Protein Kinase C Isoform-specific Differences in the Spatial-Temporal Regulation and Decoding of Metabotropic Glutamate Receptor1a-stimulated Second Messenger Responses*

Andy V. BabwahDagger §, Lianne B. DaleDagger §, and Stephen S. G. FergusonDagger ||**DaggerDagger

From the Dagger  Cell Biology Research Group, Robarts Research Institute, and the || Department of Physiology & Pharmacology, University of Western Ontario, 100 Perth Drive, London, Ontario N6A 5K8, Canada

Received for publication, October 29, 2002, and in revised form, November 18, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Metabotropic glutamate receptors (mGluRs) coupled via Gq to the hydrolysis of phosphoinositides stimulate Ca2+ and PKCbeta II oscillations in both excitable and non-excitable cells. In the present study, we show that mGluR1a activation stimulates the repetitive plasma membrane translocation of each of the conventional and novel, but not atypical, PKC isozymes. However, despite similarities in sequence and cofactor regulation by diacyglycerol and Ca2+, conventional PKCs exhibit isoform-specific oscillation patterns. PKCalpha and PKCbeta I display three distinct patterns of activity: 1) agonist-independent oscillations, 2) agonist-stimulated oscillations, and 3) persistent plasma membrane localization in response to mGluR1a activation. In contrast, only agonist-stimulated PKCbeta II translocation responses are observed in mGluR1a-expressing cells. PKCbeta I expression also promotes persistent increases in intracellular diacyglycerol concentrations in response to mGluR1a stimulation without affecting PKCbeta II oscillation patterns in the same cell. PKCbeta II isoform-specific translocation patterns are regulated by specific amino acid residues localized within the C-terminal PKC V5 domain. Specifically, Asn-625 and Lys-668 localized within the V5 domain of PKCbeta II cooperatively suppress PKCbeta I-like response patterns for PKCbeta II. Thus, redundancy in PKC isoform expression and differential decoding of second messenger response provides a novel mechanism for generating cell type-specific responses to the same signal.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The spatial-temporal patterning of Ca2+ release from intracellular stores contributes to the regulation of a diverse array of cellular responses including insulin secretion, sustained activation of mitochondrial function, and the selective activation of transcription factors required for fine-tuning gene expression during inflammatory immune responses (1-4). Receptor-stimulated oscillations in intracellular-free Ca2+ are observed in both excitable and non-excitable cells following the activation of either G protein-coupled receptors or receptor tyrosine kinases (5). The synchronization of Ca2+ oscillations at the cellular level involves at least two modes of Ca2+ signaling: repetitive baseline Ca2+ spikes and sinusoidal-type Ca2+ oscillations (6). These Ca2+ oscillations are faithfully recapitulated by the repetitive redistribution of protein kinase C (PKC)1 between the cytosol and plasma membrane (7-10). The molecular mechanism(s) underlying the repetitive baseline plasma membrane translocation of PKC are best characterized for Group I metabotropic glutamate receptors (mGluRs). PKCbeta II and PKCgamma oscillations are regulated by mGluR-stimulated oscillations in diacyglycerol (DAG), inositol 1,4,5-triphosphate (InsP3), and Ca2+ release from intracellular stores (8, 9). Glutamate-activated Ca2+ and/or PKC oscillations are observed in most cell systems including immature neuronal cultures, developing neocortex, astrocytes, and mGluR-transfected heterologous cell cultures (8, 9, 11-14).

PKC isoforms are classified into three groups based on structural properties and cofactor requirements and exhibit specific in vivo activity as well as spatial organization within the cell (15-17). The activation and plasma membrane localization of the conventional PKC isoforms (alpha , beta I, beta II, and gamma ) is regulated by Ca2+ and DAG, whereas the activity and subcellular localization of the novel PKC isoforms (delta , epsilon , eta , and theta ) is regulated by DAG, but not Ca2+ (17, 18). The atypical PKCs do not respond to either Ca2+ or DAG (17, 18). Although PKC structure/function has been studied extensively, it is unknown whether PKC subtype-specific differences in Ca2+- and/or DAG-binding affinities contribute to PKC isoform-specific activity and/or spatial-temporal distribution within cells.

In the present study, we explore whether mGluR1a-stimulated oscillations in intracellular DAG and/or Ca2+ concentrations regulate conventional and novel PKC isoform activity in an identical manner. We find that, although all conventional and novel PKC isoforms oscillate in response to mGluR1a activation, conventional PKC isoforms exhibit isozyme-specific translocation response patterns that we classify as either PKCbeta I- or PKCbeta II-like responses. Specifically, we have identified two discrete amino acid residues localized within the V5 domain of PKCbeta II that function to suppress PKCbeta I-like responses for PKCbeta II. Thus, structural differences in the PKC V5 domain allow conventional PKC isoforms to differentially decode and influence receptor-stimulated DAG and Ca2+ signals. Consequently, the expression of multiple conventional PKC isozymes in either the same cell or within different cells provides a novel mechanism by which cell type-specific responses to an identical signal may be established.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials-- Restriction enzymes were obtained from Promega and New England Biolabs Inc. The pcDNA3.1/Amp expression vector was acquired from Invitrogen. DsRed2-C1, pEGFP-C1, pEGFP-C2, and pEGFP-C3 expression vectors were purchased from Clontech. The QuikChangeTM Site-directed Mutagenesis kit was from Stratagene. The human universal Quick-CloneTM cDNA library was obtained from Clontech. Human embryonic kidney cells (HEK 293) were from American Type Culture Collection (ATCC). Fetal bovine serum was from Hyclone Laboratories Inc. Gentamicin, minimal essential medium, and 0.05% Trypsin containing 0.5 mM EDTA were acquired from Invitrogen. The calcium indicator, Oregon Green 488 BAPTA-1 AM, was obtained from Molecular Probes. Quisqualate was from Tocris Cookson Inc. All other biochemical reagents were purchased from Sigma, Fisher Scientific, and VWR.

Plasmid Constructs-- To construct EGFP-tagged PKCalpha , PKCbeta I, PKCgamma , PKCdelta , PKCepsilon , PKCeta , PKCtheta , PKCzeta , and PKCiota /lambda the cDNA for each of the PKC isoforms were first amplified by PCR from the human universal Quick-CloneTM library (Clontech). The PCR products generated were digested with the appropriate restriction enzymes and subcloned into the appropriate pEGFP-C1, pEGFP-C2, and pEGFP-C3 vectors (Clontech). The PKCbeta I cDNA was also cloned into the BglII-XbaI sites of the vector DsRed2-C1 (Clontech). The construction of EGFP-PKCbeta II (28), and EGFP-PLCdelta 1 PH domain were previously described (8). The EGFP-PKCdelta C1 domain was a generous gift from Dr. Sergio Grinstein. PKCalpha /beta II, beta II/alpha , beta I/beta II, and beta II/beta I chimeras were constructed by "two-step PCR" as described previously (19) and the resulting PCR products subcloned into either the pEGFP-C1 or pEGFP-C3 vector. PKCalpha and beta II point and deletion mutants were constructed using the QuikChangeTM Site-directed Mutagenesis kit (Stratagene). Sequence integrity of all PCR-generated products was confirmed by automated DNA sequencing.

Cell Culture and Transfection-- HEK 293 cells were maintained in minimal essential medium supplemented with 10% (v/v) fetal bovine serum and 100 µg/ml gentamicin at 37 °C in a humidified atmosphere containing 5% CO2. Cells used in each of the experiments were transfected using a modified calcium phosphate method as described previously (20). Following transfection (~18 h), cells were incubated with fresh medium and allowed to recover 8 h and allowed to grow an additional 18 h before any experimentation. In all experiments, cells were transfected with 10 µg of pcDNA3.1 plasmid cDNA containing FLAG-mGluR1a with and without 1-5 µg of each PKC construct expressed in either pEGFP or DsRed2 expression vectors.

Confocal Microscopy-- Following transfection with plasmid cDNAs encoding EGFP-PKC constructs and mGluR1a, cells were re-seeded on collagen-coated 15-mm glass-cover slips designed for use in a perfusion system (Warner Instrument Corporation). All experiments were conducted at 37 °C, and prior to visualization or additional treatments the cells were perfused with at least 5 ml of HEPES-buffered salt solution (1.2 mM KH2PO4, 5 mM NaHCO3, 20 mM HEPES, 11 mM glucose, 116 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 2.5 mM CaCl2, pH 7.4). Cellular InsP3 and DAG levels were measured indirectly through the use of the EGFP-PLCdelta 1 PH domain (8) and EGFP-PKCdelta C1 domain reporter constructs (9). Confocal-based Ca2+ imaging was performed by preloading cells for 30 min with 10 mM Oregon Green 488 BAPTA-1 AM prior to receptor activation according to the manufacturer's specifications (Molecular Probes). Confocal microscopy was performed on a Zeiss LSM-510 laser-scanning microscope using Zeiss 63× 1.4 numerical aperture oil immersion lens. Enhanced GFP and Oregon Green 488 BAPTA fluorescence was visualized with excitation at 488 nm and emission 515-540 nm emission filter set. DsRed2 fluorescence was visualized with excitation at 543 nm and emission 590-610 nm filter set. Fluorescent signals were collected sequentially every 6.8-12.5 s using the Zeiss LSM software time scan function.

Data Analysis-- PKC translocation time courses and Ca2+, DAG, and InsP3 responses were recorded as time series of 150-300 confocal images for each experiment. Image analysis was performed using the Zeiss LSM-510 physiology analysis software and was defined as the relative change in cytoplasmic fluorescence intensity over time in a 5-µm-diameter region of interest. All time course data were plotted using GraphPad Prism. The statistical significance of the data presented in Fig. 6 was analyzed using a non-parametric SIGN test.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PKC Isozyme-specific Plasma Membrane Translocation Responses-- Because individual conventional (alpha , beta I, beta II, and gamma ), novel (delta , epsilon , eta , and theta ), and atypical (iota /lambda and zeta ) PKC isoforms may exhibit differences in their receptor-activated translocation profiles, we have examined the translocation response patterns for each PKC isozyme following mGluR1a agonist activation. We find that each of the conventional PKC isoforms displays the capacity to oscillate between the cytosol to the plasma membrane in response to mGluR1a activation. Shown in Fig. 1 is an agonist-stimulated oscillatory response pattern that is exhibited by all four conventional PKC isoforms. This agonist-stimulated transient, but repetitive, translocation of enzyme to the plasma membrane is the only response pattern that is observed for PKCbeta II (PKCbeta II-like). However, PKCalpha and PKCbeta I display additional translocation responses (PKCbeta I-like) that are never observed for PKCbeta II. Although some cell to cell variability is observed, PKCbeta I-like translocation patterns can be categorized into three distinct patterns that are illustrated in Fig. 2. First, GFP-PKCalpha and GFP-PKCbeta I exhibit the capacity to oscillate between the cytosol and plasma membrane in the absence of mGluR1a activation (Fig. 2A). In these cells, mGluR1a agonist activation serves to increase the oscillation frequency of the entire cellular complement of GFP-tagged PKCalpha and PKCbeta I (Fig. 2A). The removal of the agonist results in the return of the oscillation frequency to the pre-agonist-stimulated oscillatory rate (Fig. 2A, PKCalpha ). These agonist-independent oscillations are only observed following mGluR1a expression, indicating that they occur in response to spontaneous mGluR1a activity. Second, in response to mGluR1a activation, the entire pool of either GFP-PKCalpha or GFP-PKCbeta I translocates from the cytosol to the plasma membrane where it remains persistently localized (at steady state) until the agonist is removed by perfusion (Fig. 2B). Third, mGluR1a activation stimulates the translocation of the entire GFP-PKCalpha and GFP-PKCbeta I pools to the plasma membrane, and a fraction of the enzyme returns to the cytosol and subsequently oscillates in the presence of the agonist (Fig. 2C). PKCgamma also exhibits very weak PKCbeta I-like responses (13/49 cells), but unlike what is observed for the other conventional PKC isoforms the most common PKCgamma response pattern is a single transient plasma membrane translocation (20/49 cells) (data not shown). For PKCalpha , PKCbeta I, and PKCbeta II transient translocation responses were rarely observed (< 5% of cells imaged). Taken together, these observations suggest that PKCalpha , PKCbeta I, and PKCgamma exhibit the capacity to decode subtly different changes in second messenger responses that are not recognized by PKCbeta II. Alternatively, the expression of PKCalpha , PKCbeta I, and PKCgamma may lead to multiple distinct second messenger responses to mGluR1a activation.


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Fig. 1.   Conventional GFP-tagged PKC isoform membrane translocation responses to mGluR1a activation. HEK 293 cells were transfected with cDNA encoding mGluR1a and either GFP-PKCalpha (A), GFP-PKCbeta I (B), GFP-PKCbeta II (C), or GFP-PKCgamma (D). Shown are representative images selected from a time series of 200-400 laser scanning confocal microscopic images collected at 6.8-12.5 s intervals. The images demonstrate the repetitive translocation of GFP-PKCalpha (13/33 cells), GFP-PKCbeta I (10/27 cells), GFP-PKCbeta II (62/62 cells), and GFP-PKCgamma (16/49 cells) proteins between the cytosol and plasma membrane in cells responding to persistent mGluR1a activation with 30 µM quisqualate.


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Fig. 2.   GFP-PKCalpha - and GFP-PKCbeta I-specific response patterns to mGluR1a activation. HEK 293 cells were transfected with cDNA encoding mGluR1a and either GFP-PKCalpha or GFP-PKCbeta I. Shown are GFP-PKCalpha and GFP-PKCbeta I response patterns that are not observed for GFP-PKCbeta II. These responses include the following. A, agonist-independent GFP-PKC oscillations, which increase in frequency following the treatment of cells with 30 µM quisqualate, PKCalpha (5/33 cells), and PKCbeta I (4/27 cells). B, persistent localization of GFP-PKC at the plasma membrane in response to agonist treatment, PKCalpha (8/33 cells) and PKCbeta I (11/27 cells). C, constitutive localization of GFP-PKC at the plasma membrane with concomitant oscillations of a fraction of enzymes between the cytosol and plasma membrane in response to agonist treatment, PKCalpha (7/33 cells), and PKCbeta I (2/27 cells). The micrographs shown in each panel are representative confocal micrographs taken from the GFP-PKCalpha time course illustrated in the same panel. The data are plotted as the relative GFP fluorescence intensity between the cytosol (0) and plasma membrane (1) in a single cell.

Each of the novel PKC isoforms also oscillate in response to mGluR1a activation (Fig. 3). The response for each of the novel PKC isozymes is similar to the third PKCbeta I-like response pattern outlined above (Fig. 2C). Following the agonist-dependent translocation of the entire pool of GFP-PKC, a fraction of each of the novel GFP-PKC isoforms remains localized to the plasma membrane, and the remaining enzyme oscillates in the continued presence of agonist (Fig. 3). The atypical PKC (iota /lambda and zeta ) do not respond to mGluR1a activation (data not shown). Because the conventional PKCs exhibit the greatest behavioral complexity and diversity to mGluR1a activation, we focused subsequent experimentation on the conventional PKC isoforms.


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Fig. 3.   Novel GFP-tagged PKC isoform membrane translocation responses to mGluR1a activation. HEK 293 cells were transfected with cDNA encoding mGluR1a and either GFP-PKCdelta (A), GFP-PKCepsilon (B), GFP-PKCeta (C), or GFP-PKCtheta (D). Shown are representative images selected from a time series of 200-400 laser scanning confocal microscopic images collected at 6.8-12.5 s intervals. The images demonstrate the repetitive translocation of novel GFP-PKC proteins between the cytosol and plasma membrane in response to persistent mGluR1a activation with 30 µM quisqualate. The data are representative of experiments repeated at least 6 times for each kinase.

PKCbeta I-dependent Alterations in mGluR1a-stimulated Second Messenger Responses-- It is possible that the expression of either PKCalpha or PKCbeta I leads to alterations in the patterning of mGluR1a-stimulated second messenger responses and that this may underlie the multiplicity of PKCbeta I-like response patterns. To address this possibility, we examined the patterning of red fluorescent protein tagged-PKCbeta I (DsRed2-PKCbeta I) responses at the same time as we measured changes in intracellular DAG, InsP3, and Ca2+ concentrations.

DAG responses were measured using a GFP-PKCdelta C1 domain construct, which translocates from the cytosol to the plasma membrane in response to increases in intracellular DAG concentrations (9). We found that mGluR1a-stimulated GFP-PKCdelta C1 domain translocation patterns were synchronized exactly with DsRed2-PKCbeta I membrane translocation responses (Fig. 4A). Following mGluR1a activation, the GFP-PKCdelta C1 domain either oscillated between the cytosol and plasma membrane in synchrony with DsRed2-PKCbeta I (Fig. 4A, upper panel) or accumulated with DsRed2-PKCbeta I at the plasma membrane (Fig. 4A, lower panel).


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Fig. 4.   Synchronization of DAG, InsP3, and Ca2+ responses with DsRed2-PKCbeta I translocation following mGluR1a activation. DsRed2-PKCbeta I translocation responses were measured concomitantly with alterations in intracellular DAG (A), InsP3 (B), and Ca2+ (C) concentrations. Changes in intracellular DAG concentrations were assessed by the cytosol to plasma membrane translocation of a GFP-PKCdelta C1 domain reporter construct. Changes in intracellular InsP3 concentrations were measured by the plasma membrane to cytosol translocation of a GFP-PLCdelta PH domain reporter construct. Changes in intracellular Ca2+ concentrations were measured using Oregon Green 488 BAPTA-1 AM.

InsP3 responses were measured using a GFP-PLCdelta 1 PH domain construct, which under basal conditions was localized at the plasma membrane due to its interaction with membrane phosphatidylinositol 4,5-bisphosphate (PIP2) (8). Following mGluR1a-stimulated PIP2 hydrolysis and InsP3 formation the GFP-PLCdelta 1 PH domain was released from the plasma membrane and redistributed to the cytosol (Fig. 4B). Identical to what was observed for DAG responses, the GFP-PLCdelta 1 PH domain either oscillated between the plasma membrane and cytosol at the same frequency at which DsRed2-PKCbeta I translocated from the cytosol to plasma membrane (Fig. 4B, upper panel), or the GFP-PLCdelta 1 PH domain accumulated in the cytosol with the same time course as DsRed2-PKCbeta I accumulated at the plasma membrane (Fig. 4B, lower panel). Thus, the spatial-temporal localization of PKCbeta I at the plasma membrane was coordinated with alterations in both intracellular DAG and InsP3 concentrations.

Changes in intracellular Ca2+ concentrations were measured using the Ca2+ indicator dye Oregon Green 488 BAPTA-1 AM. We found that oscillatory DsRed2-PKCbeta I responses were synchronized with Ca2+ oscillations (Fig. 4C, upper panel). However, unlike what was observed for DAG and InsP3 responses, Ca2+ oscillations persisted when DsRed2-PKCbeta I was persistently localized to the plasma membrane (Fig. 4C, lower panel).

Taken together, our observations indicate that expression of PKCbeta I alters the patterning of DAG and InsP3 formation, but not the patterning of Ca2+ release from intracellular stores in response to mGluR1a activation. This is different from cells expressing PKCbeta II where only synchronized oscillatory DAG, InsP3, and Ca2+ responses are observed following mGluR1a activation (data not shown and Ref. 8).

Effect of PKCbeta I Expression on PKCbeta II Plasma Membrane Translocation Responses-- PKCbeta I and PKCbeta II isoforms are thought to be regulated in the same manner by DAG (17). Therefore, if differences in PKCbeta I versus PKCbeta II response patterns are solely the consequence of PKCbeta I expression-dependent alterations in DAG formation or differences in mGluR1a expression levels between cells, GFP-PKCbeta II should exhibit PKCbeta I-like translocation patterns in cells co-expressing DsRed2-PKCbeta I. When co-expressed together in HEK 293 cells, we observe two distinct DsRed2-PKCbeta I and GFP-PKCbeta II responses to mGluR1a activation: 1) DsRed2-PKCbeta I and GFP-PKCbeta II exhibit synchronized oscillatory plasma membrane translocation responses (Fig. 5A); and 2) DsRed2-PKCbeta I accumulates at the plasma membrane, whereas GFP-PKCbeta II continues to oscillate between the plasma membrane and cytosol (Fig. 5B). These observations suggest that, although PKCbeta I expression alters the patterning of mGluR1a-stimulated DAG and InsP3 response patterns, PKCbeta II is apparently insensitive to PKCbeta I-induced changes in DAG formation. Moreover, the differences in PKCbeta I versus PKCbeta II membrane translocation patterns observed in the same cell indicates that differences in mGluR1a expression levels between cells cannot account for the different PKCbeta I-like translocation patterns.


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Fig. 5.   Translocation response patterns of DsRed2-PKCbeta I and GFP-PKCbeta II in the same cell. HEK 293 cells were transfected with cDNA encoding mGluR1a and both DsRed2-PKCbeta I and GFP-PKCbeta II. Shown are either synchronized DsRed2-PKCbeta I and GFP-PKCbeta II responses to 30 µM quisqualate (13/24 cells) (A) or persistent localization of DsRed2-PKCbeta I at the plasma membrane with simultaneous GFP-PKCbeta II oscillations (7/24 cells) (B). The micrographs shown in each panel are the representative confocal micrographs taken from the DsRed2-PKCbeta I and GFP-PKCbeta II time courses plotted in each panel.

Molecular Determinants for Isozyme-specific Translocation Response Patterns-- The patterning of conventional PKC isoform responses to mGluR1a activation are sub-classified as either PKCbeta I-like (agonist-independent oscillations, agonist-stimulated oscillations, and persistent plasma membrane localization) or PKCbeta II-like (only agonist-stimulated oscillations). We have used these definitions to characterize the structural determinants underlying differences in conventional PKC isozyme response patterns. PKCbeta I and PKCbeta II differ by only 53 amino acids comprising the alternatively spliced V5 domains of the kinases (Fig. 6A). Furthermore, the exchange of the last 54 amino acid residues of PKCalpha with the corresponding residues from PKCbeta II generates a PKCalpha /beta II 620-673 chimera that displays a PKCbeta II-like response pattern (Fig. 6B). Thus, PKC isoform-specific response patterns must be regulated by amino acid residues localized within V5 domains of conventional PKC isoforms.


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Fig. 6.   Structure-Function Analysis of V5 domain residues regulating PKC isoform response patterns. A, schematic representation of the domain structure for conventional PKC isoforms and the alignment of the amino acid sequences of the V5 domains from PKCalpha , PKCbeta I, and PKCbeta II. Non-conserved amino acid residues are boxed. B-D, characterization of the translocation patterns of GFP-PKCalpha , GFP-PKCbeta I and GFP-PKCbeta II chimeras, truncation and point mutations in mGluR1a-expressing cells in either the absence or presence of agonist. The response patterns assessed were plasma membrane oscillations in either the absence (-) or presence (+) of agonist and persistent plasma membrane translocation. The last 13 PKCbeta II amino acids are underlined. Asterisks indicate PKCbeta II mutants displaying differences in translocation pattern compared with the expected wild-type PKCbeta II isoform response patterns. A non-parametric SIGN test, p > 0.07 supports the null hypothesis that the asterisked PKCbeta II mutants exhibit no differences in oscillation patterns with the expected patterns observed for PKCalpha .

Sequence alignment of PKCalpha , PKCbeta I, and PKCbeta II reveals considerable carboxyl-terminal sequence conservation (Fig. 6A). The last 13 amino acids of the V5 domain exhibit the greatest sequence disparity (Fig. 6A). By swapping either the last 13 amino acids of PKCbeta II for the last 15 amino acid residues of PKCalpha (PKCbeta II/alpha 657-672) or deleting the last 13 amino acid residues from the carboxyl-terminal of PKCbeta II (PKCbeta II-S660D), we create PKCbeta II chimeras with PKCbeta I-like response patterns (Fig. 6B). Serial truncation analysis of PKCbeta II between amino acid residues 660 and 672 reveals that PKCbeta II-like responses are lost if the final six (PKCbeta II-L667Delta ) but not the final three (PKCbeta II-E670Delta ) PKCbeta II amino acids are deleted (Fig. 6C). The deletion of Lys-668-Glu-670 (PKCbeta II-KPEDelta ) from PKCbeta II also establishes a PKCalpha -like response pattern for PKCbeta II (Fig. 6C). The mutation of Lys-668, Pro-669, and Glu-670 individually to glycine residues reveals that only Lys-668 is required to maintain PKCbeta II-like responses and to suppress PKCbeta I-like response patterns (Fig. 6D). When expressed together in HEK 293 cells, we find that DsRed2-PKCbeta I and GFP-PKCbeta II-K668G exhibit identical response patterns to mGluR1a activation (Fig. 7).


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Fig. 7.   Synchronization of GFP-PKCbeta II K668G and DsRed2-PKCbeta I responses. HEK293 cells were transfected with cDNA encoding mGluR1a, DsRed2-PKCbeta I, and GFP-PKCbeta II K668G. Shown are: synchronized agonist-stimulated DsRed2-PKCbeta I and GFP-PKCbeta II translocation responses (8/14 cells) (A), synchronized agonist-independent DsRed2-PKCbeta I and GFP-PKCbeta II oscillations that increase in frequency following the treatment of cells with 30 mM quisqualate (3/14 cells) (B), and persistent localization of both DsRed2-PKCbeta I and GFP-PKCbeta II K668G at the plasma membrane in response to agonist stimulation (3/14) (C).

We found that the establishment of a PKCbeta II-like response pattern in PKCalpha required the exchange of the entire PKCbeta II V5 domain. Furthermore, neither the exchange of the last 13 amino acid residues from PKCbeta II into PKCalpha (PKCalpha /beta II 660-673) nor the introduction of the KPE motif into PKCbeta I established PKCbeta II-like responses for either PKCalpha or PKCbeta I (Fig. 6, B and C). Therefore, there must be additional amino acid residues localized within the PKCbeta II V5 domain that cooperated with Lys-668 to establish a PKCbeta II-like response pattern. Sequence alignment of PKCalpha , PKCbeta I, and PKCbeta II indicated that only 4 amino acid residues were not conserved between PKCbeta II and either PKCalpha or PKCbeta I: Asn-625, His-636, Glu-646, and Arg-649 of PKCbeta II (Fig. 6A). Therefore, we mutated each of these residues to the corresponding amino acid residue in PKCalpha and found that only PKCbeta II-N625G exhibited PKCalpha -like behavior patterns (Fig. 6D). In summary, extensive structure-function analysis identified Asn-625 and Lys-668 as essential amino acid residues within the PKCbeta II V5 domain required for the establishment of PKCbeta II translocation responses. The mutation of either residue releases the suppression of PKCalpha -like response behaviors for PKCbeta II.

    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we show that each of the conventional (alpha , beta I, beta II, and gamma ) and novel (delta , epsilon , eta , and theta ), but not atypical (zeta  and iota /lambda ) PKC isozymes respond to mGluR1a activation by repetitively translocating between the cytosol and plasma membrane. The detailed analysis of conventional PKC isoform translocation responses reveals that PKCalpha and PKCbeta I exhibit a variety of unique response patterns to mGluR1a activation that are not observed for PKCbeta II. Because PKCbeta I and PKCbeta II exhibit distinct translocation patterns to the same stimulus, even when expressed in the same cell, it is unlikely these differences can be attributed to differences in mGluR1a expression levels or G protein coupling efficiency. Rather PKCalpha and/or PKCbeta I expression alters the patterning of mGluR1a-stimulated second messenger responses. Furthermore, the sensitivity of conventional PKC isozymes to intracellular DAG and Ca2+ concentrations appears to be regulated by residues localized to the V5 domains of the kinases. Thus, we conclude that the spatial-temporal dynamics of mGluR signaling will not only be determined by the identity of the mGluR isoform that is activated but will also be controlled by the PKC isozyme that is decoding and modulating the mGluR1a-generated second messenger signals. As a consequence, the expression of multiple conventional PKC isozymes in either the same cell or within different cells provides a novel mechanism by which cell type-specific responses to an identical signal may be established.

Both PKCalpha and PKCbeta I exhibit agonist-independent oscillations in cells expressing mGluR1a, and this may be related to the observation that mGluR1a exhibits significant basal activity in the absence of agonist (21, 22). Intrinsic mGluR1a activity leading to the spontaneous activation of PLC may result in sub-threshold changes in DAG and InsP3 formation. If PKCalpha and PKCbeta I exhibit heightened sensitivity to changes in intracellular DAG concentrations as compared with PKCbeta II, this may be translated into isoform-specific agonist-independent membrane translocation. The crystal structure of the mGluR ligand-binding domain predicts that in the absence of agonist the ligand-binding domain exists in equilibrium between active and inactive conformations (23). Agonist binding likely stabilizes the active (closed) conformation of the ligand-binding domain at equilibrium, which is then translated as an increase in both the efficacy and frequency of G protein coupling. Similarly, the mutation of an aspartic acid residue at position 854 in the G protein-coupling domain of mGluR1a to an alanine residue creates a receptor that gains the capacity to drive agonist-independent PKCbeta II oscillations (8). Conversely, in cells expressing mGluR1b, a mGluR1 splice variant that exhibits reduced spontaneous G protein coupling activity (21, 24), PKCalpha and PKCbeta I do not exhibit agonist-independent oscillations, but retain the capacity to both oscillate and accumulate at the cell surface in response to mGluR1a activation (data not shown). Taken together, these observations suggest that agonist-independent PKCalpha and PKCbeta I oscillations are driven by basal mGluR1a activity.

Unlike PKCbeta II, PKCalpha and PKCbeta I exhibit the capacity to be persistently localized at the plasma membrane in response to mGluR1a activation, and this response persists until the cells are perfused with agonist-free medium. Persistent PKCalpha translocation responses have also been reported previously (25) in response to thyrotropin-releasing hormone receptor activation. Constitutive plasma membrane localization of PKCbeta II can also be achieved following the mutation of two autophosphorylated amino acid residues, Thr-641 and Ser-660, to alanine residues or by treating cells with PKC inhibitors (8, 26-28). In contrast, the autophosphorylation of equivalent residues within the carboxyl-terminal variable domain of PKCalpha prolongs its activation (29, 30), which may account in part for the persistent localization of the enzyme at the plasma membrane in response to mGluR activation. However, the persistent localization of PKCalpha and PKCbeta I at the plasma membrane cannot be completely explained by PKC subtype-regulated differences in autophosphorylation, because the spatial-temporal dynamics of DAG and InsP3 formation are also altered in these cells. One potential explanation for the observed changes in DAG and InsP3 formation in PKCalpha and PKCbeta I-expressing cells is that the enzymes participating in the regulation of intracellular DAG levels may serve as PKC isoform-specific substrates and that the V5 domain may control substrate-specificity. Consistent with this idea, the activation of endogenous conventional PKC with thymeleatoxin results in PLCbeta 3 phosphorylation and attenuation of oxytocin receptor-stimulated phosphatidylinositide turnover (31). However, it is unknown which conventional PKC isoforms contribute to the phosphorylation-dependent inactivation of PLC. An alternative explanation may involve the differential ability of conventional PKC isoforms to associate with membrane anchoring proteins due to structural differences in their V5 domains. For example, the association of PKCbeta II with receptor for activated C kinase 1 (RACK1) is regulated by three regions within the V5 domain of PKCbeta II (32), and two of these regions encompass the amino acid residues (Asn-625 and Lys-668) that regulate PKCbeta II translocation patterns. The relative contributions of PKC autophosphorylation, PLC phosphorylation, and membrane anchoring proteins to the regulation of PKC subtype-specific translocation patterns will require extensive future study.

An important observation made in the present study is that PKCbeta I and PKCbeta II exhibit distinct activation patterns even when expressed in the same cell. These differences in activity are abolished by either the mutation of Asn-625 or Lys-668 in PKCbeta II. These two residues appear to cooperate with one another to repress PKCalpha -like response patterns for PKCbeta II. This indicates the amino acid composition of the V5 domains of otherwise identical PKC isoforms regulates the relative sensitivity of the enzymes to both changes in intracellular DAG and Ca2+ concentrations. Previously, Keranen and Newton (33) demonstrated that the PKCbeta I and PKCbeta II V5 domains regulate differences in the enzymes Ca2+-dependent affinity for acidic membranes. Thus, the PKCbeta I-like versus PKCbeta II-like response patterns may reflect V5 domain-regulated differences in both DAG and Ca2+ affinity. Thus, the periodicity of activation for different conventional PKC isoforms is not only regulated by the duration and strength of DAG and Ca2+ signals, but is also determined by the relative sensitivity of the kinases to alterations in intracellular DAG and Ca2+ concentrations.

In summary, we have discovered that homologous PKC isozymes, even when expressed in the same cell, display distinct patterns of activation in response to the same receptor stimulus. This observation is of fundamental importance to our understanding of cell signaling and clearly illustrates that the expression of multiple kinase isoforms in the same cell does not result in redundancy of cellular function. Rather, our results provide concrete evidence that the stimulation of a single receptor subtype, in a single cell, has the potential to activate distinct patterns of PKC isozyme activation, which may be translated into distinct cellular responses. We speculate that this may be particularly important in the developing and adult nervous system where Ca2+ spikes and PKC translocation responses are linked to both synapse formation and synaptic plasticity required for memory and learning (34, 35).

    ACKNOWLEDGEMENTS

We thank Drs. S. J. D'Souza, L. Dagnino, R. D. Feldman, R. J. Rylett, and M. W. Salter for critical reading of the manuscript. We also thank Dr. M. Taljaard for help with statistical analysis.

    FOOTNOTES

* This work was supported by Canadian Institutes of Health Research Grant MA-15506 (to S. S. G. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

Recipient of a Canadian Hypertension Society/CIHR fellowship.

** Recipient of Heart and Stroke Foundation of Canada MacDonald Scholarship, Premier's Research Excellence Award, and Canada Research Chair in Molecular Neuroscience.

Dagger Dagger To whom correspondence should be addressed: Robarts Research Inst., 100 Perth Dr., P.O. Box 5015, London, Ontario N6A 5K8, Canada. Tel.: 519-663-3825; Fax: 519-663-3789; E-mail: ferguson@robarts.ca.

Published, JBC Papers in Press, November 25, 2002, DOI 10.1074/jbc.M211053200

    ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; DAG, diacyglycerol; InsP3, inositol 1,4,5-triphosphate; mGluR, metabotropic glutamate receptor; HEK, human embryonic kidney; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; PH, pleckstrin homology; PLC, phospholipase C.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Gilon, P., Shepherd, R. M., and Henquin, J. C. (1993) J. Biol. Chem. 268, 22265-22268[Abstract/Free Full Text]
2. Hajnoczky, G., Robb-Gaspers, L. D., Seitz, M. B., and Thomas, A. P. (1995) Cell 82, 415-424[Medline] [Order article via Infotrieve]
3. Dolmetsch, R. E., Xu, K., and Lewis, R. S. (1998) Nature 392, 933-936[CrossRef][Medline] [Order article via Infotrieve]
4. Uhlen, P., Laestadius, A., Jahnukainen, T., Soderblom, T., Backhed, F., Celsi, G., Brismar, H., Normark, S., Aperia, A., and Richter-Dahlfors, A. (2000) Nature 405, 694-697[CrossRef][Medline] [Order article via Infotrieve]
5. Berridge, M. J., Lipp, P., and Bootman, M. D. (2000) Nat. Rev. 1, 11-21[CrossRef]
6. Thomas, A. P., Bird, G. S., Hajnoczky, G., Robb-Gaspers, L. D., and Putney, J. W., Jr. (1996) FASEB J. 10, 1505-1517[Abstract/Free Full Text]
7. Oancea, E., and Meyer, T. (1998) Cell 95, 307-318[Medline] [Order article via Infotrieve]
8. Dale, L. B., Babwah, A. V., Bhattacharya, M., Kelvin, D. J., and Ferguson, S. S. G. (2001) J. Biol. Chem. 276, 35900-35908[Abstract/Free Full Text]
9. Codazzi, F., Teruel, M. N., and Meyer, T. (2001) Curr. Biol. 11, 1089-1097[CrossRef][Medline] [Order article via Infotrieve]
10. Tanimura, A., Nezu, A., Morita, T., Hashimoto, N., and Tojyo, Y. (2002) J. Biol. Chem. 277, 29054-29062[Abstract/Free Full Text]
11. Kawabata, S., Tsutsumi, R., Kohara, A., Yamaguchi, T., Nakanishi, S., and Okada, M. (1996) Nature 383, 89-92[CrossRef][Medline] [Order article via Infotrieve]
12. Kawabata, S., Kohara, A., Tsutsumi, R., Itahana, H., Hatashibe, S., Yamaguchi, T., and Okada, M. (1998) J. Biol. Chem. 273, 17381-17385[Abstract/Free Full Text]
13. Nakahara, K., Okada, M., and Nakanishi, S. (1997) J. Neurochem. 69, 1467-1475[Medline] [Order article via Infotrieve]
14. Flint, A. C., Dammerman, R. S., and Kriegstein, A. R. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12144-12149[Abstract/Free Full Text]
15. Liu, W. S., and Heckman, C. A (1998) Cell Signal 10, 529-542[CrossRef][Medline] [Order article via Infotrieve]
16. Kanashiro, C. A., and Khalil, R. A. (1998) Clin. Exp. Pharmacol. Physiol. 25, 874-985
17. Ron, D., and Kazanietz, M. G. (1999) FASEB J. 13, 1658-1676[Abstract/Free Full Text]
18. Newton, A. C. (2001) Chem. Rev. 101, 2353-2364[CrossRef][Medline] [Order article via Infotrieve]
19. Walker, S. D., Murray, N. R., Burns, D. J., and Fields, A. P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 156-160[Abstract]
20. Cullen, B. R. (1987) Methods Enzymol. 157, 684-704
21. Prezeau, L., Gomeza, J., Ahern, S., Mary, S., Galvez, T., Bockaert, J., and Pin, J. P. (1996) Mol. Pharmacol. 49, 422-429[Abstract]
22. Dale, L., Bhattacharya, M., Anborgh, P. H., Murdoch, B., Bhatia, M., Nakanishi, S., and Ferguson, S. S. G. (2000) J. Biol. Chem. 275, 38213-38220[Abstract/Free Full Text]
23. Kunishima, N., Shimada, Y., Tsuji, Y., Sato, T., Yamamoto, M., Kumasaka, T., Nakanishi, S., Jingami, H., and Morikawa, K. (2000) Nature 407, 971-977[CrossRef][Medline] [Order article via Infotrieve]
24. Kaur Dhami, G., Anborgh, P. H., Dale, L. B., Sterne-Marr, R., and Ferguson, S. S. G. (2002) J. Biol. Chem. 277, 25266-25272[Abstract/Free Full Text]
25. Vallentin, A., Prevostel, C., Fauquier, T., Bonnefont, X., and Joubert, D. (2000) J. Biol. Chem. 275, 6014-6021[Abstract/Free Full Text]
26. Keranen, L. M., Dutil, E. M., and Newton, A. C. (1995) Curr. Biol. 5, 1394-1403[Medline] [Order article via Infotrieve]
27. Feng, X., and Hannun, Y. A. (1998) J. Biol. Chem. 273, 26870-26874[Abstract/Free Full Text]
28. Feng, X., Zhang, J., Barak, L. S., Meyer, T., Caron, M. G., and Hannun, Y. A. (1998) J. Biol. Chem. 273, 10755-10762[Abstract/Free Full Text]
29. Bornancin, F., and Parker, P. J. (1996) Curr. Biol. 6, 1114-1123[Medline] [Order article via Infotrieve]
30. Bornancin, F, and Parker, P. J. (1997) J. Biol. Chem. 272, 3544-3549[Abstract/Free Full Text]
31. Yue, C., Ku, C.-Y., Liu, M., Simon, M. I., and Sanborn, B. M. (2000) J. Biol. Chem. 275, 30220-30225[Abstract/Free Full Text]
32. Stebbins, E. G., and Mochly-Rosen, D. (2001) J. Biol. Chem. 276, 29644-29650[Abstract/Free Full Text]
33. Keranen, L. M., and Newton, A. C. (1997) J. Biol. Chem. 272, 25959-25967[Abstract/Free Full Text]
34. Spitzer, N. C., Olson, E., and Gu, X. (1995) J. Neurobiol. 26, 316-324[Medline] [Order article via Infotrieve]
35. Angenstein, F., Riedel, G., Reyman, K. G., and Staak, S. (1999) Neuroscience 93, 1289-1295[CrossRef][Medline] [Order article via Infotrieve]


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