Fe3+ Facilitates Endocytic Internalization of Extracellular Aß1-42 and Enhances Aß1-42-Induced Caspase-3/Caspase-4 Activation and Neuronal Cell Death.

Amyloid ß (Aß) peptide is a critical causative factor in Alzheimer's disease (AD) and of a variety of fragmented Aß peptides Aß1-42 thought to exhibit the most neurotoxic effect. The present study investigated the effects of Fe3+ on Aß1-42 internalization and Aß1-42-induced caspase activation and neurotoxicity using mouse hippocampal slices and cultured PC-12 cells. Extracellularly applied Aß1-42 increased the cell-associated Aß1-42 levels in a concentration-dependent manner, and the effect was enhanced by adding Fe3+. Fe3+-induced enhancement of the cell-associated Aß1-42 levels was significantly inhibited by the endocytosis inhibitors dynasore and methyl-ß-cyclodextrin. Aß1-42 reduced PC-12 cell viability in a concentration-dependent manner, and further reduction of the cell viability was obtained with Fe3+. Aß1-42-induced reduction of cell viability was not affected by A187, an antagonist of amylin-3 receptor. Aß1-42 activated caspase-3, caspase-4, and caspase-8 to a variety of degrees and Fe3+ further enhanced Aß1-42-induced activation of caspase-3 and caspase-4. Taken together, these results indicate that Fe3+ accelerates endocytic internalization of extracellular Aß1-42, enhances Aß1-42-induced caspase-3/caspase-4 activation, and promotes Aß1-42-induced neuronal cell death, regardless of amylin receptor.

IL-33 suppresses GSK-3ß activation through an ST2-independent MyD88/TRAF6/RIP/PI3K/Akt pathway.

AIMS: The present study was conducted to explore the effect of interleukin-33 (IL-33) on glycogen synthase kinase-3ß (GSK-3ß) activation involving Tau phosphorylation, a critical causative factor for Alzheimer's disease (AD). MAIN METHODS: Experiments were performed using PC-12 cells. Target proteins were knocked-down by transfecting with the siRNA for each protein. The kinase activities were assessed by monitoring phosphorylation of Thr308 and Ser473 for Akt and phosphorylation of Ser9 and Tyr216 for GSK-3ß in the Western blotting. KEY FINDINGS: Exogenously applied IL-33 activated Akt and inactivated GSK-3ß. IL-33-induced Akt activation and GSK-3ß inactivation were significantly inhibited by knocking-down myeloid differentiation factor 88 (MyD88), tumor necrosis factor receptor associated factor 6 (TRAF6), receptor-interacting protein (RIP), or phosphatidylinositol 3 kinase (PI3K). IL-33 neutralized amyloid ß1-42 (Aß1-42)-induced Akt inactivation and GSK-3ß activation. SIGNIFICANCE: The results of the present study show that IL-33 inactivates GSK-3ß through an ST2-independent MyD88/TRAF6/RIP/PI3K/Akt pathway and inhibits Aß1-42-induced GSK-3ß activation. This suggests that IL-33 could restrain GSK-3ß-mediated Tau phosphorylation in AD.

Profilin facilitates PKCε activation by accelerating ATP supply.

Profilin catalyzes the exchange of actin-bound ADP to ATP. The present study investigated the role of profilin in PKCε activation. Profilin associated with PKCε in differentiated PC-12 cells under the basal conditions, which was inhibited by the PKC inhibitor GF109203X. The selective PKCε activator DCP-LA markedly increased the association, which was clearly prevented by GF109203X. The basal PKC activity in PC-12 cells was attenuated by knocking-down profilin, while the basal activities of PKA and CaMKII were not affected. DCP-LA enhanced the PKC activity to approximately 3.5 folds of the basal levels, and the effect was suppressed by knocking-down profilin. In the cell-free system, PKCε was not activated by profilin alone. DCP-LA activated PKCε in an ATP concentration (2-500 µM)-dependent manner, and addition of profilin shifted the ATP concentration/DCP-LA-induced PKCε activity relation curve to the left (the direction of lower ATP concentrations). Taken together, the results of the present study indicate that profilin binds to activated PKCε and facilitates PKCε activation by accelerating ATP supply to PKCε.

Dioleoylphosphoethanolamine Retains Cell Surface GLUT4 by Inhibiting PKCα-Driven Internalization.

BACKGROUND/AIMS: Phosphatidylethanolamine, a component of the plasma membrane, regulates diverse cellular processes. The present study investigated the role of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in the trafficking of the glucose transporter GLUT4 and the glucose homeostasis. METHODS: Monitoring of GLUT4 trafficking, GLUT4 internalization assay, and glucose uptake assay were carried out using differentiated 3T3-L1-GLUT4myc adipocytes. Akt1/2 and PKC isozymes were knocked-down by transfecting each siRNA. Cell-free PKC assay and in situ PKCα assay with a FRET probe were carried out. Oral glucose tolerance test (OGTT) was performed using BKS.Cg-+Lepdb/+Lebdb/Jcl mice, an animal model of type 2 diabetes mellitus (DM). RESULTS: DOPE increased cell surface localization of the glucose transporter GLUT4 in differentiated 3T3-L1-GLUT4myc adipocytes, regardless of Akt activation. Likewise, PKCα deficiency increased cell surface localization of GLUT4, that occludes the effect of DOPE. DOPE clearly suppressed phorbol 12-myristate 13-acetate-induced PKCα activation in the cell-free and in situ PKC assay. DOPE and PKCα deficiency cancelled endocytic internalization of GLUT4 localized on the plasma membrane after insulin stimulation. DOPE significantly enhanced glucose uptake into cells. A similar effect was obtained by knocking-down PKCα, that occludes the effect of DOPE. In OGTT, oral administration with DOPE effectively restricted an increase in the blood glucose levels after glucose loading in type 2 DM model mice. CONCLUSION: The results of the present study show that DOPE retains cell surface GLUT4 by suppressing PKCα-driven endocytic internalization of GLUT4, to enhance glucose uptake into cells and restrict an increase in the blood glucose levels after glucose loading in type 2 DM.

PKCε Increases Extracellular Elastin and Fibulin-5/DANCE in Dermal Fibroblasts.

BACKGROUND/AIMS: In the earlier study, the selective PKCε activator DCP-LA increased elastic fibres in the dermis of HR-1 hairless mice. As a process of elastic fibre formation, tropoelastin, an elastin monomer, is secreted into the extracellular space. Secreted tropoelastin is delivered to the microfibrils by fibulin-5/developmental arteries and neural crest epidermal growth factor-like (DANCE) and undergoes self-association. Then, tropoelastin assembles around the microfibrils, growing into elastin and elastic fibres by lysyl oxidase (LOX)- or LOX-like (LOXL)-mediated cross-linking. The present study was conducted to understand the mechanism underlying DCP-LA-induced increase in elastin/elastic fibre. METHODS: Western blotting, immunocytochemistory, and real-time reverse transcription-polymerase chain reaction (RT-PCR) were carried out in cultured human dermal fibroblasts. PKCε, mammalian target of rapamycin complex (mTOR), and p70 S6 kinase (S6K) were knocked-down by transfecting each siRNA. RESULTS: DCP-LA increased elastin and fibulin-5/DANCE in a treatment time (6-24 h)- and a bell-shaped concentration (1 nM-1 µM)-dependent manner in the culture medium of human dermal fibroblasts. DCP-LA markedly increased elastic fibres in the extracellular space of cultured fibroblasts. DCP-LA-induced increase in extracellular elastin and fibulin-5/DANCE was abolished by a PKC inhibitor or knocking-down PKCε. DCP-LA did not affect expression of mRNAs for tropoelastin and fiblin-5/DANCE in cultured fibroblasts. DCP-LA-induced increase in extracellular elastin and fibulin-5/DANCE was not inhibited by the protein synthesis inhibitor cycloheximide or by knocking-down mTOR and S6K. DCP-LA never increased extracellular elastin in the presence of elastase, that breaks down elastin. An inhibitor of matrix metalloproteinase 9, that degrades multiple extracellular matrix components including elastin, had no effect on the basal levels and the DCP-LA-induced increase levels of extracellular elastin. CONCLUSION: The results of the present study indicate that PKCε, activated by DCP-LA, increases elastin and fibulin-5/DANCE in the extracellular space of cultured fibroblasts by the mechanism independent of transcriptional and translational modulation or inhibition of elastolysis.

IL-33 Acts to Express Schaffer Collateral/CA1 LTP and Regulate Learning and Memory by Targeting MyD88.

Interleukin-33 (IL-33) is recognized to transmit a signal through a heterodimeric receptor complex ST2/interleukin-1 receptor accessory protein (IL-1RAcP) bearing activation of myeloid differentiation factor 88 (MyD88). High-frequency stimulation to the Schaffer collateral induced long-term potentiation (LTP) in the CA1 region of hippocampal slices from wild-type control mice. Schaffer collateral/CA1 LTP in IL-33-deficient mice was significantly suppressed, which was neutralized by application with IL-33. Similar suppression of the LTP was found with MyD88-deficient mice but not with ST2-deficient mice. In the water maze test, the acquisition latency in IL-33-deficient and MyD88-deficient mice was significantly prolonged as compared with that in wild-type control mice. Moreover, the retention latency in MyD88-deficient mice was markedly prolonged. In contrast, the acquisition and retention latencies in ST2-deficient mice were not affected. Taken together, these results show that IL-33 acts to express Schaffer collateral/CA1 LTP relevant to spatial learning and memory in a MyD88-dependent manner and that the LTP might be expressed through an IL-1R1/IL-1RAcP-MyD88 pathway in the absence of ST2.

DCP-LA Exerts an Antiaging Action on the Skin.

The present study assessed the possibility for the linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) as an antiaging compound for the skin by assaying senescence-associated ß-galactosidase (SA-ß-Gal), a biomarker of senescence and cell viability. The nitric oxide (NO) donor sodium nitroprusside (SNP) increased in SA-ß-Gal-positive cells in cultured human fibroblasts and mouse keratinocytes, and DCP-LA significantly inhibited the effect of SNP. Moreover, SNP induced cell death in cultured mouse keratinocytes, and DCP-LA significantly prevented NO stress-induced death of keratinocytes. Taken together, these results indicate that DCP-LA exerts an antiaging action on the skin.

The phosphatidylethanolamine derivative diDCP-LA-PE mimics intracellular insulin signaling.

Insulin facilitates glucose uptake into cells by translocating the glucose transporter GLUT4 towards the cell surface through a pathway along an insulin receptor (IR)/IR substrate 1 (IRS-1)/phosphatidylinositol 3 kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt axis. The newly synthesized phosphatidylethanolamine derivative 1,2-O-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-sn-glycero-3-phosphatidylethanolamine (diDCP-LA-PE) has the potential to inhibit protein tyrosine phosphatase 1B (PTP1B) and to directly activate PKCζ, an atypical isozyme, and PKCε, a novel isozyme. PTP1B inhibition enhanced insulin signaling cascades downstream IR/IRS-1 by preventing tyrosine dephosphorylation. PKCζ and PKCε directly activated Akt2 by phosphorylating at Thr309 and Ser474, respectively. diDCP-LA-PE increased cell surface localization of GLUT4 and stimulated glucose uptake into differentiated 3T3-L1 adipocytes, still with knocking-down IR or in the absence of insulin. Moreover, diDCP-LA-PE effectively reduced serum glucose levels in type 1 diabetes (DM) model mice. diDCP-LA-PE, thus, may enable type 1 DM therapy without insulin injection.

N-Ethylmaleimide Dissociates α7 ACh Receptor from a Complex with NSF and Promotes Its Delivery to the Presynaptic Membrane.

N-Ethylmaleimide (NEM)-sensitive factor (NSF) associates with soluble NSF attachment protein (SNAP), that binds to SNAP receptors (SNAREs) including syntaxin, SNAP25, and synaptobrevin. The complex of NSF/SNAP/SNAREs plays a critical role in the regulation of vesicular traffic. The present study investigated NEM-regulated α7 ACh receptor translocation. NSF associated with ß-SNAP and the SNAREs syntaxin 1 and synaptobrevin 2 in the rat hippocampus. NSF also associated with the α7 ACh receptor subunit, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1 and GluA2, and the γ-aminobutyric acid A (GABAA) receptor γ2 subunit. NEM, an inhibitor of NSF, significantly dissociated the α7 ACh receptor subunit from a complex with NSF and increased cell surface localization of the receptor subunit, but such effect was not obtained with the GluA1, GluA2 or γ2 subunits. NEM, alternatively, dissociated synaptobrevin 2 from an assembly of NSF/ß-SNAP/syntaxin 1/synaptobrevin 2. NEM significantly increased the rate of nicotine-triggered AMPA receptor-mediated miniature excitatory postsynaptic currents, without affecting the amplitude, in rat hippocampal slices. The results of the present study indicate that NEM releases the α7 ACh receptor subunit and synaptobrevin 2 from an assembly of α7 ACh receptor subunit/NSF/ß-SNAP/syntaxin 1/synaptobrevin 2, thereby promoting delivery of the α7 ACh receptor subunit to presynaptic membrane.

HUHS1015 Suppresses Colonic Cancer Growth by Inducing Necrosis and Apoptosis in Association with Mitochondrial Damage.

BACKGROUND: The newly-synthesized naftopidil analog HUHS1015 suppresses tumor growth and induces apoptosis of cells from a variety of cancer types. The present study was conduced to assess the effect of HUHS1015 on human colonic cancer cells and to clarify the underlying mechanism. RESULTS: HUHS1015 reduced cell viability of Caco-2 and CW2 human colonic cancer cell lines in a concentration (0.3-100 mM)-dependent manner. HUHS1015 increased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells in both cell lines. In flow cytometry using propidium iodide and annexin V, HUHS1015 significantly increased the populations of cells undergoing primary necrosis, early apoptosis, and late apoptosis/secondary necrosis in both cell lines. In the cell-cycle analysis, HUHS1015 increased the proportion of the sub-G1 phase of cell, which corresponds to apoptotic cells. HUHS1015 perturbed the mitochondrial membrane potential and reduced the intracellular ATP level. HUHS1015 activated caspases 3, -4, -8, and -9, particularly caspase-3. HUHS1015 promoted cytochrome c release from the mitochondria. HUHS1015 significantly inhibited tumor growth in mice inoculated with CW2 cells. CONCLUSION: HUHS1015 induces necrosis by lowering the intracellular ATP level in association with mitochondrial damage and caspase-dependent apoptosis. This occurs in part by stimulating cytochrome c release from the mitochondria to activate caspase-9 followed by the effector caspase-3, responsible for suppression of colonic cancer proliferation in the mouse xenograft model.

Combination of PKCε Activation and PTP1B Inhibition Effectively Suppresses Aß-Induced GSK-3ß Activation and Tau Phosphorylation.

Glycogen synthase kinase-3ß (GSK-3ß) is a key element to phosphorylate tau and form neurofibrillary tangles (NFTs) found in tauopathies including Alzheimer's disease (AD). A current topic for AD therapy is focused upon how to prevent tau phosphorylation. In the present study, PKCε activated Akt and inactivated GSK-3ß by directly interacting with each protein. Inhibition of protein tyrosine phosphatase 1B (PTP1B), alternatively, caused an enhancement in the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), allowing activation of Akt through a pathway along an IRS-1/phosphatidylinositol 3 kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt axis, to phosphorylate and inactivate GSK-3ß. Combination of PKCε activation and PTP1B inhibition more sufficiently activated Akt and inactivated GSK-3ß than each independent treatment, to suppress amyloid ß (Aß)-induced tau phosphorylation and ameliorate spatial learning and memory impairment in 5xFAD transgenic mice, an animal model of AD. This may represent an innovative strategy for AD therapy.

Phosphatidylinositol Derivatives Induce Gastric Cancer Cell Apoptosis by Accumulating AIF and AMID in the Nucleus.

BACKGROUND: The present study investigated the mechanism underlying the apoptosis of MKN28 human gastric cancer cells induced by the phosphatidylinositol (PI) derivative 1,2-O-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-sn-glycero-3-phosphatidyl-D-1-inositol (diDCP-LA-D-PI) and its enantiomer diDCP-LA-L-PI. MATERIALS AND METHODS: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, enzymatic caspase assay, real-time reverse transcription-polymerase chain reaction (RT-PCR), and western blotting were carried-out. RESULTS: Both diDCP-LA-D-PI and diDCP-LA-L-PI induced caspase-independent apoptosis of MKN28 cells, with the potential for diDCP-LA-L-PI being much greater than that of diDCP-LA-D-PI. diDCP-LA-D-PI and diDCP-LA-L-PI accumulated apoptosis-inducing factor (AIF) and AIF-homologous mitochondrion-associated inducer of death (AMID) in the nucleus. CONCLUSION: diDCP-LA-D-PI and diDCP-LA-L-PI induce caspase-independent apoptosis of MKN28 cells by accumulating AIF and AMID in the nucleus, with different potentials.

Diarachidonoylphosphoethanolamine induces apoptosis of malignant pleural mesothelioma cells through a Trx/ASK1/p38 MAPK pathway.

1,2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE) induces both necrosis/necroptosis and apoptosis of NCI-H28 malignant pleural mesothelioma (MPM) cells. The present study was conducted to understand the mechanism for DAPE-induced apoptosis of NCI-H28 cells. DAPE induced caspase-independent apoptosis of NCI-H28 malignant pleural mesothelioma (MPM) cells, and the effect of DAPE was prevented by antioxidants or an inhibitor of NADPH oxidase (NOX). DAPE generated reactive oxygen species (ROS) and inhibited activity of thioredoxin (Trx) reductase (TrxR). DAPE decreased an association of apoptosis signal-regulating kinase 1 (ASK1) with thioredoxin (Trx), thereby releasing ASK1. DAPE activated p38 mitogen-activated protein kinase (MAPK), which was inhibited by an antioxidant or knocking-down ASK1. In addition, DAPE-induced NCI-H28 cell death was also prevented by knocking-down ASK1. Taken together, the results of the present study indicate that DAPE stimulates NOX-mediated ROS production and suppresses TrxR activity, resulting in the decrease of reduced Trx and the dissociation of ASK1 from a complex with Trx, allowing sequential activation of ASK1 and p38 MAPK, to induce apoptosis of NCI-H28 MPM cells.

Amyloid-ß peptide increases cell surface localization of α7 ACh receptor to protect neurons from amyloid ß-induced damage.

Amyloid-ß peptide 1-42 (Aß1-42) reduced PC-12 cell viability in a concentration (1-10 µM)- and treatment time (48-72 h)-dependent manner. Nicotine prevented Aß1-42-induced PC-12 cell death, but conversely, the α7 ACh receptor antagonist α-bungarotoxin enhanced Aß1-42-induced cell toxicity. Extracellularly applied Aß1-42 significantly increased cell surface localization of α7 ACh receptor in PC-12 cells as compared with that for non-treated control cells. Cell surface localization of α7 ACh receptor in the brain of 5xFAD mouse, an animal model of Alzheimer's disease (AD), apparently increased in an age (1-12 months)-dependent manner in association with increased accumulation of Aß1-42 in the plasma membrane component. Taken together, these results indicate that Aß1-42 promotes translocation of α7 ACh receptor towards the cell surface and that α7 ACh receptor rescues neuronal cells from Aß1-42-induced damage.

Anticancer effect of adenosine on gastric cancer via diverse signaling pathways.

Extracellular adenosine induces apoptosis in a variety of cancer cells via intrinsic and extrinsic pathways. In the former pathway, adenosine uptake into cells triggers apoptosis, and in the latter pathway, adenosine receptors mediate apoptosis. Extracellular adenosine also induces apoptosis of gastric cancer cells. Extracellular adenosine is transported into cells through an adenosine transporter and converted to AMP by adenosine kinase. In turn, AMP activates AMP-activated protein kinase (AMPK). AMPK is the factor responsible for caspase-independent apoptosis of GT3-TKB gastric cancer cells. Extracellular adenosine, on the other hand, induces caspase-dependent apoptosis of MKN28 and MKN45 gastric cancer cells by two mechanisms. Firstly, AMP, converted from intracellularly transported adenosine, initiates apoptosis, regardless of AMPK. Secondly, the A3 adenosine receptor, linked to Gi/Gq proteins, mediates apoptosis by activating the Gq protein effector, phospholipase Cγ, to produce inositol 1,4,5-trisphosphate and diacylglycerol, which activate protein kinase C. Consequently, the mechanisms underlying adenosine-induced apoptosis vary, depending upon gastric cancer cell types. Understand the contribution of each downstream target molecule of adenosine to apoptosis induction may aid the establishment of tailor-made chemotherapy for gastric cancer.

Adipose-derived stem cell-conditioned medium ameliorates antidepression-related behaviors in the mouse model of Alzheimer's disease.

The present study investigated the effect of adipose-derived stem cell-conditioned medium (ASC-CM) on behavioral disorders in 5xFAD transgenic mice, a model of Alzheimer's disease (AD). The immobility time in the tail suspension and forced swim tests for 5xFAD mice was shorter than that for wild-type mice. Intravenous injection with ASC-CM restored the shortened immobility time for 5xFAD mice to the normal levels or to an extent, being still persistent 4 weeks after injection. ASC-CM significantly suppressed phosphorylation of Akt at Ser473 and glycogen synthase kinase 3ß (GSK-3ß) at Ser9 in the hypothalamus of 5xFAD mice, without affecting Tau phosphorylation, as compared with that for control 5xFAD mice without ASC-CM injection. ASC-CM did not affect cell surface localization of the N-methyl-d-aspartate (NMDA) receptor subunits NR1, -2A, and -2B both in the hippocampus and hypothalamus of 5xFAD mice. The results of the present study show that ASC-CM ameliorates antidepression-related behaviors in 5xFAD mice, perhaps by inhibiting Akt and activating GSK-3ß.

Rac1 and ROCK are implicated in the cell surface delivery of GLUT4 under the control of the insulin signal mimetic diDCP-LA-PE.

The phosphatidylethanolamine derivative 1,2-O-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-sn-glycero-3-phosphatidylethanolamine (diDCP-LA-PE) promoted GLUT4 translocation to the cell surface in differentiated 3T3-L1-GLUT4myc adipocytes through a pathway along a phosphatidylinositol 3-kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt axis, that mimics insulin signaling. Moreover, diDCP-LA-PE-induced GLUT4 translocation was suppressed by inhibitors of the Rho GTPase Rac1 and Rho-associated coiled-coil-containing protein kinase (ROCK) or knocking-down Rac1 and ROCK1. The results of the present study show that Rac1 and ROCK are critical for regulation of GLUT4 trafficking by diDCP-LA-PE as well as insulin.

DCP-LA Activates Cytosolic PKCε by Interacting with the Phosphatidylserine Binding/Associating Sites Arg50 and Ile89 in the C2-Like Domain.

BACKGROUND/AIMS: The linoleic acid derivative DCP-LA selectively and directly activates PKCε. The present study aimed at understanding the mechanism of DCP-LA-induced PKCε activation. METHODS: Point mutation in the C2-like domain on PKCε was carried out, and each kinase activity was monitored in PC-12 cells using a föerster resonance energy transfer (FRET) probe with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) at the N- and C-terminal ends of PKCε, respectively, or in the cell-free systems using a reversed phase high-performance liquid chromatography (HPLC). Intracellular PKCε mobilization was monitored in PC-12 cells using mRuby-conjugated PKCε. DCP-LA binding to PKCε was assayed using a fluorescein conjugated to DCP-LA at the carboxyl-terminal end (Fluo-DCP). Uptake of DCP-LA into cells was measured in PC-12 ells. RESULTS: In the FRET analysis, DCP-LA decreased the ratio of YFP signal intensity/CFP signal intensity in PC-12 cells and in the cell-free kinase assay, DCP-LA increased area of phosphorylated PKC substrate peptide, indicating DCP-LA-induced PKCε activation. These effects were significantly suppressed by replacing Arg50 and Ile89 by Ala or Asn in the C2-like domain of PKCε. In the fluorescent cytochemistry, DCP-LA did not affect intracellular PKCε distribution. In the cell-free binding assay, Fluo-DCP, that had no effect on the potential for PKCε activation, bound to PKCε, and the binding was inhibited only by mutating Ile89. Extracellularly applied DCP-LA was taken up into cells in a concentration-dependent manner. Although no activation was obtained in the cell-free kinase assay, the broad PKC activator PMA activated PKCε in PC-12 cells in association with translocation towards the cell surface, which was inhibited by mutating I89A. CONCLUSION: Unlike PMA DCP-LA activates cytosolic PKCε by binding to the phosphatidylserine binding/associating sites Arg50 and Ile89, possibly at the carboxyl-terminal end and the cyclopropane rings, respectively.