(Received for publication, October 22, 1996, and in revised form, November 19, 1996)
From the Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom
Serine 657 in protein kinase C- (PKC
) is a
site of phosphorylation on expression of the recombinant protein in
mammalian cells. To define the function of this phosphorylation, PKC
species with mutations of this site were investigated. The alanine
mutant, S657A PKC
, displayed slow phosphate accumulation in
pulse-chase experiments, indicating a rate-limiting role in the initial
phase of phosphorylation. Consistent with this, the aspartic acid
mutant, S657D PKC
, showed an increased rate of phosphate
accumulation. Both the S657D and S657A PKC
mutants were slow to
accumulate as fully phosphorylated forms during a second phase of
phosphorylation. This latter property is shown to correlate with an
increased phosphatase sensitivity and decreased protein kinase
activity for these two PKC
mutants. It is further shown that once
fully phosphorylated, the S657D PKC
mutant displays WT PKC
properties with respect to thermal stability and phosphatase
sensitivity in vitro and in vivo; in contrast,
the S657A PKC
mutant remains sensitive. The properties of the
Ser-657 site PKC
mutants define functional roles for this
phosphorylation in both the accumulation of phosphate on PKC
as well
as in its agonist-induced dephosphorylation. These results are
discussed in the context of a working model of PKC
behavior,
providing insight into the workings of other kinases with equivalent
sites of phosphorylation.
The phosphorylation of protein kinases has long been established in the field of protein phosphorylation. In fact, the first target of regulated phosphorylation to be elucidated (phosphorylase b) lies at the end of a protein kinase cascade, wherein the cAMP-dependent protein kinase phosphorylates and activates phosphorylase kinase (see Ref. 1). Such cascades are now relatively commonplace among the protein kinase class, and there are many examples of positive regulatory cascades (e.g. the Raf-MEK-MAPK cascade) (2) and some of negative regulatory ones (e.g. PKB-GSK-3) (3). In these contexts, the effects of phosphorylation are reflected by a "simple" loss or gain of protein kinase activity. In a number of well characterized cases, the gain of function is associated with phosphorylation within what has been termed the "activation loop" of the kinase domain (see Ref. 4).
Contrary to the kinases referred to above, the
cAMP-dependent protein kinase is regulated acutely through
the cellular production of the second messenger cAMP (see Ref. 5, and
references therein), and there is no evidence of acute regulation
through modified phosphorylation within the activation loop.
Nevertheless, it is evident that this region in its phosphorylated
state plays a key role in aligning the substrate binding site for
catalysis (6, 7). Members of the related protein kinase C
(PKC)1 family are also second
messenger-dependent protein kinases. For PKC and PKC
,
homologous sites in their activation loops have been shown to play an
essential role (8-11). Thus, while these second
messenger-dependent protein kinases may not be the subject of acute regulation through these phosphorylation sites, a similar requirement for phosphorylation pertains.
Although the activation loop phosphorylation sites in PKC and other
protein kinases share a defined, necessary role in catalysis, phosphorylation of PKC sites outside this region plays a more subtle
role in controlling function. In both PKC and PKC
,
phosphorylation of two C-terminal sites has been reported (11, 12). In
PKC
, one of these, threonine 638 (Thr-638), has been shown to
control the rate of agonist-induced dephosphorylation and inactivation of the protein in vivo but not to be required for catalytic
activity (13). This property appears to be governed by interactions
between the C-terminal region of the kinase and its catalytic core,
with phosphorylation at Thr-638 and at the activation loop (Thr-497) being required to maintain the active, phosphatase-resistant, closed
conformation of the kinase domain (13). Here, we have analyzed the role
of the serine 657 (Ser-657) phosphorylation site in PKC
. It is
established that phosphorylation of this site controls the accumulation
of phosphate at other sites on PKC
, as well as contributing to the
maintenance of the phosphatase-resistant conformation.
Bovine protein kinase C- (14) was tagged
with six histidine residues at the N terminus (His-tag PKC
) by
synthesizing oligonucleotide cassettes for substitution insertion. It
was mutagenized and sequenced using the Altered Sites in
vitro mutagenesis system (Promega) and the Sequenase Version 2.0 DNA sequencing kit (U. S. Biochemical Corp.) and finally subcloned
into the pKS1 vector according to a previously described procedure
(13). The following mutagenic oligonucleotides, which are sense
with respect to the PKC
cDNA, were used (changed bases are
underlined; an additional point mutation was designed,
introducing a snab 1 restriction site with no change at the protein
level): S657A,
CTGATTTTGAAGGCTTC
CCTACGT
AACCCCCAGTTCG; S657D,
CTGATTTTGAAGGCTTC
CTACGT
AACCCCCAGTTCG; S657E,
CTGATTTTGAAGGCTTC
TACGT
AACCCCCAGTTCG. The
mutations at the 497 and 638 sites were prepared as described previously (13).
COS-1 cells were
maintained in Dulbecco's modified Eagle's medium (DMEM) containing
10% (v/v) fetal-calf serum, 1000 units/ml penicillin, and 100 µg/ml
streptomycin. 50 µg of plasmid DNA were introduced into 0.8 ml of
60-80% confluent COS-1 cells (5 × 106cells) by
electroporation (0.45 kV for 8-10 msec). After 48 h of transient
expression, the cells were lysed in 500 µl of an ice-cold lysis
buffer containing 20 mM Tris-HCl, pH 7.5, 120 mM NaCl, 1% (v/v) Triton X-100, 2 mM
-mercaptoethanol, 5 mM benzamidine, 50 µg/ml
leupeptin, 50 µg/ml aprotinin, 50 µg/ml trypsin inhibitor, 5 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM Na3VO4, 1 mM p-nitrophenyl phosphate, and 5 mM
imidazole. The suspension was stroked 20 times in a Dounce homogenizer
and centrifuged at 12,000 × g for 30 min at 4 °C.
Recombinant mutant or WT PKC
in the Triton-soluble extract was
purified with nickel-agarose (Qiagen) using a batch procedure described
before (13) and subsequently eluted in lysis buffer (0.02% Triton
X-100) supplemented with 100 mM imidazole.
For treatment with phorbol esters, the culture medium was changed after 30 h of transient expression and 500 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) was added. At various time points, the incubation medium was removed, and cells were quickly rinsed in ice-cold phosphate-buffered saline and lysed in lysis buffer (as above but without imidazole) supplemented with 10 µM microcystin. The lysate was passed twice through a 27-gauge needle and centrifuged for 20 min at 12,000 × g at 4 °C before analysis on SDS-PAGE.
For pulse-chase experiments, the culture medium was removed after 24 h of transient expression and replaced by DMEM supplemented with 10% (v/v) dialyzed fetal calf serum. After 1 h, cells were pulse-labeled for 15 min with 100 µCi/ml of [35S]methionine (Amersham, Life Science) in DMEM plus 5% (v/v) dialyzed fetal calf serum. Chase was then started in DMEM supplemented with 10% fetal calf serum and 5 mM methionine and stopped after various time points. Cells were finally lysed, and PKC was purified with nickel-agarose as described above.
Protein Phosphatase 1 Treatment of Purified PKCThe isoform of human protein phosphatase 1 (PP1c
) (15) was obtained as a
recombinant protein after introduction of the pCW PP1 plasmid into
competent Escherichia coli DH5
. Expression and
purification were carried out as described in Refs. 13 and 16. Purified
recombinant PKC
(eluted from Ni-agarose in the absence of NaF,
Na3VO4, and p-nitrophenyl phosphate)
was treated at 22 °C for 30 min with purified recombinant PP1c
in
the presence of 1 mM MnCl2. Reactions were
stopped by adding 10 µM microcystin.
Protein kinase C activity was
assayed as described earlier (13) using Histone III-S as a substrate.
Protein concentration was determined by the bicinchoninic acid method
(Pierce) using bovine serum albumin as the standard. High resolution
SDS-PAGE was performed on samples diluted twice in 5 × Laemmli
buffer (17) using 12-cm long running gels containing 7.5% acrylamide
and 0.06% bisacrylamide. Gels were transferred onto nitocellulose and
subjected to immunoblotting using a polyclonal antibody raised against
the C terminus of PKC (18). Reproductions of autoradiographs were obtained as computer-scanned images.
Previously, it has been shown that PKC is
phosphorylated at Ser-657 (11). Using V8 protease digestion, high
pressure liquid chromatography purification, and mass
spectrometry analysis, serine 657 was found to be phosphorylated also
in recombinant 32P-labeled PKC
expressed in COS
cells.2 In order to study the role of this
phosphorylation, substitution mutants were made to prevent (S657A) or
partially mimic (S657D) phosphorylation at this site. Both Ser-657 site
PKC
mutants were expressed in COS cells, fully recovered in the
detergent-soluble fraction after lysis, and could be purified on
Ni-agarose (Fig. 1). Unlike the WT PKC
molecule that
migrates as a single 80-kDa band, a doublet was reproducibly detected
for both the 657 site mutants. It would therefore appear that
mutagenizing the 657 position in PKC
perturbs the overall processing
of the kinase as observed 48 h after transient expression in COS
cells. This contrasts with the previously studied PKC
Thr-638 site
mutants (13) that displayed complete processing under similar
conditions (Fig. 1). This "processing" is due to phosphorylation as
judged by the action of protein phosphatases on the various forms
generated (see below and Refs. 13 and 19); for simplicity, the
processing/mobility shifts observed here are referred to as
phosphorylations in the text.
In order to analyze the rate of phosphorylation of the Ser-657 mutants
directly, pulse-chase experiments using 35S-labeled
methionine were carried out after 30 h of transient expression of
either WT PKC, S657A, or S657D in COS cells. As shown in
Fig. 2A, phosphorylation at 657 clearly plays
a rate-limiting role in the accumulation of a fully phosphorylated
PKC
. Following a 15-min period in
[35S]methionine-containing medium, the subsequent rate of
accumulation of phosphorylated forms of PKC
is much reduced in the
S657A mutant. Following 40 min of chase, the WT PKC
has shifted
completely from the primary translation product, and the majority is in
a slowly migrating doublet by 80 min. By contrast, the primary
translation product of the S657A mutant is barely 50% converted to the
first more slowly migrating form by 80 min of chase. There is, also, little further phosphorylaytion during this time period. Conversely, the S657D mutant is already 50% converted to the first slowly migrating form following the 15-min labeling period. Subsequently, however, the S657D mutant is slower than WT PKC
in accumulating as a
more fully phosphorylated form.
These results imply that, in this context, there are two primary phases
of phosphorylation going on for PKC. In the first, phosphorylation
of Ser-657 plays a rate-limiting role. In the second, some property of
the Ser-657 phosphorylated protein is required that is not effectively
mimicked by an aspartic acid substitution. The aspartic acid
substitution is, however, sufficient for and indeed promotes the first
phase of phosphorylation. It can be surmised that, in the S657D mutant
(and the S657A mutant), either there is a low kinase activity in the
partially phosphorylated forms that prevents the second phase of
phosphorylation or that there is some sensitivity to protein
phosphatases that slows down the accumulation of fully phosphorylated
PKC
. The biochemical analysis of these mutants, as described below,
demonstrates that the partially phosphorylated mutant forms of PKC
are, in fact, both sensitive to protein phosphatases and of low
activity (summarized in Fig. 7). The pulse-chase data alongside the
results detailed in subsequent sections provide evidence for three
components to Ser-657 phosphorylation: (i) the phosphorylation of
Ser-657 is rate-limiting for the first phase of PKC
phosphorylation;
(ii) Ser-657 phosphorylation is required for efficient second phase phosphorylation of PKC
; and (iii) phosphorylation of Ser-657 contributes to "locking" fully phosphorylated PKC
in a closed, phosphatase-resistant conformation.
The conclusions to be drawn from these analyses are entirely consistent
with the steady-state pattern of S657D PKC expression, which
displays both fully phosphorylated and partially phosphorylated species
(see Fig. 2B; i.e. processing can go to
completion for this PKC
mutant, but it is incomplete in the steady
state). By contrast, for the WT PKC
, although fast migrating forms
can be detected by pulse labeling techniques (Fig. 2A),
these forms are rapidly phosphorylated such that they do not contribute
significantly to the steady-state level of WT PKC
. Thus, fast
migrating forms are poorly detected by Western blotting for WT PKC
(Fig. 2B).
The
slow rate of phase 2 phosphorylation of the S657D PKC mutant
indicated that the partially phosphorylated form may be functionally
compromised. In order to assess the relative specific activity and
protein phosphatase sensitivity of the S657D PKC
mutant, WT and
S657D PKC
were purified from COS cells as above and treated in
vitro with PP1c
. As shown in Fig. 3A,
the faster migrating S657D PKC
species (78 kDa) was sensitive to
PP1c
while the slower migrating (80 kDa) species was not; the 80-kDa
species behaved exactly as WT PKC
(80 kDa). There was very little
change in activity associated with the dephosphorylation of the 78-kDa S657D PKC
(Fig. 3A), implying that this species does not
contribute significantly to the determined activity. This is consistent
with calculation of the specific activity of the S657D mutant;
accounting for only the 80-kDa species yielded a specific activity for
S657D PKC
which was 103% of that of WT PKC
.
This data indicates that the fast migrating species is a low activity
form that is sensitive to dephosphorylation. Previous studies on the
PKC Thr-638 site have correlated phosphatase hypersensitivity in vitro and in vivo (13). A similar correlation
was found for the S657D PKC
mutant. Thus, on transient expression of
S657D PKC
in COS cells, TPA treatment induced a rapid (
15 min)
dephosphorylation of the fast migrating form but not the slow migrating
form (Fig. 3B). As observed in vitro (Fig.
3A), the slow migrating form behaved essentially as WT
PKC
(not shown) while the fast migrating form was hypersensitive to
phosphatases. Notably, the S657D PKC
doublet was entirely Triton
X-100-soluble in transfected cells, and only following TPA treatment
did antigen accumulate in the Triton X-100-insoluble fraction.
In PKC Thr-638 site mutants, phosphatase sensitivity parallels
thermal instability (13). This appears to be due to an alteration in
the way the C terminus (V5 domain) interacts with the catalytic core
(C3/4 domain), producing an "open" conformation. For the S657D
mutant, kinase activity is largely accounted for by a slowly migrating
form that is phosphatase-resistant, i.e. behaves like WT
PKC
. It can be predicted that this mutant would resemble the WT
protein with respect to thermal inactivation, and this is, in fact, the
case. Incubation at 25 °C for 30 min induced a 5% loss of activity
precisely that observed for WT PKC
(Fig. 4). By way
of comparison under these same conditions, the Thr-638 mutants (that do
not accumulate as partially phosphorylated forms, see Fig. 1) displayed
a much greater degree of thermal inactivation. The S657A mutant
displayed an intermediate loss of activity (see below).
The properties of the two differentially phosphorylated S657D species
indicate that once fully phosphorylated, the aspartic acid
substitution is sufficient to mimic any requirement for phosphorylation at this site. This is reflected in the full activity, phosphatase resistance (in vitro and in vivo), and thermal
stability of the kinase activity. However, it is equally evident that
the partially phosphorylated (fast migrating) form is of low activity
and is phosphatase-sensitive. It is these latter properties that would account for the slow second phase of S657D PKC phosphorylation as
discussed above.
Contrary to the S657D substitution, S657A PKC could be
dephosphorylated and inactivated in vitro by PP1c
(Fig. 5A). However, the extent of
inactivation correlated with the dephosphorylation of the slower
migrating form, indicating that, as for S657D PKC
, the faster
migrating form contributes little to the overall perceived activity.
Accounting for only the upper slow migrating S657A PKC
species
reveals a specific activity for this protein of 109% of the WT PKC
.
The results demonstrate that phosphorylation of this site is not
essential for catalysis but affects phosphatase sensitivity in
vitro.
Phosphatase hypersensitivity of S657A PKC was observed also in
vivo (Fig. 5B). Thus, on TPA treatment of S657A PKC
transfected COS cells, the mutant was dephosphorylated with virtually
complete loss of the most highly phosphorylated form by 15 min. As
described for the S657D mutant, the dephosphorylated form accumulated
in the Triton X-100-insoluble fraction; prior to TPA treatment, the S657A PKC
forms were fully Triton X-100 extractable (discussed below).
The observed increased sensitivity to phosphatases for S657A PKC,
redistribution to the cytoskeleton, and the loss of activity observed
on dephosphorylation in vitro suggests that phosphorylation at the 657 site (or an aspartic acid) normally contributes to the
"closed" conformation (V5-C3/4 interacting; see Ref. 13) of the
protein. Consistent with this view was the moderate thermal instability
of the S657A PKC
kinase activity noted above (see Fig. 4). Thus, it
would seem that phosphorylation of the 657 site not only influences the
accumulation of phosphate as described above but also contributes to
maintaining the active closed conformation (i.e. effective
V5-C3/4 interactions). Evidence that this is the case is suggested by
studies with PKC
mutants altered at the Thr-497 or Thr-497 and
Thr-638 sites. The T497E/T638E mutant retains reduced protein kinase
activity, but this mutant can still be inactivated by PP1c
similar
to the T497E mutant (Fig. 6). While not necessarily the
only other regulatory phosphorylation site on PKC
, the
phosphatase-dependent inactivation of the T497E/T638E mutant is
consistent with Ser-657 dephosphorylation. This conclusion is further
supported by the finding that an T497E/T638E/S657E mutant is both
inactive and, by migration, insensitive to TPA-induced dephosphorylation in vivo (data not shown).
A Model for the Phosphorylation Control of PKC
The
properties and phosphorylation of the S657A and S657D PKC mutants
are summarized in Fig. 7, alongside those predicted for
the WT PKC
. The presence of phosphate in the Thr-497 site in the
low/partial activity form resulting from "phase 1" is based upon
mapping of 32P-labeled peptides from the fast migrating
form of the S657D mutant (data not shown). The phosphorylation of
Thr-638 in the second phase is predicted from the dominant role this
site has in controlling the rate of dephosphorylation of PKC
(13).
For the most highly phosphorylated forms, the effects of the Ser-657
site are defined by the S657A mutant, which demonstrates no absolute
requirement for phosphorylation to yield full activity but a
requirement for phosphorylation (or an acidic residue) to enhance
thermal stability and maintain a phosphatase-resistant state. The basis
of these properties likely relates to the closed conformation
consequent to the V5-C3/4 domain interactions (13). Thus, the Ser-657
phosphorylation appears to play a role alongside that of Thr-638 in
forming this closed conformation although the Thr-638 site is dominant
in this regard. This dominance of the Thr-638 site is evidenced by the greater phosphatase sensitivity (13) and thermal instability of the
T638A compared with the S657A mutant.
The partially phosphorylated forms of both the S657A and S657D mutants
are of low activity, and this is consistent with the slow progress of
phase 2 compared with WT PKC. It can be concluded then that the
conformation/activity of the partially phosphorylated WT PKC
is more
favorable for completion of phase 2. The attainment of this partially
phosphorylated state is also dependent upon Ser-657 phosphorylation, as
evidenced by the pulse-chase experiments with the 657 site mutants.
These reveal a rate-limiting role for Ser-657 phosphorylation in phase
1. While this phase appears to lead to phosphorylation of the Ser-657
and Thr-497 sites in WT PKC
, the fact that Ser-657 phosphorylation
is rate-limiting does not define the order of these events.
A unifying hypothesis derived from these studies and those on Thr-638
site mutants (13) is that the phosphorylated Ser-657 site contributes
to contact between the C-terminal (V5) domain and the catalytic core
(C3/4). In the partially phosphorylated state (no Thr-638
phosphorylation), phosphate at Ser-657 acts alone to generate the
appropriate conformation, and aspartic acid substitution is an
insufficient surrogate. In the fully phosphorylated state, this
conformation is contributed by both the Thr-638 and Ser-657 sites;
here, aspartic acid substitution is sufficient to act in synergy with
phosphate at the Thr-638 site. Contrary to previous conclusions (11,
20), these priming phosphorylations are not involved in solubilizing
the primary translation product as evidenced by the complete solubility
of the mutants described here. In fact, the detergent-insoluble form
that can accumulate in cells is associated with dephosphorylation of
the activated enzyme. This property of the dephosphorylated enzyme
distinguishes it from the primary translation product that is soluble,
indicative of either a selective mechanism for sequestration of the
membrane-associated dephosphorylated form or the existence of a
chaperone protein operating on the primary translation product to
maintain solubility and permit appropriate folding/phosphorylation. In
view of the poor solubility of PKC expressed even at low levels in
E. coli,3 the existence of a
mammalian chaperone would seem a probable mechanism. This conclusion is
further supported by the finding that dephosphorylation of WT PKC
in vitro leads to aggregation and insolubility of the
protein.3
The results presented here and previously (13) on PKC define the
processes involved in generating and maintaining an active kinase. The
implications of these findings are broad. There are a number of protein
kinases that, like PKC
, have conserved threonine and serine sites in
locations equivalent to Thr-638 and Ser-657. For example, of particular
current interest is the control of the PKB/akt/RAC kinase. This protein
has recently been shown to be fully activated in an
agonist-dependent fashion through phosphorylation in both
its catalytic core (activation loop) site (residue T308) and in a
C-terminal site equivalent to Ser-657 in PKC
(PKB residue S473)
(21). The manner of operation of these sites in PKB is likely to be as
that described here for PKC
.
In respect of the Ser-657 site itself, this site lies within an FSF/Y
motif. In p70S6kinase, the phosphorylation of the
equivalent site within the same motif is sensitive to rapamycin (22);
no such sensitivity is observed for PKC,3 implying a
distinct mechanism of control. Interestingly, in PKC
and in the PRK
family, this equivalent site is substituted by an acidic residue
(23-25). Based upon the mutants described here, this would not affect
the fully phosphorylated form, but it may presage the existence of low
activity forms of these proteins that are subject to control by acute
phosphorylation and activation. Evidence that this may be the case
exists for PRK1(PKN) (26). The implications of the pattern and
consequence of PKC
phosphorylation thus impact on a number of
related kinases, and it will be of great interest to see how well
conserved these properties indeed are and how they integrate into the
distinct modes of regulation of these kinases.
We thank Dr. P. T. W. Cohen and coworkers for providing us with the pCW-PP1 plasmid and Louise Mansi for preparation of the manuscript. We are grateful to Drs. L. Dekker and M. Parker for constructive comments on the manuscript. We are indebted to Dr. B. Hemmings for providing data prior to publication.