(Received for publication, June 27, 1996, and in revised form, March 4, 1997)
From the Protein Phosphorylation Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom
The interaction between protein kinase C- and
its neuronal substrate, GAP-43, was studied. Two forms of protein
kinase C-
were isolated from COS cells and characterized by
differences in gel mobility, GAP-43 binding, and specific GAP-43 and
histone kinase activities. A slow migrating, low specific activity form of protein kinase C-
bound directly to immobilized GAP-43. Binding was abolished in the presence of EGTA, suggesting
Ca2+ dependence of the interaction. The free
catalytic domain of protein kinase C-
did not bind GAP-43,
suggesting the existence of a binding site in the regulatory domain.
Glutathione S-transferase-protein kinase C-
regulatory
domain fusion proteins were generated and tested for binding to GAP-43.
The V0/C2-like amino-terminal domain was defined as the GAP-43-binding
site. GAP-43 binding to this region is inhibited by EGTA and regulated
at Ca2+ levels between 10
7 and
10
6 M. The interaction between protein kinase
C-
and GAP-43 was studied in intact cells by coexpression of the two
proteins in human embryonic kidney cells followed by
immunoprecipitation. Complex formation occurred only after
treatment of the cells with the Ca2+ ionophore ionomycin,
indicating that elevation of intracellular Ca2+ is required
for interaction in vivo. It is concluded that protein kinase C-
interacts with GAP-43 through the V0/C2-like domain, outside the catalytic site, and that this interaction is modulated by
intracellular Ca2+.
The protein kinase C (PKC)1 family is
a ubiquitous and abundant kinase family involved in the transduction of
extracellular signals in a large number of different tissues (reviewed
in Refs. 1-4). PKC isotypes are activated by Ca2+,
phospholipids, and diacylglycerol (or its pharmacomimetic phorbol ester), which bind at conserved (C) regions in the regulatory domain.
Three PKC subfamilies are distinguished on the basis of the variability
in their regulatory domains and consequential differences in cofactor
dependence. The regulatory domains of PKC-, -
, and -
contain a
C1 and a C2 region, rendering them responsive to diacylglycerol/phorbol
ester, which bind at C1, as well as to Ca2+, which binds at
C2. The regulatory domains of PKC-
, -
, -
, and -
contain C1,
but lack C2, resulting in Ca2+ independence. A variable
extension (V0) is present N-terminal of the C1 region. PKC-
and
-
/
resemble the isotypes in the latter subfamily; however, their
C1 region is different and will not bind phorbol ester or
diacylglycerol (1-7).
Activation of PKC leads to phosphorylation of substrate proteins and
ultimately to a biological response. The growth-associated protein
GAP-43 (also known as neuromodulin, p57, B-50, F1, pp46, and 5) is
one of the better characterized cellular PKC substrates known (8, 9).
It has an almost exclusively neuronal localization and is present at
high levels during development or during neuronal regeneration
processes (10, 11). In early stages of development, it is not confined
to any particular part of the neuron; however, upon maturation, its
expression becomes restricted to the axon, where it remains detectable
in the axon shaft as well as in the axon terminal (12, 13). GAP-43 is a
very acidic, "rod-shaped" protein with a predicted molecular mass
of 23 kDa (9). It contains a single PKC phosphorylation site at Ser-41
and is further modified by dipalmitoylation at two adjacent Cys
residues, Cys-2 and Cys-3 (9). This allows for membrane localization of
the protein in the absence of an obvious hydrophobic domain.
Phosphorylation of GAP-43 in vivo is significantly
stimulated by phorbol ester and, in various systems, by membrane
depolarization, nerve growth factor treatment, and conditions under
which hippocampal slices show long-term potentiation (14-16).
Manipulation of GAP-43 levels suggests a role for the protein in
neurite outgrowth, possibly by modulating the adhesive properties of
the growth cone (17, 18). Furthermore, a role for GAP-43 in transmitter
release has been proposed based on observations that interference with
GAP-43 affects the release of neurotransmitter (19-23) and that
overexpression of GAP-43 changes the competence of pituitary cells to
release hormone (24). Some of the biochemical properties of GAP-43, such as its capacity to bind calmodulin and actin or to affect GTP-binding proteins, may underlie its cellular functions (8). It has
been shown that GAP-43 serves as a substrate for a number of PKC
isotypes (25, 26) and that PKC-mediated phosphorylation of GAP-43
directly modulates its calmodulin binding capacity (8, 9).
It has been postulated that in addition to being effector molecules,
PKC substrates also bind to the kinase directly, a property that may be
important for intracellular targeting of PKC. Little is known
concerning the manner in which PKC interacts with GAP-43, although the
existence of a high affinity binding site outside the phosphoacceptor
region has been proposed on the basis of enzyme kinetic studies (26).
In this study, we have addressed this issue by investigating the
molecular determinants in PKC involved in GAP-43 binding. We
demonstrate that GAP-43 interacts directly with PKC- outside the
catalytic domain at the V0/C2 region. This defines a protein
interaction domain on PKC-
and suggests new ways to evaluate the
functional relevance of the PKC/GAP-43 system.
A monoclonal antibody to GAP-43 was obtained from
Affinity Laboratories. The PKC- rabbit polyclonal antiserum was
raised against a C-terminal 10-mer peptide (27). An affinity-purified polyclonal antibody to glutathione S-transferase (GST) was
obtained from Pat Warne and Julian Downward (Imperial Cancer Research
Fund, London). The calmodulin polyclonal antibody, produced by Dr. A. Means, was obtained from Dr. E. Wood (Department of Biochemistry, University of Leeds). COS-7 cells and human embryonic kidney 293 cells
were obtained from the Cell Production Unit at the Imperial Cancer
Research Fund (London).
The PKC- expression plasmid pKS1-PKC-
(27)
was introduced into COS-7 cells by electroporation, and three 15-cm
dishes of transfected COS cells were grown. Three days
post-transfection, COS cells were washed three times in ice-cold buffer
A (137 mM NaCl, 3 mM KCl, 10 mM
Na2HPO4, 2 mM
KH2PO4, pH 7.2) and scraped in harvesting
buffer (20 mM Tris-Cl, pH 7.5, 10 mM EGTA, 5 mM EDTA, 0.3% (v/v)
-mercaptoethanol, 250 µg/ml
leupeptin, 10 mM benzamidine, 50 µg/ml
phenylmethylsulfonyl fluoride, 1% (v/v) Triton X-100). The resulting
cell suspension was homogenized in a Dounce homogenizer by 20 strokes,
incubated for 15 min at 4 °C, diluted to 10 ml in buffer B (20 mM Tris-Cl, pH 7.5, 2 mM EDTA, 10 mM benzamidine, 0.3% (v/v)
-mercaptoethanol, 0.02%
(v/v) Triton X-100), and centrifuged at 10,000 × g for
30 min at 4 °C. The supernatant was loaded onto a MonoQ column
(Pharmacia Biotech Inc.) equilibrated with buffer B. Fractions (0.5 ml)
were collected at a flow rate of 0.2 ml/min with a 25-min isocratic
wash followed by a gradient of NaCl in buffer B (0-0.5 M
in 120 min). After collection, an equal volume of glycerol was added to
each fraction, and fractions were stored at
20 °C.
DNA representing the human GAP-43 coding domain (28)
was generated by polymerase chain reaction using a human fetal brain library as template. The DNA was cloned in
BamHI-XhoI sites of pRSETb (Invitrogen) using
restriction sites engineered in the primers, and the resulting
construct was used to transform XL-1 Blue bacteria. For protein
production, 400 ml of LB medium was inoculated (1:20) with an overnight
culture of XL-1/pRSETb-GAP-43 cells and grown for 3 h at 37 °C.
Isopropyl--D-thiogalactopyranoside was then added to a
final concentration of 1 mM, and after a further incubation
of 5 h at 37 °C, cells were harvested and resuspended in 20 ml
of ice-cold buffer C (50 mM sodium phosphate, pH 8.0, 1 M NaCl, 3 mM
-mercaptoethanol, 30 mM imidazole, 10 mM benzamidine, 0.1% (v/v)
Triton X-100) containing 50 µg/ml leupeptin and 200 µg/ml
aprotinin. The cell suspension was sonicated for 1 min on ice using a
Soniprep 2000, cooled for 1 min, and sonicated twice more for 2 min
separated by 1 min of cooling. The resulting extract was centrifuged at
10,000 × g for 30 min at 4 °C, and the supernatant was taken and tumbled with Ni2+-NTA-agarose beads (QIAGEN
Inc.; 0.5-ml bed volume, washed in buffer A) for 1 h at 4 °C.
The beads were spun down and washed three times with 50 ml of buffer C
and three times with 50 ml of buffer D (50 mM sodium
phosphate, pH 6.0, 1 M NaCl, 3 mM
-mercaptoethanol, 30 mM imidazole, 10 mM
benzamidine, 0.1% (v/v) Triton X-100, 10% (v/v) glycerol) at 4 °C.
Finally, hisGAP-43 was eluted by tumbling the beads twice for 1 h
at 4 °C in 1 ml of buffer D + 350 mM imidazole. The
resulting preparation was dialyzed overnight against MilliQ H2O at 4 °C.
A BanI (filled in)-HindIII
cDNA fragment representing the regulatory domain of PKC- was
inserted in EcoRI (filled in)-HindIII-digested pGEX-KG (Pharmacia) to generate pGEX-KG-PKC-
-(1-298). A
BamHI-HindIII fragment was taken out of this
construct and inserted into pKSII(+) (Stratagene). The resulting
plasmid was digested with HindIII and ApaI and
treated with exonuclease III, resulting in random deletion of the
plasmid from the HindIII site. The digested mixture was
blunt-ended with mung bean nuclease, religated, and used to transform
XL-1 Blue bacteria. Plasmids with inserts of different lengths were
selected and verified by sequence analysis. The inserts were taken out
and reinserted into pGEX-KG. For the construction of
pGEX-KG-PKC-
-(1-121), a BamHI-BstNI fragment
was taken out of pKSII(+)-PKC-
-(1-298) and inserted into
pGEX-KG.
For protein production, 400 ml of LB medium was inoculated (1:20) with
an overnight culture of XL-1 Blue cells transformed with the
appropriate pGEX-KG-PKC- regulatory domain construct and grown for
3 h at 37 °C. Isopropyl-
-D-thiogalactopyranoside was then added to a final concentration of 0.1 mM, and
after further incubation for 3-5 h at 37 °C, cells were harvested
and resuspended in 20 ml of ice-cold buffer A. The cell suspension was
sonicated three times for 1 min on ice using a Soniprep 2000 each time, interrupted by 1 min of cooling on ice. The resulting cell extract was
centrifuged at 10,000 × g for 20 min at 4 °C, and
to the supernatant was added Triton X-100 (final concentration of 1%
(v/v)). The supernatant was tumbled with glutathione-Sepharose 4B
(Pharmacia; 0.25-ml bed volume, washed in buffer A) for 1 h at
4 °C, after which the beads were spun down and washed twice with 50 ml of ice-cold buffer A. The GST fusion proteins were eluted from the beads in 0.5 ml of buffer E (50 mM Tris-Cl, pH 8.0, 5 mM reduced glutathione).
Routinely, hisGAP-43 and GST fusion proteins were mixed and incubated in 50 µl of buffer E for 1 h at 4 °C. Then, a 20-µl bed volume of Ni2+-NTA-agarose beads in 150 µl of buffer E was added, and incubation was continued for 1 h. Subsequently, the beads were spun down, drained completely, and washed twice with 1 ml of buffer E. Proteins bound to Ni2+-NTA-agarose were eluted by shaking the beads in 30 µl of buffer E + 350 mM imidazole at 1300 rpm for 30 min at 25 °C. Beads were spun down, and the supernatant was taken. Adjustments to the incubation conditions are described below and in the figure legends and were made both before and after the addition of the beads.
Coexpression in 293 Cells and ImmunoprecipitationA GAP-43
expression construct was generated by inserting the GAP-43 coding
sequence into pcDNA3. Human embryonic kidney 293 cells were
cotransfected with pcDNA3-GAP-43 and pKS1 or pKS1-PKC- (27) by
the calcium phosphate precipitation method (29). After 3 days, the
cells were treated for 15 min with 1.3 µM ionomycin. Subsequently, the cells were homogenized in buffer A containing 1%
Triton X-100, 100 µg/ml leupeptin, 100 µg/ml aprotinin, and 10 mM benzamidine and centrifuged at 10,000 × g for 20 min at 4 °C. To the supernatants were added 50 µl of PKC-
antiserum PP084 and 100 µl of protein A-agarose (50%
bead volume in buffer A). The mixture was incubated for 16 h at
4 °C, after which the protein A beads were collected by
centrifugation and washed three times with buffer A. PKC-
was eluted
from the protein A-antibody complex by incubation for 1 h with 20 µg of peptide antigen (27) in 65 µl of buffer A. The eluate was
analyzed by SDS-PAGE and Western blotting.
PKC enzyme activity was measured as described (30) with modifications as indicated in the figure legends. GAP-43 phosphorylation was quantified by analyzing the phosphorylation mixture by SDS-PAGE and Cerenkov counting of the 32P incorporation in the GAP-43 protein band. Western analysis of proteins separated by SDS-PAGE was performed according to Towbin et al. (31). Nitrocellulose filters were incubated with antiserum (specified in the figure legends) in buffer A containing 1% fat-free skimmed milk and 0.05% Tween 20. Filters were processed using the ECL detection reagent (Amersham International, Buckinghamshire, United Kingdom). Silver staining of proteins separated by SDS-PAGE was performed according to Merril et al. (32).
It has been reported
that GAP-43 is phosphorylated by a number of PKC isotypes (25, 26). The
phosphorylation reaction may involve a complex interaction between
GAP-43 and PKC since the kinetics of phosphorylation of GAP-43
polypeptide and phosphorylation site oligopeptide differ (26). We
noticed differences among PKC- preparations in their activity toward
GAP-43, but not peptide substrates, also indicating complexity of
interaction (data not shown). To investigate this in more detail,
PKC-
was overexpressed in COS cells and partially purified by MonoQ
ion-exchange chromatography. MonoQ fractions were analyzed for kinase
activity using exogenous recombinant hisGAP-43 or histone III-S as
substrate. GAP-43 kinase activity eluted in three main peaks (peaks
1-3), two of which were dependent on cofactor, with the third being
largely cofactor-independent (Fig. 1A).
Histone kinase activity in these fractions showed an additional
cofactor-dependent activity (peak 4) eluting before peak 3 (Fig. 1B).
Fig. 1C shows a Western blot of the individual fractions
probed with an antibody recognizing the PKC- C terminus. Full-length forms of PKC-
coeluted with peaks 1 and 2, whereas a PKC-
breakdown product of ~45 kDa (representing the catalytic domain)
eluted in peak 3. Peak 1 contained a fast migrating form of PKC-
,
and peak 2 contained a slower migrating form; fraction 25, between these peaks, contained both. Neither of the forms reacted with an
antibody recognizing phosphotyrosine. Western blotting also revealed
that peak 4 represents endogenous PKC-
present in COS cells (data
not shown).
To visualize all PKC- immunoreactivity in the different fractions,
an overexposed autoradiograph is shown in Fig. 1C.
Densitometric scanning of an exposure in the linear range (Fig.
1D) revealed the amount of PKC-
in peak 1 to be 10-20%
of that in peak 2. By dividing the GAP-43 kinase activity of these
fractions by the amount of antigen, an indication (albeit a
semiquantitative one) of the specific GAP-43 kinase activity in these
PKC-
peaks was obtained. The specific GAP-43 kinase activity of peak
1 PKC-
(fraction 23) was estimated to be 7-fold that of peak 2 PKC-
(fraction 26). Specific GAP-43 kinase activity in fractions
30-33 did not differ from that in fractions 26-29. This analysis also revealed that the PKC-
catalytic domain (peak 3) had much higher specific GAP-43 kinase activity than either peak 1 or 2.
To assess the potential interaction of PKC- with
GAP-43, the individual MonoQ fractions were incubated with hisGAP-43,
after which hisGAP-43 was immobilized on Ni2+-NTA-agarose
beads, washed, and eluted from the beads by imidazole. The eluate was
analyzed by SDS-PAGE followed by Western blotting for PKC-
and
hisGAP-43. Fig. 2A shows that peak 2 PKC-
bound to immobilized GAP-43, whereas no GAP-43 binding was detected for
peak 1. PKC-
did not bind directly to the
Ni2+-NTA-agarose beads (Fig. 2B). Furthermore,
no binding of the PKC-
catalytic domain (peak 3) to GAP-43 occurred
(Fig. 2C).
PKC- is known for its Ca2+-independent catalytic
activity. Indeed, both PKC-
forms phosphorylated histone III-S in a
Ca2+-independent manner, whereas PKC-
(peak 3) showed
Ca2+ dependence under the same conditions (Fig.
3A). Surprisingly, GAP-43 binding to PKC-
(peak 2) was found to be reduced in the presence of EGTA, suggesting
Ca2+ dependence of this interaction (Fig. 3B).
In contrast to the Ca2+-independent phosphorylation of
histone by peak 2 PKC-
, phosphorylation of GAP-43 increased upon
EGTA addition (Fig. 3C). This was not the case for peak 1 PKC-
, which showed Ca2+-independent phosphorylation of
histone as well as GAP-43 (Fig. 3, A and C).
These data indicate that GAP-43 and PKC- interact directly with each
other in a Ca2+-dependent fashion at a site
outside the catalytic domain. Furthermore, PKC-
is functionally
heterogeneous since only the later eluting, slow migrating form (peak
2) bound to GAP-43. (It should be noted that since GAP-43 is not
expressed in COS cells, the lack of binding to peak 1 is not due to a
pre-existing GAP-43·PKC-
complex.)
In view of the fact that full-length
PKC- bound to GAP-43, but the the catalytic domain of PKC-
did
not, we investigated where the interaction occurs in the regulatory
domain. PKC-
regulatory domain fragments of different length were
produced as recombinant GST fusion proteins. Fig. 4
shows the relative yield of the various fragments after extraction of
the proteins and purification using glutathione-Sepharose 4B. Of four
regulatory domain fragments, GST-PKC-
-(1-121) showed the highest
levels of expression and the highest yield. PKC-
-(1-121) represents
the region of PKC-
that previously has been identified as V0 (1, 2)
and that shares structural homology with the C2 region of the classical PKC isotypes (33). GST-PKC-
-(1-298) and GST-PKC-
-(1-165)
showed, in addition to proteins of the predicted size, a breakdown
product of ~40 kDa. GST-PKC-
-(1-121) and GST-PKC-
-(1-61) were
homogeneous proteins of the predicted molecular mass.
To establish whether the regulatory domain of PKC- was able to bind
GAP-43, the various GST-PKC-
fusion proteins were assayed for
binding to hisGAP-43 as described above. Fig.
5A shows that both GST-PKC-
-(1-298) and
GST-PKC-
-(1-121) co-immobilized with hisGAP-43 on
Ni2+-NTA beads. GST-PKC-
-(1-61) and GST did not
associate with hisGAP-43. In the absence of hisGAP-43,
GST-PKC-
-(1-298) and GST-PKC-
-(1-121) were not present in the
eluate, indicating that they do not bind directly to the beads. Fig.
5B shows the concentration dependence of the binding of
GST-PKC-
-(1-121) to hisGAP-43. At 0.5 µM GAP-43, half-maximum binding of GST-PKC-
-(1-121) was estimated to occur at
50 nM.
The binding of GST-PKC--(1-121) to hisGAP-43 was not affected by
high concentrations of NaCl (Fig. 6A).
Similarly, 2 mM CaCl2 did not change binding,
but it was completely lost in the presence of 1 mM EGTA
(Fig. 6A). The interaction between PKC-
-(1-121) and
hisGAP-43 may therefore be supported by trace calcium ions present in
the incubation buffers. Analysis of the Ca2+ dependence of
GAP-43 binding to PKC-
-(1-121) in an EGTA-buffered system showed
binding to occur at Ca2+ concentrations higher than
10
7 M (Fig. 6B). At a
concentration of 10
7 M or lower, binding was
dramatically reduced. The binding of hisGAP-43 to the
Ni2+-NTA-agarose beads was not affected by these
Ca2+ concentrations (data not shown).
Characterization of the PKC-
To
refine the binding of PKC--(1-121) to GAP-43, we generated a
C-terminal truncation mutant of GAP-43 (hisGAP-43-(1-146)) and
analyzed its capacity to bind the various GST-PKC-
regulatory domain
constructs. In all aspects, hisGAP-43-(1-146) behaved in the same way
as full-length hisGAP-43 both in terms of the binding preferences and
concentration dependences of binding (Fig. 7). Thus, the
binding of PKC-
to GAP-43 occurs in the N-terminal part of the
protein between residues 1 and 146.
Since GAP-43 is a calmodulin-binding protein, we investigated whether
the binding of GST-PKC--(1-121) would preclude calmodulin binding
to GAP-43. Calmodulin was identified on SDS gel by silver stain (Fig.
8B) based on its migration at the appropriate
size (17 kDa) and by Western blotting using a calmodulin antibody (Fig. 8C). In line with existing literature, calmodulin bound to
hisGAP-43 (immobilized on Ni2+-NTA-agarose beads) in the
absence, but not in the presence, of CaCl2 (Fig. 8,
B and C). GST-PKC-
-(1-121) binding to
hisGAP-43 occurred whether or not calmodulin was associated, and
similarly, calmodulin binding was unaffected when increasing amounts of
GST-PKC-
-(1-121) were bound to hisGAP-43 (Fig. 8). hisGAP-43
association with the Ni2+-NTA-agarose beads was identical
under all conditions (Fig. 8A). Thus, PKC-
binding to
hisGAP-43 occurs at a site that is different from the
calmodulin-binding site.
Co-immunoprecipitation of GAP-43 and PKC-
To test whether
GAP-43 and PKC- are able to form a productive complex in intact
cells, co-immunoprecipitation assays were performed. A GAP-43 and a
PKC-
expression construct were transfected into 293 cells; PKC-
was immunoprecipitated; and the immunoprecipitate was analyzed for the
presence of GAP-43 by Western blotting. Since GAP-43 binds to PKC-
in a Ca2+-dependent manner in vitro,
transfected cells were treated with the Ca2+ ionophore
ionomycin to elevate intracellular Ca2+ levels. Fig.
9 shows GAP-43 to be present in PKC-
immunoprecipitates from 293 cells cotransfected with GAP-43 and PKC-
and treated with ionomycin. In the absence of ionomycin treatment, very
little GAP-43 was detected. As a control, immunoprecipitations were
carried out on cell lysates from cells that were transfected with
GAP-43, but not PKC-
. In these immunoprecipitates, background levels of GAP-43 were detected comparable to the level detected in the immunoprecipitates from cells not treated with ionomycin. We conclude that GAP-43 and PKC-
form a complex in cells in response to
elevation of intracellular Ca2+ as predicted on the basis
of the in vitro binding studies.
Evidence has been presented for the formation of a
GAP-43·PKC- complex in intact cells and for the direct binding of
GAP-43 at the regulatory domain of PKC-
. Within the regulatory
domain, the V0 region presents a minimal binding site. Little is known about the structure and function of this region, which is present in
novel as well as atypical PKC isotypes. Recently, it was proposed that
it may share structural homology with the C2 region in PKC-
, -
,
and -
(33). Our observation that GAP-43 binding occurs at the V0/C2
region of PKC-
indicates that it may serve as a protein-protein
interaction domain. Furthermore, the Ca2+ dependence of the
interaction shows that circumstances may exist in which
"Ca2+-independent" PKCs, such as PKC-
, may still
respond to changes in Ca2+ levels.
The PKC- employed in this study was partially purified from COS
cells and separated into two forms, characterized by differences in
mobility on SDS gels. The slow migrating form of PKC-
(peak 2) binds
GAP-43, and binding of GAP-43 to this form was abolished by EGTA
treatment, suggesting Ca2+ dependence of the interaction.
No GAP-43 binding to the fast migrating form of PKC-
(peak 1) was
detected. The difference in migration of the two PKC-
forms is
likely to be a result of phosphorylation, as was previously shown for
many other PKC isotypes (34, 35). Although tyrosine phosphorylation of
PKC-
is a well established phenomenon (36-38), we could find
no evidence that one of the forms in this study is
tyrosine-phosphorylated.
Under all circumstances, we observed a reciprocal correlation between
the ability of PKC- to bind GAP-43 and its specific GAP-43 kinase
activity. The high binding form shows low specific activity and
vice versa. EGTA treatment of the binding form (abolishing GAP-43 binding) results in an increase in GAP-43 kinase activity. Under
these conditions, histone kinase activity is not affected, indicating
that EGTA impinges on a component in the reaction specifically related
to GAP-43. Phosphorylation of GAP-43 by peak 1, the non-binding form,
is not affected by EGTA, indicating that the effect of EGTA on GAP-43
phosphorylation is restricted to the binding form. The free catalytic
domain of PKC-
, which does not bind GAP-43, shows high activity. A
unifying explanation for our observations is that GAP-43 binding may
affect the specific GAP-43 kinase activity of PKC-
, measured in the
in vitro phosphorylation reaction. In addition to GAP-43
binding, other properties of PKC-
play a role in the phosphorylation
reaction. We observed that differences occur in histone kinase activity
between the PKC-
forms, although there is no complete quantitative
agreement between the histone kinase activity of the two PKC-
forms
and their respective GAP-43 kinase activities (Fig. 1, A and
B). In this respect, the difference in histone kinase
activity may be a reflection of the intrinsic catalytic capacity of the
two PKC-
forms since histone is essentially a non-physiological
substrate. Other determinants become relevant when a physiological
substrate such as GAP-43 is used to assay kinase activity, as
exemplified here by the non-catalytic interaction between the PKC-
V0/C2 domain and GAP-43. Although the exact mechanism by which binding
of GAP-43 to V0/C2 would contribute to specific activity is not
formally demonstrated here, one possibility is that it affects the
off-rate of phosphorylated product and hence the turnover of the
phosphorylation reaction. Earlier work showing a low catalytic rate of
phosphorylation of full-length GAP-43 versus GAP-43
phosphorylation site oligopeptide also implied rate-limiting
interactions outside the direct site of catalysis (26), consistent with
the above conclusion.
Binding of V0/C2 to GAP-43 is not prevented by binding of calmodulin to GAP-43. Therefore, calmodulin and V0/C2 have different binding sites on GAP-43. This binding site is not in the C-terminal part of the molecule since the binding characteristics of GAP-43-(1-146) are identical to those of full-length GAP-43. The binding site may therefore lie between the calmodulin-binding site (residues 43-51) and residue 146 or in the extreme N terminus of GAP-43.
Binding of GAP-43 to the regulatory domain of PKC- indicates that
this domain is not just a target region for cofactor, but in fact
serves as a protein-protein interaction domain. As such, this
observation falls within the more general pattern of data suggesting
such a role for the regulatory domain (39, 40). For instance, several
phospholipid-binding proteins have been shown to interact with the
regulatory domain of PKC in a phospholipid-dependent way.
This binding occurs, at least in part, at the pseudosubstrate site,
which itself has phospholipid binding capacity (39, 41). Although
GAP-43 has been shown to bind phospholipid (9), phospholipids were not
present in the binding studies here. The binding of GAP-43 to PKC-
at the V0/C2 region is reminiscent of the binding of RACK1 to PKC-
,
which takes place at the C2 region (40, 42). However, in contrast to
GAP-43, RACK1 is not a PKC substrate. Furthermore, PKC-
does not
need to be in an active conformation to bind to GAP-43. It was
suggested that RACK1 binding to the C2 region is important for the
subcellular redistribution of PKC-
, but not of PKC-
and -
,
upon phorbol ester stimulation of cells (42). Such specificity is
intriguing in light of our observations, in that the interaction
between PKC-
and GAP-43 at the V0/C2 region may be important for the
subcellular localization of PKC-
. The finding that, on coexpression
of GAP-43 and PKC-
, a complex of the two proteins can be
immunoprecipitated indicates that interaction can occur
physiologically. The demonstration that this complex formation is
dependent on the pretreatment of cells with the Ca2+
ionophore ionomycin indicates that the interaction is regulated by
Ca2+ in a manner consistent with that determined in
vitro for the binding of GAP-43 to the V0/C2 region in PKC-
.
C2-like regions are present not only in PKC, but also in a number of
other proteins (33), and in these contexts appear to be involved in
protein-protein interaction as well. The C2-like regions in
synaptotagmin, for example, have been shown to bind syntaxin and AP-2
and also to dimerize (43-45). Many of these interactions are
Ca2+-dependent, and structural analysis of the
C2A region of synaptotagmin I has revealed a Ca2+-binding
site involving four aspartate residues forming a "cup-like cavity"
to accommodate a Ca2+ ion (46). Our results show that
Ca2+-dependent interactions can take place at a
C2-like region that does not contain these four aspartate residues,
indicating that the basis of the Ca2+ responses of the C2
regions may be more complex than initially thought. This can be
concluded also from the recent molecular cloning of a large number of
synaptotagmin isotypes, which revealed that at least two of them, while
containing the same four aspartate residues in their C2A region
as synaptotagmin I, did not show Ca2+-dependent
syntaxin binding (43).
The data presented demonstrate that PKC- interacts with GAP-43 in a
manner controlled by two regulatory devices. First, the binding is
Ca2+-dependent both in vitro and
in vivo. Second, fractionation of PKC-
reveals the
presence of binding and non-binding forms. Since this heterogeneity is
stable to fractionation, it is likely to be a consequence of PKC-
post-translational modification. In vivo, the combined
effects of these two regulatory devices will serve to determine whether
or not PKC-
and GAP-43 interact. Future efforts will need to address
this interaction in situ to assess its physiological role.
We thank Drs. Clive Dickson and Enrique Rozengurt for critical comments on this manuscript.