PIKE-mediated PI3-kinase activity is required for AMPA receptor surface expression

AMPAR (α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor) is an ion channel involved in the formation of synaptic plasticity. However, the molecular mechanism that couples plasticity stimuli to the trafficking of postsynaptic AMPAR remains poorly understood. Here, we show that PIKE (phosphoinositide 3-kinase enhancer) GTPases regulate neuronal AMPAR activity by promoting GluA2/GRIP1 association. PIKE-L directly interacts with both GluA2 and GRIP1 and forms a tertiary complex upon glycine-induced NMDA receptor activation. PIKE-L is also essential for glycine-induced GluA2-associated PI3K activation. Genetic ablation of PIKE (PIKE(-/-)) in neurons suppresses GluA2-associated PI3K activation, therefore inhibiting the subsequent surface expression of GluA2 and the formation of long-term potentiation. Our findings suggest that PIKE-L is a critical factor in controlling synaptic AMPAR insertion.     

Matrine attenuates high‐fat diet‐induced in vivo and ox‐LDL‐induced in vitro vascular injury by regulating the PKCα/eNOS and PI3K/Akt/eNOS pathways

Lipid metabolism disorders lead to vascular endothelial injury. Matrine is an alkaloid that has been used to improve obesity and diabetes and for the treatment of hepatitis B. However, its effect on lipid metabolism disorders and vascular injury is unclear. Here, we investigated the effect of matrine on high‐fat diet fed mice and oxidized low‐density lipoprotein (ox‐LDL)‐induced human umbilical vein endothelial cells (HUVECs). Computational virtual docking analyses, phosphoinositide 3‐kinase (PI3K) and protein kinase C‐α (PKCα) inhibitors were used to localize matrine in vascular injuries. The results showed that matrine‐treated mice were more resistant to abnormal lipid metabolism and inflammation than vehicle‐treated mice and exhibited significantly alleviated ox‐LDL‐stimulated dysfunction of HUVECs, restored diminished nitric oxide release, decreased reactive oxygen species generation and increased expression phosphorylation of AKT‐Ser473 and endothelial nitric oxide synthase (eNOS)‐Ser1177. Matrine not only up‐regulates eNOS‐Ser1177 but also down‐regulates eNOS‐Thr495, a PKCα‐controlled negative regulator of eNOS. Using computational virtual docking analyses and biochemical assays, matrine was also shown to influence eNOS/NO via PKCα inhibition. Moreover, the protective effects of matrine were significantly abolished by the simultaneous application of PKCα and the PI3K inhibitor. Matrine may thus be potentially employed as a novel therapeutic strategy against high‐fat diet‐induced vascular injury.     

C-5 Substituted heteroaryl 3-pyridinecarbonitriles as PKCθ inhibitors: Part I

We earlier reported that 3-pyridinecarbonitriiles with a 4-methylindolyl-5-amino group at C-4 and a phenyl group at C-5 were inhibitors of PKCθ. Keeping the group at C-4 of the pyridine core constant, we varied the water solubilizing group on the phenyl ring at C-5 and then replaced the C-5 phenyl ring with several monocyclic heteroaryl rings, including furan, thiophene and pyridine. Analog 6e with a 4-methylindol-5-ylamino group at C-4 and a 5-[(4-methylpiperazin-1-yl)methyl]-2-furyl group C-5 had an IC₅₀ value of 4.5 nM for the inhibition of PKCθ.     

Recognition of an intra‐chain tandem 14‐3‐3 binding site within PKCε

The phosphoserine/threonine binding protein 14‐3‐3 stimulates the catalytic activity of protein kinase C‐ε (PKCε) by engaging two tandem phosphoserine‐containing motifs located between the PKCε regulatory and catalytic domains (V3 region). Interaction between 14‐3‐3 and this region of PKCε is essential for the completion of cytokinesis. Here, we report the crystal structure of 14‐3‐3ζ bound to a synthetic diphosphorylated PKCε V3 region revealing how a consensus 14‐3‐3 site and a divergent 14‐3‐3 site cooperate to bind to 14‐3‐3 and so activate PKCε. Thermodynamic data show a markedly enhanced binding affinity for two‐site phosphopeptides over single‐site 14‐3‐3 binding motifs and identifies Ser 368 as a gatekeeper phosphorylation site in this physiologically relevant 14‐3‐3 ligand. This dual‐site intra‐chain recognition has implications for other 14‐3‐3 targets, which seem to have only a single 14‐3‐3 motif, as other lower affinity and cryptic 14‐3‐3 gatekeeper sites might exist.     

PLCγ1–PKCγ Signaling‐Mediated Hsp90α Plasma Membrane Translocation Facilitates Tumor Metastasis

The 90‐kDa heat shock protein (Hsp90α) has been identified on the surface of cancer cells, and is implicated in tumor invasion and metastasis, suggesting that it is a potentially important target for tumor therapy. However, the regulatory mechanism of Hsp90α plasma membrane translocation during tumor invasion remains poorly understood. Here, we show that Hsp90α plasma membrane expression is selectively upregulated upon epidermal growth factor (EGF) stimulation, which is a process independent of the extracellular matrix. Abrogation of EGF‐mediated activation of phospholipase (PLCγ1) by its siRNA or inhibitor prevents the accumulation of Hsp90α at cell protrusions. Inhibition of the downstream effectors of PLCγ1, including Ca²⁺ and protein kinase C (PKCγ), also blocks the membrane translocation of Hsp90α, while activation of PKCγ leads to increased levels of cell‐surface Hsp90α. Moreover, overexpression of PKCγ increases extracellular vesicle release, on which Hsp90α is present. Furthermore, activation or overexpression of PKCγ promotes tumor cell motility in vitro and tumor metastasis in vivo, whereas a specific neutralizing monoclonal antibody against Hsp90α inhibits such effects, demonstrating that PKCγ‐induced Hsp90α translocation is required for tumor metastasis. Taken together, our study provides a mechanistic basis for the role for the PLCγ1–PKCγ pathway in regulating Hsp90α plasma membrane translocation, which facilitates tumor cell motility and promotes tumor metastasis.     

Signaling Defect in the Activation of Caspase-3 and PKCδ in Human TUR Leukemia Cells Is Associated with Resistance to Apoptosis

Exposure of the two related human leukemic cell lines U937 and TUR to chemotherapeutic compounds resulted in opposite effects on induction and resistance to apoptosis. Incubation of U937 cells with 1-β- -arabinofuranosylcytosine or the etoposide VP-16 was accompanied by growth arrest in G₀/G₁of the cell cycle and an accumulation of a population in the sub-G₁phase which exhibited characteristics typical for the apoptotic pathway. In contrast, human TUR leukemia cells demonstrated no significant effects after a similar treatment with Ara-C and VP-16. Thus, TUR cells continued to proliferate in the presence of these anti-cancer drugs and the number of apoptotic cells as evaluated by propidium iodide staining and the detection of internucleosomal DNA fragmentation was significantly reduced when compared to the parental U937 cells. Similar effects were observed upon serum-starvation demonstrating resistance to apoptosis in TUR cells. Whereas induction of apoptosis is regulated by a network of distinct factors including the activation of proteolytically active caspases, we investigated these pathways in both cell lines. U937 cells demonstrated activation of the 32-kDa caspase-3 upon drug treatment by cleavage into the 20-kDa activated form. However, there was no 20-kDa caspase-3 fragment detectable in TUR cells. Simultaneously, the enzymatic activity of caspase-3 was significantly increased in drug-treated U937 cells as measuredin vitroby enhanced metabolization of a fluorescence substrate andin vivoby cleavage of an appropriate substrate for caspase-3, namely, protein kinase Cδ. In contrast, there was little if any caspase-3 activation detectable in drug-treated TUR cells. Taken together, these data suggest a signaling defect in the activation of the caspase-3 proteolytic system in TUR cells upon treatment with chemotherapeutic compounds which is associated with resistance to apoptosis in these human leukemia cells.     

Role of PLD−PKCζ signaling axis in p47phox phosphorylation for activation of NADPH oxidase by angiotensin II in pulmonary artery smooth muscle cells

We sought to determine the mechanism by which angiotensin II (ANGII) stimulates NADPH oxidase‐mediated superoxide (O₂^.?) production in bovine pulmonary artery smooth muscle cells (BPASMCs). ANGII‐induced increase in phospholipase D (PLD) and NADPH oxidase activities were inhibited upon pretreatment of the cells with chemical and genetic inhibitors of PLD2, but not PLD1. Immunoblot study revealed that ANGII treatment of the cells markedly increases protein kinase C‐α (PKC‐α), ‐δ, ‐ε, and ‐ζ levels in the cell membrane. Pretreatment of the cells with chemical and genetic inhibitors of PKC‐ζ, but not PKC‐α, ‐δ, and ‐ε, attenuated ANGII‐induced increase in NADPH oxidase activity without a discernible change in PLD activity. Transfection of the cells with p47phox small interfering RNA inhibited ANGII‐induced increase in NADPH oxidase activity without a significant change in PLD activity. Pretreatment of the cells with the chemical and genetic inhibitors of PLD2 and PKC‐ζ inhibited ANGII‐induced p47phox phosphorylation and subsequently translocation from cytosol to the cell membrane, and also inhibited its association with p22phox (a component of membrane‐associated NADPH oxidase). Overall, PLD?PKCζ?p47phox signaling axis plays a crucial role in ANGII‐induced increase in NADPH oxidase‐mediated O₂^.? production in the cells.     

Regulation of protein kinase Cδ downregulation by protein kinase Cε and mammalian target of rapamycin complex 2

Phosphorylation and dephosphorylation of PKCs can regulate their activity, stability and function. We have previously shown that downregulation of PKCδ by tumor promoting phorbol esters was compromised when HeLa cells acquired resistance to cisplatin (HeLa/CP). In the present study, we have used these cells to understand the mechanism of PKCδ downregulation. A brief treatment of HeLa cells with phorbol 12,13-dibutyrate (PDBu) induced phosphorylation of PKCδ at the activation loop (Thr505), turn motif (Ser643), hydrophobic motif (Ser662) and Tyr-311 sites to a greater extent in HeLa/CP cells compared to HeLa cells. Prolonged treatment with PDBu led to downregulation of PKCδ in HeLa but not in HeLa/CP cells. The PKC inhibitor Gö 6983 inhibited PDBu-induced downregulation of PKCδ, decreased Thr505 phosphorylation and increased PKCδ tyrosine phosphorylation at Tyr-311 site. However, knockdown of c-Abl, c-Src, Fyn and Lyn had little effect on PKCδ downregulation and Tyr311 phosphorylation. Pretreatment with the phosphatidylinositol 3-kinase inhibitor Ly294002 and mTOR inhibitor rapamycin restored the ability of PDBu to downregulate PKCδ in HeLa/CP cells. Knockdown of mTOR and rictor but not raptor facilitated PKCδ downregulation. Depletion of PKCε also enhanced PKCδ downregulation by PDBu. These results suggest that downregulation of PKCδ is regulated by PKCε and mammalian target of rapamycin complex 2 (mTORC2).     

α‐tocopherol affects neuronal plasticity in adult rat dentate gyrus: The possible role of PKCδ

Hippocampus dentate gyrus (DG) is characterized by neuronal plasticity processes in adulthood, and polysialylation of NCAM promotes neuronal plasticity. In previous investigations we found that α‐tocopherol increased the PSA‐NCAM‐positive granule cell number in adult rat DG, suggesting that α‐tocopherol may enhance neuronal plasticity. To verify this hypothesis, in the present study, structural remodeling in adult rat DG was investigated under α‐tocopherol supplementation conditions. PSA‐NCAM expression was evaluated by Western blotting, evaluation of PSA‐NCAM‐positive granule cell density, and morphometric analysis of PSA‐NCAM‐positive processes. In addition, the optical density of synaptophysin immunoreactivity and the synaptic profile density, examined by electron microscopy, were evaluated. Moreover, considering that PSA‐NCAM expression has been found to be related to PKCδ activity and α‐tocopherol has been shown to inhibit PKC activity in vitro, Western blotting and immunohistochemistry followed by densitometry were used to analyze PKC. Our results demonstrated that an increase in PSA‐NCAM expression and optical density of DG molecular layer synaptophysin immunoreactivity occurred in α‐tocopherol‐treated rats. Electron microscopy analysis showed that the increase in synaptophysin expression was related to an increase in synaptic profile density. In addition, Western blotting revealed a decrease in phospho‐PKC Pan and phospho‐PKCδ, demonstrating that α‐tocopherol is also able to inhibit PKC activity in vivo. Likewise, immunoreactivity for the active form of PKCδ was lower in α‐tocopherol‐treated rats than in controls, while no changes were found in PKCδ expression. These results demonstrate that α‐tocopherol is an exogenous factor affecting neuronal plasticity in adult rat DG, possibly through PKCδ inhibition. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Synthesis, biological evaluation and molecular modelling of sulfonohydrazides as selective PI3K p110alpha inhibitors

A series of 2-methyl-5-nitrobenzenesulfonohydrazides were prepared and evaluated as inhibitors of PI3K. An isoquinoline derivative shows good selectivity for the p110α isoform over p110β and p110δ, and also demonstrates good in vitro activity in a cell proliferation assay. Molecular modelling provides a rationalisation for the observed SAR.     

The role of the Escherichia coli λ receptor in the transport of maltose and maltodextrins

The λ receptor is a peptidoglycan-associated integral protein that spans the outer membrane. Beside its function in phage λ adsorption it participates in transport. The latter function can be summarized as follows: (1) Receptor allows the nonspecific permeation of small molecules other than maltose and maltodextrins (in close analogy to a molecular sieve). Here the only criterion for selectivity is size and it has the properties of an unspecific pore. In this respect, it is similar to the outer membrane proteins Ia, Ib, and Ic, the porins. (2) It is a binding protein for maltodextrins. Binding affinity is low but increases by a factor of 500 as the chain length of the maltodextrins increases. In contrast, the affinity of the periplasmic maltose-binding protein for maltose and maltodextrins is similarly high (in the μM range). (3) In the in vitro system of liposomes, the λ receptor facilitates specifically the diffusion of maltodextrins that exceed the size limit given by its porin function. This clearly demonstrates that the λ receptor alone is able to specifically overcome the permeability barrier of the outer membrane for maltodextrins. (4) From the genetic and kinetic analysis of maltose and maltodextrin transport, it can be concluded that the λ receptor interacts with the periplasmic maltose-binding protein. (5) Electron microscopic studies indicate a location for the maltose-binding protein in the outer cell envelope. This location is dependent on the presence of the λ receptor.     

Metformin attenuates the effect of Staphylococcus aureus on airway tight junctions by increasing PKCζ‐mediated phosphorylation of occludin

Airway epithelial tight junction (TJ) proteins form a resistive barrier to the external environment, however, during respiratory bacterial infection TJs become disrupted compromising barrier function. This promotes glucose flux/accumulation into the lumen which acts as a nutrient source for bacterial growth. Metformin used for the treatment of diabetes increases transepithelial resistance (TEER) and partially prevents the effect of bacteria but the mechanisms of action are unclear. We investigated the effect of metformin and Staphylococcus aureus on TJ proteins, zonula occludins (ZO)‐1 and occludin in human airway epithelial cells (H441). We also explored the role of AMP‐activated protein kinase (AMPK) and PKCζ in metformin‐induced effects. Pretreatment with metformin prevented the S. aureus‐induced changes in ZO‐1 and occludin. Metformin also promoted increased abundance of full length over smaller cleaved occludin proteins. The nonspecific PKC inhibitor staurosporine reduced TEER but did not prevent the effect of metformin indicating that the pathway may involve atypical PKC isoforms. Investigation of TJ reassembly after calcium depletion showed that metformin increased TEER more rapidly and promoted the abundance and localization of occludin at the TJ. These effects were inhibited by the AMPK inhibitor, compound C and the PKCζ pseudosubstrate inhibitor (PSI). Metformin increased phosphorylation of occludin and acetyl‐coA‐carboxylase but only the former was prevented by PSI. This study demonstrates that metformin improves TJ barrier function by promoting the abundance and assembly of full length occludin at the TJ and that this process involves phosphorylation of the protein via an AMPK‐PKCζ pathway.     

Mitochondrial PKC‐ε deficiency promotes I/R‐mediated myocardial injury via GSK3β‐dependent mitochondrial permeability transition pore opening

Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)‐induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase‐2 (ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross‐clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout (ALDH2^?/?) and wild‐type (WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C (PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin‐related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N‐acetylcysteine (NAC) or PKC‐δ shRNA treatment on glycogen synthase kinase‐3β (GSK‐3β) activity and mitochondrial permeability transition pore (mPTP) opening were also detected. The results showed that ALDH2^?/? mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC‐ε translocation was lower in ALDH2^?/? mice than in WT mice, and PKC‐δ was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species (ROS) level, which could be reversed by NAC pre‐treatment under I/R injury. In addition, PKC‐ε inhibition caused activation of caspase9, caspase3 and Drp1Ser⁶¹⁶ in response to I/R stress. Importantly, expression of phosphorylated GSK‐3β (inactive form) was lower in ALDH2^?/? mice than in WT mice, and both were increased by NAC pre‐treatment. I/R‐induced mitochondrial translocation of GSK‐3β was inhibited by PKC‐δ shRNA or NAC pre‐treatment. In addition, mitochondrial membrane potential (?Ψ_m) was reduced in ALDH2^?/? mice after I/R, which was partly reversed by the GSK‐3β inhibitor (SB216763) or PKC‐δ shRNA. Collectively, our data provide the evidence that abnormal PKC‐ε/PKC‐δ ratio promotes the activation of Drp1 signalling, caspase cascades and GSK‐3β‐dependent mPTP opening, which results in mitochondrial injury‐triggered cardiomyocyte apoptosis and myocardial dysfuction in ALDH2^?/? mice following I/R stress.     

Structural basis of conformational variance in phosphorylated and non‐phosphorylated states of PKCβII

PKCβII activation is achieved by primary phosphorylation at three phosphorylation sites, followed by the addition of secondary messengers for full activation. Phosphorylation is essential for enzyme maturation, and the associated conformational changes are known to modulate the enzyme activation. To probe into the structural basis of conformational changes on phosphorylation of PKCβII, a comprehensive study of the changes in its complexes with ATP and ruboxistaurin was performed. ATP is a phosphorylating agent in its phosphorylation reaction, and ruboxistaurin is its specific inhibitor. This study provides insight into the differences in the important structural features in phosphorylated and non‐phosphorylated states of PKCβII. Less conformational changes when PKCβII is bound to inhibitor in comparison to when it is bound to its phosphorylating agent in both states were observed. The interactions of ruboxistaurin significant in restricting PKCβII to attain the conformational state competent for full activation are reported. Proteins 2014; 82:1332–1347. © 2013 Wiley Periodicals, Inc.     

Activation of D1 dopamine receptors increases surface expression of AMPA receptors and facilitates their synaptic incorporation in cultured hippocampal neurons

Considerable evidence indicates that neuroadaptations leading to addiction involve the same cellular processes that enable learning and memory, such as long-term potentiation (LTP), and that psychostimulants influence LTP through dopamine (DA)-dependent mechanisms. In hippocampal CA1 pyramidal neurons, LTP involves insertion of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors into excitatory synapses. We used dissociated cultures to test the hypothesis that D1 family DA receptors influence synaptic plasticity in hippocampal neurons by modulating AMPA receptor trafficking. Brief exposure (5 min) to a D1 agonist increased surface expression of glutamate receptor (GluR)1-containing AMPA receptors by increasing their rate of externalization at extrasynaptic sites. This required the secretory pathway but not protein synthesis, and was mediated mainly by protein kinase A (PKA) with a smaller contribution from Ca2+-calmodulin-dependent protein kinase II (CaMKII). Prior D1 receptor stimulation facilitated synaptic insertion of GluR1 in response to subsequent stimulation of synaptic NMDA receptors with glycine. Our results support a model for synaptic GluR1 incorporation in which PKA is required for initial insertion into the extrasynaptic membrane whereas CaMKII mediates translocation into the synapse. By increasing the size of the extrasynaptic GluR1 pool, D1 receptors may promote LTP. Psychostimulants may usurp this mechanism, leading to inappropriate plasticity that contributes to addiction-related behaviors.     

Different regulation of Purkinje cell dendritic development in cerebellar slice cultures by protein kinase Cα and ‐β

Activity of protein kinase C (PKC), and in particular the PKCγ‐isoform, has been shown to strongly affect and regulate Purkinje cell dendritic development, suggesting an important role for PKC in activity‐dependent Purkinje cell maturation. In this study we have analyzed the role of two additional Ca²⁺‐dependent PKC isoforms, PKCα and ‐β, in Purkinje cell survival and dendritic morphology in slice cultures using mice deficient in the respective enzymes. Pharmacological PKC activation strongly reduced basal Purkinje cell dendritic growth in wild‐type mice whereas PKC inhibition promoted branching. Purkinje cells from mice deficient in PKCβ, which is expressed in two splice forms by granule but not Purkinje cells, did not yield measurable morphological differences compared to respective wild‐type cells under either experimental condition. In contrast, Purkinje cell dendrites in cultures from PKCα‐deficient mice were clearly protected from the negative effects on dendritic growth of pharmacological PKC activation and showed an increased branching response to PKC inhibition as compared to wild‐type cells. Together with our previous work on the role of PKCγ, these data support a model predicting that normal Purkinje cell dendritic growth is mainly regulated by the PKCγ‐isoform, which is highly activated by developmental processes. The PKCα isoform in this model forms a reserve pool, which only becomes activated upon strong stimulation and then contributes to the limitation of dendritic growth. The PKCβ isoform appears to not be involved in the signaling cascades regulating Purkinje cell dendritic maturation in cerebellar slice cultures. © 2003 Wiley Periodicals, Inc. J Neurobiol 57: 95–109, 2003     

Impaired AMPA signaling and cytoskeletal alterations induce early synaptic dysfunction in a mouse model of Alzheimer's disease

Alzheimer's disease (AD) is a devastating neurodegenerative disorder that impairs memory and causes cognitive and psychiatric deficits. New evidences indicate that AD is conceptualized as a disease of synaptic failure, although the molecular and cellular mechanisms underlying these defects remain to be elucidated. Determining the timing and nature of the early synaptic deficits is critical for understanding the progression of the disease and for identifying effective targets for therapeutic intervention. Using single‐synapse functional and morphological analyses, we find that AMPA signaling, which mediates fast glutamatergic synaptic transmission in the central nervous system (CNS), is compromised early in the disease course in an AD mouse model. The decline in AMPA signaling is associated with changes in actin cytoskeleton integrity, which alters the number and the structure of dendritic spines. AMPA dysfunction and spine alteration correlate with the presence of soluble but not insoluble Aβ and tau species. In particular, we demonstrate that these synaptic impairments can be mitigated by Aβ immunotherapy. Together, our data suggest that alterations in AMPA signaling and cytoskeletal processes occur early in AD. Most important, these deficits are prevented by Aβ immunotherapy, suggesting that existing therapies, if administered earlier, could confer functional benefits.