From the Department of Neurology and Neurosurgery, McGill University, Montreal Neurological Institute, Montreal, Quebec H3A 2B4, Canada
Received for publication, July 18, 2000, and in revised form, November 7, 2000
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ABSTRACT |
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Ca2+-independent or novel
protein kinase Cs (nPKCs) contain an N-terminal C2 domain of unknown
function. Removal of the C2 domain of the Aplysia nPKC Apl
II allows activation of the enzyme at lower concentrations of
phosphatidylserine, suggesting an inhibitory role for the C2 domain in
enzyme activation. However, the mechanism for C2 domain-mediated
inhibition is not known. Mapping of the autophosphorylation sites for
protein kinase C (PKC) Apl II reveals four phosphopeptides in the
regulatory domain of PKC Apl II, two of which are in the C2 domain at
serine 2 and serine 36. Unlike most PKC autophosphorylation sites,
these serines could be phosphorylated in trans.
Interestingly, phosphorylation of serine 36 increased binding of the C2
domain to phosphatidylserine membranes in vitro. In cells,
PKC Apl II phosphorylation at serine 36 was increased by PKC
activators, and PKC phosphorylated at this position translocated more
efficiently to membranes. Moreover, mutation of serine 36 to alanine
significantly reduced membrane translocation of PKC Apl II. We suggest
that translocation of nPKCs is regulated by phosphorylation of the C2 domain.
Protein kinase Cs
(PKCs)1 are a family of
lipid-activated enzymes that play important roles in many cellular
processes including regulation of synaptic strength in the nervous
system (1). We have studied the regulation of PKC in
Aplysia, an important model system for synaptic plasticity,
where PKC plays several important roles in regulating synaptic strength
(2-6). The structure of PKCs is conserved in Aplysia.
Ca2+-activated PKCs (cPKCs) in vertebrates ( In cPKCs, the C2 domain mediates Ca2+-dependent
binding to the membrane lipid phosphatidylserine (PS) (8, 9). This
binding is believed to be the primary step in kinase activation. First, it transiently recruits the enzyme to the membrane where its
physiological activator, diacylglycerol (DAG), resides. Second, in
conjunction with the C1 domain interacting with DAG, binding of the C2
domain to PS induces a conformational change that activates the enzyme (10, 11). Finally, the C2 domain binds to the receptor for activated
protein kinase C (RACK), and this binding is important for the location
of PKC translocation and for cellular functions of PKC (12).
In contrast to cPKCs, a detailed model for the role of the N-terminal
C2 domain in nPKC activation is not available. C2 domains of nPKCs do
not bind constitutively to lipids and are not regulated by calcium
(13). However, similar to cPKCs, C2 domain-derived peptides from nPKCs
act as isoform-specific inhibitors and activators in cells, implicating
the C2 domain in the regulation of enzymatic activity (14-16). Removal
of the C2 domain of PKC In vitro, PKC Apl II is activated more poorly than PKC Apl
I, even when calcium is removed from PKC Apl I, suggesting a
requirement of additional activators for stimulation of PKC Apl II
in vivo (13, 19). Comparisons between PKC PKCs are regulated by phosphorylation. Phosphorylation in the
activation loop by phosphoinositide-dependent protein
kinase I (PDK-1) is required for subsequent PKC "maturation" by
autophosphorylation at two residues critical for enzyme stability and
activity (8, 23, 24). Phosphorylation of PKC In this paper we identify two autophosphorylation sites, serine 2 and
serine 36, in the C2 domain of the nPKC Apl II. Phosphorylation at
serine 36 increases lipid binding to the C2 domain and increases translocation of PKCs in cells. We suggest that phosphorylation of C2
domains in novel PKCs is important for regulating their translocation.
Reagents--
4- Plasmid Construction of PKCs, Fusion Proteins, and
Mutants--
Full-length clones of PKC Apl II were present in
Bluescript SK and the baculovirus vector BB3 (19). Initially, PKC Apl
II was excised from baculovirus BB3 using
SacI/SalI and inserted into the new vector BB4
(Invitrogen) at SacI/SalI. The C2 domain S36A and
S36E mutations were generated with a two-step mutagenic procedure using
the polymerase chain reaction (PCR). First round PCR used the C2 domain
of PKC Apl II in the pMALC-C2 plasmid (New England Biolabs) as a
template and either the outside 5' primer (O5) and the inside 3' primer
(I3) or the inside 5' primer (I5) and the outside 3' primer (O3) (see
Table I for primers). The products
from the first round synthesis were combined and used as the template
for second round synthesis using O5 and O3. The resultant product was
cut with appropriate enzymes (Table I) and inserted into PKC Apl II in
the BB4 vector. A new site was formed by the mutagenesis (Table I) and
was used to confirm the cloning. The mutations were also made in the
pMAL-C2 vector encoding the MBP-C2 fusion protein using a similar
procedure. The final product was cut with appropriate enzymes (Table I)
and inserted into the MBP-C2 domain at the indicated cloning sites
(Table I). Ser-68 Baculovirus Construction--
Spodoptera frugiperda
(Sf9, Sf21; Invitrogen) cells were grown in suspension
cultures with supplemented Grace's media (Life Technologies, Inc.) and
10% fetal bovine serum (Life Technologies, Inc.). The baculovirus
transfer vectors were cotransfected with linearized baculovirus
(Invitrogen), and the resultant blue colonies were plaque-purified.
Positive colonies were confirmed by PCR and by immunoblotting with
anti-PKC Apl II or anti-PKC PKC Purification from Baculovirus--
Biochemical purification
of all Aplysia PKCs, human PKC Fusion Protein Synthesis--
Both a maltose-binding protein
(MBP) and a glutathione S-transferase (GST) fusion protein
were expressed in bacterial DH5 Antibody Production and Immunoblotting--
We synthesized a
peptide (CRLQKG(pS)TKEK; where pS is phosphoserine)
corresponding to amino acids 30-40 of the C2 domain of PKC Apl II with
Ser-36 converted to phosphoserine (Quality Controlled Biochemicals, MA)
to generate a phosphopeptide antibody. The peptide was conjugated to
bovine serum albumin maleimide (Pierce) via the cysteine and was
injected intramuscularly into rabbits along with TitreMax Gold (Cytrx,
Norcross, GA) three times at 4-week intervals. Serum was affinity
purified and used in Western blots as described (32) with the
phosphorylated peptide antibody at 1 µg/ml dilution and goat
anti-rabbit, horseradish peroxidase-conjugated secondary antibody at 1 µg/ml. The phosphopeptide antibody was preabsorbed using the
nonphosphorylated form of the peptide at 1 µg/µl for 30 min prior
to its addition to the immunoblot. Results were visualized by enhanced
chemiluminescence (PerkinElmer Life Sciences).
Lipid Preparation--
Sucrose-loaded large unilamellar vesicles
containing trace [3H]PC were prepared. Briefly, lipid
mixtures in chloroform were dried under a stream of nitrogen and
resuspended in buffer (20 mM HEPES, pH 7.5, 170 mM sucrose). They were then subjected to 5 freeze-thaw
cycles in liquid nitrogen and extruded through two stacked 0.1-µm
polycarbonate filters using a Liposofast microextruder (Avestin, Inc.,
Ontario, Canada) as described (33). Phosphatidylserine liposomes were
prepared by resuspending dried lipids in buffer and vortexing (19).
PKC Assays--
Protein kinase C (5 nM) activity was
assayed using phorbol esters or the mixed micelle assay as described
(19, 34).
PKC and Fusion Protein in Vitro Phosphorylation--
PKCs
(200-500 nM) or PKC-derived C2 domain fusion proteins (2 µM) were incubated in reaction buffer (50 mM
Tris-HCl, pH 7.5, 10 mM MgCl2, 5 mM
EGTA) with 50 µg/ml dioleoyl phosphatidylserine, 20 nM
PMA, and [ C2 Domain Membrane-binding Assay--
Membrane binding of C2
domain constructs was determined by measuring the binding to
sucrose-loaded vesicles (19, 33, 35). MBP fusion proteins on amylose
beads were phosphorylated by PKC as described above. PKC, ATP, and
lipids were removed by sedimenting the beads at low speeds (3000 × g for 1 min) followed by three washes with sedimentation
buffer (0.3 mg ml Cell Fractionation--
Recombinant baculovirus encoding wild
type, S36A, or S36E PKC Apl II was incubated with Sf-21 cells in media
at 27 °C. After 72 h of infection, cells were incubated with
vehicle (control) or 4- Phosphorylation and Partial Tryptic Digestion--
Purified PKC
Apl II or PKC Apl II Analysis of Tryptic Peptides by Two-dimensional Thin Layer
Chromatography--
Analysis was performed as described with several
modifications (32, 36). Membrane fragments containing protein bands
were preblocked in 0.5% PVP-360 in 100 mM acetic acid,
washed, and subsequently digested overnight with 10 µg of
TPCK-treated trypsin in 10 mM Tris, pH 7.5, and 5%
acetonitrile. An additional 10 µg of trypsin was added to each band
for 3 h the following day, and the supernatant solution containing
desired peptides was lyophilized to completion. Peptides were
resuspended in pH 1.9 buffer (0.6 M formic and 1.4 M acetic acid) and analyzed by two-dimensional TLC using a
Hunter-3000 HTLC. Labeled peptides were visualized by autoradiography
using Kodak BioMax MS film on the plate. Predicted migrations of
phosphopeptides were calculated as described (36).
Phosphoamino Acid Analysis--
After tryptic digestion and
lyophilization, PKC Apl II or C2 fusion protein peptides derived from
in vitro phosphorylations were hydrolyzed to constituent
amino acids using constant boiling 6 N HCl for 2.5 h
at 110 °C. Sample amino acids were mixed with phosphoserine,
phosphothreonine, and phosphotyrosine standards prior to running the
mixture in the primary TLC dimension using standard buffers (36).
Phosphoamino acid standards were stained with 0.25% ninhydrin in
acetone, and autoradiography was performed on the completed plate.
Data Analysis--
Immunoblots and autoradiograms were scanned,
and analysis was performed using the public domain NIH Image program
(developed at the National Institutes of Health and available on the
Internet). We calibrated the program with the uncalibrated OD feature
of NIH Image, which transforms the data using the formula
y = log10 (255/(255 The C2 Domain of PKC Apl II Contains Two Autophosphorylation
Sites--
To determine whether the C2 domain of PKC Apl II is
regulated by autophosphorylation, we compared tryptic phosphopeptide
maps generated after autophosphorylation of purified PKC Apl II or a
purified mutant lacking the C2 domain (PKC Apl II The Autonomous PKC Apl II Is Not Autophosphorylated at Site
4--
Purified PKC Apl II contains both regulated and autonomous
activities (22). The autonomous activity generated by purification is
similar to the autonomous form of PKC Apl II that is found in the
nervous system (22). To determine differences between autonomous and
regulated kinase activities, we compared tryptic phosphopeptide maps of
PKC Apl II that were autophosphorylated in the absence of activators
(autonomous activity) or after activation by phorbol esters (Fig.
3A). Autophosphorylation of
peptide 4 was greatly decreased in the autonomous kinase (Fig.
3B). Quantitation of the relative phosphorylation of site 4 in three separate experiments from two different purified preparations
confirmed this result (Fig. 3C). This could be due to
existing phosphorylation of the site corresponding to peptide 4 in this
kinase or to a conformational change in the regulatory domain that
reduces phosphorylation of this site.
Fatty Acids Do Not Preferentially Autophosphorylate the C2
Domain--
PKC Apl II also can be stimulated by fatty acids.
Autophosphorylation induced by fatty acids can be distinguished from
that induced by phorbol esters since removal of the C2 domain greatly decreases fatty acid-induced autophosphorylation without affecting phosphorylation induced by phorbol esters (19). To determine whether
this difference is due to selective fatty acid-induced phosphorylation
of the C2 domain, we compared PKC that was autophosphorylated in the
presence of oleic acid or in the presence of phorbol esters (Fig.
3A). Phosphorylation of phosphopeptides 2 and 3 by oleic acid and PS-PMA was comparable (Fig. 3B). Quantitation of
the relative phosphorylation in three separate experiments showed no
significant difference between oleic acid and phorbol ester stimulation
(Fig. 3C). Thus, the selective requirement of the C2 domain
for fatty acid-induced phosphorylation is not due to preferential
autophosphorylation of this domain.
Sites in the C2 Domain Can Be Trans-phosphorylated--
Incubation
of PKC Apl II or PKC Apl I (data not shown) with fusion proteins
containing the C2 domain led to trans-phosphorylation of the C2 domain
(Fig. 4A). Phosphopeptide
mapping results of both trans-phosphorylated GST- and MBP-C2 domain
fusion proteins indicated two major C2 domain phosphorylation sites
(Fig. 4, B and C). In contrast, phosphorylation
of GST or MBP alone did not generate these sites (data not shown).
Coapplication of phosphopeptides derived from autophosphorylated PKC
Apl II and the trans-phosphorylated GST-C2 domain fusion protein
demonstrated that phosphopeptide 3 was identical in the
autophosphorylated kinase and in the trans-phosphorylated C2 domain
(Fig. 4D). An additional phosphopeptide (2') was also observed in both fusion proteins, whose migration was slightly altered
from phosphopeptide 2 in autophosphorylated PKC Apl II.
Site 2 Is Serine 2--
We used a series of C2 domain deletion
constructs (19) to attempt to delineate the sites of
trans-phosphorylation that corresponded to the various phosphopeptides.
However, deleting any area of the C2 domain prevented phosphorylation
of peptide 3 suggesting that phosphorylation of this site is sensitive
to conformation of the C2 domain (data not shown). In contrast, the
site that produced phosphopeptide 2' was removed by an N-terminal
deletion but not a C-terminal deletion (data not shown). If site 2 was at the N terminus of the C2 domain, the tryptic peptide is predicted to
be altered by addition of two amino acids from the linker region of the
fusion protein (MSR The Major Autophosphorylation Site of PKC Apl II Is Serine
36--
Phosphoamino acid analysis of phosphopeptides 2, 3, and 5 revealed them all to be serines (data not shown). By using a
hypothetical phosphopeptide map of regulatory domain tryptic peptides,
and assuming phosphopeptide 2 is M(pS)R, there were only two possible serines (serine 36 and serine 68) that were consistent with the migration of phosphopeptide 3 (Fig.
5A). Conversion of serine 68 to alanine did not affect peptide 3 phosphorylation (data not shown).
In contrast conversion of serine 36 to alanine prevented peptide 3 phosphorylation in both the trans-phosphorylated fusion protein (Fig.
5, B-D) and the intact kinase (Fig. 5,
E-G).
C2 Domain Phosphorylation at Serine 36 Promotes Lipid
Binding--
The C2 domain of PKC Apl II does not bind to
phosphatidylserine membrane preparations using vesicles (19), lipid
micelles (13), or extruded sucrose-loaded vesicles (Fig.
6A; quantitated in Fig.
6B). In contrast, the C2 domain showed significant binding to extruded sucrose-loaded vesicles (60% PS, 40% PC) after
trans-phosphorylation by PKC Apl II (Fig. 6A; quantitated in Fig.
6B). This result was also observed using PS vesicles (data
not shown). In these experiments less than 5% of the fusion protein
was phosphorylated (see "Experimental Procedures"), and thus
phosphorylated protein did not contribute significantly to the measured
translocation of total fusion protein. The binding was reduced if the
vesicles contained only PC (Fig. 6A; quantitated in Fig.
6B). Conversion of serine 36 to alanine significantly
decreased binding of the phosphorylated fusion protein to the lipid
vesicles (Fig. 6A; quantitated in Fig. 6B),
whereas conversion of serine 68 to alanine did not decrease binding
(data not shown). In contrast, conversion of serine 36 to glutamic acid significantly increased binding of the fusion protein to lipids, even
in the absence of phosphorylation. These results suggest that
phosphorylation of serine 36 increased lipid binding. Phosphorylation of serine 2 may also be involved in binding since the phosphorylated form of both the S36E and S36A fusion proteins bound lipids
better than did their nonphosphorylated form (Fig. 6A,
quantitated in Fig. 6B).
The conversion of serine 36 to glutamic acid caused a small shift in
the position of the protein on SDS-PAGE gels (Fig. 6A). This
band comigrated with a phosphorylated form of the wild type fusion
protein (Fig. 6A). The form of the fusion protein migrating at this position bound better to the lipid vesicles than did the lower
band (Fig. 6A). These results suggest that phosphorylation caused a conformational change involved in lipid binding. However, phosphorylation was neither necessary nor sufficient to cause the
shift, since (i) phosphorylated wild type protein incubated with PC
vesicles alone rarely exhibited the shift in molecular weight (Fig.
6A) and (ii) in experiments using higher concentrations of
fusion proteins and sucrose-loaded vesicles, the shifted band was seen
in the nonphosphorylated wild type protein and even in the
nonphosphorylated S36A fusion protein (data not shown). These results
are consistent with a model whereby a conformational shift is required
for lipid binding, and phosphorylation of the fusion protein enhances
the stability of this conformation.
Conversion of Serine 36 to Alanine or Glutamic Acid Does Not Affect
Enzyme Activation--
Removal of the C2 domain allows kinase
activation at lower concentrations of PS using the mixed micelle assay.
To determine whether mutations at serine 36 could mimic this effect, we
assayed PKC Apl II S36A and S36E and wild type PKC Apl II in the mixed micelle assay. No difference was seen in the concentration of PS
required for activation of PKC Apl II S36A or S36E compared with the
wild type control (Fig. 7). The lack of
an effect of S36E suggests that phosphorylation of this site is not
important for removing C2 domain inhibition or that the glutamic acid
does not mimic phosphorylation of serine 36. Whereas the conversion to
glutamic acid does allow for lipid binding of the isolated C2 domain,
there may be differences between the phosphorylated residue and the
glutamic acid in the context of the whole protein.
Characterization of a Phosphospecific Antibody to Serine
36--
To examine phosphorylation of serine 36 in cells, we generated
a phosphopeptide antibody to serine 36 (phospho-Ser-36). We tested the
specificity of the antibody using autophosphorylated, purified PKC Apl
II and PKC Apl II S36A (Fig.
8A). Purified PKC Apl II
showed some reactivity with the phosphopeptide antibody, and
stimulation of PKC Apl II by either PS-PMA or oleic acid resulted in a
large increase in immunoreactivity with the antibody (Fig. 8A,
upper panel). In contrast, no immunoreactivity was seen with Apl
II S36A either before or after stimulation despite the ability of this
enzyme to autophosphorylate itself as measured using incorporation of
[ Phosphorylation of Serine 36 in Cells--
The phosphopeptide
antibody to serine 36 recognized PKC Apl II overexpressed in
Sf21 cells (Fig. 8B). Immunoreactivity was specific
for phosphorylated proteins as no immunoreactivity is seen when PKC Apl
II S36A was expressed to the same levels (Fig. 8, B and
C). Immunoreactivity increased after addition of PDBu to the
cells (150 ± 30%, n = 3, S.E.), consistent with
serine 36 being autophosphorylated in cells (Fig. 8B). PDBu
also translocated serine 36-phosphorylated PKC (Fig. 8B).
The relative percent translocation of serine 36-phosphorylated PKC was
increased compared with total PKC Apl II (690 ± 300% compared
with 90 ± 30%, n = 3, S.E.), consistent with a
role for phosphorylation in translocation. However, this large increase
in percentage translocation is probably also related to a pool of
misfolded PKC after overexpression in SF21 cells. Misfolded PKC would
neither translocate nor be autophosphorylated at serine 36, thus
decreasing the relative translocation of the nonphosphorylated pool of
PKC Apl II. Phosphorylation was not sufficient for translocation, since
in the absence of PDBu phosphorylated protein was found mainly in the
supernatant. Similarly, the distribution of Apl II S36E was not
different than that of the wild type enzyme (data not shown), and
translocation of Apl II S36E was similar to wild type PKC Apl II
(60 ± 18%, n = 3, S.E.). In contrast, the
relative percent translocation of PKC Apl II S36A was impaired compared
with wild type PKC Apl II (20 ± 20% compared with 90 ± 30%, n = 3, S.E.), suggesting that phosphorylation of
the wild type protein is required for proper translocation of PKC.
Thus, even though only a small percentage of wild type enzyme is
phosphorylated at any one point in time, a larger percentage of the
translocated wild type protein may have been assisted in translocation
by transient phosphorylation, and this would lead to an increased
translocation of wild type protein compared with the Ser Autophosphorylation of PKC Apl II--
A major site for
autophosphorylation in PKC Apl II is serine 36 in the C2 domain, and
PKC Apl II is probably also autophosphorylated at serine 2 in the C2
domain. Although these autophosphorylations are likely due to
cis-autophosphorylation (initial studies suggest single order
kinetics), unlike many PKC phosphorylations, serines 2 and 36 can be
phosphorylated in trans. Furthermore, serine 36 is contained
in a consensus PKC phosphorylation site (Fig.
9A). At this time, we do not
know whether serine 2 and serine 36 are cis- or trans-phosphorylated in
cells. The site was trans-phosphorylated by PKC Apl I in
vitro; however, we did not see phosphorylation of a kinase-dead
mutant of PKC Apl II when coexpressed in Sf21 cells with PKC Apl
I (data not shown).
The autonomous kinase was not autophosphorylated at peptide 4. We have
not directly determined the sequence of peptide 4, but based on partial
tryptic digests it is likely to be in the regulatory domain.
Furthermore, based on predicted tryptic phosphopeptide mobilities, both
peptides 4 and 5 are in the hinge domain (Fig. 5A). It seems
unlikely that phosphorylation in the hinge domain would be sufficient
to produce an autonomous kinase, and thus we favor a model for
autonomous kinase formation where a conformational change in the
regulatory region no longer presents the site in peptide 4 for
autophosphorylation. This is consistent with experiments using
proteolysis as an assay for hinge domain conformation that found
discrete constraints on sensitivity of proteolytic sites in the hinge
domain. These constraints changed with conformational changes of the
whole enzyme (38). We could consider this as additional evidence that
autonomous enzyme formation is a result of conformational changes in
PKC Apl II.
Mechanistic Model for C2 Domain Phosphorylation--
The C2
domains of nPKCs contain an extended region in what would be the
calcium binding region loop 1 as defined in cPKCs. In PKC
This hypothesis is consistent with earlier results measuring lipid
binding to C2 domain deletion constructs of PKC Apl II. In these
studies, removal of the C terminus of the C2 domain allows for lipid
binding, whereas removal of the N terminus of the C2 domain (containing
the phosphorylation site and proposed lipid-binding site) does not
(19). This suggests that the N-terminal region of the C2 domain
contains a site for lipid binding that is normally inhibited by the
C-terminal of the C2 domain. A model for the actions of the
PKC Role of C2 Domain Phosphorylation in Translocation by
PDBu--
Phorbol esters translocate PKC Apl II to the membrane in
cells, and this translocation may be partially dependent on C2 domain phosphorylation at serine 36. Although one might expect PDBu-mediated translocation to depend only on the C1 domain, mutations in the C2
domain of cPKCs can decrease PDBu-mediated translocation, suggesting that both domains are required for translocation (11, 42). Moreover,
the C2 domain is required for translocation of cPKCs by natural stimuli
(43). Since there are few differences in lipid and/or phorbol
ester-binding between the C1 domains of cPKCs and nPKCs (13), it is
likely that the C2 domain is also required for nPKC translocation
in vivo. Deletions in the C2 domain can block PDBu binding,
and the presence of the C2 domain decreases the affinity of the C1
domain for PDBu in a PS-dependent fashion (44, 45). The
phosphorylation at serine 36 may not only increase lipid binding to the
C2 domain but decrease C2 domain-mediated inhibition of the C1 domain.
However, whereas conversion of serine 36 to glutamic acid was
sufficient to increase binding of the C2 domain to lipids, it did not
appear to decrease C2 domain-mediated inhibition of the C1 domain in
kinase assays and did not have a dramatic effect in the translocation
of PKC. The conformational change in C2 domain that allows binding to
lipid may not affect the C2-C1 domain interaction. Alternatively, the
conformations may not be identical in the phosphorylated protein and
the S36E fusion protein. Thus, whereas S36E is sufficient to induce a
lipid binding conformation, it is not sufficient to remove the C2-C1 domain interaction. Future work will be needed to address this issue.
It has also been demonstrated that the C1 and C2 domains work in a
concerted fashion to facilitate prolonged activation of cPKCs (10, 11,
20, 42). The transient translocation of PKC is mediated by the binding
of the C2 domain of the soluble kinase to the membrane in the presence
of Ca2+ ions (11). However, a more stable and prolonged
translocation was associated with the DAG binding to the C1 domain in
the presence of C2 domain binding. Thus, there exist two distinct
states of cPKC membrane association as follows: a low affinity state
mediated by the C2 domain alone and a high affinity state mediated by
both the C1 and C2 domains acting together. We propose that where
calcium recruits the aid of conventional C2 domains to translocate
cPKCs, phosphorylation may recruit the aid of novel C2 domains to bind lipids and perpetuate PKC membrane association (Fig.
10). This requirement of the C2 domain
to perpetuate PKC membrane association would act to help rather than
hinder kinase activity and may indeed be a mechanism for persistent
kinase activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
,
)
and invertebrates (PKC Apl I in Aplysia) have two C1 domains
and a C2 domain. Ca2+-independent or novel PKCs (nPKCs) in
vertebrates (
,
,
, and
) and invertebrates (PKC Apl II in
Aplysia) also have two C1 domains and a C2 domain. However,
the C2 domain in these kinases is N-terminal to the C1 domains and
lacks several of the critical aspartic acids necessary for the
coordination of Ca2+ ions (7).
does not perturb the substrate specificity
or response of the enzyme to phosphatidylinositol 3-kinase activity
(17, 18). However, removal of the C2 domain of PKC Apl II decreases the
amount of PS necessary for kinase activity, implicating the C2 domain
in kinase regulation (19).
and PKC
also
indicate that nPKCs require additional factors to bind efficiently to
lipid membranes (20). PKC Apl II can be activated under a number of physiological conditions in cells, usually after prolonged activation of signal transduction pathways (6, 21). Prolonged activation also
induces autonomous activity of PKC Apl II (22). This activation is
specific, as PKC Apl I does not become autonomous under these conditions. The autonomous activity does not result from proteolysis to
form a protein kinase M but probably results from post-translational modifications in the regulatory domain of PKC Apl II (22).
on tyrosine residues is
thought to be important for activation of the kinase (25-27). Upon
activation, PKCs additionally phosphorylate themselves on several
residues. The roles for these phosphorylations are still unknown.
Autophosphorylation at a conserved site in the C-terminal domain has
been associated with persistent activation of PKC and may be involved
in removal of the enzyme from the membrane (28, 29). A phosphorylation site has also been identified in the C2 domain of vertebrate PKC
that is correlated with PKC activation (30). Autophosphorylation of the
-
family of novel PKCs, which includes the invertebrate kinases
PKC Apl II in Aplysia, Drosophila PKC98F, and
Caenorhabditis elegans PKCIB, has not been investigated.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Phorbol 12,13-dibutyrate was from LC
Services; dioleoyl phosphatidylserine (PS) and dioleoyl
phosphatidylcholine (PC) were from Avanti Polar Lipids Inc., Alabaster,
AL; Triton X-100 was from Avanti; prestained molecular weight markers
were from Amersham Pharmacia Biotech; and
N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin (TPCK) was from Worthington. All other reagents
were of the highest grade available.
Ala and Ser-2
Ala were made with a similar
strategy using different inside primers and the same outside primers
(Table I). A clone of human PKC
was a kind gift of A. Toker (Harvard Medical School). The construct was amplified with PCR using VENT DNA
polymerase (New England Biolabs, Beverly, MA) and inserted into BB4 at
KpnI and BamHI. The construct lacking the C2 domain used an
alternative 5' primer containing the initiating methionine of PKC
followed by amino acid 145. GST and MBP-C2 fusion proteins and MBP-C2
deletion constructs were made previously (13, 21).
Wild type and mutant PKC primers and cloning sites
antibodies ((31) Calbiochem). High titer
viral stocks (108-1010 plaque-forming units
(pfu)/ml) were generated as described (19).
, and mutants was performed
as described previously (19) with the following modifications. Briefly,
250 ml of Sf21 cells (2 × 106 cells/ml) were
infected with 1 × 109 pfus of baculovirus and
incubated for 64 h for optimal PKC expression. Cells were
pelleted, resuspended, sonicated, and centrifuged as described. The
supernatant solution was passed through a 0.22-µm filter, diluted to
50 ml with buffer A (20 mM Tris-HCl, pH 8.0, 0.5 mM EGTA, 0.5 mM EDTA, 10 mM
2-mercaptoethanol, 10% glycerol), and loaded onto a Mono-Q column
(Bio-Rad) previously equilibrated in buffer A. The remaining steps were
performed following previous protocols (19). All baculovirus expressed
enzymes were aliquoted and stored frozen at
70 °C until needed.
cells from either a pMAL-C2 plasmid
(New England Biolabs) or a pGEX 5x.1 plasmid (Amersham Pharmacia
Biotech). These plasmids contained an insert of PKC Apl II amino acids
1-155 corresponding to the C2 domain (13, 21). The MBP-C2 domain and
GST-C2 domain fusion proteins were purified by affinity chromatography
on amylose (New England Biolabs) or GST resin columns (Amersham
Pharmacia Biotech), respectively.
-32P]ATP (4 µCi in 50 µM
ATP) for 30 min at room temperature. Phosphorylation was quenched by
the addition of 5× Laemmli sample buffer and boiled.
1 ovalbumin, 100 mM KCl, 1 mM dithiothreitol, 5 mM
MgCl2, 20 mM HEPES, pH 7.5). Fusion proteins
were eluted from the beads with sedimentation buffer containing 10 µM maltose. The purified fusion proteins were prespun to
pellet aggregates for 30 min at 100,000 × g and
15 °C in a TLA-100 ultracentrifuge (Beckman Instruments, Palo Alto,
CA). Soluble fusion proteins (1-2 µM) were incubated with sucrose-loaded vesicles (20 µM lipid) for 10 min at
15 °C. The proteins associated with the sucrose-loaded vesicles were then separated from free protein by ultracentrifugation at 100,000 × g for 30 min at 15 °C. Pelleting of lipids was
confirmed using scintillation counting of
[3H]PC which was included as a tracer in the
sucrose-loaded vesicles or liposomes. Membrane-bound (pellet fraction)
and free (supernatant fraction) C2 domain fusion protein was separated
by SDS-PAGE and transferred to nitrocellulose membranes. Total protein
was visualized by Ponceau-S staining and phosphorylated protein by
autoradiography using Kodak BioMax MS film on the membrane. In some
cases quantitation of the Ponceau-S staining was confirmed by
immunoblotting using an antibody to the maltose-binding protein. In
some experiments, the fusion protein was excised from the gels, and the
amount of radioactivity incorporated was calculated by scintillation
counting with correction for quenching. This experiment demonstrated
that less than 5% of the total fusion protein was phosphorylated under these conditions.
-PDBu (experimental) for 1 h at
27 °C. Cells were then harvested and lysed in homogenization buffer
(50 mM Tris, pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2.6 mM 2-mercaptoethanol,
20 mg/ml aprotinin, 5 mM benzamadine, 0.1 mM
leupeptin, 50 mM NaF, 5 mM sodium
pyrophosphate, pH 8.5, and 1 µM microcystin). The lysate
was centrifuged at 100,000 × g for 30 min at 4 °C
to separate the cytoplasmic fraction (supernatant) from the
membrane/cytoskeleton fraction (pellet). Equal fractions of the
supernatant and pellet fractions were analyzed by SDS-PAGE (9%
acrylamide gels), transferred to nitrocellulose membranes, and
immunoblotted with the PKC Apl II antibody and the phospho-Ser-36 antibody.
C2 was autophosphorylated as described (19). The
phosphorylation reaction contained 10 pmol of PKC Apl II or PKC Apl
II
C2, 50 µM ATP, reaction buffer (80 mM
Tris, pH 7.5, 10 mM EGTA, 20 mM
MgCl2, 40 nM PMA, and 150 µg/ml dioleoyl
phosphatidylserine) and proceeded for 30 min at room temperature in the
presence of [
-32P]ATP. Phosphorylation was quenched by
the addition of 5 µM inhibitory peptide. The 75-µl
proteolysis reaction contained 10 pmol of either phosphorylated PKC Apl
II or PKC Apl II
C2 and 0.1% Triton X-100. The phosphorylated kinase
component was incubated for 3 min at 30 °C, and proteolysis was
initiated by addition of 2 µg/ml of N-tosyl-L-phenylalanine chloromethyl
ketone-treated trypsin and incubated for an additional 5 min at
30 °C. Reactions were quenched by addition of 20 µl of Laemmli
sample buffer and boiled. Proteins were separated by SDS-PAGE on 7%
gels and electrophoretically transferred to nitrocellulose. Membranes
were subjected to autoradiography to visualize phosphate incorporation
into proteins and immunoblot with the anti-C2 domain antibody for
identification of C2 domain containing protein fragments (13).
x)),
where x is the pixel value (0-254). Control experiments demonstrated that after this calibration, values were linear with respect to the amount of protein over a wide range of values (37). To
measure translocation, the relative percentage was calculated by first
determining the percentage on the pellet in control and experimental
(PDBu-treated) conditions. The percentage change between these two
conditions (experimental
control/control)·100 was determined.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C2). PKC Apl II had
5 reproducible phosphopeptides (Fig.
1A). Two of these phosphopeptides (2 and 3) were not found in the enzyme lacking the C2 domain (Fig. 1B). Removing the C2 domain generated no
additional or modified sites (Fig. 1C). The absence of
phosphopeptides 2 and 3 could be due to their location in the C2 domain
or conformational changes due to the loss of the C2 domain. To obtain
further evidence these sites were actually located in the C2 domain, we
obtained tryptic phosphopeptide maps of C2 domain-containing regions of PKC Apl II after partial tryptic digests of autophosphorylated PKC Apl
II and PKC Apl II
C2 (Fig.
2A). The C2 domain containing fragments were identified based on immunoreactivity to a C2
domain-specific antibody (Fig. 2B) and by their absence in
partial tryptic digests of PKC Apl II
C2 (Fig. 2B, lane
2). Three bands were isolated from the gel and completely digested
with trypsin for phosphopeptide mapping analysis (Fig. 2C).
The largest C2 domain fragment, corresponding to the size of the intact
regulatory domain contained phosphopeptides 2-5 suggesting that all
four of these sites are present in the regulatory domain (Fig.
2C, upper panel). Phosphopeptide 1 was not further
investigated but based on predicted mobility it may correspond to the
C-terminal fragment that contains two predicted phosphorylated sites
(28). The C2 domain core itself is quite resistant to tryptic digestion
(13). Phosphopeptide mapping of the smallest C2 domain fragment
corresponding to the size of the core exhibited only phosphopeptide 3 (Fig. 2C, lower panel). Thus, phosphopeptide 3 is contained
in the C2 domain, whereas phosphopeptide 2 is either near the end of
the C2 domain and is sensitive to tryptic digestion or is outside the
C2 domain, and its phosphorylation is decreased by C2 domain
removal.
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Fig. 1.
PKC Apl II autophosphorylates on five primary
peptides (peptides 1-5), two of which are in the C2 domain.
Proteins were phosphorylated, digested, and analyzed as described under
"Experimental Procedures." A, phosphopeptide map of wild
type PKC Apl II. B, phosphopeptide map of the mutant PKC Apl
II lacking its C2 domain (PKC Apl II C2)
illustrating that it does not phosphorylate peptides 2 and 3. C, phosphopeptide map of coapplied PKC Apl II wild type and
PKC Apl II
C2 illustrating that the spots from wild type PKC Apl II
are the same as those from PKC Apl II
C2 with the exception of
peptides 2 and 3. + and
refer to the polarity of the primary
electrophoresis dimension in buffer pH 1.9; vertical arrows
indicate direction of liquid chromatography; S signifies
origin of sample application or coapplication.
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Fig. 2.
Phosphopeptide 3 originates in the core of
the C2 domain. Proteins were phosphorylated, digested, and
analyzed as described under "Experimental Procedures."
A, PKC Apl II wild type (lane 1) and PKC Apl
II C2 (lane 2) were phosphorylated in vitro for
30 min in the presence of [
-32P]ATP. The samples were
partially digested with 2 µg/ml trypsin for 5 min at 30 °C,
separated by SDS-PAGE, transferred to nitrocellulose, and exposed to
x-ray film (Autoradiogram). B, Western blot of
the membrane in A using an antibody (Ab) directed
to the C2 domain of PKC Apl II (13) confirming that the bands positive
for 32P incorporation in A are C2
domain-containing fragments. C, phosphopeptide maps of C2
domain containing bands from B, lane 1 (upper to
lower), illustrating that peptide 3 is isolated as the C2
domain core is isolated. + and
refer to the polarity of the
primary electrophoresis dimension in buffer pH 1.9; vertical
arrows indicate direction of liquid chromatography; S
signifies origin of sample application.
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Fig. 3.
Autophosphorylation of peptide 4 is absent in
the autonomous kinase. Proteins were phosphorylated, digested, and
analyzed as described under "Experimental Procedures."
A, wild type PKC Apl II was in vitro
autophosphorylated in the presence of [ -32P]ATP and
either no activators, PS/TPA, or oleic acid for stimulation.
The samples were separated by SDS-PAGE, transferred to nitrocellulose,
and exposed to x-ray film (Autoradiogram). B,
phosphopeptide mapping analysis of bands from A. + and
refer to the polarity of the primary electrophoresis dimension
in buffer, pH 1.9; vertical arrows indicate direction of
liquid chromatography; S signifies origin of sample
application. C, quantitation of NIH image analyzed data.
Values are means ± S.E. (n = 3).
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Fig. 4.
C2 domain fusion proteins are
transphosphorylated on phosphopeptides 2' and 3. Proteins were
phosphorylated, digested, and analyzed as described under
"Experimental Procedures." A, GST-C2 and MBP-C2 domain
fusion proteins were transphosphorylated by PKC Apl II in the presence
of [ -32P]ATP. The samples were separated by SDS-PAGE,
transferred to nitrocellulose, and exposed to x-ray film
(Autoradiogram). B, phosphopeptide mapping
analysis of the GST-C2 domain fusion protein in A
illustrating that the GST-C2 domain fusion protein yields
phosphopeptides 2' and 3 as well another phosphopeptide (see
A in panel B) which is also seen after
phosphorylation of GST alone (data not shown). C,
phosphopeptide mapping analysis of the MBP-C2 domain fusion protein in
A indicating that the MBP-C2 domain fusion protein also
yields phosphopeptides 2' and 3. D, phosphopeptide analysis
of coapplied wild type PKC Apl II and the GST-C2 domain fusion protein
illustrating that phosphopeptide 3 is identical in the
autophosphorylated kinase and the fusion protein. + and
refer
to the polarity of the primary electrophoresis dimension in buffer pH
1.9; vertical arrows indicate direction of liquid
chromatography; S signifies origin of sample
application.
ASMSR) consistent with the change from site 2 to the new site 2'. An N-terminal location would also be consistent
with the loss of phosphopeptide 2' from the C2 domain by the partial
tryptic digestion (Fig. 2C, lower panel). Indeed, site 2 was
not observed when serine 2 was mutated to alanine in the MBP-C2 domain
fusion protein (data not shown).
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Fig. 5.
Serine 36 is the primary phosphorylated
residue in the C2 domain of PKC Apl II. A, hypothetical
phosphopeptide mapping of the regulatory domain of PKC Apl II
identifies probable peptide 3. The regulatory region of PKC Apl II was
divided into constituent tryptic peptides and their mass/charge ratio
calculated based on singular phosphorylations. The relative
hydrophobicity of each peptide was then calculated in the
phospho-chromatography buffer (36). The resulting values for regulatory
domain peptides (black circles), C2 domain-specific peptides
(gray circles), and C2 domain peptide TTTK (white
circle) were plotted against each other to generate a
phosphopeptide "map" where the location of each peptide is
relative. B-D, the residue within peptide 3 responsible for
its heavy phosphorylation is serine 36. Proteins were phosphorylated,
digested, and analyzed as described under "Experimental
Procedures." B, phosphopeptide analysis of the wild type
(wt) MBP-C2 domain. C, phosphopeptide analysis of
the MBP-C2 domain fusion protein with a Ser-36 Ala mutation (MBP-C2
S36A). D, phosphopeptide analysis of coapplied wild type
MBP-C2 domain and MBP-C2 S36A. E-G, PKC Apl II
autophosphorylates at serine 36. E, phosphopeptide analysis
of wild type PKC Apl II. F, phosphopeptide map of the mutant
PKC Apl II containing the Ser-36
Ala mutation (PKC Apl II S36A).
G, phosphopeptide map of coapplied PKC Apl II wild type and
PKC Apl II S36A. + and
refer to the polarity of the primary
electrophoresis dimension in buffer pH 1.9; vertical arrows
indicate direction of liquid chromatography; S signifies
origin of sample application or coapplication.
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Fig. 6.
Sucrose-loaded lipid vesicle assay for C2
domain membrane binding. Proteins were phosphorylated on beads,
washed, eluted, precentrifuged to remove aggregates, and then incubated
with sucrose-loaded vesicles as described under "Experimental
Procedures." A, purified MBP-Apl II C2 (WT),
MBP-Apl II C2 with serine 36 converted to alanine (S36-A),
or MBP-Apl II C2 with serine 36 converted to glutamic acid
(S36-E) were incubated with sucrose-loaded vesicles (20 µM lipid) consisting of either 60% PS, 40% PC
(PS/PC) or 100% PC (PC) for 10 min at 15 °C.
The sucrose vesicles were subsequently sedimented in a Beckman TLA-100
ultracentrifuge at 100,000 × g for 30 min, and the
supernatant (S) and pellet (P) fractions were
separated and analyzed by PAGE and membrane transfer. Total protein was
visualized by Ponceau-S staining (bottom gel), whereas
phosphorylated protein was measured by autoradiography (top
gel). Although very little of the nonphosphorylated wild type
fusion protein domain is sedimented, a considerable amount of the
phosphorylated protein does sediment. The serine 36 to alanine mutation
reduced sedimentation of the phosphorylated protein significantly,
whereas converting serine 36 to glutamic acid increased sedimentation
of the nonphosphorylated protein. B, graphic illustration of
quantitated results from experiments similar to those in A.
Data are displayed as the percentage of total protein found in the
bound (P) fraction for both total protein (T,
white bars) and phosphorylated protein (black
bars). Values are means ± S.E. (n = 6).
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Fig. 7.
Substrate phosphorylation is unaffected by
the S36A or S36E mutations. Protein kinase C activity assays were
performed as described under "Experimental Procedures." PKC assays
were performed for wild type (wt) PKC Apl II (white
circles), PKC Apl II S36A (black circles), and PKC Apl
II S36E (white squares) illustrating no significant change
in phosphatidylserine-dependent substrate phosphorylation
in the mutant kinases.
-32P]ATP (Fig. 8A, middle panel). Thus,
this antibody was specific for phosphorylation of serine 36.
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Fig. 8.
Serine 36 is phosphorylated in
vivo and is necessary for PKC membrane translocation.
Proteins were phosphorylated and kinase assays performed as described
under "Experimental Procedures." A, purified wild type
(wt) PKC Apl II and PKC Apl II S36A were autophosphorylated
in vitro. Enzymes were stimulated by no activators
(BL), 50 µg/ml dioleoyl phosphatidylserine and 20 nM PMA (PS/TPA), or 20 µM oleic
acid (Oleic) in the absence ( ) or presence (+) of 50 µM ATP and [
-32P]ATP. A, upper
panel, immunoblot using the phospho-Ser-36 antibody illustrating
the increase in immunoreactivity with PS/PMA and oleic acid stimulation
of the wild type PKC that is absent in the mutant, PKC Apl II S36A.
A, middle panel, autoradiogram of the upper panel
illustrating an increase in 32P incorporation for the
stimulated wild type PKC that parallels that seen in the
anti-phospho-Ser-36 Western blot. A, lower panel, immunoblot
of the upper panel using the PKC Apl II antibody indicating
that approximately equal protein is loaded in each lane and between PKC
constructs. B, cells were fractionated as described under
"Experimental Procedures." Anti-PKC Apl II immunoblot of
fractionated Sf-21 cells infected with baculovirus coding for wild type
PKC Apl II (1st to 4th lanes) and PKC Apl II S36A
(5th to 8th lanes) after treatment in the absence
(
) or presence (+) of 4-
-PDBu. C, anti-phospho-Ser-36
immunoblot of the experiment in B illustrating serine 36 phosphorylation in vivo of only wild type PKC Apl II and the
preferential PDBu-induced membrane translocation of serine
36-phosphorylated PKC.
Ala mutation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Sequence alignment of novel C2 domains and
model of phosphorylation-induced lipid binding. A,
comparison of the C2 domains of PKC Apl II and novel vertebrate PKCs.
Loop positions are based on those described previously (7, 41).
Shaded residues are conserved in all isoforms. Darkly
shaded residues represent known phosphorylation sites in PKC Apl
II (serine 2 and 36) and phosphorylatable residues in loop 1 of C2
domains of other novel PKCs. Sites of the Rack inhibitory peptide and
pseudo-Rack peptide are from Ref. 16. B, model of
phosphorylation-induced lipid binding. B, left panel, a
hypothetical representation of the C2 domain of PKC Apl II with a
positively charged lipid binding surface on loop 1 whose access is
restricted by the position of loop 3. B, right panel,
phosphorylation of serine 36 in the putative -helix of loop 1 shifts
the position of loop three to expose the lipid binding surface on loop
1. C2 domain structure adapted from Ref. 41.
this
region forms an
-helix (40), but there is little sequence
conservation in this region in the
/
family (7), and without a
crystal structure of an
-like C2 domain the organization of the
loops is still speculative. Serine 36 is contained in the extended
C-terminal region of loop 1 (Fig. 9A). Phosphorylation of
serine 36 increases in vitro lipid binding and cellular
translocation of PKC Apl II. However, since phosphorylation increases
negative charge, it would not be expected to increase binding to acidic lipids by electrostatic interactions directly. We propose a model (Fig.
9B) where phosphorylation of this residue leads to a
conformational shift in the loops of the C2 domain that expose a
cryptic lipid-binding site. The N-terminal portion of loop 1 is highly
conserved in PKC
-like PKCs and contains a peptide used as a specific
inhibitor of PKC
binding to RACK (14-16; Fig. 9A). This
region might act as the cryptic lipid binding/RACK-binding domain for
these nPKCs. In contrast, the extended region of the loop that contains
serine 36 is not as well conserved. However, all isoforms contain a
phosphorylatable site in this region (Fig. 9A), and PKC
contains at least one autophosphorylation site in the C2
domain.2
-activating peptide also predicts intra-C2 domain interactions
(16). This peptide leads to increased isoform-specific membrane
translocation of PKC
(16). The peptide is made from a conserved
sequence around loop 3 (Fig. 9A) and is believed to interact
with loop 1 to increase access of PKC
to RACKS (16). We suggest that
phosphorylation of the C2 domain serves the same purpose as the
peptide, inhibiting intra-loop interactions to expose a cryptic lipid
or RACK-binding site. Indeed loop 3 is negatively charged suggesting
that introducing a negative charge in loop 1 would inhibit loop 1-loop
3 interactions. Intra-C2 domain interactions have also been shown to be
important in regulating the C2-B domain of synaptotagmin (39).
Different synaptotagmin isoforms show distinct abilities to bind to
inositol polyphosphates through a region in the C2 domain. However,
differences in binding are not due to the actual inositol polyphosphate
binding sequence, which is well conserved in all isoforms. Instead
isoform-specific differences map to the C terminus of the C2 domain
where some isoforms have residues that interact with the inositol
polyphosphate binding domain and inhibit binding, whereas other
isoforms lack these residues and exhibit binding (39). Deletion of the
C terminus of the C2 domain of synaptotagmin allows inositol
polyphosphate binding to all the isoforms (39).
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Fig. 10.
Model of the role for C2 domain
phosphorylation in novel PKC membrane translocation and
activation. A, fully mature PKC Apl II (phosphorylated
on its activation loop Thr-561 by PDK1 and autophosphorylated on
its two C-terminal residues resides in the cytosol awaiting activation.
The pseudosubstrate (PS) is occupying the catalytic pocket,
and the C2 domain restricts activator binding to the C1 domain.
B, PKC Apl II can become activated through production of DAG
by PLC or PLC
and high levels of phosphatidylserine binding to
its C1 domain. Solely the C1 domain of PKC Apl II mediates this
membrane translocation and as a result its membrane affinity would be
low and lead to only transient kinase activation. C,
alternatively, PKC Apl II can become autophosphorylated (in
cis or perhaps in trans) on serine 36 in loop 1 of its C2 domain. D, this phosphorylation may expose a
cryptic C2 domain lipid binding site allowing phosphatidylserine
binding, and now both the C1 and C2 domains are responsible for
recruiting the kinase to the membrane. This C1- and C2-mediated
activation produces a high membrane affinity and may be responsible for
persistent activation of novel PKCs like PKC Apl II.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Peter McPherson for helpful comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant MT-12046 from the Medical Research Council of Canada.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.
Recipient of a Medical Research Council of Canada Studentship.
§ Recipient of a Chercheur-Boursier from the Fonds de la Recherche en Santé du Québec. To whom correspondence be addressed: Dept. of Neurology and Neurosurgery, McGill University, Montreal Neurological Institute, Rm. 776, 3801 Rue University, Montreal, Quebec H3A 2B4, Canada. Tel.: 1-514-398-1486; Fax: 1-514-398-8106; E-mail: mdws@musica.mcgill.ca.
Published, JBC Papers in Press, November 9, 2000, DOI 10.1074/jbc.M006339200
2 A. M. Pepio and W. Sossin, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PKC, protein kinase C; cPKC, classical PKCs; nPKC, novel PKC; PS, phosphatidylserine, PC, phosphatidylcholine; pfu, plaque-forming units; MBP, maltose-binding protein; GST, glutathione S-transferase; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin; PDBu, phorbol dibutyrate; PMA, phorbol 12-myristate 13-acetate; RACK, receptor for activated protein kinase C; PCR, polymerase chain reaction; O5, outside 5' primer; I3, inside 3' primer; I5, inside 5' primer; O3, outside 3' primer; DAG, diacylglycerol.
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