| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 18, 15277-15285, May 3, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Activity by C1-C2 Domain Interactions*
From the Department of Pathology, Cell Biology and Anatomy, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Received for publication, December 20, 2001, and in revised form, February 14, 2002
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
In this study, the role of interdomain
interactions involving the C1 and C2 domains in the mechanism of
activation of PKC was investigated. Using an in vitro assay
containing only purified recombinant proteins and the phorbol ester,
4 The 10 closely related isozymes that constitute the protein kinase
C (PKC)1 family of
serine/threonine kinases each occupy critical nodes in the complex
cellular signal transduction networks that regulate diverse cellular
processes, including: secretion, proliferation, differentiation,
apoptosis, permeability, migration, and hypertrophy (1-7). In common
with many signaling proteins, the structure of PKC is modular,
consisting of a C-terminal catalytic region containing the active site,
and a regulatory region with conserved domains that mediate membrane
association and activation. PKC isozymes are classified according to
the structural and functional differences in these conserved domains
(8, 9). In the case of the "conventional" PKC The mechanism by which the conventional PKC isozymes become membrane
associated, and thus activated, involves two sequential steps. The
first involves an initial Ca2+- and anionic
phospholipid-dependent interaction of the C2 domain with
the membrane, which is then followed by binding of diacylglycerol or
phorbol esters to the C1 domains (12-15). Mutagenesis studies have
identified a number of critical hydrophobic residues within the C1
domains of PKC There is increasing evidence supporting the notion that the existence
of two C1 domains affords a complex modulatory role in the
regulation of PKC activity. Phorbol esters have been shown to interact
with both of the C1A and C1B domains, with distinct low and
high affinities (29-33). Furthermore, we have shown that diacylglycerol inhibits the low affinity phorbol ester interaction while enhancing high affinity phorbol ester binding,
indicating that the diacylglycerol has a higher affinity for the low
affinity phorbol ester-binding site than does phorbol ester itself (30, 31). Additional support for the non-equivalence of the interaction of
diacylglycerol, phorbol esters, and also other activators with the two
C1 domains has been provided by other studies that have observed
non-equivalent roles of the domains with respect to membrane association and activation (34-37). Studies from this laboratory showed that the increased level of phorbol ester binding that results
from interaction of diacylglycerol with the low affinity phorbol
ester-binding site on conventional PKC isozymes corresponded to an
elevated level of enzyme activity that was greater than that induced by
either phorbol ester or diacylglycerol alone (30, 31, 38). The above,
and recent data suggesting that the C1A of PKC Inter-domain interactions are important in PKC regulation, although
detailed features of these interactions remain obscure. The
conformational change that leads to the displacement of the pseudosubstrate of membrane-associated PKC isozymes involves pronounced rearrangements of the individual domains that constitute the enzyme molecule. In a recent study, it was shown that the rate of
translocation of GFP-tagged PKC The aim of the present study was to determine the role of interdomain
interactions in the mechanism of activation of PKC Materials--
Adenosine 5'-triphosphate (ATP) was from Roche
Molecular Biochemicals (Indianapolis, IN). [ Expression and Purification of PKC Isozymes, the D55A Mutant of
PKC Measurements of PKC Activity--
PKC isozyme activities were
assayed by measuring the rate of phosphate incorporation into a peptide
substrate as previously described (30). For the "conventional" PKC
isoforms and the their catalytic subunits, a peptide corresponding to
the phosphorylation site domain of myelin basic protein (QKRPSQRSKYL,
MBP4-14) was used as the substrate, whereas assays of
novel PKC and atypical PKC Measurements of PKC-C1-domain Binding Using Surface Plasmon
Resonance (SPR)--
Binding of C1 domain peptides to PKC isozymes was
determined using SPR from measurements of the accompanying increase in
refractive index as a function of time using a BiacoreTM
2000 (Biacore, Inc., Piscataway, NJ). All measurements were performed at 25 °C. Initially, the To investigate the role of domain-domain intramolecular
interactions in the mechanism of activation of PKC, the assumption was
made that if such interactions occur within the isozyme molecule, and
are involved in the activating conformational change, then peptides
corresponding to the isolated domains might compete for these
interactions and modulate the activity of the isozyme. To address this,
various fusion proteins containing regions of the regulatory domain
spanning the C1A and C1B domains were prepared, as shown in Fig.
1. Binding of these domains to PKC, and
the effects on PKC isozyme activities, were determined using assay
systems that contained only purified proteins along with the required peptide substrates, cofactors, and activators. By excluding membranes from the assay systems, effects could be unambiguously ascribed to
direct protein-protein interactions between these domains and PKC.
-12-O-tetradecanoylphorbol-13-acetate (TPA), but
lacking lipids, it was found that PKC
bound specifically, and with
high affinity, to a C1A-C1B fusion protein of the same isozyme. The
C1A-C1B domain also potently activated the isozyme in a phorbol
ester- and diacylglycerol-dependent manner. The level of
this activity was comparable with that resulting from membrane association induced under maximally activating conditions. Furthermore, it was found that C1A-C1B bound to a peptide containing the C2 domain of PKC. The C1A-C1B domain also activated conventional PKCI, -II, and -
isoforms, but not novel PKC
or -
.
PKC and - were each activated by their own C1 domains, whereas
PKC, -I, -II, or - activities were unaffected by the C1
domain of PKC and only slightly activated by that of PKC. PKC
activity was unaffected by its own C1 domain and those of the other PKC isozymes. Based on these findings, it is proposed that the activating conformational change in PKC results from the dissociation of intra-molecular interactions between the C1A-C1B domain
and the C2 domain. Furthermore, it is shown that PKC forms dimers
via inter-molecular interactions between the C1
and C2 domains of two neighboring molecules. These mechanisms may also
apply for the activation of the other conventional and novel PKC isozymes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, -I/II, and
- isozymes, these include the activator-binding C1 domains, and the
Ca2+-binding C2 domain. The C1 domains consist of a tandem
C1A and C1B arrangement, each of which can potentially bind the
endogenous activator, diacylglycerol and exogenous activators including
phorbol esters. The "novel" PKC, -, -
, -
, and -µ
isozymes, contain C2 domains that lack Ca2+ binding
ability, while retaining functional C1A and C1B domains. The
"atypical" PKC, -
, and -
regulatory domains also lack a functional C2 domain and contain a single C1 domain that lacks the
ability to bind activators, the function of which remains obscure. Each
isozyme becomes catalytically competent by undergoing multiple
serine/threonine and tyrosine phosphorylations that are either
autocatalytic, or catalyzed by "PKC kinases" such as the phosphoinositide-dependent kinase, PDK1 (7, 10, 11).
and PKC that appear to have distinct roles in
ligand binding and membrane association (16-18). Based on the x-ray
crystallographic structure of the C1B domain of PKC, it has been
suggested that activator binding to the C1 domain facilitates membrane-insertion by "capping" a hydrophilic groove to form a contiguous hydrophobic surface that can interact with the membrane interior (19). However, it would appear from our studies that the
interaction of the phorbol ester induces a conformational change in the
C1 domain, the extent of which is reflected in the activity of the
enzyme, implying a more active role for phorbol ester-C1 domain
interactions beyond that of presenting a hydrophobic surface to the
interior of the membrane (20, 21). The interaction of diacylglycerol
with the C1 domains also results in an increased stereo- and
regiospecificity of both membrane association and activation for PS
(13, 22-24). The combined interactions of the C1 and C2 domains with
the membrane provides the free energy required for structural
rearrangements that lead to the dissociation of the N-terminal
pseudosubstrate from the active site to allow substrate binding
(25-27). This process is thought to be further facilitated by a weak
interaction of the released pseudosubstrate with anionic head groups at
the membrane surface (28).
is the
diacylglycerol-binding site, while the C1B domain binds phorbol ester
(16, 24), are compatible with the C1A and C1B domains containing the
low and high affinity phorbol ester-binding sites, respectively.
in RBL cells was markedly slower
when compared with that of a truncation mutant lacking the N-terminal
V1 region (14). Based on this, it was suggested that membrane
association and activation resulting from binding of diacylglycerol to
the C1 domains of this isozyme first requires the release of a "V1 clamp," which holds the C1 domain between the catalytic and V1 region, due to an interaction of the pseudosubstrate with the active
site. In another study, the mutation of a single aspartate (Asp55) residue in the C1A domain of PKC was
shown to result in a marked reduction in both the PS and
Ca2+ concentration requirements for membrane association
and activation, implicating a tethering of the C1A domain to a
neighboring region within the PKC molecule (15, 24). It was
suggested that such an interaction might retain the isozyme in an
inactive conformation by preventing the penetration of the C1A domain
into the membrane and thus its interaction with diacylglycerol
(24).
. Using in
vitro activity and binding assays, it was found that PKC
engaged in a phorbol ester-dependent, high affinity and
specific interaction with a fusion protein that contained its own C1A
and C1B domains (C1A-C1B). The level of activity induced by
interaction with the C1A-C1B domain was found to be comparable with
that resulting from membrane association induced under maximally
activating conditions. Also, the C1A-C1B domain interacted with a
fusion protein containing the C2 domain of PKC. Taken together,
these findings provide evidence for the existence of an interdomain interaction between the C1 and C2 domains, the dissociation of which is
a rate-determining step in the mechanism of activation of PKC.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP
was from PerkinElmer Life Science (Boston, MA).
1-Palmitoyl-2-oleoylphosphatidylcholine (POPC) and bovine brain
phosphatidylserine (BPS) were each from Avanti Polar Lipids, Inc.
(Alabaster, AL). Peptide substrates were custom synthesized by the
Kimmel Cancer Center peptide synthesis facility of Thomas Jefferson
University.
4-12-O-Tetradecanoylphorbol-13-acetate (TPA)
and the soluble diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DiC8), were each obtained from Sigma. A mixed preparation
of the catalytic subunits of the conventional PKC isozymes was
purchased from Calbiochem (La Jolla, Ca). All other chemicals were
of analytical grade and obtained from Fisher Scientific
(Pittsburgh, PA).
, and C1/C2 Domains--
Recombinant PKC, -I, -II,
-, and - (rat brain) were prepared using the baculovirus
Spodoptera frugiperda (Sf9) insect cell expression
system as originally described (39), with modifications (40).
Purification procedures were as previously described (30, 40).
Baculovirus encoding the PKC mutant, D55A, in which the aspartate 55 of the C1A domain was mutated to an alanine (24), was a kind gift from
Dr. Wonhwa Cho, and was purified using the same procedure as that used
for wild-type PKC (30, 40). The isoforms PKC, PKC, and PKC
were overexpressed in Sf9 cells as fusion proteins containing a
(His)6 attached to the C terminus (40) and were purified as
described (32, 40). Fusion proteins containing fragments of PKC
encompassing the C1A, C1B, and C2 (C1A-C1B-C2), the C1A and C1B
(C1A-C1B), the separate C1A (C1A) and C1B (C1B) domains, and
also the C1A and C1B domains of PKC (C1A-C1B), were each prepared
as described previously (32, 40) (and see Fig. 1). To provide
structural stability, solubility, and to aid purification, fusions
peptides were tagged with glutathione S-transferase (GST) at
the N terminus and with (His)6 at the C terminus. An
expression vector containing the GST-tagged C1A and C1B domains of
PKC (C1A-C1B) was a kind gift from Dr. Peter M. Blumberg. The
isolation and purification of each tagged protein was performed
previously described (32, 40).
activity used a peptide
corresponding to the pseudosubstrate region of novel PKC
(-peptide), in which the single alanine residue was replaced by
serine (25, 41, 42). Briefly, the assay (75 µl) consisted of 50 mM Tris-HCl (pH 7.40), 0.1 mM EGTA or
CaCl2, 50 µM MBP4-14, or 50 µM -peptide, TPA (500 nM or as indicated)
or varying levels of DiC8, and fusion proteins containing
the appropriate PKC domains present at a fixed concentration of 10 nM unless otherwise indicated. Where added, POPC and BPS were present at a total concentration of 150 µM as large
unilamellar vesicles, prepared as described previously (43). After
thermal equilibration to 30 °C, assays were initiated by the
simultaneous addition of the required PKC isoform (0.3 nM)
along with 5 mM Mg2+, 15 µM ATP,
0.3 µCi of [-32P]ATP (3000 Ci/mmol) and terminated
after 30 min with 100 µl of 175 mM phosphoric acid.
Following this, 100 µl was transferred to P81 filter papers, which
were washed three times in 75 mM phosphoric acid.
Phosphorylated peptide was determined by scintillation counting.
C1A-C1B or C1A-C1B domains were
captured through the respective (His)6 tag to the
nickel/NTA surface of an NTA sensor chip, prepared according to the
manufacturers instructions (Biacore, Inc., Piscataway, NJ), to a level
of 75 response units. Solutions containing either PKC isozymes or
fusion peptides at the required concentrations, in the presence or
absence of 500 nM TPA were then injected over this surface
and the response was measured as a function of time. The surface was
regenerated after each injection by two 10-s injections of 100 mM NaOH, followed by a single 10-s injection of 10 mM HCl. After subtraction of the contribution of bulk
refractive index changes and nonspecific interactions of PKC isozymes
with the nickel-NTA surface, which were typically less than 1% of the
total signal, the individual association (ka) and
dissociation (kd) rate constants were obtained by
global fitting of data to a 1:1 Langmuir binding model using
BIAevaluationTM (Biacore, Inc.). These values were then
used to calculate the dissociation constant (KD).
The values of average squared residual (
2) obtained were
not found to be significantly improved by fitting data to models that
assumed bivalent or heterogeneous interactions between PKC isozymes and
C1 domain peptides. In a separate control experiment (results not
shown), it was found that the contribution of mass transport to the
observed values of KD was negligible, based on the
observation that these values were independent of flow rate within a
range encompassing that used (10 to 50 µl min
1).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (12K):
[in a new window]
Fig. 1.
Domains used in this study. Each domain
was expressed as a fusion protein tagged at the C-terminal end with GST
and at the N-terminal end with (His)6. See "Experimental
Procedures" for details.
PKC Binds Directly to the C1A-C1B Domain--
Based on
measurements of SPR, it was found that, in the presence of TPA, PKC
bound to an immobilized fusion peptide containing the isolated
C1A-C1B domain in a reversible, concentration-dependent manner (Fig. 2A). The
interaction was phorbol ester-dependent since it was found
that the level of binding in the absence of TPA was negligible. The
value of KD, calculated from the ratio of the
association and dissociation rate constants, indicated a high affinity
interaction (Table I). Furthermore, the
value of the maximal level of PKC binding at equilibrium (Rmax) is consistent with a PKC-C1A-C1B
binding stoichiometry of 1:1, assuming maximal occupancy of
ligand-binding sites. PKC was also found to interact with the
C1A-C1B domain (Fig. 2B), although with a markedly
reduced affinity, as shown by the ~500-fold increase in the value of
KD (Table I).
|
|
It has been suggested in a previous study that the C1A of PKC may
interact with residues within the C2 domain, and thereby impede the
activating conformational change (24). Consistent with this, the
results shown in Fig. 2C indicate that in the presence of
TPA, the C1A-C1B domain binds to a fusion protein containing the C2
domain of PKC (C1A-C1B-C2). Similar to the interaction of PKC
with the C1A-C1B, the binding of the C1A-C1B-C2 peptide to the
C1A-C1B domain was found to be TPA-dependent. Note that in this experiment the C1A-C1B domain was initially bound to the
sensor chip surface through the (His)6 tag to a level
corresponding to saturation. Under these conditions, any interactions
of the C1A-C1B with itself were also saturated, ruling out the
possibility that the immobilized C1A-C1B domain may have interacted
with the C1A-C1B portion of the C1A-C1B-C2 fusion protein.
Importantly, the value of KD for the interaction was
similar to that calculated for the interaction of the C1A-C1B domain
with intact PKC (Table I). Furthermore, it was found that
C1A-C1B-C2 bound to the immobilized C1A-C1B (Fig. 2D),
although with markedly reduced affinity (Table I).
Interaction of PKC C1A and C1B Domains with PKC Results in
Activation--
PKC was found to be activated by low nanomolar
levels of the C1A-C1B domain in the presence of a fixed
concentration of TPA (500 nM) and in the absence of
membrane lipids (Fig. 3A,
). This effect was phorbol ester-dependent since the
C1A-C1B domain negligibly affected PKC activity in the absence of
TPA (Fig. 3A, 
). The concentration of C1A-C1B
corresponding to a half-maximal increase in PKC activity was ~1
nM, which is consistent with the value of
KD for the interaction of PKC with the C1A-C1B
domain, determined from the SPR binding data (Table I), and again
indicates that the activation resulted from a high-affinity interaction. It should be noted that the maximal specific activity of
PKC induced by the C1A-C1B domain was comparable with that determined for PKC associated with membranes composed of BPS and
POPC in the presence of Ca2+ and TPA, each activator being
at saturating levels (Fig. 3A, 
).
|
To rule out the possibility that the observed activation of PKC by
the C1A-C1B domain may have resulted from an interaction with a site
contained within the catalytic region of PKC, the effects of the
C1A-C1B domain on the activity of catalytic subunits derived from
limited proteolysis of a mixed PKC, -I/II, and - preparation
was determined (Fig. 3A, 
). Consistent with the reported
independence of the activity of the catalytic subunit from cofactor and
activator requirements (44, 45), the specific activity of the
preparation was found to be similar to that observed for PKC induced
by association with BPS/POPC vesicles in the presence of
Ca2+ and TPA (Fig. 3A, ). Whereas PKC was
activated by low nanomolar levels of the C1A-C1B domain, the
activity of the catalytic subunit preparation was found to be
unaffected by the domain within a similar concentration range (Fig.
3A, ). This is consistent with the site of interaction of
PKC with the C1A-C1B domain that mediates the activation being
contained within the regulatory domain. However, similar to the effects
on intact PKC activity, the presence of the C1A-C1B domain
concentrations greater than 10 nM also resulted in an
inhibition of catalytic subunit activity. This observation indicates
that the inhibitory effects of high levels of the C1A-C1B domain may
involve interactions with the catalytic domain, which is consistent
with the findings of a recent study that showed that GST fusion
proteins containing the regulatory domains of PKC and PKC each
inhibited the activity of the catalytic subunit of PKC (46). The
possibility that the activation of PKC by the C1A-C1B domain may
have involved GST or (His)6 was ruled out by the finding
that PKC activity was unaffected by a fusion peptide containing
these moieties alone (Fig. 3A, 
).
The dependence of PKC activity on the concentration of TPA,
determined in the presence or absence of C1A-C1B, is shown in Fig.
3B. In the absence of C1A-C1B, a small increase in the
level of PKC activity was observed within a high TPA concentration range (). This is consistent with the results of previous studies that have indicated that PKC isozymes bind phorbol esters in the absence of membranes, although with relatively low affinity, and that
this results in partial activation (47-50). Similar to the effect of
membrane association, the addition of the C1A-C1B domain resulted in
a >1000-fold decrease in the concentration dependence for TPA induced
activity, consistent with a dramatic increase in phorbol ester binding
affinity (Fig. 3B, 
). Thus, the calculated value of the
TPA concentration corresponding to a half-maximal increase in PKC
activity of 5 ± 2 nM is within a phorbol ester concentration range shown previously to be sufficient to elicit activation of the membrane-associated isozyme (31). Similar to TPA, the
soluble diacylglycerol, DiC8, also activated PKC in the
presence of the C1A-C1B domain, in a
concentration-dependent manner (Fig. 3B, ).
The concentration of DiC8 required to induce a half-maximal
increase in PKC activity (875 ± 55 nM) was
~150-fold greater than that observed for TPA, which is consistent
with the reduced affinity of diacylglycerols for binding to PKC
compared with phorbol esters (51, 52).
The concentration-dependent effects of the individual C1A
and C1B domains on the activity of PKC induced in the presence of a
saturating level of TPA, are shown in Fig. 3C. Both the
C1A () and C1B () domains activated PKC with similar
concentration dependence. However, the C1A and C1B concentrations
required to induce a half-maximal increase in activity were in each
case ~200-fold greater than the value obtained for the full-length C1A-C1B domain. Furthermore, although the C1A domain induced a
greater level of activity than the C1B domain, this activity was
~3-fold lower than that induced by the C1A-C1B domain.
The Effect of C1A-C1B on the Activity of the PKC C1A Domain
Mutant, D55A--
Recently, it was observed that the mutation of
aspartate 55 to an alanine in the C1A domain of PKC (D55A) resulted
in an increase in membrane binding affinity and an increased level of Ca2+ and PS independent activity. From this, it was
proposed that PKC might, in part, be restrained in an inactive
conformation by an inhibitory intramolecular interaction involving this
residue (24). Here, we further examined the intrinsic activity of D55A, first in the absence of the C1A-C1B domain, membranes,
Ca2+, and TPA, and showed it was higher than that of
wild-type PKC (Fig. 4), as reported
previously (24). Consistent with the higher intrinsic activity of D55A,
the addition of TPA alone resulted in significant D55A activity,
relative to the small effect on wild-type PKC activity. The
concentration-response curves for TPA-induced activation of D55A and
wild-type PKC, shown in Fig. 4 (inset), indicate that
this corresponds to a marked decrease in the TPA concentration range
required for D55A activation. In the absence of TPA, the activities of
D55A and PKC were both negligibly affected by the addition of the
C1A-C1B or C1A domain. Importantly, contrasting with the
TPA-induced activation of wild-type PKC by the C1A-C1B or C1A
domains, neither domain activated D55A in the presence of TPA.
|
Effects of C1A-C1B Domains of PKC, -, -, and - on the
Activities of a Panel of PKC Isoforms--
To determine the isozyme
specificity of the activating effect of the C1A-C1B domain, the
concentration-dependent effects of C1A-C1B, and also the
C1A-C1B, C1A-C1B, and C1 domains, on the activities of PKC,
-I, -II, -, -, -, and - were each determined in the
presence of TPA (Fig. 5). Consistent with the results shown in Fig. 3A, PKC activity was
potentiated ~100-fold by the C1A-C1B domain in a
concentration-dependent manner (Fig. 5, ). The
C1A-C1B domain also activated conventional PKCI, -II, and to
reduced extent, PKC. However, the activities of novel PKC or -
were each unaffected. Consistent with the observation that the
C1A-C1B domain bound to PKC, albeit with lower affinity (Fig.
2B and see Table I), the C1A-C1B domain was found to
induce a ~10-fold increase in PKC activity, while having
negligible effects on the activities of PKCI and -II (Fig. 5,
). This suggests that it may also share with the C1A-C1B domain
some limited ability to interact with PKC. By contrast, each of the conventional PKC, -I, -II, and -
activities were unaffected by the C1A-C1B domain (Fig. 5, ), and
also by the C1 domain (Fig. 5, 
). Similar to PKC, the
activities of PKC and - were each potentiated by their respective
C1A-C1B domains, while being unaffected by the C1 domains of the other
isozymes. Furthermore, as found for PKC, the level of activity
induced by the respective C1 domain was close to that induced by
membrane association in the presence of TPA and Ca2+, each
activator being present at a maximally activating concentration. By
contrast to the conventional and novel PKC isozymes, the activity of
atypical PKC was unaffected by its own C1 domain and those of the
other PKC isozymes.
|
Intermolecular Interactions Involving the C1A-C1B Domain Mediate
in the Self-association of PKC--
The possibility exists that
once disengaged from the intramolecular interaction, the C1 and C2
domains may participate in an intermolecular interaction
with their counterparts on a neighboring PKC molecule, which would
therefore be expected to result in the formation of dimers. To address
this possibility, the activity of PKC was measured as a function of
its concentration, as shown in Fig. 6.
The double-log plot of PKC activity against concentration, obtained
in the presence of a fixed level of TPA but in the absence of membrane
lipids (Fig. 6, ), contained an inflection at a PKC concentration
of ~4 nM, which corresponded to a change in gradient from
0.83 ± 0.5 to 2.23 ± 0.3. This result is consistent with a
change from 1 to 2 active sites per active PKC complex and suggests
the isozyme undergoes a monomer-dimer equilibrium, the dimer having a
relatively higher activity. Furthermore, consistent with the notion
that the dimerization of PKC may be mediated by intermolecular C1-C2
domain interactions, the presence of a fixed concentration of the
C1A-C1B domain (Fig. 6, ) yielded a linear relationship of slope
0.89 ± 0.3 within the same PKC-concentration range. To address
the question whether PKC associated with membranes may also engage
in a monomer-dimer equilibrium, the concentration dependence of PKC
activity was measured in the presence of BPS/POPC vesicles,
Ca2+, and TPA (Fig. 6, ). The concentration of BPS (20 mol % of the total lipid concentration), Ca2+ (0.1 mM), and TPA (500 nM) used each corresponded to
those previously shown to induce complete membrane association of
PKC and a maximal level of activation of the isozyme (38). It was
found that the activity of membrane-associated PKC again displayed
positive cooperativity with respect to PKC concentration. Thus,
a double-log plot of PKC activity against concentration again
contained an inflection corresponding to a change in the slope of the
regression line from 0.78 ± 0.6 to 2.02 ± 0.4, which is
consistent with PKC-PKC dimerization. Furthermore, the PKC
concentration corresponding to the inflection point was ~10-fold
lower than that observed for PKC in the absence of membranes,
indicating that in this case dimerization occurred at a lower PKC
concentration, consistent with an increased monomer-dimer association
constant.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
The activation of PKC isozymes by association with membranes
ultimately hinges on a conformational change induced by diacylglycerol- or phorbol ester binding to the C1 domains and PS/Ca2+
binding to the C2 domain (12, 15), that results in the removal of the
pseudosubstrate from the active site to allow binding of a substrate
(25-27). This contribution provides evidence supporting the existence
of an additional intramolecular interaction between the C1 and C2
domains of PKC, the dissociation of which is a requirement for the
activating conformational change to proceed.
The observation from SPR experiments, showing that the C1A-C1B
domain peptide interacted with both intact PKC and with the C2
portion of the C1A-C1B-C2 peptide, suggests a critical and functional interaction between the C1A, C1B, and C2 domains of the
isozyme. This is further reinforced by the observation that the
interaction of PKC with the C1A-C1B domain resulted in pronounced enzyme activity, consistent with the activating conformational change
requiring the dissociation of a C1-C2 domain interaction that otherwise
holds the enzyme in a "closed" inactive state. We therefore propose
that the activating conformational change in PKC corresponds to an
equilibrium between closed and "open" states that is regulated by a
C1-C2 interaction; the open active state being stabilized by
interaction with a membrane surface, or with filamentous actin (53).
This provides experimental evidence for an interaction involving the C2
domain that was suggested could occur in a recent study for PKC
(24), and also was alluded to in an earlier study for a novel PKC
isozyme from Aplysia (54).
The finding that phorbol ester or diacylglycerol was absolutely
required for PKC activation by the C1A-C1B domain (see Fig. 3A), suggests that a transient dissociation of the C1-C2
interaction may be initially required to expose the phorbol
ester- or diacylglycerol-binding sites within the C1 domains. This
would be consistent with the low level of PKC activity that is induced
by phorbol ester alone in the absence of membranes (see Fig.
3B and Refs. 47-50). The observation that the interaction
of PKC with the C1A-C1B resulted in a dramatic reduction in the
TPA concentration dependence of activity (see Fig. 3B) is
also consistent with the proposal that the phorbol ester-binding sites
within the C1 domains are exposed by the formation of the open state.
In this model, the binding of the C1A-C1B domain peptide to the C2
domain combined with phorbol ester or diacylglycerol binding to the C1
domains, would then "lock" the PKC molecule in an open state by
blocking the formation of the C1-C2 interaction. This open state
appears to correspond to the fully active conformation of PKC, based
on the observation that the level of PKC activity induced by
interaction with TPA and the C1A-C1B domain approached that
resulting from membrane association induced under maximally activating
conditions. The finding that the interaction of the C1A-C1B domain
with the C1A-C1B-C2 peptide was also TPA-dependent, suggests that the properties of the C1A-C1B-C2 peptide may reflect those of the intact PKC molecule, in that it may also be engaged in
a transient equilibrium between closed and open states; the latter
being stabilized by phorbol ester binding to its C1 domains.
The finding that the dose-response curves for the activation of PKC
by the C1A and C1B domains were each shifted to the right by ~2
orders of magnitude relative to that for the full-length C1A-C1B
domain (compare Fig. 3, A with C), suggests that
residues in both C1A and C1B domains may participate in
interdomain interactions with the C2 domain that stabilizes the closed
conformation of PKC. The observation that the activity of D55A was
still to some extent potentiated by phorbol ester (see Fig. 4),
although the C1A-C2 domain interaction is absent in this mutant, also
indicates that the dissociation of other interactions in addition to
that between the C1A and C2 domains may be involved.
Investigations using the PKC C1A domain mutant, D55A, reveal further
features of the unfolding mechanism that leads to activation. The
intrinsic activator-independent activity of the D55A mutant was
observed to be markedly increased compared with that of wild type
PKC (24) (and see Fig. 4), while the TPA concentration dependence of
the activity of D55A was dramatically reduced compared with that
obtained for wild-type PKC (see Fig. 4, inset).
Furthermore, unlike the wild type PKC, the activity of D55A was not
affected by the addition of the C1A-C1B domain.
Earlier studies have suggested that the conformation of the
"cytosolic" isozyme may be retained in an inactive or closed state by a "strong interaction" between the pseudosubstrate and the active site that "clamps" the C1 domains between the V1 and
catalytic domain (14), although binding data and assignment of the
regions of the regulatory domain involved were not
determined. Nevertheless, this V1 clamp model, which was suggested to
prevent the binding of diacylglycerol or phorbol ester to the C1
domains, is entirely compatible with our observations, and is extended
to show that additional C1-C2 interactions are required for the
unfolding of the pseudosubstrate from the active site. While in the
previous study it was suggested that an electrostatic interaction
between of the V1 region and the membrane surface was required to
stabilize the open state of PKC (14), it was found here that PKC
was fully activated by interaction with the C1A-C1B domain in the presence of TPA, even though membranes were absent. This
suggests that the reversible electrostatic interaction of the
pseudosubstrate with lipid head groups favors, but may not alone,
induce the formation of the open active state.
With regard to isozyme specificity, the interaction with the C1A-C1B
domain and the ensuing activation appears to be confined to
conventional PKC, I, II, and to a lesser extent PKC, based on the observation that the activities of novel PKC, -, and atypical PKC were unaffected by the domain (see Fig. 5). This apparent specificity was also suggested by the observation that the
activities of PKC, -I, and -II were, to a lesser degree, activated by the C1A-C1B domain and were unaffected by the
C1A-C1B and C1 domains. The finding that the C1A-C1B and
C1A-C1B domains also potentiated the activities of PKC and
PKC, respectively, suggests that intramolecular interactions
involving the C1 domains of these isozymes may also mediate the
activating conformational change. Although, it is possible that these
interactions may be similar to those between the C1 and C2 domains of
PKC, the organization of these domains and also the pseudosubstrate
within the structures of PKC and - differ markedly from that of
PKC.
The observation that measurements of PKC activity as a function of
PKC yielded curves that contained an inflection point corresponding
to a change in gradient from ~1 to ~2 indicates a change in the
reaction kinetics from being first-order to second-order, with respect
to the enzyme concentration. This is consistent with a
concentration-dependent equilibrium between monomeric and
dimeric forms of PKC. Evidence supporting the notion that PKC may be active in PKC-PKC and/or PKC-substrate complexes has also been provided
elsewhere (55-60). Furthermore, evidence that PKC may become active
upon self-association in the cellular environment was presented
recently based on the observation of PKC dimers in lysates derived
from murine B82L fibroblasts treated with calcium ionophore, phorbol
ester, or epidermal growth factor (60). The self-association of PKC
might be mediated by the same intramolecular interactions between the
C1 and C2 domains discussed above but now between two different PKC
molecules, since the presence of the C1A-C1B peptide restored a
linear relationship between enzyme concentration and activity. The
observation that the interaction of the C1A-C1B domain was specific
for PKC, 
I, II, and to a lesser extent PKC, and
that the activity of PKC was relatively less affected by the C1
domains of PKC, -, and -, suggests that PKC can dimerize
with itself, and potentially also with other conventional PKC isozymes.
However, in the cellular environment, whether PKC isozymes form homo-
or heterodimers would depend on whether these isozymes are
co-localized. The roles of intermolecular PKC-PKC interactions, and the
balance between inter- and intramolecular C1-C2 interactions in the
mechanism of activation of PKC remain to be investigated.
Conclusion--
The existence of C1-C2 domain interactions that
retain the PKC molecule in an inactive conformation was directly
demonstrated by the observation that the domains involved bind directly
to one another and that this leads to activation. The current
observations support a model in which PKC activation corresponds to
a transition from a closed inactive to an open active state that is
governed by the dissociation of the C1-C2 interaction.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. Wonhwa Cho (University of
Chicago) for the gift of the D55A mutant of PKC and Dr.
Peter M. Blumberg (National Cancer Institute) for the C1A-C1B
domain peptide. We also thank Shawn K. Milano, Jeffrey P. Curry, and Kevin J. Gergich for technical contributions in some of the work.
| |
FOOTNOTES |
|---|
* This work was supported by United States Public Health Service Grants AA08022, AA07215, AA07186, and AA07465.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.
To whom correspondence should be addressed: Rm. 271 JAH, Dept. of
Pathology, Cell Biology and Anatomy, Thomas Jefferson University, Philadelphia, PA 19107. Tel.: 215-503-5019; Fax: 215-923-2218; E-mail:
Chris.Stubbs@mail.tju.edu.
Published, JBC Papers in Press, February 15, 2002, DOI 10.1074/jbc.M112207200
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
PKC, protein kinase
C;
BPS, bovine brain phosphatidylserine;
DiC8, 1,2-dioctanoyl-sn-glycerol;
MBP4-14, myelin
basic protein peptide substrate;
-peptide, peptide substrate based
on the PKC pseudosubstrate;
POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine;
TPA, 4-12-O-tetradecanoylphorbol-13-acetate;
GST, glutathione S-transferase;
SPR, surface plasmon
resonance.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. |
Mellor, H.,
and Parker, P. J.
(1998)
Biochem. J.
332,
281-292[Medline]
[Order article via Infotrieve] |
| 2. |
Nishizuka, Y.
(1995)
FASEB J.
9,
484-496[Abstract] |
| 3. |
Jaken, S.
(1996)
Curr. Opin. Cell Biol.
8,
168-173[CrossRef][Medline]
[Order article via Infotrieve] |
| 4. |
Naruse, K.,
and King, G. L.
(2000)
Circ. Res.
86,
1104-1106 |
| 5. |
Clarke, H.,
Marano, C. W.,
Peralta Soler, A.,
and Mullin, J. M.
(2000)
Adv. Drug Deliv. Rev.
41,
283-301[CrossRef][Medline]
[Order article via Infotrieve] |
| 6. |
Musashi, M.,
Ota, S.,
and Shiroshita, N.
(2000)
Int. J. Hematol.
72,
12-19[Medline]
[Order article via Infotrieve] |
| 7. |
Dempsey, E. C.,
Newton, A. C.,
Mochly-Rosen, D.,
Fields, A. P.,
Reyland, M. E.,
Insel, P. A.,
and Messing, R. O.
(2000)
Am. J. Physiol. Lung Cell. Mol. Physiol.
279,
L429-438 |
| 8. |
Newton, A. C.
(1996)
J. Biol. Chem.
48,
28495-28498 |
| 9. |
Newton, A. C.
(1997)
Curr. Opin. Cell Biol.
9,
161-167[CrossRef][Medline]
[Order article via Infotrieve] |
| 10. |
Parekh, D. B.,
Ziegler, W.,
and Parker, P. J.
(2000)
EMBO J.
19,
496-503[CrossRef][Medline]
[Order article via Infotrieve] |
| 11. |
Li, W.,
Mischak, H., Yu, J. C.,
Wang, L. M.,
Mushinski, J. F.,
Heidaran, M. A.,
and Pierce, J. H.
(1994)
J. Biol. Chem.
269,
2349-2352 |
| 12. |
Newton, A. C.,
and Johnson, J. E.
(1998)
Biochim. Biophys. Acta
1376,
155-172[Medline]
[Order article via Infotrieve] |
| 13. | Johnson, J. E., Giorgione, J., and Newton, A. C. (2000) Biochemistry 39, 11360-11369[CrossRef]< |
