Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

D(3) dopamine receptor (D(3)R) is expressed mainly in parts of the brain that control the emotional behaviors. It is believed that the improper regulation of D(3)R is involved in the etiology of schizophrenia. Desensitization of D(3)R is weakly associated with G protein-coupled receptor kinase (GRK)/beta-arrestin-directed internalization. This suggests that there might be an alternative pathway that regulates D(3)R signaling. This report shows that D(3)R undergoes robust protein kinase C (PKC)-dependent sequestration that is accompanied by receptor phosphorylation and the desensitization of signaling. PKC-dependent D(3)R sequestration, which was enhanced by PKC-beta or -delta, was dynamin dependent but independent of GRK, beta-arrestin, or caveolin 1. Site-directed mutagenesis of all possible phosphorylation sites within the intracellular loops of D(3)R identified serine residues at positions 229 and 257 as the critical amino acids responsible for phorbol-12-myristate-13-acetate (PMA)-induced D(3)R phosphorylation, sequestration, and desensitization. In addition, the LxxY endocytosis motif, which is located between residues 252 and 255, was found to play accommodating roles for PMA-induced D(3)R sequestration. A continuous interaction with the actin-binding protein 280 (filamin A), which was previously known to interact with D(3)R, is required for PMA-induced D(3)R sequestration. In conclusion, the PKC-dependent but GRK-/beta-arrestin-independent phosphorylation of D(3)R is the main pathway responsible for the sequestration and desensitization of D(3)R. Filamin A is essential for both the efficient signaling and sequestration of D(3)R.
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PMID:Roles of protein kinase C and actin-binding protein 280 in the regulation of intracellular trafficking of dopamine D3 receptor. 1753 8

Platelet-activating factor (PAF) is a potent phospholipid mediator involved in several diseases such as allergic asthma, atherosclerosis and psoriasis. The human PAF receptor (PAFR) is a member of the G-protein-coupled receptor family. Following stimulation, PAFR becomes rapidly desensitized; this refractory state is dependent on PAFR phosphorylation, internalization and down-regulation. In this report, we show that the PAFR inverse agonist, WEB2086, can induce phosphorylation and down-regulation of PAFR. Using selective inhibitors, we determined that the agonist, PAF, and WEB2086 could induce phosphorylation of PAFR by PKC. Moreover, dominant-negative (DN) mutant of PKC isoforms beta inhibited WEB2086-stimulated PAFR phosphorylation, whereas PAF-stimulated phosphorylation was inhibited by DN PKCalpha and delta. WEB2086 also induced PAFR down-regulation which could be blocked by PKC inhibitors and by DN PKCbeta. WEB2086-induced down-regulation was dynamin-dependent but arrestin-independent. Unlike PAF, WEB2086-stimulated intracellular trafficking of PAFR was independent of Rab5. Specific inhibitors of lysosomal proteases and of proteasomes were both effective in reducing WEB2086-induced PAFR down-regulation, indicating the importance of receptor targeting to both lysosomes and proteasomes in long-term cell desensitization to WEB2086. These results indicate that although both agonists and inverse agonists induce receptor PAFR down-regulation, this may be accomplished through different signal transduction and trafficking pathways.
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PMID:Inverse agonist-induced signaling and down-regulation of the platelet-activating factor receptor. 1760 20

Little is known regarding the mechanism(s) by which glycine receptors are endocytosed. Here we examined the endocytosis of homomeric alpha1 glycine receptors expressed in HEK 293 cells using immunofluorescence/confocal microscopy and whole-cell patch-clamp recordings. Our studies demonstrate that constitutive endocytosis of glycine receptors is blocked by the dominant negative dynamin construct K44A and that intracellular dialysis with peptide P4, a dynamin/amphiphysin-disrupting peptide, increased whole-cell glycine-gated chloride currents. To examine whether receptor endocytosis could be regulated by PKC, experiments with the PKC activator PMA (phorbol 12-myristate 13-acetate) were performed. PMA, but not its inactive analogue PMM (phorbol 12-monomyristate), stimulated receptor endocytosis and inhibited glycine-gated chloride currents. Similar to constitutive endocytosis, PKC-stimulated endocytosis was blocked by dynamin K44A. Mutation of a putative AP2 adaptin dileucine motif (L314A, L315A) present in the receptor cytoplasmic loop blocked PMA-stimulated receptor endocytosis and also prevented PMA inhibition of glycine receptor currents. In patch-clamp experiments, intracellular dialysis of a 12-amino acid peptide corresponding to the region of the receptor containing the dileucine motif prevented PKC modulation of wild-type glycine receptors. Unlike PKC modulation of the receptor, constitutive endocytosis was not affected by mutation of this dileucine motif. These results demonstrate that PKC activation stimulates glycine receptor endocytosis, that both constitutive endocytosis and PKC-stimulated endocytosis are dynamin-dependent, and that PKC-stimulated endocytosis, but not constitutive endocytosis, occurs via the dileucine motif (L314A, L315A) within the cytoplasmic loop of the receptor.
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PMID:Mechanisms of homomeric alpha1 glycine receptor endocytosis. 1788 75

Transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry pathway for transepithelial Ca(2+) reabsorption in kidney. Many hormones alter renal Ca(2+) reabsorption at least partly by regulating TRPV5. The mechanism for acute regulation of TRPV5 by phospholipase C-coupled hormones is largely unknown. Here, we found that protein kinase C (PKC) activator 1-oleoyl-acetyl-sn-glycerol (OAG) increased TRPV5 current density and surface abundance in cultured cells. The OAG-mediated increase of TRPV5 was prevented by preincubation with specific PKC inhibitors. Coexpression with a dominant-negative dynamin increased the basal TRPV5 current density and prevented the increase by OAG. Knockdown of caveolin-1 by small interference RNA (siRNA) prevented the increase of TRPV5 by OAG. In contrast, knockdown of clathrin heavy chain had no effects. OAG had no effect on TRPV5 expressed in caveolin-1 null cells derived from caveolin-1 knockout mice. Forced expression of recombinant caveolin-1 restored the regulation of TRPV5 by OAG in caveolin-1 knockout cells. Mutations of serine-299 and/or serine-654 of TRPV5 (consensus residues for phosphorylation by PKC) abolished the regulation by OAG. Parathyroid hormone (PTH) increased TRPV5 current density in cells coexpressing TRPV5 and type 1 PTH receptor. The increase caused by PTH was prevented by PKC inhibitor, mutation of serine-299/serine-654, or by knockdown of caveolin-1. Thus, TRPV5 undergoes constitutive caveolae-mediated endocytosis. Activation of PKC increases cell surface abundance of TRPV5 by inhibiting the endocytosis. This mechanism of regulation by PKC may contribute to the acute stimulation of TRPV5 and renal Ca(2+) reabsorption by PTH.
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PMID:Protein kinase C inhibits caveolae-mediated endocytosis of TRPV5. 1830 97

Endocytosis supports cell communication, growth, and pathogen infection. The species B human adenovirus serotype 3 (Ad3) is associated with epidemic conjunctivitis, and fatal respiratory and systemic disease. Here we show that Ad3 uses dynamin-independent endocytosis for rapid infectious entry into epithelial and haematopoietic cells. Unlike Ad5, which uses dynamin-dependent endocytosis, Ad3 endocytosis spatially and temporally coincided with enhanced fluid-phase uptake. It was sensitive to macropinocytosis inhibitors targeting F-actin, protein kinase C, the sodium-proton exchanger, and Rac1 but not Cdc42. Infectious Ad3 macropinocytosis required viral activation of p21-activated kinase 1 (PAK1) and the C-terminal binding protein 1 of E1A (CtBP1), recruited to macropinosomes. These macropinosomes also contained the Ad3 receptors CD46 and alpha v integrins. CtBP1 is a phosphorylation target of PAK1, and is bifunctionally involved in membrane traffic and transcriptional repression of cell cycle, cancer, and innate immunity pathways. Phosphorylation-defective S147A-CtBP1 blocked Ad3 but not Ad5 infection, providing a direct link between PAK1 and CtBP1. The data show that viruses induce macropinocytosis for infectious entry, a pathway used in antigen presentation and cell migration.
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PMID:Subversion of CtBP1-controlled macropinocytosis by human adenovirus serotype 3. 1832 76

The antidepressant and cocaine sensitive plasma membrane monoamine transporters are the primary mechanism for clearance of their respective neurotransmitters and serve a pivotal role in limiting monoamine neurotransmission. To identify molecules in pathways that regulate dopamine transporter (DAT) internalization, we used a genetic complementation screen in Xenopus oocytes to identify a mitogen-activated protein (MAP) kinase phosphatase, MKP3/Pyst1/DUSP6, as a molecule that inhibits protein kinase C-induced (PKC) internalization of transporters, resulting in enhanced DAT activity. The involvement of MKP3 in DAT internalization was verified using both overexpression and shRNA knockdown strategies in mammalian cell models including a dopaminergic cell line. Although the isolation of MKP3 implies a role for MAP kinases in DAT internalization, MAP kinase inhibitors have no effect on internalization. Moreover, PKC-dependent down-regulation of DAT does not correlate with the phosphorylation state of several well-studied MAP kinases (ERK1/2, p38, and SAPK/JNK). We also show that MKP3 does not regulate PKC-induced ubiquitylation of DAT but acts at a more downstream step to stabilize DAT at the cell surface by blocking dynamin-dependent internalization and delaying the targeting of DAT for degradation. These results indicate that MKP3 can act to enhance DAT function and identifies MKP3 as a phosphatase involved in regulating dynamin-dependent endocytosis.
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PMID:Genetic complementation screen identifies a mitogen-activated protein kinase phosphatase, MKP3, as a regulator of dopamine transporter trafficking. 1843 1

Vascular ATP-sensitive K(+) (K(ATP)) channels are critical regulators of arterial tone and, thus, blood flow in response to local metabolic needs. They are important targets for clinically used drugs to treat hypertensive emergency and angina. It is known that protein kinase C (PKC) activation inhibits K(ATP) channels in vascular smooth muscles. However, the mechanism by which PKC inhibits the channel remains unknown. Here we report that caveolin-dependent internalization is involved in PKC-epsilon-mediated inhibition of vascular K(ATP) channels (Kir6.1 and SUR2B) by phorbol 12-myristate 13-acetate or angiotensin II in human embryonic kidney 293 cells and human dermal vascular smooth muscle cells. We showed that Kir6.1 substantially overlapped with caveolin-1 at the cell surface. Cholesterol depletion with methyl-beta-cyclodextrin significantly reduced, whereas overexpression of caveolin-1 largely enhanced, PKC-induced inhibition of Kir6.1/SUR2B currents. Importantly, we demonstrated that activation of PKC-epsilon caused internalization of K(ATP) channels, the effect that was blocked by depletion of cholesterol with methyl-beta-cyclodextrin, expression of dominant-negative dynamin mutant K44E, or knockdown of caveolin-1 with small interfering RNA. Moreover, patch-clamp studies revealed that PKC-epsilon-mediated inhibition of the K(ATP) current induced by PMA or angiotensin II was reduced by a dynamin mutant, as well as small interfering RNA targeting caveolin-1. The reduction in the number of plasma membrane K(ATP) channels by PKC activation was further confirmed by cell surface biotinylation. These studies identify a novel mechanism by which the levels of vascular K(ATP) channels could be rapidly downregulated by internalization. This finding provides a novel mechanistic insight into how K(ATP) channels are regulated in vascular smooth muscle cells.
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PMID:Protein kinase C-epsilon induces caveolin-dependent internalization of vascular adenosine 5'-triphosphate-sensitive K+ channels. 1866 57

Organic anion transporter 1 (OAT1) mediates the body disposition of a diverse array of environmental toxins and clinically important drugs. Therefore, understanding the regulation of this transporter has profound clinical significance. We previously demonstrate that OAT1 activity was down-regulated by activation of protein kinase C (PKC), kinetically revealed as a decrease in the maximum transport velocity V(max) without significant change in the substrate affinity K(m) of the transporter. In the current study, we showed that OAT1 constitutively internalized from and recycled back to the plasma membrane, and PKC activation accelerated OAT1 internalization without affecting OAT1 recycling. We further showed that treatment of OAT1-expressing cells with concanavalin A, depletion of K(+) from the cells, or transfection of dominant negative mutants of dynamin-2 or Eps15 into the cells, all of which block the clathrin-dependent endocytotic pathway, significantly blocked constitutive and PKC-regulated OAT1 internalization. We finally showed that OAT1 colocalized with transferrin, a marker for clathrin-dependent endocytosis, at the cell surface and in the EEA1-positive early endosomes. Together, our findings demonstrated for the first time that (i) OAT1 constitutively traffics between plasma membrane and recycling endosomes, (ii) PKC activation down-regulates OAT1 activity by altering already existent OAT1 trafficking, and (iii) OAT1 internalization occurs partly through a dynamin- and clathrin-dependent pathway.
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PMID:Organic anion transporter OAT1 undergoes constitutive and protein kinase C-regulated trafficking through a dynamin- and clathrin-dependent pathway. 1881 1

The dopamine transporter (DAT) mediates reuptake of dopamine from the synaptic cleft and is a target for widely abused psychostimulants such as cocaine and amphetamine. Nonetheless, little is known about the cellular distribution and trafficking of natively expressed DAT. Here we use novel fluorescently tagged cocaine analogs to visualize DAT and DAT trafficking in cultured live midbrain dopaminergic neurons. The fluorescent tags were extended from the tropane N-position of 2beta-carbomethoxy-3beta-(3,4-dichlorophenyl)tropane using an ethylamino-linker. The rhodamine-, OR Green-, or Cy3-labeled ligands had high binding affinity for DAT and enabled specific labeling of DAT in live neurons and visualization by confocal imaging. In the dopaminergic neurons, DAT was uniformly distributed in the plasma membrane of the soma, the neuronal extensions, and varicosities along these extensions. FRAP (fluorescence recovery after photobleaching) experiments demonstrated bidirectional movement of DAT in the extensions and indicated that DAT is highly mobile both in the extensions and in the varicosities (immobile fraction less than approximately 30%). DAT was constitutively internalized into vesicular structures likely representing intracellular transporter pools. The internalization was blocked by lentiviral-mediated expression of dominant-negative dynamin and internalized DAT displayed partial colocalization with the early endosomal marker EGFP-Rab5 and with the transferrin receptor. DAT internalization and function was not affected by activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or by inhibition with staurosporine or GF109203X. These data are in contrast to findings for DAT in transfected heterologous cells and challenge the paradigm that trafficking and cellular distribution of endogenous DAT is subject to regulation by PKC.
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PMID:Visualization of dopamine transporter trafficking in live neurons by use of fluorescent cocaine analogs. 1947 7

Cell polarity is crucial for the generation of cell diversity. Recent evidence suggests that the actin cytoskeleton plays a key role in establishment of embryonic polarity, yet the mechanisms that maintain polarity cues in particular membrane domains during development remain unclear. Dynamin, a large GTPase, functions in both endocytosis and actin dynamics. Here, the Caenorhabditis elegans dynamin ortholog, DYN-1, maintains anterior polarity cues. DYN-1-GFP foci are enriched in the anterior cortex in a manner dependent on the anterior polarity proteins, PAR-6 and PKC-3. Membrane internalization and actin comet formation are enriched in the anterior, and are dependent on DYN-1. PAR-6-labeled puncta are also internalized from cortical accumulations of DYN-1-GFP. Our results demonstrate a mechanism for the spatial and temporal regulation of endocytosis in the anterior of the embryo, contributing to the precise localization and maintenance of polarity factors within a dynamic plasma membrane.
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PMID:Dynamin participates in the maintenance of anterior polarity in the Caenorhabditis elegans embryo. 1953 59


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