Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M309894200 on October 15, 2003

J. Biol. Chem., Vol. 279, Issue 3, 2254-2261, January 16, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
279/3/2254    most recent
M309894200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Uchino, M.
Right arrow Articles by Saito, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Uchino, M.
Right arrow Articles by Saito, N.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Isoform-specific Phosphorylation of Metabotropic Glutamate Receptor 5 by Protein Kinase C (PKC) Blocks Ca2+ Oscillation and Oscillatory Translocation of Ca2+-dependent PKC*

Motoi Uchino{ddagger}§, Norio Sakai{ddagger}, Kaori Kashiwagi{ddagger}, Yasuhito Shirai{ddagger}, Yoshiaki Shinohara¶, Kenzo Hirose||, Masamitsu Iino||, Takehira Yamamura§, and Naoaki Saito{ddagger}**

From the {ddagger}Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan, the §Second Department of Surgery, Hyogo College of Medicine, Nishinomiya 663-8501, Japan, the Department of Biological Sciences, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan, and the ||Department of Pharmacology, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan

Received for publication, September 5, 2003 , and in revised form, October 14, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Prolonged activation of metabotropic glutamate receptor 5a (mGluR5a) causes synchronized oscillations in intracellular calcium, inositol 1,4,5-trisphosphate production, and protein kinase C (PKC) activation. Additionally, mGluR5 stimulation elicited cyclical translocations of myristoylated alanine-rich protein kinase C substrate, which were opposite to that of {gamma}PKC (i.e. from plasma membrane to cytosol) and dependent on PKC activity, indicating that myristoylated alanine-rich protein kinase C substrate is repetitively phosphorylated by oscillating {gamma}PKC on the plasma membrane. Mutation of mGluR5 Thr840 to aspartate abolished the oscillation of {gamma}PKC, but the mutation to alanine (T840A) did not. Cotransfection of {gamma}PKC with {beta}IIPKC, another Ca2+-dependent PKC, resulted in synchronous oscillatory translocation of both classical PKCs. In contrast, cotransfection of {delta}PKC, a Ca2+-independent PKC, abolished the oscillations of both {gamma}PKC and inositol 1,4,5-trisphosphate. Regulation of the oscillations was dependent on {delta}PKC kinase activity but not on {gamma}PKC. Furthermore, the T840A-mGluR5-mediated oscillations were not blocked by the {delta}PKC overexpression. These results revealed that activation of mGluR5 causes translocation of both {gamma}PKC and {delta}PKC to the plasma membrane. {delta}PKC, but not {gamma}PKC, phosphorylates mGluR5 Thr840, leading to the blockade of both Ca2+ oscillations and {gamma}PKC cycling. This subtype-specific targeting proposes the molecular basis of the multiple functions of PKC.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Metabotropic glutamate receptors (mGluRs)1 are the most abundant receptors in the brain and are involved in neuronal differentiation and synaptic plasticity (1, 2). mGluRs are coupled to G proteins and exert their effects via various signal transduction pathways. Among the eight different subtypes of mGluRs, activation of mGluR1 and mGluR5 causes an increase in inositol 1,4,5-triphosphate (IP3) and diacylglycerol through activation of phospholipase C, which leads to mobilization of Ca2+ from intracellular Ca2+ stores and the activation of protein kinase C (PKC) (3, 4). Although both mGluR1 and mGluR5 are coupled to the IP3/Ca2+ cascade, the patterns of Ca2+ mobilization by these two mGluRs are distinctly different (5, 6). Stimulation of mGluR1 induces transient and low frequency Ca2+ oscillations, whereas high frequency oscillations are stimulated by mGluR5 (5). Activation of mGluR5 also induces the repetitive translocation of PKC between the cytoplasm and plasma membrane, which is synchronized with the Ca2+ oscillations. It has been suggested that feedback phosphorylation of mGluR5, but not of mGluR1, by PKC results in Ca2+ oscillation through Ca2+ mobilization from endoplasmic reticulum (5). However, subsequent studies provided controversial results regarding the involvement of PKC in the Ca2+ oscillation induced by mGluRs (7-9).

PKC plays a pivotal role in many signaling pathways, and the existence of multiple subtypes suggests that different isoforms have varied functions. The PKC superfamily consists of at least 10 subtypes and is divided into three groups, classical PKC (cPKC; {alpha}, {beta}I, {beta}II, {gamma}), novel PKC (nPKC; {delta}, {epsilon}, {eta}, {theta}), and atypical PKC ({zeta}, {iota}/{lambda}), based on their structures (10-13). Each PKC subtype has differential sensitivity to activators and shows tissue-specific and cell-specific expression, suggesting that each subtype plays a subtype-specific role in various signal transduction pathways and in the regulation of numerous cellular processes (10, 11, 14). The subtype-specific function of PKC, however, has not been clarified by conventional biochemical techniques due to the low substrate specificity among family members. Recent technical developments enabled us to monitor the movement of PKC in living cells using PKC fused with green fluorescent protein (GFP) (15, 16). Reports using GFP-tagged PKC subtypes have demonstrated that PKC translocation varies, depending on the PKC subtype and on stimulation (16-19). These results suggest that correct spatial and temporal translocation of each subtype is necessary for its PKC activation and phosphorylation of the specific substrates. Analysis of subtype- and stimulus-specific PKC translocation provides a way to study their individual role in cell signaling pathways.

In the present study, we explore the modulation of mGluR5a-induced Ca2+ oscillation by different PKC subtypes in living cells. We find that fast Ca2+ oscillations and {gamma}PKC translocation by mGluR5 are blocked by the nPKC-dependent phosphorylation of Thr840 in the C-terminal intracellular loop of mGluR5a.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—L-Glutamate, thapsigargin, O,O'-bis(2-aminophenyl)ethylene glycol-N,N,N,N-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM), cytochalasin D, and colchicine were purchased from Nacalai Tesque (Kyoto, Japan). Aristolochic acid, compound 48/80, and arachidonyl trifluoromethyl ketone were obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA). Bromoenol lactone and D609 were obtained from Cayman Chemical (Ann Arbor, MI) and Kamiya Biomedical Co. (Seattle, WA), respectively. Methylphenylethynylpyridine hydrochloride was from Tocris (Bristol, UK). Rottlerin, Gö6983, and Gö6976 were from Calbiochem. All other chemicals were of analytical grade.

Cell Culture—HEK293 cells were obtained from the Riken cell bank (Tukuba, Japan). The HEK293 cells, stably expressing metabotropic glutamate receptor 5 (HEK/mGluR5), were a gift from Dr. Nakanishi (Department of Biological Science, Kyoto University Faculty of Medicine, Kyoto, Japan). HEK293 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37 °C. All media were supplemented with penicillin (100 units/ml) and streptomycin (100 µg/ml), and the fetal bovine serum used was not heat-inactivated.

Preparation of Plasmids to Express GFP- or DsRed-tagged PKC Subtypes and Their Mutants—The expression plasmids bearing cDNA of {alpha}PKC-GFP, {beta}IPKC-GFP, {beta}IIPKC-GFP, {gamma}PKC, {gamma}PKC lacking the C1 domain ({gamma}PKC {Delta}C1-GFP) or C2 domain ({gamma}PKC {Delta}C2-GFP), {delta}PKC-GFP, {delta}PKC-KN-GFP, {epsilon}PKC-GFP, and myristoylated alanine-rich protein kinase C substrate (MARCKS)-GFP were prepared as described previously (16-18, 20). An expression plasmid encoding {gamma}PKC-DsRed or MARCKS-DsRed was constructed according to the method previously described (16). A cDNA fragment encoding DsRed with a 5' HindIII and 3' EcoRI site was obtained by PCR using pDsRed1-C1 (Clontech, Palo Alto, CA) as a template. The sense and antisense primers used were 5'-TTAAGCTTATGGTGCGCTCCTCCAAGAAC-3' and 5'-TTGAATTCCTACAGGAACAGGTGGTGGCG-3', respectively. cDNAs for {gamma}PKC or MARCKS were obtained from BS186 (16) or BS555 (21), respectively. cDNAs of {gamma}PKC or MARCKS were subcloned into pTB701 together with PCR products for DsRed. We also constructed a plasmid encoding the C1 and C2 domains of {gamma}PKC fused with GFP (C1C2-GFP) by PCR using BS354 (17). The sense and antisense primers used for PCR were 5'-TTGAATTCGCCATGGTGAAGAGCCACAAGTTCACC-3' and 5'-TTAGATCTCGGTACATTGTAATACTCGC-3', respectively. All PCR products were verified by sequencing. The expression plasmids encoding the pleckstrin homology domain (PHD) of PLC-{delta}1 fused with GFP (GFP-PHD) and the mGluRs were constructed as described previously (22, 23).

Transfection of Plasmids into Cultured Cells—Transfection of plasmids was performed by lipofection using FugeneTM 6 Transfection Reagent (Roche Applied Science), according to the manufacturer's standard protocol. For co-expression of plasmids, the same amount of each plasmid was mixed and transfected. Experiments were performed 24-48 h after the transfection.

Observation of Translocation and Oscillation of GFP- or DsRed-fused Protein—Transfected cells were spread onto the glass bottom culture dishes (MatTek Corp., Ashland, MA) and cultured at 37 °C for at least 24 h before the observation. The culture medium was replaced with normal HEPES buffer composed of 135 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, 10 mM glucose, pH 7.3. Translocation of fusion proteins was triggered by a direct application of various stimulants at a 10 times higher concentration into the buffer to obtain the appropriate final concentration. The fluorescence of the fusion proteins was monitored by confocal laser-scanning fluorescence microscopy (model LSM 510 invert; Carl Zeiss, Jena, Germany). GFP-fused protein was monitored at 488-nm argon excitation using a 510-535-nm band pass barrier filter. DsRed-fused protein was monitored at 543-nm HeNe1 excitation using a 590-nm band pass barrier filter. DsRed and GFP were monitored simultaneously using multitracking software, which alternately detects each fluorescence by switching the laser and filter system quickly. Images were collected sequentially every 3 s.

Data Analysis—The time course of translocation was recorded as a time series of 100-200 images for each experiment. Image analysis was performed using the Zeiss LSM 510 software, and the membrane fluorescence ratio was calculated. This was defined as the plasma membrane fluorescence intensity/cytoplasm fluorescence intensity in a 1-7-µm2 region of interest. Regions of interest were circles for cytoplasm and rectangles for the plasma membrane (see Fig. 1A). For each time point, the membrane fluorescence was calculated from at least five different regions of interest. These values were averaged and plotted to generate a time course of translocation.



View larger version (37K):
[in this window]
[in a new window]
  		FIG. 1.
L-Glutamate-induced oscillatory translocation of {gamma}PKC-DsRed and GFP-PHD via mGluR5 receptor activation. A, simultaneous imaging of {gamma}PKC-DsRed and GFP-PHD after mGluR5 stimulation. HEK cells expressing mGluR5 and both {gamma}PKC-DsRed and GFP-PHD (PLC{delta}1) were stimulated with 100 µM L-glutamate, and then fluorescence of both GFP and DsRed was simultaneously detected by confocal microscopy. Images were collected sequentially every 3 s. The average of the fluorescence intensity in at least five different regions in the cytosol or on the plasma membrane was calculated as indicated (1-7 µm2 of the circle for the cytosol or rectangle for the plasma membrane). The images in Fig. 1A show the fluorescence of {gamma}PKC-DsRed and GFP-PHD at the time points indicated in B (a-c). B, synchronized translocation of {gamma}PKC-DsRed and GFP-PHD. The ratio (mean fluorescent intensity on the plasma membrane/intensity in the cytosol) was plotted at each time point in Fig. 1B. The time scale bar represents 60 s. Data shown are representative of at least five experiments.

 
Knockdown of Endogenous {delta}PKC by Short Interfering RNA—Knockdown of endogenous {delta}PKC in HEK293 cells by using short interfering RNAs (siRNA) was performed as described previously (24). Briefly, 21-nucleotide RNAs with 3' overhangs of 2'-deoxythymidine were chemically synthesized by Nihon Bio Service Co. (Asaka, Japan). The siRNA sequences of human {delta}siRNA corresponded to the coding region 603-621 relative to the first nucleotide of the start codon. To generate a duplex of siRNAs, 20 µM sense and antisense single-strand siRNAs were annealed by incubating the mixed siRNAs in annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate) for 1 min at 90 °C followed by a 1-h incubation at 37 °C. Transfection of duplex siRNA was performed by using LipofectAMINE 2000 (Invitrogen) according to the method described by Elbashir et al. (25). Annealed siRNA was transfected at a final concentration of 250 nM into 90-100% confluent HEK/mGluR5 by lipofection, and the efficacy of knockdown was assessed by immunoblotting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
L-Glutamate Induced Translocation and Oscillation of {gamma}PKC DsRed and GFP-PHD—The high frequency oscillatory translocation of Ca2+-dependent PKC, {beta}II-PKC, in mGluR5a-expressing HEK293 (HEK/mGluR5) cells was reported by Dale et al. (8). We here focused on the neuron-specific {gamma} isoform of PKC, {gamma}PKC, and examined its translocation upon activation of mGluR5. Fig. 1 shows that the application of L-glutamate (100 µM final concentration) to HEK/mGluR5 cells induced repetitive translocation of {gamma}PKC-DsRed between the cytoplasm and the plasma membrane. Simultaneously, we examined the translocation of GFP-fused pleckstrin homology domain (GFP-PHD) of PLC-{delta}1, which is released from the plasma membrane into cytoplasm when PIP2 is hydrolyzed and the amount of cytoplasmic IP3 is increased, perhaps due to its higher affinity for IP3 than PIP2 (22, 23). During continuous stimulation of mGluR5, GFP-PHD also showed an oscillatory translocation from the plasma membrane to cytoplasm. The oppositely directed oscillations for GFP-PHD and {gamma}PKC-DsRed were synchronized with a frequency of 3-5 times/min (43-90 mHz, mean = 72.3 ± 5.3 mHz, n = 21) and lasted at least 10 min. Application of (1S,3R)-trans-aminocyclopentane-1,3-dicarboxylic acid, an agonist of type I mGluRs, also induced the same oscillation of {gamma}PKC-DsRed (data not shown). Glutamate-induced oscillation of {gamma}PKC-DsRed via mGluR5a is also shown in Video 1 (Supplemental Material). The frequency of oscillation in Video 1 was 66.7 mHz.

Characteristics of mGluR5-mediated Oscillations of {gamma}PKCDsRed—Activation of mGluR5 leads to the hydrolysis of PIP2 and the subsequent increase in two second messengers, DAG and Ca2+. DAG and Ca2+ translocate cPKCs to the plasma membrane by binding to their C1 and C2 domains, respectively (10, 26, 27). To investigate the source of these two second messengers, we first examined the effects of inhibitors for phospholipases on the oscillation of {gamma}PKC through mGluR5. PLC inhibitors (Compound 48/80) but not inhibitors for PLA2 (aristilochic acid, bromoenol lactone, and arachidonyl trifluoromethyl ketone) or phospholipase D (ethanol) completely blocked the translocation of {gamma}PKC, indicating that DAG from PIP2 through PLC is indispensable for the mGluR5-mediated translocation of {gamma}PKC and that fatty acids or lysophosphates metabolized by PLA2 or DAG produced from phosphatidyl choline by phospholipase D are not involved in the generation of {gamma}PKC oscillations. The dependence of mGluR5-induced {gamma}PKC oscillation on intracellular or extracellular free Ca2+ was further studied by treating with EGTA alone, BAPTA-AM, and thapsigargin. Chelating extracellular Ca2+ with EGTA during the continuous stimulation of mGluR5 did not block the {gamma}PKC oscillation but slightly decreased the frequency of {gamma}PKC oscillation from 76.0 ± 4.0 to 61.0 ± 1.0 mHz. (Table I). In contrast, blocking the intracellular Ca2+ rise with 5 µM thapsigargin (Table I) by inhibiting endoplasmic reticulum Ca2+-ATPase or by chelating both extra- and intracellular Ca2+ with 2.5 mM EGTA, 15 µM BAPTA-AM (data not shown) completely abolished the oscillation. Oscillations were not altered by the deletion of the C1/DAG binding domain of {gamma}PKC but were abolished by deletion of the C2/Ca2+-binding domain (data not shown). These observations strongly suggested that the mGluR5-mediated oscillations of {gamma}PKC are dependent on the activation of PLC and Ca2+ release from intracellular Ca2+-stores and do not require DAG production. However, the repetitive oscillations required the continuous stimulation of mGluR5, as the glutamate-induced oscillation of {gamma}PKC-DsRed was quickly abolished by the application of 50 µM methylphenylethynylpyridine, a selective antagonist of mGluR5. Neither inhibition of microtubule polymerization (colchicine) nor actin polymerization (cytochalasin D) altered the repetitive distribution of PKC (Table I).


View this table:
[in this window]
[in a new window]
  		TABLE I
Effects of various inhibitors on oscillation frequencies of {gamma}PKC-GFP or its mutants by mGluR5 stimulation

Inhibitors were treated 15 min before (Pretreatment 1) the application of 100 µM glutamate to mGluR5/HEK cells. Some inhibitors were applied during glutamate-induced oscillation (Post-treatment 2). Oscillation frequencies are shown the mean ± S.E. (n = 5).

 
nPKC but Not cPKC Inhibits the mGluR5-mediated Oscillation—It is still controversial whether or not the PKC-dependent phosphorylation of Thr840 in the G protein-interacting domain of mGluR5 is necessary for the mGluR5-stimulated Ca2+ oscillations (5, 6). Thr840 of mGluR5 can be phosphorylated by PKC in vivo and in vitro, and phosphorylation of mGluR5 at Thr840 abolishes the fast oscillation (30-60 mHz) but not the slower oscillation (10 mHz) through mGluR1a or mGluR5a (8). To determine which isoform of PKC is involved in the regulation of Ca2+ and PKC oscillation in our system, we investigated the effects of overexpression of other PKC isoforms on glutamate-induced oscillation of {gamma}PKC. As shown in Fig. 2, when {beta}IIPKC-GFP, which is also a member of DAG- and Ca2+-dependent PKC (cPKC), and {gamma}PKC-DsRed were co-transfected into HEK/mGluR5 cells, synchronous oscillation of {gamma}PKC-DsRed and {beta}IIPKC-GFP was observed (Fig. 2A and Supplemental Material, Video 2). The same result was obtained when either {alpha}PKC-GFP or {beta}IPKC-GFP was co-transfected with {gamma}PKC (Table II). However, as shown in Fig. 2B, when {delta}PKC-GFP, which is a member of DAG-dependent and Ca2+-independent PKC (nPKC), was co-expressed, mGluR5 stimulated a single translocation of {gamma}PKC-DsRed and {delta}PKC-GFP to the plasma membrane, but subsequent oscillatory translocations were not observed. Overexpression of {epsilon}PKC-GFP also blocked the oscillation; however, {zeta}PKC, an atypical PKC, did not respond to mGluR5 activation and did not influence {gamma}PKC oscillation (Table II). The inhibitory effect of {delta}PKC-GFP was due to the kinase activity of {delta}PKC-GFP, because the treatment with rottlerin, a specific inhibitor of {delta}PKC, restored the {gamma}PKC oscillation even in the HEK/mGluR5 cells overexpressing {delta}PKC-GFP (Table II). Furthermore, overexpression of the kinase-dead mutant of {delta}PKC ({delta}PKC-KN-GFP) with {gamma}PKC-DsRed did not alter the oscillation of {gamma}PKC, and {delta}PKC-KN-GFP itself showed a synchronous oscillation to {gamma}PKC-DsRed (Fig. 2, C and D, and Table II). These results suggest that DAG is repetitively produced by the hydrolysis of PIP2 and translocates {delta}PKC to the plasma membrane and that the DAG production is synchronous to IP3 and Ca2+ mobilization.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 2.
Effects of co-expression with {beta}22 or {delta}PKC-GFP on mGluR5-induced oscillatory translocation of {gamma}PKC-DsRed. A, effect of co-expression of {beta}22PKC-GFP on the translocation of {gamma}PKC-DsRed induced by mGluR5 stimulation. Glutamate stimulation induced synchronous oscillation of both {beta}22PKC and {gamma}PKC. Data shown are representative of at least five experiments. B, effect of co-expression of {delta}PKC-GFP on the translocation of {gamma}PKC-DsRed induced by mGluR5 stimulation. Glutamate stimulation induced a transient translocation of both {delta}PKC and {gamma}PKC, and oscillatory translocation of {gamma}PKC was not observed. Data shown are representative of at least five experiments. C, simultaneous imaging of {gamma}PKC-DsRed and {delta}PKC-KN-GFP after mGluR5 activation. Both {gamma}PKC-DsRed and {delta}PKC-KN-GFP showed repetitive translocation. Data shown are representative of at least five experiments. D, synchronized translocation of {gamma}PKC-DsRed and {delta}PKC-KN-GFP by mGluR5 activation. Overexpression of the kinase-negative mutant of {delta}PKC-GFP ({delta}PKC-KN-GFP) did not block the oscillation of {gamma}PKC-DsRed. {delta}PKC-KN-GFP itself showed repetitive translocation, which is synchronized with that of PKC-DsRed. Data shown are representative of at least five experiments.

 


View this table:
[in this window]
[in a new window]
  		TABLE II
Oscillatory translocation of various PKC subtypes and their mutants induced by mGluR5 stimulation

Frequencies are shown as mean ± S.E. (n = 5).

 
{gamma}PKC Activation Is Not Necessary for the Ca2+ or {gamma}PKC Oscillation—Previous reports demonstrated that the phosphorylation/dephosphorylation of a single amino acid of mGluR5 is critical for Ca2+ oscillations. It was suggested that PKC-dependent phosphorylation of mGluR5 interferes with signal transduction between receptor and G protein, but subsequent dephosphorylation of the receptors restores signal transduction by hydrolyzing PIP2 (5). If this model is true, it is possible that a Ca2+-dependent PKC such as {gamma}PKC repetitively interferes with signal transduction by phosphorylating Thr840 of mGluR5. Thus, to examine whether kinase activity of {gamma}PKC is necessary for the generation of mGluR5-mediated Ca2+ oscillation, we tested the translocation of {gamma}PKC lacking its kinase domain. After stimulation of mGluR5, the GFP-tagged C1C2 domain of {gamma}PKC showed oscillatory translocation, which was synchronous to that of full-length {gamma}PKC (Fig. 3A). The oscillation of C1C2 domain was also observed in the presence of Gö6983, a broad PKC inhibitor (Fig. 3B), indicating that kinase activity of {gamma}PKC is not necessary for the generation of mGluR5-mediated oscillation of Ca2+.



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 3.
Oscillatory translocation of {gamma}PKC lacking catalytic domain. A, mGluR5 HEK cells expressing both {gamma}PKC-DsRed and the GFP-tagged C1C2 domain of {gamma}PKC, which lacks catalytic activity, were stimulated with 100 µM L-glutamate. Both PKCs revealed oscillatory translocation. Data shown are representative of at least five experiments. B, effects of Gö6983 (a cPKC inhibitor) on oscillation of C1C2-GFP. The application of 6 nM Gö6983 (bar) did not affect oscillation of {gamma}PKC C1C2-GFP. Data shown are representative of at least five experiments.

 
Both {delta}PKC and {gamma}PKC Are Translocated/Activated by mGluR5 Stimulation, but Only {delta}PKC Is Responsible for Phosphorylating Thr840 of mGluR5—The inhibitory effect of {delta}PKC-GFP on {gamma}PKC-translocation is in good agreement with previous findings that phosphorylation of Thr840 of mGluR5 inhibits the fast oscillation of Ca2+ (6). This putative PKC phosphorylation site is not conserved in mGluR1a. Rather, the acidic amino acid Asp is present in the analogous position. The substitution of the Asp in mGluR1a with Thr restored the PKC oscillation (Fig. 4A). In contrast, mutation of mGluR5 (Thr840 -> Asp) abolished the oscillation, consistent with the previous reports (6) (Fig. 4B). Interestingly, substitution of Thr840 to Ala, an unphosphorylatable amino acid, protected mGluR5-mediated oscillation from the inhibitory effects of {delta}PKC, consistent with a model in which {delta}PKC is translocated to the plasma membrane and phosphorylates Thr840, which abolishes the oscillatory activation of PLC and {gamma}PKC.



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 4.
Oscillatory translocation of {gamma}PKC and its attenuation by {delta}PKC in response to the stimulation of mutants of group I mGluRs. Various constructs containing mutations in the C-terminal intracellular loop of group I mGluRs were transiently co-transfected into HEK293 cells with {gamma}PKC-DsRed and {delta}PKC-GFP or {delta}PKC-KN GFP. All data shown are representative of at least five experiments. A, translocation by the stimulation of mGluR1 and its mutant (mGluR1T). An Asp residue at position 854 of mGluR1 was substituted for threonine (mGluR1T, D854T). B, translocation by the stimulation of mGluR5 and its mutant (mGluR5D, mGluR5A). A threonine residue at position 840 of mGluR5 (Thr840) was substituted to aspartate (mGluR5D, T840D) or alanine (mGluR5A, T840A).

 
It is still unclear why {delta}PKC, but not {gamma}PKC, is responsible for Thr840 phosphorylation, although both are translocated to the plasma membrane by mGluR5 stimulation. All cPKCs translocated to the plasma membrane as {delta}PKC does, but only {delta}PKC altered signaling (Table II). One possibility is that cPKCs cannot phosphorylate Thr840 because the translocation is not accompanied by activation. To investigate whether {gamma}PKC is active at the plasma membrane during the oscillation, we used MARCKS-GFP. MARCKS, a major PKC substrate, is constitutively present on the plasma membrane and translocates from the plasma membrane to the cytoplasm upon phosphorylation by PKC. We reasoned that, if membrane-associated PKC was active, PKC translocation would be accompanied by the reverse (i.e. plasma membrane to cytosol) translocation of MARCKS-GFP. After the application of glutamate to the HEK-mGluR5 cells expressing both MARCKS-GFP and {gamma}PKC-DsRed, MARCKS-GFP was translocated from the plasma membrane to cytoplasm and was redistributed to the plasma membrane. The oscillation in MARCKS-GFP was synchronized with that of {gamma}PKC-DsRed, although in the reverse direction (Fig. 5, A and B, and Supplemental Material, Video 3). MARCKS, however, did not show this oscillatory movement when co-expressed with the C1C2 domain of {gamma}PKC, which lacks kinase activity (Fig. 5C). This finding suggests that {gamma}PKC but not endogenous PKC can phosphorylate MARCKS, although it is possible that overexpressed C1C2-{gamma}PKC buffers DAG or Ca2+ and prevents activation of endogenous PKCs. Treatment with the Ca2+-dependent PKC inhibitor Gö6976 inhibited the oscillation of MARCKS-GFP but not that of {gamma}PKC-DsRed, consistent with our hypothesis that the oscillation of MARCKS is due to its repetitive phosphorylation by active {gamma}PKC (Fig. 5D). Oscillation of the C1C2 domain was not altered by Gö6976 (data not shown). These data provided evidence that {gamma}PKC is activated when translocated to the plasma membrane but fails to phosphorylate Thr840 of mGluR5.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 5.
mGluR5-mediated translocation and oscillation of {gamma}PKC-DsRed and MARCKS-GFP. mGluR5 HEK cells expressing both {gamma}PKC-DsRed and MARCKS-GFP were stimulated with 100 µM L-glutamate. Fluorescence of GFP and DsRed was simultaneously monitored by confocal microscopy. A, fluorescence images of {gamma}PKC-DsRed and GFP-PHD at the time point (0-24 s). B, time course of oscillations of {gamma}PKC-DsRed and MARCKS-GFP. The time scale bar represents 60 s. Data shown are representative of at least five experiments. C, translocation of MARCKS in cells co-expressing MARCKS-DsRed and {gamma}PKC C1C2-GFP. D, effects of Gö6976 on the oscillation of MARCKS-GFP. Data shown are representative of at least three experiments.

 
siRNA-induced Suppression of Endogenous {delta}PKC and Its Effect on Spontaneous {gamma}PKC Oscillation—Spontaneous oscillatory translocation of {gamma}PKC could be observed without activation of mGluR5 receptors. 11% of HEK cells expressing {gamma}PKC-GFP showed spontaneous oscillations of {gamma}PKC. We therefore examined the effects of {delta}PKC knockdown on this spontaneous activity by using siRNA for human {delta}PKC. 48 h after transfection of siRNA, endogenous {delta}PKC in HEK cells was undetectable, as shown previously (24). Since the amounts of endogenous nPKCs including {epsilon}PKC are much lower than that of {delta}PKC, siRNA of {delta}PKC is effective enough to knock down most of the endogenous nPKCs. Treatment with siRNA significantly increased the number of cells from 11 to 72%, which showed spontaneous {gamma}PKC oscillation without glutamate stimulation. Knockdown of {delta}PKC slightly increased the number of the cells showing glutamate-induced oscillation of {gamma}PKC: 56% (without siRNA) and 86% (with siRNA).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Ca2+ oscillation is involved in various cellular responses including the control of gene expression and sustained activation of mitochondrial function (28, 29). The frequency of the Ca2+ oscillations is critically important for efficacy and specificity of gene transcription (e.g. the transcription via NF-{kappa}B is enhanced by low frequency Ca2+ oscillation, whereas high frequency Ca2+ oscillations activate NF-AT) (29). In differentiating neurons, specific neuronal characteristics, including channel maturation and neurotransmitter expression, are altered depending on the frequency of Ca2+ oscillations (30). In the present study, we examined the oscillatory translocation of Ca2+ and the neuron-specific {gamma}PKC.

Transient or prolonged stimulation of mGluR1a or mGluR5a with glutamate triggers different patterns of increase in intracellular Ca2+ (5-7, 31). Continuous Ca2+ oscillations at ~50 mHz were observed by the stimulation of mGluR5, whereas stimulation of mGluR1 resulted in a single transient peak of Ca2+ mobilization without high frequency Ca2+ oscillation. Although mGluR1-mediated Ca2+ oscillations were sometimes observed after the initial peak, the frequency was much lower (less than 25 mHz) than that elicited by mGluR5, and the oscillation through mGluR1 was due to Ca2+ influx through the Ca2+ channel (5). In contrast, mGluR5-mediated Ca2+ oscillations were induced by cyclical mobilization of Ca2+ from intracellular stores. To analyze the subtype-specific translocation of PKC and its functional role in Ca2+ oscillation, we focused on the fast Ca2+ oscillation that is coupled to the IP3/DAG pathway and observed only upon mGluR5 stimulation.

The continuous oscillatory translocation of {gamma}PKC at a frequency of 72.3 mHz was induced by prolonged application of glutamate to HEK cells expressing mGluR5a. The repetitive translocation of {gamma}PKC was dependent on PLC activation but independent of PLA2 and phospholipase D (Table I) and synchronized with the oscillatory translocation of PHD-GFP (Fig. 1). Deletion of the DAG-binding C1 domain from {gamma}PKC did not alter its ability to oscillate, indicating that DAG binding is not critical for cyclical association of {gamma}PKC with the membrane. In contrast, the loss of Ca2+ binding C2 domain abolished {gamma}PKC localization, suggesting that repetitive binding of Ca2+ to the C2 domain causes the oscillatory translocation and that PKC translocation to the plasma membrane reflects an increase in intracellular Ca2+.

Previous studies revealed that Thr840 of mGluR5 is phosphorylated by PKC and that phosphorylation of this Thr residue is responsible for mGluR5-controlled Ca2+ oscillations (6). Our results are in good agreement with these reports. Specifically, we show that phosphorylation of Thr840 by {delta}PKC prevents {gamma}PKC oscillation and that substitution of Thr840 with the acidic amino acid, Asp, also blocks the oscillation. It is noteworthy that mutation of Thr840 to Ala resulted in {delta}PKC-insensitive mGluR5 (T840A)-mediated oscillation of {gamma}PKC. This suggests that phosphorylation of mGluR5 Thr840 is responsible for the blockade but not necessary for the generation of Ca2+ and PKC oscillation. Dale et al. (8) also reported that oscillatory mGluR signaling does not involve the repetitive feedback phosphorylation by PKC and desensitization of mGluR activity. They showed that oscillatory translocations of {beta}IIPKC at different frequencies are stimulated by mGluR5 and mGluR1, and the frequencies are determined by the charge of a single amino acid residue in the C-terminal intracellular loop of mGluRs (8). In the present studies, we could not detect an mGluR1-induced oscillation of {gamma}PKC. It may be the differences between PKC subtypes. Indeed, Babwah et al. (9) reported that {beta}IIPKC but not {gamma}PKC displays oscillation in response to mGluR1 activation. Several reports have shown that both Ca2+ and PKC oscillations are attenuated by PKC inhibitors (5, 6, 8, 23), and these findings apparently contradict the model in which Ca2+ and cPKC oscillations are inhibited by the nPKC-dependent phosphorylation of Thr840. However, it is possible that, in addition to Thr840, other amino acid residues are involved in control of the oscillations and that phosphorylation of this as yet unidentified amino acid may be necessary for the oscillations.

GFP-tagged PKC rapidly and reversibly translocates in response to the activation of G-protein-coupled receptors in living cells (16-18, 32, 33). Several reports have demonstrated that PKC translocation is subtype- and stimulus-specific (17, 18). Phorbol ester slowly translocates all cPKC and nPKC members to the plasma membrane, whereas arachidonic acid induces translocation of {gamma}PKC to the plasma membrane and {epsilon}PKC to the Golgi complex (17, 20). Furthermore, activation of {delta}PKC by three different activators induced distinct patterns of {delta}PKC translocation and resulted in different cellular responses (18-20). These findings suggest that the variety of PKC functions contributing to signal transduction may be due to these multiple targeting mechanisms for each PKC subtype.

The key finding of the current work is that, although both {delta}PKC and {gamma}PKC translocate to the plasma membrane in response to mGluR5 stimulation, their requirements for translocation and the results of that localization are different. Specifically, {delta}PKC, but not {gamma}PKC, phosphorylates Thr840 of mGluR5 and blocks repetitive activation of PLC. Additionally, {gamma}PKC translocation to the plasma membrane can be initiated by a rise in intracellular Ca2+ and is independent of DAG formation (15, 16), whereas DAG formation is indispensable for {delta}PKC translocation. It should be noted, however, that the simultaneous increase in both DG and Ca2+ is necessary for maximal activation of {gamma}PKC (3, 16). We tested the possibility that the membrane-associated {gamma}PKC could not phosphorylate mGluR5 because it was not catalytically active. Therefore, we examined the activity of the oscillating {gamma}PKC using GFP-tagged MARCKS, a PKC substrate. MARCKS is a major PKC substrate that is distributed in various cell types (34-36). It is localized on the plasma membrane by insertion of its myristate chain into the bilayer and by the electrostatic interaction of the cluster of the basic residues in the effector domain with acidic lipids (21, 37). PKC-mediated phosphorylation of this basic cluster neutralizes the charge and facilitates MARCKS movement from the membrane into the cytosol (38). MARCKS-GFP showed mGluR5-mediated oscillatory translocation from the plasma membrane to cytoplasm, which was synchronized with the translocation of {gamma}PKC. This mGluR5-mediated oscillation of MARCKS was blocked by a cPKC inhibitor, and exogenous {gamma}PKC lacking its catalytic domain (C1C2-GFP) failed to translocate MARCKS, indicating that membrane-associated {gamma}PKC is active and repetitively phosphorylates MARCKS in response to mGluR5 stimulation. Thus, although both {delta}PKC and {gamma}PKC are activated and translocated to the plasma membrane, Thr840 of mGluR5 is subtype-specifically phosphorylated by {delta}PKC. This difference may be due to subtly different membrane targeting of cPKC versus nPKC, or PKC subtypes might have substrate specificity under physiological conditions. The knockdown of {delta}PKC using siRNA increased the number of HEK/mGluR5 cells that exhibited spontaneous oscillations of {gamma}PKC, suggesting that the phosphorylation/dephosphorylation of mGluR5 by endogenous {delta}PKC and unknown phosphatases regulates intracellular Ca2+ mobilization. Further studies are necessary to clarify the molecular mechanism of this subtype-specific regulation of mGluR5-mediated oscillation.


   FOOTNOTES
 
* This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology in Japan; a grant-in-aid for Scientific Research on Priority Areas (C) Advanced Brain Science Project from the Ministry of Education, Culture, Sports, Science and Technology in Japan; and the Uehara Memorial Foundation and Sankyo Foundation of Life Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains three videos. Back

** To whom correspondence should be addressed: 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan. Tel.: 81-78-803-5961; Fax: 81-78-803-5971; E-mail: naosaito{at}kobe-u.ac.jp.

1 The abbreviations used are: mGluR, metabotropic glutamate receptor; PKC, protein kinase C; cPKC, classical PKC; nPKC, novel PKC; DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; PIP2, phosphotidylinositol 4,5-bisphosphate; PHD, pleckstrin homology domain; PLC, phospholipase C; PLA2, phospholipase A2; MARCKS, myristoylated alanine-rich C-kinase substrate; GFP, green fluorescent protein; DsRED, DiscoSoma red protein; KN, kinase-negative; siRNA, short interfering RNA; BAPTA-AM, O,O'-bis(2-aminophenyl)ethylene glycol-N,N,N,N-tetraacetic acid tetraacetoxymethyl ester. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Michelle R. Lennartz of The Albany Medical College for helpful discussions of this work.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Nakanishi, S., and Masu, M. (1994) Annu. Rev. Biophys. Biomol. Struct. 23, 319-348[Medline] [Order article via Infotrieve]
  2. Hollmann, M., and Heinemann, S. (1994) Annu. Rev. Neurosci. 17, 31-108[CrossRef][Medline] [Order article via Infotrieve]
  3. Nishizuka, Y. (1984) Nature 308, 693-698[CrossRef][Medline] [Order article via Infotrieve]
  4. Berridge, M. J., and Irvine, R. F. (1984) Nature 312, 315-321[CrossRef][Medline] [Order article via Infotrieve]
  5. Kawabata, S., Kohara, A., Tsutsumi, R., Itahana, H., Hayashibe, S., Yamaguchi, T., and Okada, M. (1998) J. Biol. Chem. 273, 17381-17385[Abstract/Free Full Text]
  6. Kawabata, S., Tsutsumi, R., Kohara, A., Yamaguchi, T., Nakanishi, S., and Okada, M. (1996) Nature 383, 89-92[CrossRef][Medline] [Order article via Infotrieve]
  7. Nash, M. S., Schell, M. J., Atkinson, P. J., Johnston, N. R., Nahorski, S. R., and Challiss, R. A. (2002) J. Biol. Chem. 277, 35947-35960[Abstract/Free Full Text]
  8. Dale, L. B., Babwah, A. V., Bhattacharya, M., Kelvin, D. J., and Ferguson, S. S. (2001) J. Biol. Chem. 276, 35900-35908[Abstract/Free Full Text]
  9. Babwah, A. V., Dale, L. B., and Ferguson, S. S. G. (2003) J. Biol. Chem. 278, 5419-5426[Abstract/Free Full Text]
  10. Nishizuka, Y. (1988) Nature 334, 661-665[CrossRef][Medline] [Order article via Infotrieve]
  11. Nishizuka, Y. (1992) Science 258, 607-614[Abstract/Free Full Text]
  12. Osada, S., Mizuno, K., Saido, T. C., Suzuki, K., Kuroki, T., and Ohno, S. (1992) Mol. Cell. Biol. 12, 3930-3938[Abstract/Free Full Text]
  13. Ono, Y., Fujii, T., Ogita, K., Kikkawa, U., Igarashi, K., and Nishizuka, Y. (1988) J. Biol. Chem. 263, 6927-6932[Abstract/Free Full Text]
  14. Nishizuka, Y. (1995) FASEB J. 9, 484-496[Abstract]
  15. Oancea, E., Teruel, M., Quest, A., and Meyer, T. (1998) J. Cell Biol. 140, 485-498[Abstract/Free Full Text]
  16. Sakai, N., Sasaki, K., Ikegaki, N., Shirai, Y., and Saito, N. (1997) J. Cell Biol. 139, 1465-1476[Abstract/Free Full Text]
  17. Shirai, Y., Kashiwagi, K., Yagi, K., Sakai, N., and Saito, N. (1998) J. Cell Biol. 143, 511-521[Abstract/Free Full Text]
  18. Ohmori, S., Shirai, Y., Sakai, N., Fujii, M., Konishi, H., Kikkawa, U., and Saito, N. (1998) Mol. Cell. Biol. 18, 5263-5271[Abstract/Free Full Text]
  19. Kajimoto, T., Ohmori, S., Shirai, Y., Sakai, N., and Saito, N. (2001) Mol. Cell. Biol. 21, 1769-1783[Abstract/Free Full Text]
  20. Kashiwagi, K., Shirai, Y., Kuriyama, M., Sakai, N., and Saito, N. (2002) J. Biol. Chem. 277, 18037-18045[Abstract/Free Full Text]
  21. Ohmori, S., Sakai, N., Shirai, Y., Yamamoto, H., Miyamoto, E., Shimizu, N., and Saito, N. (2000) J. Biol. Chem. 275, 26449-26457[Abstract/Free Full Text]
  22. Hirose, K., Kadowaki, S., Tanabe, M., Takeshima, H., and Iino, M. (1999) Science 284, 1527-1530[Abstract/Free Full Text]
  23. Nakahara, K., Okada, M., and Nakanishi, S. (1997) J. Neurochem. 69, 1467-1475[Medline] [Order article via Infotrieve]
  24. Irie, N., Sakai, N., Ueyama, T., Kajimoto, T., Shirai, Y., and Saito, N. (2002) Biochem. Biophys. Res. Commun. 298, 738-743[CrossRef][Medline] [Order article via Infotrieve]
  25. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Nature 411, 494-498[CrossRef][Medline] [Order article via Infotrieve]
  26. Ono, Y., Fujii, T., Igarashi, K., Kuno, T., Tanaka, C., Kikkawa, U., and Nishizuka, Y. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4868-4871[Abstract/Free Full Text]
  27. Nishizuka, Y. (1986) Science 233, 305-312[Abstract/Free Full Text]
  28. Hajnoczky, G., Robb-Gaspers, L. D., Seitz, M. B., and Thomas, A. P. (1995) Cell 82, 41-424
  29. Dolmetsch, R. E., Xu, K., and Lewis, R. S. (1998) Nature 392, 933-936[CrossRef][Medline] [Order article via Infotrieve]
  30. Gu, X., and Spitzer, N. C. (1995) Nature 375, 784-787[CrossRef][Medline] [Order article via Infotrieve]
  31. Codazzi, F., Teruel, M. N., and Meyer, T. (2001) Curr. Biol. 11, 1089-1097[CrossRef][Medline] [Order article via Infotrieve]
  32. Shirai, Y., Kashiwagi, K., Sakai, N., and Saito, N. (2000) J. Cell Sci. 113, 1335-1343[Abstract]
  33. Oancea, E., and Meyer, T. (1998) Cell 95, 307-318[CrossRef][Medline] [Order article via Infotrieve]
  34. Aderem, A. A., Albert, K. A., Keum, M. M., Wang, J. K., Greengard, P., and Cohn, Z. A. (1988) Nature 332, 362-364[CrossRef][Medline] [Order article via Infotrieve]
  35. Aderem, A. (1992) Cell 71, 713-716[CrossRef][Medline] [Order article via Infotrieve]
  36. Blackshear, P. J. (1993) J. Biol. Chem. 268, 1501-1504[Free Full Text]
  37. George, D., and Blackshear, P. (1992) J. Biol. Chem. 267, 24879-24885[Abstract/Free Full Text]
  38. Kligman, D., and Patel, J. (1986) J. Neurochem. 47, 298-303[Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.