(Received for publication, July 9, 1995)
From the
RACK1 is a protein kinase C (PKC)-binding protein that fulfills
the criteria previously established for a receptor for activated
C-kinase (RACK). If binding of PKC to RACK anchors the activated enzyme
near its protein substrates, then inhibition of this binding should
inhibit translocation and function of the enzyme in vivo.
Here, we have identified such inhibitors that mimic the RACK1-binding
site on PKC. We first found that a C2-containing fragment, but not
a C1-containing fragment of
PKC, bound to RACK1 and inhibited
subsequent
PKC binding. The RACK1-binding site was further mapped;
peptides
C2-1 (
PKC(209-216)),
C2-2
(
PKC(186-198)), and
C2-4
(
PKC(218-226), but not a number of control peptides, bound
to RACK1 and inhibited the C2 fragment binding to RACK1. Peptides
C2-1,
C2-2, and
C2-4 specifically
inhibited phorbol ester-induced translocation of the C2-containing
isozymes in cardiac myocytes and insulin-induced
PKC translocation
and function in Xenopus oocytes. Therefore, peptides
corresponding to amino acids 186-198, and 209-226 within
the C2 region of the
PKC are specific inhibitors for functions
mediated by
PKC.
Protein kinase C (PKC) ()isozymes are
phosphatidylserine (PS)- and diacylglycerol (DG)-dependent kinases (1) that translocate from the soluble fraction to the cell
particulate fraction following activation(2, 3) .
Several PKC isozymes are present in each cell type. The isozymes are
localized to different subcellular
compartments(4, 5, 6) , and following
stimulation, each translocates to distinct intracellular
structures(7) . The translocation and binding of PKC isozymes
to different intracellular structures suggests distinct physiological
roles for individual isozymes. We previously demonstrated that
inhibition of PKC translocation inhibits its
function(8, 9) . Therefore, inhibitors that prevent
the translocation of specific isozymes, can provide pharmacological
tools to determine the function of each isozyme.
The PKC isozymes can be divided into three subfamilies: conventional cPKC, novel nPKC, and atypical aPKC(10) . Each PKC isozyme contains unique (V) regions. In addition, there are regions common to all the isozymes. The subfamilies differ from each other in the common (C) regions within their regulatory domains. The regulatory domain of the cPKC isozymes contains two common regions, C1 and C2. The C1 region consists of two cysteine-rich loops that mediate DG and phorbol ester binding(11, 12, 13) . C1 is also found in the nPKC subfamily and one of the cysteine loops is found in the aPKC subfamily(14, 15, 16) . The C2 region is present only in the cPKC subfamily and mediates calcium binding; all the C2-containing isozymes require calcium for their activity(10) . This region may also serve as a low affinity calcium sensor(17) . In addition, the C2 region mediates PS binding(18) . However, recent studies indicate that the V1 and C1 regions also mediate calcium and PS binding(19, 20) . Finally, the C2 regions of other C2-containing proteins were proposed to mediate direct binding of these translocating proteins to lipids at the plasma membrane(21) .
Translocation of PKC to the cell particulate fraction was thought to
reflect direct binding of the enzyme to lipids at the plasma membrane.
However, data from several laboratories including our own indicate that
translocated PKC interacts with proteins at the site of translocation (2, 22, 23, 24, 25, 26, 27, 28) .
We have identified several proteins from the cell particulate fraction
that bind PKC only in the presence of its activators(24) .
Binding of PKC to these proteins was concentration dependent,
saturable, and specific, suggesting that these binding proteins are
receptors for activated C-kinase, or RACKs(24) . Recently, we
cloned RACK1, a gene encoding for a 36-kDa homolog of the
subunit of G proteins (accession number U03390) that fulfills the
criteria for RACKs(25) . RACK1 is neither a PKC substrate nor
an inhibitor(25) . Rather, it increases PKC phosphorylation of
substrates presumably by stabilizing the active form of
PKC(25) .
The RACK1-binding site on PKC is unknown. If this
site is identified, peptides that mimic the binding site could serve as
specific inhibitors of PKC translocation and function. Our previous
studies suggested that the C2 region of cPKC contains at least part of
the RACK-binding site on the enzyme; other C2-containing proteins such
as synaptotagmin (29) and phospholipase C (30) also bind to a mixture of RACKs prepared from the cell
particulate fraction. In addition, recombinant fragments of
synaptotagmin containing the C2 homologous region bind to RACKs and
inhibit PKC binding to RACKs(29) . However, these studies were
carried out with heterologous C2-containing fragments from
synaptotagmin and a preparation containing a mixture of RACKs. Here, we
used recombinant RACK1, recombinant fragments of
PKC containing
the C1 or the C2 regions, and short synthetic peptides derived from the
C2 region, to identify the RACK1-binding site on
PKC. We found
that the C2 region contains at least part of the RACK1-binding site on
PKC and that some C2-derived peptides act as specific inhibitors of
hormone-induced translocation and functions of the C2-containing
PKC isozymes.
PMA-induced translocation of PKC
isozymes was determined by immunofluorescence studies as described
before(32) . The cells were then washed with PBS, fixed with
cold acetone for 3 min, and washed twice with cold PBS. The cells were
incubated for 1 h with 1% normal goat serum in PBS containing 0.1%
Triton X-100 followed by overnight incubation with anti-I,
II, or
PKC polyclonal antibodies (Research and Diagnostic
Antibodies; 1:100), anti-
PKC polyclonal antibodies (Santa Cruz
Biotechnology; 1:100), or anti-RACK1 (Transduction Laboratories; 1;100)
diluted in PBS containing 0.1% Triton X-100 and 2 mg/ml bovine serum
albumin. The cells were washed three times with PBS containing 0.1%
Triton, incubated for 2 h with fluorescein-conjugated anti-rabbit IgG
antibodies (to detect binding of anti-PKC antibodies; Organon Teknika;
1:1000) or anti-mouse IgM antibodies (to detect binding of anti-RACK1
antibodies; Boehringer Mannheim; 1:1000) and washed again three times
with PBS containing 0.1% Triton. After mounting with Miowiol 4-88
(Calbiochem), the slides were viewed with a Zeiss IM35 microscope using
a 40X water immersion objective. Multiple fields of cells for each
treatment group and each PKC isozyme were monitored, and the number of
cells showing the localization of activated isozyme (32) was
recorded. Data are presented as the percentage of cells having the
tested isozyme at the activated site. Images of RACK1 and PKC
localization from the Zeiss microscope were recorded on Kodak TMax 400
film, and the exposure time was 30 s for these micrographs.
Previous studies suggested that at least part of the
RACK-binding site on PKC lies within the C2-region of the
enzyme(29) . In addition these studies suggested that the
activators of PKC (PS, DG, and calcium) are required to expose the
RACK-binding site on
PKC rather than for the interaction of the C2
region with RACKs(29) . If the RACK1-binding site on
PKC
is within the C2 region, a C2-containing fragment of
PKC should
bind to RACK1 in a PS-, DG-, and calcium-independent manner. We used
two fragments of
PKC expressed as fusion proteins with GST. One of
the fragments, L9, includes the V1 region, the pseudosubstrate
sequence, and the C1 and V2 regions (amino acids
3-182)(20) . The second fragment, L10, includes the V1
region, the pseudosubstrate sequence, and the first cysteine repeat
from the C1 region, as well as the entire C2 and V3 regions (amino
acids 3-76 and 143-339)(20) . In an overlay assay,
L10, the C2-containing fragment, but not L9, the C1-containing
fragment, bound to RACK1 (Fig. 1). Saturation of binding of L10
to RACK1 was obtained at
1 µM. In contrast, the
binding of L9 to RACK1 (Fig. 1, lanes 2 and 4)
was minimal, not saturable and similar to the nonspecific binding of
GST carrier protein alone (not shown). The V1 region, the
pseudosubstrate site, and the first cysteine-loop of C1 are present in
both L10 and L9. However, L10 also contains the C2 and V3 regions of
PKC. Therefore, these results suggest that the C2 and/or the V3 regions
bound to RACK1. In addition, PKC activators PS and calcium did not
increase the binding of L10 to RACK1 (Fig. 1, lanes 1versus3). Because these activators are required
for the binding of intact PKC to RACK1(25) , the data are
consistent with our previous studies (29) suggesting that the
PKC activators are required only to expose the RACK-binding site in the
intact PKC and that this site is already exposed in the C2-containing
fragment L10.
Figure 1:
Binding of
PKC fragments L9 and L10 to RACK1, and effects of calcium and
phosphatidylserine. L10 (lanes 1 and 3) and L9 (lanes 2 and 4) (
10 µM) were
incubated with nitrocellulose blots of SDS-PAGE loaded with RACK1
(
0.5 µg-1 µg RACK1/strip) in an overlay assay in the
presence (lanes 1 and 2) and absence (lanes 3 and 4) of phosphatidylserine and calcium (PS/Ca). After washing, binding of the fragments was detected
with anti-GST antibodies (1:5,000). An autoradiograph, representative
of three independent experiments is shown. The arrow indicates
the position of RACK1, and the intensity of the bands are proportional
to the amount of L9 or L10 bound to RACK1.
If the RACK1-binding site is within the C2 and/or V3
regions of PKC, then L10 (which contains these regions) should
inhibit the binding of intact
PKC to RACK1. RACK1 was immobilized
on an amylose column, and
PKC binding in the presence of PS and
calcium and L10 or L9 was determined (Fig. 2). In the presence
of L10 (Fig. 2, lanes 2versus1),
but not L9 (Fig. 2, lane 3),
PKC binding to RACK1
was completely inhibited. Similar results were also obtained when the
effect of the fragments on
PKC binding to RACK1 was determined
using the overlay assay (not shown). Since L10 inhibited
PKC
binding to RACK1, the C2 region and/or the V3 regions, present in L10
and not in L9, are likely to contain at least part of the RACK1-binding
site on
PKC.
Figure 2:
PKC binding to RACK1 in the presence
of L9 and L10. PKC (10 nM) together with L10 (
10
µM, lane 2), L9 (
10 µM, lane 3), or buffer (lane 1) was incubated with
RACK1-maltose binding fusion protein in the presence of
phosphatidylserine and calcium for 30 min at room temperature, as
described under ``Experimental Procedures.'' After extensive
washing, the proteins were eluted from the column with maltose and
applied to SDS-PAGE.
PKC levels were then detected by Western blot
analysis with anti-
PKC antibodies. (This antibody also recognizes
fragment L10, but not L9.) The autoradiograph is representative of
three independent experiments.
We found that synaptotagmin fragments that contain
the C2 homologous region bind to purified RACKs (29) and to
recombinant RACK1 (n = 3, data not shown) and inhibit
PKC binding to RACKs (29) . We have therefore reasoned that
homologous sequences within the C2 region of PKC and synaptotagmin
may mediate their binding to RACK1. Three
PKC-derived peptides
derived from the homologous sequences of
PKC and synaptotagmin
were prepared (Fig. 3A):
C2-1 (KQKTKTIK)
(
PKC(209-216),
C2-2 (MDPNGLSDPYVKL)
(
PKC(186-198), and
C2-4 (SLNPEWNET)
(
PKC(218-226). In addition, another C2-derived peptide,
C2-3 (IPDPKSE) (
PKC(201-207), that shares no
homology with synaptotagmin (Fig. 3A), was also
synthesized. As seen in Fig. 3B, immobilized peptides
C2-1,
C2-2, and
C2-4, but not
C2-3, bound directly to RACK1. Similar binding of these
peptides to RACK1 in the absence of PKC activators was observed (not
shown). An additional control peptide corresponding to
PKC (amino
acids 266-273 in the C2 domain) did not bind to RACK1 (data not
shown; n = 3).
Figure 3:
A, amino acid alignment of homologous
sequences within the C2-region of PKC and synaptotagmin (p65).
Alignment of part of the C2 region was carried out according to Clark et al.(21) . Boxed areas are the peptides
C2-1,
C2-2,
C2-3, and
C2-4. Capital letters denote conserved amino acids; lowercase
letters denote unique sequences; and -`` denotes a gap. Numbers on the right are the position of the carboxy
amino acid in the protein. The percent homology for synaptotagmin and
PKC in the area indicated is 67%. B, selective RACK1
binding of the C2-derived peptides. The C2-derived peptides
C2-1,
C2-2,
C2-3, and
C2-4
(
1 nmol/slot) were immobilized on nitrocellulose using a slot-blot
apparatus, and the immobilized peptides were overlaid with RACK1
(
20 nM). Bound RACK1 was detected using anti-Flag
antibodies (1:10,000). Shown are average results of four independent
experiments for
C2-1 and
C2-2 and two independent
experiments for
C2-3 and
C2-4. C,
selective inhibition of L10 binding to RACK1 by the C2-derived
peptides. The peptides (10 µM) were incubated with
immobilized RACK1 as described under ''Experimental
Procedures`` and subsequent binding of L10 to RACK1 determined in
an overlay assay. Binding is expressed as percent of that obtained in
the absence of peptide and is representative of two independent
experiments
If the C2-1,
C2-2, and
C2-4 peptides represent the binding
site for RACK1 in the C2 region, these peptides should inhibit C2
binding to RACK1. As expected, using the overlay assay, we found that
when RACK1 was preincubated with
C2-1,
C2-2, and
C2-4 subsequent binding of L10 to RACK1 was inhibited (Fig. 3C). However, minimal or no inhibition of this
binding occurred by incubation with
C2-3 or scrambled
C2-1 (Fig. 3C). Therefore, amino acids
186-198 and 209-226 of
PKC appear to be at least part
of the RACK1-binding site in the C2 region.
Using primary rat
neonatal cardiac myocytes in culture, we have previously demonstrated
that activation of PKC by PMA or by norepinephrine causes
isozyme-specific translocation to distinct subcellular
sites(7, 32) . If these C2-derived peptides mimic the
RACK1-binding site on the C2-containing isozymes, they should inhibit
stimulation-induced translocation and binding of these isozymes to
their RACKs, but not the translocation of the C2-less isozymes. To test
this prediction, we first determined whether RACK1 is present in
cardiac myocytes. Using anti-RACK1 antibodies, we found RACK1
immunostaining at perinuclear structures and diffusely in the cytosol (Fig. 4A). RACK1 location was not altered by PMA or
norepinephrine (3-100 nM and 2 µM,
respectively; not shown). However, following activation with
norepinephrine or PMA, IIPKC immunoreactivity translocated and was
co-localized with RACK1 (Fig. 4, A and B,
respectively, and not shown). Partial co-localization of activated
IPKC with RACK1 was also noted (not shown, see also (32) ). In contrast, RACK1 immunoreactivity did not co-localize
with inactive or activated C2-less isozymes,
or
PKC (see in
the following and (32) ), suggesting that RACK1 may be a
specific anchoring protein for activated
PKC in cardiac myocytes.
Figure 4:
RACK1 and IIPKC are co-localized in
cardiac myocytes treated with norepinephrine. Cells were treated with 2
µM norepinephrine for 5 min and stained with anti-RACK1
antibodies (A) or anti-
IIPKC antibodies (B) as
described under ''Experimental Procedures.`` Strong
perinuclear staining and diffuse cytosolic staining is observed with
the two antibodies. Preabsorption of anti-RACK1 with recombinant RACK1
abolishes this immunostaining (not shown). Controls for anti-
IIPKC
antibodies were carried out as described elsewhere (32) .
We next determined whether the C2-derived peptides, that inhibit
PKC binding to RACK1 in vitro, inhibit activation-induced
translocation of these C2-containing isozymes. The peptides were
introduced into the cells by transient permeabilization with saponin
(50 µg/ml), which has been successfully used to introduce various
peptides and other compounds into different cell
types(33, 34, 35) . As was demonstrated in
cardiac myocytes
and other
cells(33, 36, 37, 38) ,
permeabilization with saponin does not affect the viability of the
cells nor other cellular functions including contraction rate, gene
expression, and cell growth. After permeabilization, the subcellular
localization of different PKC isozymes following stimulation with PMA
was determined by immunofluoresence as described
previously(32) .
Transient permeabilization of these cells
in the absence of any peptide did not affect the localization of
I,
II,
, or
PKC isozymes before or after
stimulation.
IPKC in non-stimulated cells was found on cytosolic
structures. After exposure to 100 nM PMA for 15 min,
antibodies against this isozyme showed localization to perinuclear and
intranuclear structures in
80% of the cells (Fig. 5, vehicle).
IIPKC was also cytosolic before stimulation,
and in
80% of the cells it translocated to perinuclear structures
after PMA treatment (Fig. 5, vehicle). In contrast,
permeabilization in the presence of peptides
C2-1,
C2-2, or
C2-4 (10 µM extracellular
concentration) resulted in inhibition of the PMA-stimulated
translocation of the
I and
IIPKC isozymes by 65-95%,
with
C2-4 causing the largest inhibition (Fig. 5).
Other peptides, including scrambled
C2-1, a control peptide
derived from the C2-region outside the synaptotagmin-C2 homology region
(
PKC(266-272)), and the C2-derived peptide
C2-3
that did not inhibit L10 binding to RACK1 (Fig. 3C) did
not affect PMA-induced translocation of
I and
IIPKC in
cardiac myocytes (Fig. 5).
Figure 5:
PMA-induced subcellular distribution of
PKC isozymes in neonatal cardiac myocytes. Cardiac myocytes were
permeabilized in the presence or absence of peptides (10
µM). After removal of the permeabilization buffer, PMA
(100 nM) was added for 15 min, and the cells were fixed with
acetone as described under ''Experimental Procedures.`` The
subcellular localization of PKC isozymes was then determined with
anti-I,
II, and
PKC polyclonal antibodies (Research and
Diagnostic Antibodies; 1:100), and anti-
PKC polyclonal antibodies
(Santa Cruz Biotechnology; 1:100). Results are expressed as percentage
of cells out of all scored cells with PKC isozyme localized at the
activated site (intranuclear for
IPKC, perinuclear for
IIPKC,
perinuclear and fibrillar for
PKC, and on cross-striated
structures for
PKC(32) ). For each condition,
100-150 cells were scored. Results for the subcellular
localization of
I,
II, and
PKC are mean ± S.E. of
seven independent experiments for
C2-1, eight for
C2-2, three for
C2-3, two for
C2-4,
five for
C2-1 scrambled, and two for the control peptide
(
PKC(266-273)). Results for the subcellular localization of
PKC are representative of two independent
experiments.
Because the C2 region is present
in PKC, but not in
or
PKC, for
example(1, 16) , the C2-derived peptides should only
affect the translocation of the C2-containing isozymes, but not that of
the C2-less isozymes. Similar to non-permeabilized cells, we found that
treatment with 100 nM PMA resulted in the translocation of
PKC from the nucleus to cross-striated structures in 80% of the
cells, whereas
PKC translocated from the nucleus to perinuclear
and fibrillar cytosolic structures in 90% of the cells. Moreover, as
predicted, the translocation of these C2-less isozymes was not affected
by introduction of any of the C2-derived peptides into the cells (Fig. 5). These results indicate that the
C2-1,
C2-2, and
C2-4 peptides are specific inhibitors
of translocation for the C2-containing cPKC isozymes such as
I and
IIPKC, but not for the C2-less nPKC isozymes such as
and
PKC.
If translocation of PKC is required for its function,
peptides that inhibit
PKC translocation should also inhibit
PKC-mediated function. The function of
PKC in cardiac
myocytes has not yet been determined. Therefore, we used another assay
system, insulin-induced maturation of Xenopus oocytes. We
previously demonstrated that oocyte maturation is mediated in part by
PKC; insulin treatment results in translocation of
PKC (but
not other PKC isozymes) from the cytosol to the cell particulate
fraction (9) and maturation is delayed by the PKC-specific
inhibitor pseudosubstrate peptide(8, 9) . Furthermore,
this insulin-induced response is also inhibited when PKC translocation
is blocked by injection of purified RACKs (8) or a peptide
corresponding to the PKC-binding site on RACKs(9) . Therefore,
inhibition of translocation inhibits PKC-mediated function.
If
C2-containing isozymes regulate oocyte maturation, microinjection of
C2-derived peptides that inhibit the translocation of C2-containing
isozymes should delay insulin-induced oocyte maturation. The C2-derived
peptides were microinjected into intact oocytes. C2-1,
C2-2, and
C2-4 (5 µM-500
µM) significantly delayed oocyte maturation in a
dose-dependent manner (Fig. 6, A and B, and
not shown). When 50% of the vehicle-injected oocytes reached maturation
only 3-8% of the oocytes injected with
C2-1,
C2-2 and
C2-4 have responded (Fig. 6C). In contrast, microinjection of
C2-3 (Fig. 6, B and C), or a number
of other control peptides including scrambled
C2-1 (Fig. 6C), did not affect insulin-induced oocyte maturation (see
also (8) ). Therefore,
C2-1,
C2-2, and
C2-4 peptides derived from the RACK1-binding site on
PKC specifically inhibited insulin-induced regulation of oocyte
maturation.
Figure 6:
Xenopus oocyte maturation after
microinjection of the C2-derived peptides. Time course of
insulin-induced Xenopus oocyte maturation at the indicated
times after microinjection of vehicle (control, 20 mM NaCl)
(),
C2-1 (
),
C2-2 (
) (50
µM). Xenopus oocyte maturation at the indicated
time after microinjection of vehicle (control, 20 mM NaCl)
(
),
C2-3 (
) and
C2-4 (
) (50
µM). In each experiment, 10-15 oocytes were
microinjected. Results are expressed as percentage of oocytes that
reached germinal vesicle breakdown and are representative of at least
three independent experiments. Percentage of oocytes reaching
maturation after microinjection of tested peptides at a time that 50%
of vehicle-injected oocytes reached maturation (indicated by a dashed line in A and B). Results are
expressed as average ± S.E. and n denotes the number of
independent experiments.
We then determined whether this inhibition of PKC
function in oocytes was due to prevention of insulin-induced PKC
translocation to the cell particulate fraction. Since
immunofluorescence studies in Xenopus oocytes are not
possible, we determined
PKC translocation by cell fractionation.
The distribution of
PKC between the soluble and particulate
fractions of oocytes (100,000
g supernatant and
pellet, respectively) was determined in oocytes injected with vehicle
or
C2-1 using anti-
PKC antibodies (Fig. 7).
Microinjection of
C2-1 to non-stimulated oocytes did not
affect
PKC distribution (not shown) and was similar to control
non-stimulated oocytes (Fig. 7, lanes 1 and 2). In vehicle-injected oocytes, insulin treatment resulted in
a decrease in the level of the 80-kDa
PKC from the cytosol and a
corresponding increase in the particulate fraction level (Fig. 7, lanes 4 and 3versus2 and 1). However, no insulin-induced translocation of
PKC was observed following microinjection of
C2-1;
rather, there was a decrease in the
PKC level in the particulate
fraction (Fig. 7, lanes 5versus1),
suggesting degradation of
PKC. Similar results were also observed
following microinjection of
C2-2 (not shown). Therefore,
C2-1 and
C2-2-inhibition of PKC-mediated function
following insulin-induced stimulation appears to be due to inhibition
of
PKC translocation.
Figure 7:
Effect of C2-1 microinjection
on the subcellular distribution of
PKC in Xenopus oocytes. Oocytes were microinjected with vehicle (20 mM
NaCl) (lanes 1-4) or
C2-1 (50
µM; lanes 5 and 6) and the distribution
of
PKC in the particulate (p) and cytosolic (c)
fractions was determined 60 min after incubation without (lanes 1 and 2) or with insulin (lanes 3-6). The
cell particulate fraction (lanes 1, 3, and 5) and
cytosolic fractions (lanes 2, 4, and 6) of
the oocytes were prepared as described under ''Experimental
Procedures,`` using 100 oocytes for each treatment and PKC was
detected using anti-
PKC antibodies (1:1000) in Western blot
analysis. The antibodies reacted with an
80-kDa protein that
corresponds with
PKC. The identity of the two other immunoreactive
bands in the particulate fraction of control and insulin-treated
oocytes is unknown. (This antibody recognizes both
I and
IIPKC isozymes. However, only
IIPKC appears to translocate on
insulin treatment in these cells). The figure is a representative of
results obtained in three independent
experiments.
Using the L9 and L10 recombinant fragments of PKC and
short peptides derived from the C2 region, we have mapped at least part
of the RACK1-binding site on
PKC to amino acids 186-198 and
209-226 within the C2 region. Furthermore, peptides corresponding
to these sequences inhibited the translocation of C2-containing
isozymes but not the translocation of C2-less isozymes in neonatal
cardiac myocytes. Finally, these peptides inhibited PKC-mediated
function in Xenopus oocytes. Since RACK1 immunoreactivity was
found in cardiac myocytes (Fig. 4A) and Xenopus oocytes, (
)it appears likely that the C2-derived
peptides inhibited PKC function by binding to RACK1 and blocking
subsequent binding of the intact enzyme.
The PKC fragment L10
also contains the V3 region of the enzyme, and therefore, our study
cannot rule out a role for V3 in binding of
PKC to RACK1. Since
each of the C2-containing isozymes in cardiac myocytes are localized to
a different subcellular site(39) , it is likely that isozyme
unique sequences (e.g. V1, V3, and V5) also contain
isozyme-specific RACK-binding sites in addition to the site within the
common C2 region. Other studies suggested that binding of PKC to
proteins different from RACK1 is mediated by the pseudosubstrate
sequence (via a phospholipid bridge(40) ) or by the catalytic
domain of PKC(41, 42) . However, the role of the
interaction of PKC with these PKC-binding proteins in vivo has
not yet been determined.
Very recently, the role of the C1 region in
localizing PKC to the Golgi apparatus has been
reported(43) . Golgi functions were inhibited by overexpression
of both intact
PKC and the C1 fragment of
PKC, leading the
authors to suggest that the C1 region may mediate subcellular
localization. Our studies with the C1 fragment of
PKC cannot
exclude the possibility that this domain may also participate in
localizing the enzyme. However, the combined in vitro and in vivo studies indicate that the C2 domain is required for
this interaction; inhibitors of the C2 domain binding to RACK1 prevent
PKC translocation and function.
The C2 region of other translocating enzymes also appears to be required for their translocation and function. Recent data indicate that the C2 region of cytosolic phospholipase A2 associates with membranes, whereas a mutant of this lipase lacking the C2 region does not(44) . In addition, a fusion protein containing 43 amino acids from the C2 region of Ras GTPase (GAP) confers calcium-dependent interaction with cellular membranes, whereas a GAP mutant lacking this region does not(45) . Finally, the binding of synaptotagmin to membranes is abolished by protease treatment of the membrane(46) , and peptides derived from the C2 region of synaptotagmin inhibited calcium-induced neurotransmitter release from the giant squid axon (47) . Therefore, the C2 region appears to mediate translocation for a number of translocating proteins.
It appears
that the inhibitory effects of the C2-derived peptides presented here
are due to the inhibition of translocation of PKC rather than of other
C2-containing translocating proteins. PMA is not thought to induce
translocation of phospholipase C or GTPase activating protein, and
synaptotagmin immunoreactivity was not found in cardiac myocytes (not
shown). Similarly, insulin treatment does not induce translocation of
phospholipase C
or GTPase activating protein in Xenopus oocytes, nor is there synaptotagmin immunoreactivity in oocytes
(data not shown). In addition, progesterone-induced oocyte maturation
that does not involve PKC activation (8, 48) was not
affected by the C2-derived peptides (n = 3, data not
shown). Therefore, the effects of the
C2-1,
C2-2,
and
C2-4 peptides are most likely specific for PKC. Because
we do not have antibodies that distinguish between
I and
IIPKC for Western blot analysis, we could not determine whether
one or both mediate the insulin-induced effect.
The inhibitory
effects of the C2 peptides were sequence-specific.
C2-1,
C2-2, and
C2-4, but not
C2-3 or a number of control peptides, inhibited
translocation of
PKC. Since
C2-1 is highly basic (Fig. 3A), it was previously proposed to mediate direct
binding of the C2-containing proteins to the negatively charged PS in
the membrane(21) . However, we found that its inhibitory
activity cannot be attributed to charge only, since a scrambled
C2-1 peptide was inactive in inhibiting either the
translocation of C2-containing
PKC isozymes (Fig. 5) or
their function (Fig. 6).
Why was there a decrease in the
levels of PKC in activated oocytes injected with peptides
C2-1 or
C2-2? Activated PKC has been previously
demonstrated to be more sensitive to proteolysis (49) and
subsequent inactivation, and we found that co-incubation with RACKs in vitro partially protects from this inactivation. (
)Since the levels of
PKC in the absence of insulin
treatment did not decrease following microinjection of
C2-1
and
C2-2 peptides (not shown), the data are consistent with
increased sensitivity of activated
PKC to proteolysis in the
presence of these translocation inhibitors in vivo. Taken
together, the data suggest that
C2-1,
C2-2, and
C2-4 bound to the
PKC-specific RACK in Xenopus oocytes and inhibited
PKC translocation and function.
Relatively high concentrations (50 µM) of the
C2-derived peptides were required to produce inhibition of
insulin-induced oocyte maturation. This may be due to degradation of
the peptides in the oocytes during the course of the experiments
(hours), the effect of the yolk on the distribution of the peptide in
the oocytes, and/or because of competition of the endogenous intact PKC
for binding to RACKs. In cardiac myocytes, the intracellular
concentration of the peptides has not been determined directly.
However, using a tracer radioactive probe, the final concentration of
the peptide was estimated to be 1 µM when the applied
concentration of the peptide is 10 µM.
Furthermore, since the peptides are likely to be sensitive to
proteolysis, the intracellular concentration in cardiac myocytes is
likely to be much lower.
Because the C2-derived peptides
specifically inhibited translocation and function of PKC in
vivo, they belong to a new class of PKC inhibitors, translocation
inhibitors. This term was introduced to describe an inhibitor of
5-lipoxygenase, MK0866. MK0866 inhibits the binding of 5-lipoxygenase
to its intracellular receptor, FLAP, a binding that is required for
activation of the enzyme(50) . We have recently identified
additional translocation inhibitors for PKC; a 15 amino acid peptide
(peptide I) from other PKC-binding proteins inhibited the interaction
between PKC and RACK1 and the function of
PKC in
vivo(9, 51) . Therefore, peptides that mimic the
interaction sites on either PKC or RACK are inhibitors of PKC-mediated
function.
How do short peptides from the C2 region of PKC
inhibit PKC translocation and function? Many small ligands are thought
to interact with their receptor or enzyme via a ``greasy
pocket.'' In contrast, the interaction of two proteins in the cell
may reflect multiple binding sites on relatively large surface areas of
these proteins. Peptides derived from the interacting areas are likely
to have lower affinity for their binding sites as compared to the
intact proteins. Yet, if these peptides act as a wedge between the two
interacting proteins, they may serve as specific and effective
inhibitors.
The structure of the C2 domain of p65 has recently been
determined (52) . Examination of that structure demonstrates
that C2-1,
C2-2, and
C2-4 but not
C2-3 are localized on adjacent exposed regions in the C2
domain. Two of the biologically active peptides (
C2-1 and
C2-2) correspond to sequences that contain both loops and
strand structures. In contrast,
C2-4, the peptide with
the highest biological activity (Fig. 5), corresponds to
sequences from only a surface
strand structure (the fifth
strand(52) ). It is possible that the interaction surfaces
between PKC and RACK1 are constituted only by
strands. Future
experiments utilizing shorter peptides will address this possibility.
It is interesting to note that the co-crystal structure of the
Ras-binding domain of c-Raf-1 with the small GTP-binding protein Rap1A
suggests that the interaction between the two proteins is mediated by
single anti-parallel
strands at the edge of the
molecules(53) . The authors also predicted that such a small
interaction site between the two proteins provides an opportunity to
design drugs that inhibit this interaction. Indeed, our data on
PKC and RACK1 interaction demonstrate that such inhibitors have
the predicted biological activity.
Finally, PKC translocation
inhibitors can be used to elucidate the cellular role of specific PKC
isozymes. Our finding that the C2-1,
C2-2, and
C2-4 peptides caused a delay in insulin-induced oocyte
maturation indicates that C2-containing isozymes, most likely
PKC,
mediate this function in oocyte maturation. Since in cardiac myocytes,
inhibition of translocation of C2-less isozymes was not observed, the
inhibitory peptides can be used as tools to identify the PKC isozymes
that mediate specific cellular functions in cells in which multiple
isozymes are activated by a single stimulus. The role of C2-containing
isozymes in the PMA-induced regulation of cardiac myocyte function is
currently under investigation using these peptides.