Regulation of CB1 cannabinoid receptor internalization by a promiscuous phosphorylation-dependent mechanism

Agonists stimulate cannabinoid 1 receptor (CB₁R) internalization. Previous work suggests that the extreme carboxy-terminus of the receptor regulates this internalization – likely through the phosphorylation of serines and threonines clustered within this region. While truncation of the carboxy-terminus (V460Z CB₁) and consequent removal of these putative phosphorylation sites prevents endocytosis in AtT20 cells, the residues necessary for CB₁R internalization remain elusive. To determine the structural requirements for internalization, we evaluated endocytosis of carboxy-terminal mutant CB₁Rs stably expressed in HEK293 cells. In contrast to AtT20 cells, V460Z CB₁R expressed in HEK293 cells internalized to the same extent and with similar kinetics as the wild-type receptor. However, mutation of serine and/or threonine residues within the extreme carboxy-terminal attenuated internalization when these receptors were expressed in HEK293 cells. These results establish that the extreme carboxy-terminal phosphorylation sites are not required for internalization of truncated receptors, but are required for internalization of full-length receptors in HEK293 cells. Analysis of β-arrestin-2 recruitment to mutant CB₁R suggests that putative carboxy-terminal phosphorylation sites mediate β-arrestin-2 translocation. This study indicates that the local cellular environment affects the structural determinants of CB₁R internalization. Additionally, phosphorylation likely regulates the internalization of (full-length) CB₁Rs.     

Phosphorylation of the norepinephrine transporter at threonine 258 and serine 259 is linked to protein kinase C-mediated transporter internalization

Recently, we have demonstrated the phosphorylation- and lipid raft-mediated internalization of the native norepinephrine transporter (NET) following protein kinase C (PKC) activation (Jayanthi, L. D., Samuvel, D. J., and Ramamoorthy, S. (2004) J. Biol. Chem. 279, 19315-19326). Here we tested an hypothesis that PKC-mediated phosphorylation of NET is required for transporter internalization. Phosphoamino acid analysis of 32P-labeled native NETs from rat placental trophoblasts and heterologously expressed wild type human NET (WT-hNET) from human placental trophoblast cells revealed that the phorbol ester (beta-PMA)-induced phosphorylation of NET occurs on serine and threonine residues. Beta-PMA treatment inhibited NE transport, reduced plasma membrane hNET levels, and stimulated hNET phosphorylation in human placental trophoblast cells expressing the WT-hNET. Substance P-mediated activation of the G alpha(q)-coupled human neurokinin 1 (hNK-1) receptor coexpressed with the WT-hNET produced effects similar to beta-PMA via PKC stimulation. In striking contrast, an hNET double mutant harboring T258A and S259A failed to show NE uptake inhibition and plasma membrane redistribution by beta-PMA or SP. Most interestingly, the plasma membrane insertion of the WT-hNET and hNET double mutant were not affected by beta-PMA. Although the WT-hNET showed increased endocytosis and redistribution from caveolin-rich plasma membrane domains following beta-PMA treatment, the hNET double mutant was completely resistant to these PKC-mediated effects. In addition, the PKC-induced phosphorylation of hNET double mutant was significantly reduced. In the absence of T258A and S259A mutations, alanine substitution of all other potential phosphosites within the hNET did not block PKC-induced phosphorylation and down-regulation. These results suggest that Thr-258 and Ser-259 serve as a PKC-specific phospho-acceptor site and that phosphorylation of this motif is linked to PKC-induced NET internalization.     

Syntaxin 1A and Receptor for Activated C Kinase Interact with the N-Terminal Region of Human Dopamine Transporter

The dopamine transporter (DAT) regulates the extent and duration of dopamine receptor activation through sodium-dependant reuptake of dopamine into presynaptic neurons, resulting in termination of dopaminergic neurotransmission. Using the yeast two-hybrid system, we have identified novel interactions between DAT, the SNARE protein syntaxin 1A, and the receptor for activated C kinases (RACK1). This association involves the intracellular N-terminal domain of human DAT (hDAT). Our data suggest that hDAT may exist as dimers or oligomers and that its protein-protein interactions with syntaxin 1A and RACK1 form functional regulatory complexes that may mediate DAT trafficking through modulation of hDAT phosphorylation by PKC.     

Protein kinase C mediates phosphorylation, desensitization, and trafficking of the D2 dopamine receptor

Previously, D2 dopamine receptors (D2 DARs) have been shown to undergo G-protein-coupled receptor kinase phosphorylation in an agonist-specific fashion. We have now investigated the ability of the second messenger-activated protein kinases, protein kinase A (PKA) and protein kinase C (PKC), to mediate phosphorylation and desensitization of the D2 DAR. HEK293T cells were transiently transfected with the D2 DAR and then treated with intracellular activators and inhibitors of PKA or PKC. Treatment with agents that increase cAMP, and activate PKA, had no effect on the phosphorylation state of the D2 DAR, suggesting that PKA does not phosphorylate the D2 DAR in HEK293T cells. In contrast, cellular treatment with phorbol 12-myristate 13-acetate (PMA), a PKC activator, resulted in an approximately 3-fold increase in D2 DAR phosphorylation. The phosphorylation was specific for PKC as the PMA effect was mimicked by phorbol 12,13-dibutyrate, but not by 4alpha-phorbol 12,13-didecanoate, active and inactive, phorbol diesters, respectively. The PMA-mediated D2 DAR phosphorylation was completely blocked by co-treatment with the PKC inhibitor, bisindolylmaleimide II, and augmented by co-transfection with PKCbetaI. In contrast, PKC inhibition had no effect on agonist-promoted phosphorylation, suggesting that PKC is not involved in this response. PKC phosphorylation of the D2 DAR was found to promote receptor desensitization as reflected by a decrease in agonist potency for inhibiting cAMP accumulation. Most interestingly, PKC phosphorylation also promoted internalization of the D2 DAR through a beta-arrestin- and dynamin-dependent pathway, a response not usually associated with PKC phosphorylation of G-protein-coupled receptors. Site-directed mutagenesis experiments resulted in the identification of two domains of PKC phosphorylation sites within the third intracellular loop of the receptor. Both of these domains are involved in regulating sequestration of the D2 DAR, whereas only one domain is involved in receptor desensitization. These results indicate that PKC can mediate phosphorylation of the D2 DAR, resulting in both functional desensitization and receptor internalization.     

Modulation of a Recombinant Glycine Transporter (GLYT1b) by Activation of Protein Kinase C

Abstract: Treatment of human embryonic kidney cells (HEK 293 cells) expressing the mouse glycine transporter 1 (GLYT1b) with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) decreased specific [³H]glycine uptake. This down-regulation resulted from a reduction of the maximal transport rate and was blocked by the PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporine. The inhibitory effect of PMA treatment was also observed after removing all five predicted phosphorylation sites for PKC in GLYT1b by site-directed mutagenesis. These data indicate that glycine transport by GLYT1b is modulated by PKC activation; however, this regulation may involve indirect phosphorylation mechanisms.     

Protein kinase C-mediated functional regulation of dopamine transporter is not achieved by direct phosphorylation of the dopamine transporter protein

Dopaminergic neurotransmission is terminated by the action of the presynaptic dopamine transporter (DAT). It mediates Na(+)/Cl(-) -dependent re-uptake of extracellular dopamine (DA) into the cell, and is regarded as a major regulatory mechanism for synaptic transmission. Previous works have documented that protein kinase C (PKC) activator or inhibitor alters DA uptake by DAT, suggesting that PKC phosphorylation plays an important regulatory mechanism in DAT function. Based on the existence of consensus amino acid sequences for PKC phosphorylation, it has been postulated that PKC regulation of DAT is mediated by the direct phosphorylation of DAT protein. In this study, we try to discover whether the functional regulation of DAT by PKC is due to direct phosphorylation of DAT. The PKC null mutant hDAT, where all putative PKC phosphorylation sites are eliminated, has been constructed by the replacement of serine/threonine residues with glycines. The mutation itself showed no effect on the functional activities of DAT. The DA uptake activity of PKC null mutant was equivalent to those of wild-type hDAT (80-110% of wild-type). Phorbol ester activation of PKC inhibited DA uptake of wild-type hDAT by 35%, and staurosphorine blocked the effect of phorbol ester on DA uptake. The same phenomena was observed in PKC null mutant DAT, although no significant phosphorylation was observed by PKC activation. Confocal microscopic analysis using EGFP-fused DAT revealed that the activation of PKC by phorbol ester elicited fluorescent DAT to be internalized into the intracellular space both in wild-type and PKC null mutant DAT in a similar way. These results suggest that PKC-mediated regulation of DAT function is achieved in an indirect manner, such as phosphorylation of a mediator protein or activation of a clathrin-mediated pathway.     

Protein kinase C-dependent ubiquitination and clathrin-mediated endocytosis of the cationic amino acid transporter CAT-1

Cationic amino acid transporter 1 (CAT-1) is responsible for the bulk of the uptake of cationic amino acids in most mammalian cells. Activation of protein kinase C (PKC) leads to down-regulation of the cell surface CAT-1. To examine the mechanisms of PKC-induced down-regulation of CAT-1, a functional mutant of CAT-1 (CAT-1-HA-GFP) was generated in which a hemagglutinin antigen (HA) epitope tag was introduced into the second extracellular loop and GFP was attached to the carboxyl terminus. CAT-1-HA-GFP was stably expressed in porcine aorthic endothelial and human epithelial kidney (HEK) 293 cells. Using the HA antibody internalization assay we have demonstrated that PKC-dependent endocytosis was strongly inhibited by siRNA depletion of clathrin heavy chain, indicating that CAT-1-HA-GFP internalization requires clathrin-coated pits. Internalized CAT-1-HA-GFP was accumulated in early, recycling, and late endosomes. PKC activation also resulted in ubiquitination of CAT-1. CAT-1 ubiquitination and endocytosis in phorbol ester-stimulated porcine aorthic endothelial and HEK293 cells were inhibited by siRNA knockdown of NEDD4-2 and NEDD4-1 E3 ubiquitin ligases, respectively. In contrast, ubiquitination and endocytosis of the dopamine transporter was dependent on NEDD4-2 in all cell types tested. Altogether, our data suggest that ubiquitination mediated by NEDD4-2 or NEDD4-1 leading to clathrin-mediated endocytosis is the common mode of regulation of various transporter proteins by PKC.     

Inhibition of the High-Affinity Brain Glutamate Transporter GLAST-1 via Direct Phosphorylation

Abstract: Neurotransmission at excitatory glutamatergic synapses is terminated by the reuptake of the neurotransmitter by high-affinity transporters, which keep the extracellular glutamate concentration below excitotoxic levels. The amino acid sequence of the recently isolated and cloned brain-specific glutamate/aspartate transporter (GLAST-1) of the rat reveals three consensus sequences of putative phosphorylation sites for protein kinase C (PKC). The PKC activator phorbol 12-myristate 13-acetate (PMA) decreased glutamate transport activity in Xenopus oocytes and human embryonic kidney cells (HEK293) expressing the cloned GLAST-1 cDNA, within 20 min, to 25% of the initial transport activity. This down-regulation was blocked by the PKC inhibitor staurosporine. GLAST-1 transport activity remains unimpaired by phorbol 12-monomyristate. Removal of all putative PKC sites of wild-type GLAST-1 by site-directed mutagenesis did not abolish inhibition of glutamate transport. [³²P]Phosphate-labeled wild-type and mutant transport proteins devoid of all predicted PKC sites were detected by immunoprecipitation after stimulation with PMA. Immunoprecipitation of [³⁵S]methionine-labeled transporter molecules indicates a similar stability of phosphorylated and nonphosphorylated GLAST-1 protein. Immunofluorescence staining did not differentiate surface staining of HEK293 cells expressing GLAST-1 with and without PMA treatment. These data suggest that the neurotransmitter transporter activity of GLAST-1 is inhibited by phosphorylation at a non-PKC consensus site.     

Opposing effects of phorbol-12-myristate-13-acetate, an activator of protein kinase C, on the signaling of structurally related human dopamine D1 and D5 receptors

The ‘cross‐talk’ between different types of neurotransmitters through second messenger pathways represents a major regulatory mechanism in neuronal function. We investigated the effects of activation of protein kinase C (PKC) on cAMP‐dependent signaling by structurally related human D1‐like dopaminergic receptors. Human embryonic kidney 293 (HEK293) cells expressing D1 or D5 receptors were pretreated with phorbol‐12‐myristate‐13‐acetate (PMA), a potent activator of PKC, followed by analysis of dopamine‐mediated receptor activation using whole cell cAMP assays. Unpredictably, PKC activation had completely opposite effects on D1 and D5 receptor signaling. PMA dramatically augmented agonist‐evoked D1 receptor signaling, whereas constitutive and dopamine‐mediated D5 receptor activation were rapidly blunted. RT–PCR and immunoblotting analyses showed that phorbol ester‐regulated PKC isozymes (conventional: α, βI, βII, γ; novel: δ, ?, η, θ) and protein kinase D (PKCµ) are expressed in HEK293 cells. PMA appears to mediate these contrasting effects through the activation of Ca²⁺‐independent novel PKC isoforms as revealed by specific inhibitors, bisindolylmaleimide I, Gö6976, and Gö6983. The finding that cross‐talk between PKC and cAMP pathways can produce such opposite outcomes following the activation of structurally similar D1‐like receptor subtypes is novel and further strengthens the view that D1 and D5 receptors serve distinct functions in the mammalian nervous and endocrine systems.     

Protein kinase C activation promotes the internalization of the human cationic amino acid transporter hCAT-1. A new regulatory mechanism for hCAT-1 activity

The human cationic amino acid transporter hCAT-1 is almost ubiquitously expressed and probably the most important entity for supplying cells with extracellular arginine, lysine, and ornithine. We have previously shown that hCAT-1-mediated transport is decreased after protein kinase C (PKC) activation by phorbol 12-myristate 13-acetate (PMA) (Gr?f, P., Forstermann, U., and Closs, E. I. (2001) Br. J. Pharmacol. 132, 1193-1200). In the present study, we examined the mechanism of this down-regulation. In both Xenopus laevis oocytes and U373MG glioblastoma cells, PMA treatment promoted the internalization of hCAT-1 (fused to the enhanced green fluorescence protein (EGFP)) as visualized by fluorescence microscopy. Biotinylation of cell surface proteins and subsequent Western blot analyses confirmed that the cell surface expression of hCAT-1.EGFP was significantly reduced upon PMA treatment. Pretreatment with the PKC inhibitor bisindolylmaleimide I prevented the reduction by PMA of both hCAT-1.EGFP-induced arginine transport and the internalization of the transporter. Similar results were obtained with hCAT-1 expressed endogenously in DLD-1 colon carcinoma cells. Inhibition of protein synthesis did not augment the PMA effect. In addition, the PMA effect was reverted in washout experiments without changing the hCAT-1 protein expression, suggesting that the PMA effect is reversible in these cells. PKC did not phosphorylate hCAT-1 directly as evidenced by in vivo phosphorylation experiments and mutational analysis, indicating an indirect action of PKC on hCAT-1.     

Amphetamine regulation of dopamine transport. Combined measurements of transporter currents and transporter imaging support the endocytosis of an active carrier

Dopaminergic neurotransmission is fine-tuned by the rate of removal of dopamine (DA) from the extracellular space via the Na(+)/Cl(-)-dependent DA transporter (DAT). DAT is a target of psychostimulants such as amphetamine (AMPH) and cocaine. Previously, we reported that AMPH redistributes the human DAT away from the cell surface. This process was associated with a reduction in transport capacity. This loss of transport capacity may result either from a modification of the function of DAT that is independent of its cell surface redistribution and/or from a reduction in the number of active transporters at the plasma membrane that results from DAT trafficking. To discriminate between these possibilities, we stably transfected HEK-293 cells with a yellow fluorescent protein (YFP)-tagged human DAT (hDAT cells). In hDAT cells, acute exposure to AMPH induced a time-dependent loss of hDAT activity. By coupling confocal imaging with patch-clamp whole-cell recordings, we have demonstrated for the first time that the loss of AMPH-induced hDAT activity temporally parallels the accumulation of intracellular hDAT. In addition, presteady-state current analysis revealed a cocaine-sensitive, voltage-dependent capacitance current that correlated with the level of transporter membrane expression and in turn served to monitor the AMPH-induced trafficking of hDAT. We found that the decrease in hDAT cell surface expression induced by AMPH was not paralleled by changes in the ability of the single transporter to carry charges. Quasi-stationary noise analysis of the AMPH-induced hDAT currents revealed that the unitary transporter current remained unaltered during the loss of hDAT membrane expression. Taken together, these data strongly suggest that the AMPH-induced reduction of hDAT transport capacity results from the removal of active hDAT from the plasma membrane.     

Conventional protein kinase C isoforms regulate human dopamine transporter activity in Xenopus oocytes

The hypothesis that specific protein kinase C (PKC) isoforms regulate dopamine transporter (DAT) function was tested in Xenopus laevis oocytes expressing human (h)DAT. Activation of conventional PKCs (cPKCs) and novel PKCs (nPKCs) using 10 nM phorbol 12-myristate 13-acetate (PMA) significantly inhibited DAT-associated transport currents. This effect was reversed by isoform-non-selective PKC inhibitors, selective inhibitors of cPKCs and deltaPKC, and by Ca2+ chelation. By contrast, the epsilonPKC translocation inhibitor peptide had no effect on PMA-induced inhibition of hDAT transport-associated currents. Thus, the primary mechanism by which PMA regulates hDAT expressed in oocytes appears to be by activating cPKC(s).     

Protein kinase C activation stimulates the phosphorylation and internalization of the sst2A somatostatin receptor

The sst2A receptor is expressed in the endocrine, gastrointestinal, and neuronal systems as well as in many hormone-sensitive tumors. This receptor is rapidly internalized and phosphorylated in growth hormone-R2 pituitary cells following somatostatin binding (Hipkin, R. W., Friedman, J., Clark, R. B., Eppler, C. M., and Schonbrunn, A. (1997) J. Biol. Chem. 272, 13869-13876). The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), also stimulates sst2A phosphorylation. Here we examine the mechanisms and consequences of PMA and agonist-induced sst2A phosphorylation. Like somatostatin, both PMA and bombesin increased sst2A receptor phosphorylation within 2 min. The PKC inhibitor GF109203X blocked PMA- and bombesin- stimulated sst2A phosphorylation, whereas stimulation by the somatostatin analog SMS 201-995 was unaffected. Agonist and PMA each stimulated phosphorylation in two receptor domains, the third intracellular loop and the C-terminal tail. Functionally, PMA dramatically increased the internalization of the sst2A receptor-ligand complex. This PMA stimulation was blocked by GF109203X, whereas basal internalization was unaffected. However, neither basal nor PMA-stimulated internalization was altered by pertussis toxin, whereas both were blocked by hypertonic sucrose. Therefore PKC activation and agonist binding stimulate sst2A phosphorylation by distinct mechanisms, and PKC potentiates internalization of the sst2A receptor via clathrin-coated pits. Thus, hormonal stimulation of PKC-coupled receptors may provide a mechanism for regulating the inhibitory actions of somatostatin in target tissue.     

Activation and nuclear translocation of PKCdelta,Pyk2 and ERK1/2 by gonadotropin releasing hormone in HEK293 cells

The mechanism of agonist-induced activation of Pyk2 and its relationship with ERK1/2 phosphorylation was analyzed in HEK293 cells stably expressing the gonadotropin releasing hormone (GnRH) receptor. GnRH stimulation caused rapid and sustained phosphorylation of ERK1/2 and Pyk2 that was accompanied by their nuclear translocation. Pyk2 was also localized on cell membranes and at focal adhesions. Dominant negative Pyk2 (PKM) had no effect on GnRH-induced ERK1/2 phosphorylation and c-fos expression. These actions of GnRH on ERK1/2 and Pyk2 were mimicked by activation of protein kinase C (PKC) and were abolished by its inhibition. GnRH caused translocation of PKC and δ, but not of , ι and λ, to the cell membrane, as well as phosphorylation of Raf at Ser338, a major site in the activation of MEK/ERK1/2. Stimulation of HEK293 cells by EGF caused marked ERK1/2 phosphorylation that was attenuated by the selective EGFR receptor (EGF-R) kinase inhibitor, AG1478. However, GnRH-induced ERK1/2 activation was independent of EGF-R activation. These results indicate that activation of PKC is responsible for GnRH-induced phosphorylation of both ERK1/2 and Pyk2, and that Pyk2 activation does not contribute to GnRH signaling. Moreover, GnRH-induced phosphorylation of ERK1/2 and expression of c-fos in HEK293 cells is independent of Src and EGF-R transactivation, and is mediated through the PKC/Raf/MEK cascade.     

Bradykinin-induced internalization of the human B2 receptor requires phosphorylation of three serine and two threonine residues at its carboxyl tail

The binding of bradykinin (BK) to B2 receptor triggers the internalization of the agonist-receptor complex. To investigate the mechanisms and the receptor structures involved in this fundamental process of receptor regulation, the human B2 receptor was mutated within its cytoplasmic tail by complementary strategies of truncation, deletion, and amino acid substitution. Ligand binding, signal transduction, internalization as well as phosphorylation were studied for the mutated receptors expressed in COS, CHO, and HEK 293 cells. Truncation of 44 out of 55 amino acid residues of the receptor's cytoplasmic tail corresponding to positions 321-364 did not alter the kinetics of BK binding and the receptor coupling to phospholipase C and phospholipase A2. By contrast, truncations after positions 320 and 334, deletions within the segment covering positions 335-351, as well as alanine substitution of serine and threonine residues within segment 335-351 diminished the internalization capacity of the mutant receptors. Mutants with a markedly reduced internalization potential failed to produce BK-induced receptor phosphorylation suggesting that phosphorylation may be involved in receptor internalization. The mutagenesis approaches converged at the conclusion that three serines in positions 339, 346, and 348 and two threonines in positions 342 and 345, contained in a sequence segment that is highly conserved between species, have a critical role in the ligand-dependent internalization and phosphorylation of kinin receptors and can intervene in these processes in an alternative manner. However, mutants lacking these residues were still sensitive to dominant-negative forms of beta-arrestin and dynamin, suggesting the existence of additional receptor structure(s) involved in the receptor sequestration through clathrin-coated vesicles.     

Up-regulation of the Pit-2 phosphate transporter/retrovirus receptor by protein kinase C epsilon

The membrane receptors for the gibbon ape leukemia retrovirus and the amphotropic murine retrovirus serve normal cellular functions as sodium-dependent phosphate transporters (Pit-1 and Pit-2, respectively). Our earlier studies established that activation of protein kinase C (PKC) by treatment of cells with phorbol 12-myristate 13-acetate (PMA) enhanced sodium-dependent phosphate (Na/Pi) uptake. Studies now have been carried out to determine which type of Na/Pi transporter (Pit-1 or Pit-2) is regulated by PKC and which PKC isotypes are involved in the up-regulation of Na/Pi uptake by the Na/Pi transporter/viral receptor. It was found that the activation of short term (2-min) Na/Pi uptake by PMA is abolished when cells are infected with amphotropic murine retrovirus (binds Pit-2 receptor) but not with gibbon ape leukemia retrovirus (binds Pit-1 receptor), indicating that Pit-2 is the form of Na/Pi transporter/viral receptor regulated by PKC. The PKC-mediated activation of Pit-2 was blocked by pretreating cells with the pan-PKC inhibitor bisindolylmaleimide but not with the conventional PKC isotype inhibitor G? 6976, suggesting that a novel PKC isotype is required to regulate Pit-2. Overexpression of PKCepsilon, but not of PKCalpha, -delta, or -zeta, was found to mimic the activation of Na/Pi uptake. To further establish that PKCepsilon is involved in the regulation of Pit-2, cells were treated with PKCepsilon-selective antisense oligonucleotides. Treatment with PKCepsilon antisense oligonucleotides decreased the PMA-induced activation of Na/Pi uptake. These results indicate that PMA-induced stimulation of Na/Pi uptake by Pit-2 is specifically mediated through activation of PKCepsilon.     

Heterologous activation of protein kinase C stimulates phosphorylation of delta-opioid receptor at serine 344, resulting in beta-arrestin- and clathrin-mediated receptor internalization

The purpose of the current study is to investigate the effect of opioid-independent, heterologous activation of protein kinase C (PKC) on the responsiveness of opioid receptor and the underlying molecular mechanisms. Our result showed that removing the C terminus of delta opioid receptor (DOR) containing six Ser/Thr residues abolished both DPDPE- and phorbol 12-myristate 13-acetate (PMA)-induced DOR phosphorylation. The phosphorylation levels of DOR mutants T352A, T353A, and T358A/T361A/S363S were comparable to that of the wild-type DOR, whereas S344G substitution blocked PMA-induced receptor phosphorylation, indicating that PKC-mediated phosphorylation occurs at Ser-344. PKC-mediated Ser-344 phosphorylation was also induced by activation of G(q)-coupled alpha(1A)-adrenergic receptor or increase in intracellular Ca(2+) concentration. Activation of PKC by PMA, alpha(1A)-adrenergic receptor agonist, and ionomycin resulted in DOR internalization that required phosphorylation of Ser-344. Expression of dominant negative beta-arrestin and hypertonic sucrose treatment blocked PMA-induced DOR internalization, suggesting that PKC mediates DOR internalization via a beta-arrestin- and clathrin-dependent mechanism. Further study demonstrated that agonist-dependent G protein-coupled receptor kinase (GRK) phosphorylation sites in DOR are not targets of PKC. Agonist-dependent, GRK-mediated receptor phosphorylation and agonist-independent, PKC-mediated DOR phosphorylation were additive, but agonist-induced receptor phosphorylation could inhibit PKC-catalyzed heterologous DOR phosphorylation and subsequent internalization. These data demonstrate that the responsiveness of opioid receptor is regulated by both PKC and GRK through agonist-dependent and agonist-independent mechanisms and PKC-mediated receptor phosphorylation is an important molecular mechanism of heterologous regulation of opioid receptor functions.     

Truncation of human dopamine transporter by protease calpain

It has been shown recently that the N-terminal domain of the dopamine transporter (DAT) plays a role in several transporter functions. Here we provide evidence for a possible cellular mechanism of how the N-terminus of dopamine transporter might be removed in vivo. We isolated a recombinant N-terminal protein region of human dopamine transporter and cleaved it with calpain protease. Peptide fragment analysis revealed the existence of two calpain cleavage sites at positions Thr43/Ser44 and Leu71/Ser72 of the DATN-terminus. We show that calpain activation in rat striatal synaptosomes leads to a rapid decrease of dopamine transporter N-terminal epitopes corresponding to the protein sequences removed by a calpain cleavage at Thr43/Ser44 and that the process is totally blocked by a calpain inhibitor. Calpain truncation of the DATN-terminus abolishes its interaction with the receptor of activated protein kinase C, RACK1 and removes protein sequences previously implicated in amphetamine-induced dopamine release, PKC-dependent endocytosis and the interaction of DAT with the dopamine D2 receptor. The above suggests that cleavage of DAT by calpain may significantly modify dopamine homeostasis under pathological or physiological conditions.     

Protein kinase C inhibits autophagy and phosphorylates LC3

During autophagy, the microtubule-associated protein light chain 3 (LC3), a specific autophagic marker in mammalian cells, is processed from the cytosolic form (LC3-I) to the membrane-bound form (LC3-II). In HEK293 cells stably expressing FLAG-tagged LC3, activation of protein kinase C inhibited the autophagic processing of LC3-I to LC3-II induced by amino acid starvation or rapamycin. PKC inhibitors dramatically induced LC3 processing and autophagosome formation. Unlike autophagy induced by starvation or rapamycin, PKC inhibitor-induced autophagy was not blocked by the PI-3 kinase inhibitor wortmannin. Using orthophosphate metabolic labeling, we found that LC3 was phosphorylated in response to the PKC activator PMA or the protein phosphatase inhibitor calyculin A. Furthermore, bacterially expressed LC3 was directly phosphorylated by purified PKC in vitro. The sites of phosphorylation were mapped to T6 and T29 by nanoLC-coupled tandem mass spectrometry. Mutations of these residues significantly reduced LC3 phosphorylation by purified PKC in vitro. However, in HEK293 cells stably expressing LC3 with these sites mutated either singly or doubly to Ala, Asp or Glu, autophagy was not significantly affected, suggesting that PKC regulates autophagy through a mechanism independent of LC3 phosphorylation.     

Overexpression of thioredoxin reductase 1 inhibits migration of HEK-293 cells

Background information. TrxR (thioredoxin reductase), in addition to protecting against oxidative stress, plays a role in the redox regulation of intracellular signalling pathways controlling, among others, cell proliferation and apoptosis. The aim of the present study was to determine whether TrxR1 is involved in the regulation of cell migration. Results. Stably transfected HEK‐293 (human embryonic kidney) cells which overexpress cytosolic TrxR1 (HEK‐TrxR15 and HEK‐TrxR11 cells) were used in the present study. We found that the stimulation of cell motility induced by PKC (protein kinase C) activators, PMA and DPhT (diphenyltin), was inhibited significantly in the HEK‐TrxR15 and HEK‐TrxR11 cells compared with control cells. The overexpression of TrxR1 also inhibited characteristic morphological changes and reorganization of the F‐actin cytoskeleton induced by PMA and DPhT. In addition, the selective activation of PKCδ by DPhT was inhibited in cells that overexpressed cytosolic TrxR1. Furthermore, rottlerin, a selective inhibitor of PKCδ, and PKCδ siRNA (small interfering RNA), suppressed the morphological changes induced by DPhT in the control cells. Conclusions. The overexpression of TrxR1 inhibits migration of HEK‐293 cells stimulated with PMA and DPhT. Moreover, our observations suggest that this effect is mediated by the inhibition of PKCδ activation.