From the 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
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ABSTRACT |
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Metabotropic glutamate receptors (mGluRs) coupled
via Gq to the hydrolysis of phosphoinositides stimulate
Ca2+ and PKC 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).
PKC 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 ( 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 PKC 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 PKC 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-PLC 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.
PKC Isozyme-specific Plasma Membrane Translocation Responses--
Because individual conventional (
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 PKC PKC
DAG responses were measured using a GFP-PKC
InsP3 responses were measured using a GFP-PLC
Changes in intracellular Ca2+ concentrations were
measured using the Ca2+ indicator dye Oregon Green 488 BAPTA-1 AM. We found that oscillatory DsRed2-PKC
Taken together, our observations indicate that expression of PKC Effect of PKC Molecular Determinants for Isozyme-specific Translocation Response
Patterns--
The patterning of conventional PKC isoform responses to
mGluR1a activation are sub-classified as either PKC
Sequence alignment of PKC
We found that the establishment of a PKC In the present study, we show that each of the conventional ( Both PKC Unlike PKC An important observation made in the present study is that PKC 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).
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. PKC
and PKC
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 PKC
II translocation responses are observed in
mGluR1a-expressing cells. PKC
I expression also promotes persistent
increases in intracellular diacyglycerol concentrations in
response to mGluR1a stimulation without affecting PKC
II oscillation
patterns in the same cell. PKC
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 PKC
II cooperatively suppress
PKC
I-like response patterns for PKC
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
II and PKC
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).
,
I,
II, and
) is regulated by Ca2+ and DAG, whereas the activity and subcellular
localization of the novel PKC isoforms (
,
,
, and
) 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.
I- or PKC
II-like responses.
Specifically, we have identified two discrete amino acid residues
localized within the V5 domain of PKC
II that function to suppress
PKC
I-like responses for PKC
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
PKC
I, PKC
, PKC
, PKC
, PKC
, PKC
, PKC
, and PKC
/
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 PKC
I cDNA was also cloned
into the BglII-XbaI sites of the vector DsRed2-C1
(Clontech). The construction of EGFP-PKC
II
(28), and EGFP-PLC
1 PH domain were previously
described (8). The EGFP-PKC
C1 domain was a generous gift from Dr.
Sergio Grinstein. PKC
/
II,
II/
,
I/
II, and
II/
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. PKC
and
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.
1 PH domain
(8) and EGFP-PKC
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
I,
II, and
),
novel (
,
,
, and
), and atypical (
/
and
) 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 PKC
II (PKC
II-like).
However, PKC
and PKC
I display additional translocation responses
(PKC
I-like) that are never observed for PKC
II. Although some cell
to cell variability is observed, PKC
I-like translocation patterns
can be categorized into three distinct patterns that are illustrated in
Fig. 2. First, GFP-PKC
and GFP-PKC
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
PKC
and PKC
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,
PKC
). 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-PKC
or GFP-PKC
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-PKC
and GFP-PKC
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). PKC
also exhibits very weak PKC
I-like responses
(13/49 cells), but unlike what is observed for the other conventional
PKC isoforms the most common PKC
response pattern is a single
transient plasma membrane translocation (20/49 cells) (data not shown).
For PKC
, PKC
I, and PKC
II transient translocation responses
were rarely observed (< 5% of cells imaged). Taken together, these
observations suggest that PKC
, PKC
I, and PKC
exhibit the
capacity to decode subtly different changes in second messenger
responses that are not recognized by PKC
II. Alternatively, the
expression of PKC
, PKC
I, and PKC
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-PKC
(A), GFP-PKC
I (B), GFP-PKC
II
(C), or GFP-PKC
(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-PKC
(13/33 cells),
GFP-PKC
I (10/27 cells), GFP-PKC
II (62/62 cells), and GFP-PKC
(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-PKC - and
GFP-PKC
I-specific response patterns to mGluR1a
activation. HEK 293 cells were transfected with cDNA encoding
mGluR1a and either GFP-PKC
or GFP-PKC
I. Shown are GFP-PKC
and
GFP-PKC
I response patterns that are not observed for GFP-PKC
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,
PKC
(5/33 cells), and PKC
I (4/27 cells). B, persistent
localization of GFP-PKC at the plasma membrane in response to agonist
treatment, PKC
(8/33 cells) and PKC
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, PKC
(7/33
cells), and PKC
I (2/27 cells). The micrographs shown in
each panel are representative confocal micrographs taken
from the GFP-PKC
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.
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
(
/
and
) 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-PKC
(A), GFP-PKC
(B), GFP-PKC
(C),
or GFP-PKC
(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.
I-dependent Alterations in mGluR1a-stimulated
Second Messenger Responses--
It is possible that the expression of
either PKC
or PKC
I leads to alterations in the patterning of
mGluR1a-stimulated second messenger responses and that this may
underlie the multiplicity of PKC
I-like response patterns. To address
this possibility, we examined the patterning of red fluorescent protein
tagged-PKC
I (DsRed2-PKC
I) responses at the same time as we
measured changes in intracellular DAG, InsP3, and
Ca2+ concentrations.
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-PKC
C1 domain translocation patterns were
synchronized exactly with DsRed2-PKC
I membrane translocation
responses (Fig. 4A). Following
mGluR1a activation, the GFP-PKC
C1 domain either oscillated between
the cytosol and plasma membrane in synchrony with DsRed2-PKC
I (Fig.
4A, upper panel) or accumulated with
DsRed2-PKC
I at the plasma membrane (Fig. 4A, lower
panel).
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Fig. 4.
Synchronization of DAG, InsP3,
and Ca2+ responses with
DsRed2-PKC I translocation following mGluR1a
activation. DsRed2-PKC
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-PKC
C1 domain reporter construct. Changes in intracellular
InsP3 concentrations were measured by the plasma membrane
to cytosol translocation of a GFP-PLC
PH domain reporter construct.
Changes in intracellular Ca2+ concentrations were measured
using Oregon Green 488 BAPTA-1 AM.
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-PLC
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-PLC
1 PH domain either
oscillated between the plasma membrane and cytosol at the same
frequency at which DsRed2-PKC
I translocated from the cytosol to
plasma membrane (Fig. 4B, upper panel), or the GFP-PLC
1 PH domain accumulated in the cytosol with the same time course as DsRed2-PKC
I accumulated at the plasma membrane (Fig. 4B, lower panel). Thus, the spatial-temporal
localization of PKC
I at the plasma membrane was coordinated with
alterations in both intracellular DAG and InsP3 concentrations.
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-PKC
I was persistently localized to the plasma
membrane (Fig. 4C, lower panel).
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
PKC
II where only synchronized oscillatory DAG, InsP3,
and Ca2+ responses are observed following mGluR1a
activation (data not shown and Ref. 8).
I Expression on PKC
II Plasma Membrane
Translocation Responses--
PKC
I and PKC
II isoforms are thought
to be regulated in the same manner by DAG (17). Therefore, if
differences in PKC
I versus PKC
II response patterns are
solely the consequence of PKC
I expression-dependent
alterations in DAG formation or differences in mGluR1a expression
levels between cells, GFP-PKC
II should exhibit PKC
I-like
translocation patterns in cells co-expressing DsRed2-PKC
I. When
co-expressed together in HEK 293 cells, we observe two distinct
DsRed2-PKC
I and GFP-PKC
II responses to mGluR1a activation: 1)
DsRed2-PKC
I and GFP-PKC
II exhibit synchronized oscillatory plasma
membrane translocation responses (Fig.
5A); and 2) DsRed2-PKC
I
accumulates at the plasma membrane, whereas GFP-PKC
II continues to
oscillate between the plasma membrane and cytosol (Fig. 5B).
These observations suggest that, although PKC
I expression alters the
patterning of mGluR1a-stimulated DAG and InsP3 response
patterns, PKC
II is apparently insensitive to PKC
I-induced changes
in DAG formation. Moreover, the differences in PKC
I
versus PKC
II membrane translocation patterns observed in
the same cell indicates that differences in mGluR1a expression levels
between cells cannot account for the different PKC
I-like translocation patterns.
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Fig. 5.
Translocation response patterns of
DsRed2-PKC I and
GFP-PKC
II in the same cell. HEK 293 cells
were transfected with cDNA encoding mGluR1a and both DsRed2-PKC
I
and GFP-PKC
II. Shown are either synchronized DsRed2-PKC
I and
GFP-PKC
II responses to 30 µM quisqualate (13/24 cells)
(A) or persistent localization of DsRed2-PKC
I at the
plasma membrane with simultaneous GFP-PKC
II oscillations (7/24
cells) (B). The micrographs shown in each
panel are the representative confocal micrographs taken from
the DsRed2-PKC
I and GFP-PKC
II time courses plotted in each
panel.
I-like
(agonist-independent oscillations, agonist-stimulated oscillations, and
persistent plasma membrane localization) or PKC
II-like (only
agonist-stimulated oscillations). We have used these definitions to
characterize the structural determinants underlying differences in
conventional PKC isozyme response patterns. PKC
I and PKC
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 PKC
with the corresponding residues from PKC
II generates a PKC
/
II
620-673 chimera that displays a PKC
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 PKC , PKC
I, and PKC
II. Non-conserved amino acid
residues are boxed. B-D, characterization of the
translocation patterns of GFP-PKC
, GFP-PKC
I and GFP-PKC
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 PKC
II amino acids are
underlined. Asterisks indicate PKC
II mutants
displaying differences in translocation pattern compared with the
expected wild-type PKC
II isoform response patterns. A non-parametric
SIGN test, p > 0.07 supports the null hypothesis that
the asterisked PKC
II mutants exhibit no differences in oscillation
patterns with the expected patterns observed for PKC
.
, PKC
I, and PKC
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 PKC
II for the last 15 amino acid residues
of PKC
(PKC
II/
657-672) or deleting the last 13 amino acid
residues from the carboxyl-terminal of PKC
II (PKC
II-S660D), we
create PKC
II chimeras with PKC
I-like response patterns (Fig.
6B). Serial truncation analysis of PKC
II between amino
acid residues 660 and 672 reveals that PKC
II-like responses are lost
if the final six (PKC
II-L667
) but not the final three
(PKC
II-E670
) PKC
II amino acids are deleted (Fig.
6C). The deletion of Lys-668-Glu-670
(PKC
II-KPE
) from PKC
II also establishes a PKC
-like response
pattern for PKC
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 PKC
II-like responses and to suppress
PKC
I-like response patterns (Fig. 6D). When expressed
together in HEK 293 cells, we find that DsRed2-PKC
I and
GFP-PKC
II-K668G exhibit identical response patterns to mGluR1a
activation (Fig. 7).
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Fig. 7.
Synchronization of
GFP-PKC II K668G and
DsRed2-PKC
I responses. HEK293 cells were
transfected with cDNA encoding mGluR1a, DsRed2-PKC
I, and
GFP-PKC
II K668G. Shown are: synchronized agonist-stimulated
DsRed2-PKC
I and GFP-PKC
II translocation responses (8/14 cells)
(A), synchronized agonist-independent DsRed2-PKC
I and
GFP-PKC
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-PKC
I
and GFP-PKC
II K668G at the plasma membrane in response to agonist
stimulation (3/14) (C).
II-like response pattern in
PKC
required the exchange of the entire PKC
II V5 domain. Furthermore, neither the exchange of the last 13 amino acid residues from PKC
II into PKC
(PKC
/
II 660-673) nor the introduction of the KPE motif into PKC
I established PKC
II-like responses for
either PKC
or PKC
I (Fig. 6, B and C).
Therefore, there must be additional amino acid residues localized
within the PKC
II V5 domain that cooperated with Lys-668 to establish
a PKC
II-like response pattern. Sequence alignment of PKC
,
PKC
I, and PKC
II indicated that only 4 amino acid residues were
not conserved between PKC
II and either PKC
or PKC
I: Asn-625,
His-636, Glu-646, and Arg-649 of PKC
II (Fig. 6A).
Therefore, we mutated each of these residues to the corresponding amino
acid residue in PKC
and found that only PKC
II-N625G exhibited
PKC
-like behavior patterns (Fig. 6D). In summary,
extensive structure-function analysis identified Asn-625 and Lys-668 as
essential amino acid residues within the PKC
II V5 domain required
for the establishment of PKC
II translocation responses. The mutation
of either residue releases the suppression of PKC
-like response
behaviors for PKC
II.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
I,
II, and
) and novel (
,
,
, and
), but not
atypical (
and
/
) 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 PKC
and PKC
I exhibit a variety of unique
response patterns to mGluR1a activation that are not observed for
PKC
II. Because PKC
I and PKC
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
PKC
and/or PKC
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.
and PKC
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 PKC
and PKC
I exhibit heightened
sensitivity to changes in intracellular DAG concentrations as compared
with PKC
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 PKC
II oscillations (8).
Conversely, in cells expressing mGluR1b, a mGluR1 splice variant that
exhibits reduced spontaneous G protein coupling activity (21, 24),
PKC
and PKC
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 PKC
and PKC
I oscillations are driven by basal mGluR1a activity.
II, PKC
and PKC
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 PKC
translocation responses have also been reported previously (25) in response to
thyrotropin-releasing hormone receptor activation. Constitutive plasma
membrane localization of PKC
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 PKC
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
PKC
and PKC
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
PKC
and PKC
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
PLC
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 PKC
II with receptor for activated C kinase 1 (RACK1)
is regulated by three regions within the V5 domain of PKC
II (32),
and two of these regions encompass the amino acid residues (Asn-625 and
Lys-668) that regulate PKC
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.
I and
PKC
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 PKC
II. These two residues
appear to cooperate with one another to repress PKC
-like response
patterns for PKC
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 PKC
I and PKC
II V5 domains regulate
differences in the enzymes Ca2+-dependent
affinity for acidic membranes. Thus, the PKC
I-like versus
PKC
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.
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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.
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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.
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
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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.
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