (hDgat1(-/-)) mice. hDgat1(-/-) mouse hearts had 95% increased DAG and 85% increased ceramides compared with floxed controls. 50% of these mice died by 9 months of age. The heart failure marker brain natriuretic peptide increased 5-fold in hDgat1(-/-) hearts, and fractional shortening (FS) was reduced. This was associated with increased expression of peroxisome proliferator-activated receptor α and cluster of differentiation 36. We crossed hDgat1(-/-) mice with previously described enterocyte-specific Dgat1 knock-out mice (hiDgat1(-/-)). This corrected the early mortality, improved FS, and reduced cardiac ceramide and DAG content. Treatment of hDgat1(-/-) mice with the glucagon-like peptide 1 receptor agonist exenatide also improved FS and reduced heart DAG and ceramide content. Increased fatty acid uptake into hDgat1(-/-) hearts was normalized by exenatide. Reduced activation of protein kinase Cα (PKCα), which is increased by DAG and ceramides, paralleled the reductions in these lipids. Our mouse studies show that loss of DGAT1 reproduces the lipid abnormalities seen in severe human heart failure.

Molecular interplays involved in the cellular uptake of octaarginine on cell surfaces and the importance of syndecan-4 cytoplasmic V domain for the activation of protein kinase Cα

Arginine-rich cell-penetrating peptides (CPPs) are promising carriers for the intracellular delivery of various bioactive molecules. However, many ambiguities remain about the molecular interplays on cell surfaces that ultimately lead to endocytic uptake of CPPs. By treatment of cells with octaarginine (R8), enhanced clustering of syndecan-4 on plasma membranes and binding of protein kinase Cα (PKCα) to the cytoplasmic domain of syndecan-4 were observed; these events potentially lead to the macropinocytic uptake of R8. The cytoplasmic V domain of syndecan-4 made a significant contribution to the cellular uptake of R8, whereas the cytoplasmic C1 and C2 domains were not involved in the process.

Novel nootropic drug sunifiram enhances hippocampal synaptic efficacy via glycine‐binding site of N‐methyl‐D‐aspartate receptor

Sunifiram is a novel pyrrolidone nootropic drug structurally related to piracetam, which was developed for neurodegenerative disorder like Alzheimer's disease. Sunifiram is known to enhance cognitive function in some behavioral experiments such as Morris water maze task. To address question whether sunifiram affects N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic function in the hippocampal CA1 region, we assessed the effects of sunifiram on NMDAR-dependent long-term potentiation (LTP) by electrophysiology and on phosphorylation of synaptic proteins by immunoblotting analysis. In mouse hippocampal slices, sunifiram at 10-100 nM significantly enhanced LTP in a bell-shaped dose-response relationship which peaked at 10 nM. The enhancement of LTP by sunifiram treatment was inhibited by 7-chloro-kynurenic acid (7-ClKN), an antagonist for glycine-binding site of NMDAR, but not by ifenprodil, an inhibitor for polyamine site of NMDAR. The enhancement of LTP by sunifilam was associated with an increase in phosphorylation of α-amino-3-hydroxy-5-methylisozazole-4-propionate receptor (AMPAR) through activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and an increase in phosphorylation of NMDAR through activation of protein kinase Cα (PKCα). Sunifiram treatments at 1-1000 nM increased the slope of field excitatory postsynaptic potentials (fEPSPs) in a dose-dependent manner. The enhancement was associated with an increase in phosphorylation of AMPAR receptor through activation of CaMKII. Interestingly, under the basal condition, sunifiram treatments increased PKCα (Ser-657) and Src family (Tyr-416) activities with the same bell-shaped dose-response curve as that of LTP peaking at 10 nM. The increase in phosphorylation of PKCα (Ser-657) and Src (Tyr-416) induced by sunifiram was inhibited by 7-ClKN treatment. The LTP enhancement by sunifiram was significantly inhibited by PP2, a Src family inhibitor. Finally, when pretreated with a high concentration of glycine (300 μM), sunifiram treatments failed to potentiate LTP in the CA1 region. Taken together, sunifiram stimulates the glycine-binding site of NMDAR with concomitant PKCα activation through Src kinase. Enhancement of PKCα activity triggers to potentiate hippocampal LTP through CaMKII activation.

Branched polyethylenimine-based PKCα-responsive gene carriers

We examined in vitro performance of the branched polyethylenimine (bPEI)-based gene carriers which respond to cancer-specific activation of protein kinase Cα (PKCα) to express plasmid DNA. The carriers were synthesized straightforward by using amide bond formation between a peptide terminal carboxyl and a primary amine group of bPEI. To examine the effect of the peptide contents in the carrier, we prepared several carriers with various peptide contents. The obtained polymers form polyplexes with tighter condensation of plasmid DNA than our previous gene carriers. After internalization of the polyplexes via endocytosis, the polyplexes effectively escaped from the endosome into cytosol. Then, the polyplexes showed a clear-cut response to PKCα to release plasmid DNA for gene expression. We determined the optimum contents of the peptides in carriers as 5 mol% to achieve the clear-cut response to PKCα.

The Syndecan-4/Protein Kinase Cα Pathway Mediates Prostaglandin E2-induced Extracellular Regulated Kinase (ERK) Activation in Endothelial Cells and Angiogenesis in Vivo

Prostaglandin E2 (PGE2) is regarded as the main mediator of inflammatory symptoms. In addition, it also plays an important role in tumor growth and angiogenesis. In this study, we examined the mechanism of PGE2-induced angiogenic response. We show that in the absence of proteoglycan syndecan-4 (Sdc4), PGE2-induced ERK activation is decreased significantly, as is endothelial cell migration and cord formation in a two-dimensional Matrigel assay. In vivo, PGE2-induced angiogenesis is reduced dramatically in Sdc4(-/-) mice. The mechanism was traced to Sdc4-dependent activation of protein kinase Cα (PKCα). Transduction of an Sdc4 S183E mutant (a cytoplasmic domain mutation that blocks Sdc4-dependent PKCα activation) into Sdc4(-/-) endothelial cells was not able to rescue the loss of PGE2-induced ERK activation, whereas a transduction with full-length Sdc4 resulted in full rescue. Furthermore, PGE2-induced angiogenesis was also reduced in PKCα(-/-) mice. Taken together, these results demonstrate that PGE2-induced activation of angiogenesis is mediated via syndecan-4-dependent activation of PKCα.

Cryptococcus neoformans Activates RhoGTPase Proteins Followed by Protein Kinase C, Focal Adhesion Kinase, and Ezrin to Promote Traversal across the Blood-Brain Barrier

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis. Previous studies have demonstrated that Cryptococcus binding and invasion of human brain microvascular endothelial cells (HBMEC) is a prerequisite for transmigration across the blood-brain barrier. However, the molecular mechanism involved in the cryptococcal blood-brain barrier traversal is poorly understood. In this study we examined the signaling events in HBMEC during interaction with C. neoformans. Analysis with inhibitors revealed that cryptococcal association, invasion, and transmigration require host actin cytoskeleton rearrangement. Rho pulldown assays revealed that Cryptococcus induces activation of three members of RhoGTPases, e.g. RhoA, Rac1, and Cdc42, and their activations are required for cryptococcal transmigration across the HBMEC monolayer. Western blot analysis showed that Cryptococcus also induces phosphorylation of focal adhesion kinase (FAK), ezrin, and protein kinase C α (PKCα), all of which are involved in the rearrangement of host actin cytoskeleton. Down-regulation of FAK, ezrin, or PKCα by shRNA knockdown, dominant-negative transfection, or inhibitors significantly reduces cryptococcal ability to traverse the HBMEC monolayer, indicating their positive role in cryptococcal transmigration. In addition, activation of RhoGTPases is the upstream event for phosphorylation of FAK, ezrin, and PKCα during C. neoformans-HBMEC interaction. Taken together, our findings demonstrate that C. neoformans activates RhoGTPases and subsequently FAK, ezrin, and PKCα to promote their traversal across the HBMEC monolayer, which is the critical step for cryptococcal brain infection and development of meningitis.

Deficiency of Capn4 Gene Inhibits Nuclear Factor-κB (NF-κB) Protein Signaling/Inflammation and Reduces Remodeling after Myocardial Infarction

Calpain has been implicated in acute myocardial injury after myocardial infarction (MI). However, the causal relationship between calpain and post-MI myocardial remodeling has not been fully understood. This study examined whether deletion of Capn4, essential for calpain-1 and calpain-2 activities, reduces myocardial remodeling and dysfunction following MI, and if yes, whether these effects of Capn4 deletion are associated with NF-κB signaling and inflammatory responses in the MI heart. A novel mouse model with cardiomyocyte-specific deletion of Capn4 (Capn4-ko) was employed. MI was induced by left coronary artery ligation. Deficiency of Capn4 dramatically reduced the protein levels and activities of calpain-1 and calpain-2 in the Capn4-ko heart. In vivo cardiac function was relatively improved in Capn4-ko mice at 7 and 30 days after MI when compared with their wild-type littermates. Deletion of Capn4 reduced apoptosis, limited infarct expansion, prevented left ventricle dilation, and reduced mortality in Capn4-ko mice. Furthermore, cardiomyocyte cross-sectional areas and myocardial collagen deposition were significantly attenuated in Capn4-ko mice, which were accompanied by down-regulation of hypertrophic genes and profibrotic genes. These effects of Capn4 knock-out correlated with restoration of IκB protein and inhibition of NF-κB activation, leading to suppression of proinflammatory cytokine expression and inflammatory cell infiltration in the Capn4-ko heart after MI. In conclusion, deficiency of Capn4 reduces adverse myocardial remodeling and myocardial dysfunction after MI. These effects of Capn4 deletion may be mediated through prevention of IκB degradation and NF-κB activation, resulting in inhibition of inflammatory responses.

Advanced Oxidation Protein Products Activate Intrarenal Renin–Angiotensin Systemviaa CD36-Mediated, Redox-Dependent Pathway

Activation of intrarenal renin-angiotensin system (RAS) has a detrimental effect on the progression of chronic kidney diseases (CKDs), although the regulation of intrarenal RAS remains unclear. The aim of the present study was to evaluate the role of advanced oxidation protein products (AOPPs) in intrarenal RAS activation. AOPPs upregulated the expression of almost all components of RAS and increased activity of angiotensin-converting enzyme in cultured proximal tubular epithelial cells. The triggering effect of AOPP-albumin was 100-times stronger than that of unmodified albumin. The effect of AOPP-albumin was mainly mediated by a CD36-dependent, redox-sensitive signaling involving activation of protein kinase Cα, NADPH oxidase, and nuclear factor-κB/activation protein-1. Chronic AOPP-albumin loading in unilateral nephrectomy rats resulted in deposition of AOPPs in renal tubular cells accompanied with local RAS activation and functional perturbations such as increase in urinary albumin excretion. Accumulation of AOPPs was also detected in human renal tubular cells and correlated with expression of angiotensin II in renal biopsies from 19 patients with IgA nephropathy. This study demonstrated for the first time that AOPPs modified albumin functions as a strong trigger of intrarenal RAS via a CD36-mediated, redox-dependent pathway. Given the fact that accumulation of AOPPs is prevalent in diabetes and CKD, targeting AOPPs could be a strategy for the therapeutic intervention of CKD. Antioxid. Redox Signal. 18, 19-35.

Fluorometric detection of protein kinase Cα activity based on phosphorylation-induced dissociation of a polyion complex

Here, we report the fluorometric detection of protein kinase Cα (PKCα) activity in a cancerous cell lysate using a polyion complex (PIC) composed of a quencher (BHQ3)-modified chondroitin sulfate [CS(X)] and a dendrimer modified with a cationic peptide substrate (FKKQGSFAKKK-NH2) and a near infrared (NIR) fluorophore (Cy5.5) (polymer 1). When polymer 1 formed the PIC with CS(X) through electrostatic interactions, the NIR fluorescence was quenched effectively via Förster resonance energy transfer (FRET) between Cy5.5 and BHQ3. However, this quenched fluorescence was recovered when the pendant peptides in polymer 1 were phosphorylated with PKCα due to the dissociation of the PIC. When PKCα was added to the PIC dispersion, a significant increase in fluorescence intensity was observed, whereas its fluorescence increase was inhibited with a PKCα inhibitor in a concentration-dependent manner. Furthermore, our PIC was robust enough to measure PKCα activity in a cancerous cellular lysate without purification. The PKCα-responsive PIC offers a simple, rapid, sensitive, and robust approach to detect PKCα activity in crude cellular lysates that would be suitable for drug screening formats and cancer diagnosis using crude cellular lysates.

Protein kinase Cα activity is important for contraction-induced FXYD1 phosphorylation in skeletal muscle

Exercise-induced phosphorylation of FXYD1 is a potential important regulator of Na(+)-K(+)-pump activity. It was investigated whether skeletal muscle contractions induce phosphorylation of FXYD1 and whether protein kinase Cα (PKCα) activity is a prerequisite for this possible mechanism. In part 1, human muscle biopsies were obtained at rest, after 30 s of high-intensity exercise (166 ± 31% of Vo(2max)) and after a subsequent 20 min of moderate-intensity exercise (79 ± 8% of Vo(2max)). In general, FXYD1 phosphorylation was increased compared with rest both after 30 s (P < 0.05) and 20 min (P < 0.001), and more so after 20 min compared with 30 s (P < 0.05). Specifically, FXYD1 ser63, ser68, and combined ser68 and thr69 phosphorylation were 26-45% higher (P < 0.05) after 20 min of exercise than at rest. In part 2, FXYD1 phosphorylation was investigated in electrically stimulated soleus and EDL muscles from PKCα knockout (KO) and wild-type (WT) mice. Contractile activity caused FXYD1 ser68 phosphorylation to be increased (P < 0.001) in WT soleus muscles but to be reduced (P < 0.001) in WT extensor digitorum longus. In contrast, contractile activity did not affect FXYD1 ser68 phosphorylation in the KO mice. In conclusion, exercise induces FXYD1 phosphorylation at multiple sites in human skeletal muscle. In mouse muscles, contraction-induced changes in FXYD1 ser68 phosphorylation are fiber-type specific and dependent on PKCα activity.

Up-Regulation of A-Type Potassium Currents Protects Neurons Against Cerebral Ischemia

Excitotoxicity is the major cause of many neurologic disorders including stroke. Potassium currents modulate neuronal excitability and therefore influence the pathological process. A-type potassium current (I(A)) is one of the major voltage-dependent potassium currents, yet its roles in excitotoxic cell death are not well understood. We report that, following ischemic insults, the I(A) increases significantly in large aspiny (LA) neurons but not medium spiny (MS) neurons in the striatum, which correlates with the higher resistance of LA neurons to ischemia. Activation of protein kinase Cα increases I(A) in LA neurons after ischemia. Cultured neurons from transgenic mice lacking both Kv1.4 and Kv4.2 subunits exhibit an increased vulnerability to ischemic insults. Increase of I(A) by recombinant expression of Kv1.4 or Kv4.2 is sufficient in improving the survival of MS neurons against ischemic insults both in vitro and in vivo. These results, taken together, provide compelling evidence for a protective role of I(A) against ischemia.

ATP release due to Thy-1–integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation

Thy-1, an abundant mammalian glycoprotein, interacts with αvβ3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Cα upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between αvβ3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion.

Vitamin E isoforms directly bind PKCα and differentially regulate PKCα activity. (112.8)

Abstract Tocopherol isoforms of vitamin E differentially regulate inflammation and vascular cell adhesion molecule (VCAM)-1 signaling during leukocyte transendothelial migration. During VCAM-1 signaling, tocopherols modulate oxidative activation of protein kinase Cα (PKCα). Although tocopherols are antioxidants, it is not known whether tocopherols also directly regulate PKCα; therefore, we determined whether α-tocopherol and γ-tocopherol directly regulate non-oxidative or oxidative activation of purified PKCα. In a non-oxidative environment, phosphatidylserine (PS)/calcium cofactor-induced PKCα activity was increased by γ-tocopherol but not by α-tocopherol. In contrast, PS-independent oxidative-activation of PKCα was inhibited by α-tocopherol and by 10-fold higher concentrations of γ-tocopherol. In binding studies, lipid vesicles containing α-tocopherol or γ-tocopherol increased binding to the C2 domain (the PS binding domain), but not the C1b domain, of PKCα. To determine direct binding of tocopherols to PKCα in the absence of other lipids, we measured binding of a fluorescent α-tocopherol (NBD-α-tocopherol) to PKCα. Consistent with vesicle binding studies, NBD-α-tocopherol and PS competed for binding to PKCα, indicating that α-tocopherol binds the C2 domain. In summary, α-tocopherol and γ-tocopherol bind the C2 domain of PKCα, but differentially regulate PKCα activity. Therefore, α-tocopherol and γ-tocopherol can function as agonists or antagonists at the C2 domain of PKCα.