From the Departments of Pharmacology and Chemistry
and Biochemistry, University of California at San Diego,
La Jolla, California 92093-0640 and § Howard Hughes
Medical Institute, Vollum Institute, Portland, Oregon 97201-3098
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ABSTRACT |
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Protein kinase C is processed by three
phosphorylation events before it is competent to respond to second
messengers. Specifically, the enzyme is first phosphorylated at the
activation loop by another kinase, followed by two ordered
autophosphorylations at the carboxyl terminus (Keranen, L. M.,
Dutil, E. M., and Newton, A. C. (1995) Curr.
Biol. 5, 1394-1403). This study examines the role of negative charge at the first conserved carboxyl-terminal phosphorylation position, Thr-641, in regulating the function and subcellular localization of protein kinase C The protein kinase C family of enzymes transduce the myriad of
extracellular signals that promote phospholipid hydrolysis (1).
Generation of diacylglycerol, typically in the plasma membrane,
activates most members of this class of kinases by recruiting them to
membranes, where they are activated by interaction with the
phospholipid, phosphatidylserine (2). Binding of both diacylglycerol and phosphatidylserine to the protein results in a conformational change that removes an autoinhibitory pseudosubstrate domain from the
active site, thus allowing substrate binding and catalysis (3).
Despite 2 decades of studying the regulation of protein kinase C, it
has only recently been appreciated that the enzyme must be
phosphorylated before it is competent to respond to second messengers.
Pulse-chase experiments by Fabbro and co-workers (4) in the late 1980s
provided the first evidence that protein kinase C is phosphorylated
in vivo. Specifically, they showed that protein kinase C is
first synthesized as an inactive, dephosphorylated precursor with an
apparent molecular mass of 74 kDa that associates with the
detergent-insoluble fraction of cells; this species was chased to a
transient 77-kDa phospho-form and then to the final 80-kDa mature form
that localized to the cytosol (4). Comparison with protein kinase A
suggested that one of the phosphorylation sites was a conserved Thr on
the activation loop that is located near the entrance to the active
site; phosphorylation on this loop controls the function of a large
number of kinases (5). In support of this, replacement of the potential
phosphorylated Thr on the activation loop of protein kinase C Mass spectrometric analysis later established that protein kinase C The first phosphorylation event of protein kinase C Dephosphorylation studies implicate Thr-641 as being the key switch for
locking protein kinase C in a catalytically competent conformation; for
mature enzyme previously processed by phosphorylation, phosphate at this position is required and is sufficient for maximal catalysis (8, 10). However, mutagenesis studies have provided conflicting results on the role of this residue. Mutagenesis studies with protein kinase C Protein kinase C has also been shown to autophosphorylate by an
intramolecular reaction at a number of non-conserved residues in
vitro (21, 22). One such residue in protein kinase C This study explores the role of phosphorylation of Thr-641 in
regulating protein kinase C Bovine brain L- Mutagenesis--
Expression vectors encoding the cDNA
sequence of protein kinase C Expression of Protein Kinase C in COS-7 Cells and Subcellular
Fractionation--
DNA (5-10 µg per 10-cm plate) encoding wild-type
protein kinase C Expression of Mutant Protein Kinase Cs in Sf21
Cells--
Sf21 insect cells were infected with baculovirus
encoding wild-type protein kinase C Electrophoresis and Western Blots--
Aliquots of cell lysate,
cytosol, detergent-soluble supernatant (membrane), or
detergent-insoluble pellet from COS-7 cells expressing wild-type
protein kinase C Cell-free Translation--
cDNA encoding wild-type protein
kinase C Protein Kinase C Activity--
Protein kinase C activity in 3-5
µl of detergent-soluble fractions was assayed by measuring the rate
of phosphorylation of a synthetic peptide in the presence or absence of
brain phosphatidylserine, diacylglycerol, and Ca2+,
essentially as described (25). The reaction mixture (80 µl) contained
50 µM protein kinase C-selective peptide (see
"Materials and Methods") in 20 mM HEPES (pH 7.5 at
30 °C), 1 mM DTT, 0.1 mM
[ Immunocytochemistry--
COS-7 cells were grown to confluency on
glass coverslips and transfected with 1-2 µg of the indicated
protein kinase C Phosphorylation Site Mutant Mimicking Dephosphorylated
Thr-641--
A series of mutants at each of the two carboxyl-terminal
phosphorylation sites in protein kinase C
Initial studies focused on analyzing a protein kinase C mutant in which
Thr-641 (in vivo autophosphorylation site) and Thr-634 (in vitro autophosphorylation site) were mutated to Ala
(Fig. 1). However, biochemical analysis
revealed that this mutant did not mimic protein dephosphorylated on
Thr-641. First, the double mutant T634A/T641A displayed the same
electrophoretic mobility as wild-type enzyme. Because phosphorylation
of Thr-641 causes an approximately 2-kDa shift in electrophoretic
mobility (8), this result indicated that incorporation of a phosphate
at an additional (compensating) site was causing an equivalent mobility shift (8). Second, phosphatase treatment caused the same effects on the
T634A/T641A mutant and wild-type enzyme. Specifically, protein
phosphatase 1 caused a large electrophoretic mobility shift and
complete loss of activity; in wild-type enzyme, this large shift and
loss of activity result from dephosphorylation at two carboxyl-terminal
positions, Thr-641 and Ser-660. In addition, protein phosphatase 2A
caused a small electrophoretic mobility shift with no loss of activity;
in wild-type enzyme this smaller shift results from loss of phosphate
at one carboxyl-terminal position, Ser-660 (10). These data revealed
that mutation of position 634 and 641 to Ala resulted in a compensating
phosphorylation that mimicked the effect of phosphorylation of Thr-641
in all parameters tested. Analysis of the sequence around Thr-641
indicated a possible candidate for such a compensating phosphorylation, Ser-654. To eliminate any potential compensating phosphorylation at
residues near Thr-641, we constructed a triple mutant
T634A/T641A/S654A. This mutant is referred to as T641AAA
henceforth.
Lack of Negative Charge at Carboxyl-terminal Phosphorylation Sites
Results in Association of Protein Kinase C with the Detergent-insoluble
Cell Fraction--
Wild-type protein kinase C and four phosphorylation
site mutants, T641E, T641AAA, S660A, and S660E, were expressed in COS-7 cells, and their subcellular partitioning and electrophoretic mobilities were compared in Fig. 2. As
reported previously, wild-type enzyme from cell lysate migrated as two
major species (lane 1): the slowest migrating species
partitioned in the detergent-soluble fraction (lane 2, **)
and the fastest migrating species partitioned in the
detergent-insoluble fraction (lane 3,
Replacement of Thr-641 with Glu resulted in expression of protein whose
electrophoretic mobility and subcellular fractionation pattern was
similar to that of wild-type enzyme except that the fastest migrating
form was absent. Specifically, T641E in cell lysate migrated as two
species (lane 4), with the slowest species co-migrating with
fully phosphorylated wild-type enzyme (**) and, like mature wild-type
enzyme, partitioned in the detergent-soluble fraction (lane
5). The fastest migrating form had an apparent mass of 78 kDa (*),
co-migrating with wild-type enzyme containing a single phosphate on its
carboxyl terminus and partitioned in the detergent-insoluble fraction
(lane 6). These data reveal that Glu at position 641 mimics
phosphate in causing a decrease in the electrophoretic mobility of the enzyme.
The T641AAA mutant migrated as a single band corresponding to
completely dephosphorylated protein kinase C (
Expression of S660A in COS-7 cells resulted in the appearance of two
bands in the lysate. Fig. 2, lane 10, shows a major band co-migrating with dephosphorylated wild-type enzyme (
In summary, the T641E and S660E mutants did not contain species
co-migrating with the lowest 76-kDa band of wild-type enzyme, consistent with negative charge at either of these positions causing an
apparent 2 kDa in electrophoretic mobility. Both T641E and S660E
mutants contained species that co-migrated with the 78- and 80-kDa
bands of wild-type protein, representing forms with negative charge at
one (78 kDa) or both (80 kDa) carboxyl-terminal sites. The alanine
mutants, on the other hand, lacked species co-migrating with the
uppermost 80-kDa band, consistent with lack of negative charge at the
indicated phosphorylation site. Furthermore, the T641AAA mutant
migrated as only one band which corresponded to the fastest migrating
form of protein kinase C. Thus, the T641AAA was not phosphorylated on
Ser-660.
Lack of Negative Charge at Carboxyl-terminal Phosphorylation Sites
Does Not Cause Protein Insolubility--
The fractionation studies
above revealed that lack of negative charge at Thr-641 caused protein
kinase C to partition with the detergent-insoluble fraction of cells.
One possibility is that this insolubility reflects an intrinsic
property of the protein (i.e. misfolded and aggregated).
Alternatively, it could result from a specific interaction of the
dephosphorylated protein kinase C with its environment (i.e.
binding to component in detergent-insoluble fraction). To distinguish
between these possibilities, we asked whether dephosphorylated protein
kinase C was soluble when expressed in a cell-free in vitro
transcription/translation system. Protein kinase C expressed in this
system is not phosphorylated; it co-migrates with completely
dephosphorylated protein kinase C and is not recognized by
phosphorylation-specific antibodies (data not shown).
Wild-type protein kinase C and T641AAA were expressed in a cell-free
transcription/translation system, and the reaction mixtures were then
centrifuged at 100,000 × g for 20 min. Fig.
3 shows that both wild-type protein
kinase C (corresponding to dephosphorylated precursor enzyme;
lane 1) and T641AAA mutant (lane 3) remained in
the supernatant. Thus, differences in the milieu of where protein kinase C is expressed, rather than intrinsic properties of the protein,
appear to dictate its solubility. This suggests that the detergent
insolubility of the in vivo expressed proteins lacking phosphate at the carboxyl terminus results from interaction with cellular components and does not reflect an intrinsic property of the
isolated protein.
Requirement of Negative Charge at Position 641 for Catalytic
Function--
The effect of negative charge at position 641 on
catalytic activity of protein expressed in COS-7 cells was examined in
Fig. 4. For the data presented, the
activity contributed by endogenous protein kinase C Phorbol Ester-induced Membrane Translocation Depends on
Carboxyl-terminal Phosphorylation--
The effect of phorbol ester
treatment on the subcellular localization of wild-type and
phosphorylation site mutants of protein kinase C was examined both
biochemically and immunocytochemically. Fig.
5 shows the effect of PDBu treating COS-7
cells expressing wild-type protein kinase C or phosphorylation site
mutants on the partitioning of the enzyme in the cytosolic fraction
(S), the membrane fraction (M), or the
detergent-insoluble pellet fraction (P). As discussed above,
the mature wild-type enzyme partitions in the detergent-soluble
fraction (e.g. Fig. 2, lane 2). Further fractionation of the detergent-soluble fraction into the cytosolic and
membrane fractions revealed that, under the conditions of cell culture,
half the mature protein partitioned in the cytosol (Fig. 5, lane
1) and half was associated with the membrane (Fig. 5, lane
2). As observed in Fig. 2, faster migrating species of protein
kinase C partitioned in the detergent-insoluble fraction (lane
3). Phorbol ester treatment caused the wild-type protein kinase C
in the cytosolic fraction to redistribute to the membrane (lanes
4 and 5); in addition, a slight increase in the amount of partially phosphorylated/dephosphorylated species in the pellet was
observed (lane 6). In marked contrast, the T641AAA mutant was refractory to phorbol ester treatment. Although approximately 10%
of the protein was present in the cytosolic fraction, no significant change was noted upon phorbol ester treatment. The majority of the
protein was associated with the detergent-insoluble fraction, and this
distribution did not change upon phorbol ester treatment (compare
lanes 9 and 12).
Unlike the T641AAA mutant, the S660A mutant responded detectably to
phorbol ester treatment. In unstimulated cells, approximately half the
protein was in the cytosolic fraction (lane 13) and half in
the membrane fraction (lane 14). Phorbol ester treatment
caused protein kinase C to disappear from the cytosol and, for the
slower mobility species and also the membrane, redistribute to the
detergent-insoluble fraction (lane 18). The subcellular
partitioning of T641E and S660E, in the presence or absence of phorbol
esters, was indistinguishable from that of wild-type enzyme (data not shown).
The above biochemical studies revealed that the T641AAA and S660A
mutants exhibited different subcellular fractionation patterns from
wild-type protein kinase C
Fig. 6A shows that wild-type
protein kinase C
The immunostaining pattern of both T641AAA (Fig. 6D) and
S660A (Fig. 6G) differed from that of wild-type enzyme in
two aspects: staining of the plasma membrane was absent, and the
cytoplasmic staining appeared less diffuse and more punctate. The
punctate staining did not result from protein kinase C binding the
actin-based cytoskeleton, because the phalloidin stain was distinct
from that of the protein kinase C stain, and co-distribution was not
apparent in composite pictures (Figs. 6, F and
I). Although the protein kinase C staining pattern shared
some features in common with that of the endoplasmic reticulum
staining, composite analysis revealed only minimal co-distribution of
the two proteins (not shown). Thus, the phosphorylation site mutants
associated with a detergent-insoluble component that was neither
actin-based nor a result of association with the endoplasmic reticulum.
In response to PMA, wild-type protein kinase C redistributed to the
plasma membrane, as evidenced by the stronger staining of the cell
periphery (Fig. 6B). Phorbol ester treatment also promoted
changes in cell morphology, as evidenced by induction of filopodia and
actin distribution; no changes in the endoplasmic reticulum were
apparent (data not shown). Composite analysis suggested that the
altered distribution of protein kinase C resulting from PMA treatment
did not reflect association of the enzyme with actin or the endoplasmic reticulum.
PMA treatment of both T641AAA and S660A-expressing cells caused a
marked difference in the staining pattern of the protein kinase C
mutants (Fig. 6, E and H). Specifically, staining
became considerably more punctate for both the S660A and T641AAA
mutants. The punctate staining did not represent co-distribution of
protein kinase C with either actin or the endoplasmic reticulum (not
shown), as illustrated by composite analysis. COS-7 cells expressing
the phosphorylation site mutants did not undergo significant phorbol ester-dependent changes in cell morphology.
In summary, biochemical fractionation studies and immunocytochemical
analysis revealed that negative charge at the carboxyl-terminal phosphorylation sites is required for the cytosolic localization of
protein kinase C. Mutants lacking negative charge at these positions
associate with a detergent-insoluble fraction that does not appear to
be the endoplasmic reticulum or to be actin-based. Although phorbol
esters do not alter the detergent insolubility of these mutants, they
do cause the proteins to redistribute in this detergent-insoluble
fraction, suggesting that the phorbol-binding site of the mutants
remains exposed and able to bind ligand. However, the strength of the
interaction with the detergent-insoluble fraction dominates over the
binding energy provided by phorbol esters in recruiting protein kinase
C to the plasma membrane.
This study establishes that phosphorylation of Thr-641 in protein
kinase C This study also establishes that mutation of the key residue, Thr-641,
is not adequate to examine the role of its phosphorylation in protein
kinase C function because the enzyme compensates by incorporating
phosphate at adjacent residues that are functionally similar. This
finding underscores the need for careful analysis of phosphorylation
site mutants.
Carboxyl-terminal Phosphorylation Is Required for
Catalysis--
Analysis of phosphorylation site mutants reveals that
negative charge at position 641 is required for the catalytic
competence of protein kinase C. Replacing Thr-641 as well as potential
compensating phosphorylation sites with Ala results in expression of
protein that is inactive both in COS-7 cells (data not shown) and in
insect cells. This inactivity is unlikely to result from unusually high protein instability, since addition of protein stabilizers such as
glycerol are unable to promote activity. The finding that negative charge at Thr-641 is required for catalysis is consistent with data
from phosphatase sensitivity experiments which revealed that protein
phosphorylated at Thr-641, but not Thr-500 and Ser-660, is
catalytically active. In contrast, dephosphorylation at all three
positions inactivates protein kinase C (8, 10).
Parker and co-workers (20) recently mutated Thr-638 in protein kinase C
In protein kinase C
The crystal structure of protein kinase A reveals that its carboxyl
terminus is tethered to the small, ATP-binding lobe of the kinase core
by a phosphorylated residue, Ser-338 (27, 28); this residue corresponds
to that of Thr-641 in protein kinase C Lack of Negative Charge on Thr-641 Prevents Phosphorylation of
Ser-660--
Previously we showed that protein kinase C effectively
autophosphorylates on Ser-660 in vitro; specifically, pure
protein kinase C that has been quantitatively dephosphorylated on
Thr-500 and Ser-660 by treatment with protein phosphatase 2A
stoichiometrically re-autophosphorylates on Ser-660 upon addition of
MgATP (8, 10). The finding that the catalytically incompetent T641AAA mutant is not phosphorylated on Ser-660 in COS-7 cells indicates that
this modification is also an autophosphorylation in vivo. In
support of Ser-660 being an autophosphorylation event, other kinase-dead constructs of protein kinase C are also not phosphorylated at Ser-660.3
The inability of the T641AAA mutant to become phosphorylated on Ser-660
in vivo supports the hypothesis that phosphorylation of
Thr-641 precedes that of Ser-660 (8). This hypothesis was based on the
finding that Ser-660 is not phosphorylated in any of the partially
phosphorylated forms that associate with the detergent-insoluble
fraction, indicating that its phosphorylation coincides with release of
the fully phosphorylated enzyme into the cytosol.
Lack of Negative Charge at Carboxyl Terminus Tethers Protein
Kinase C to Detergent-insoluble Cell Fraction--
Subcellular
fractionation studies reveal that lack of negative charge at the
carboxyl-terminal phosphorylation positions causes protein kinase C to
associate with the detergent-insoluble cell fraction. Both non- or
partially phosphorylated forms of wild-type enzyme and Ala mutants
(T641AAA and S660A) partition with the detergent-insoluble cell
fraction. This insolubility is unlikely to reflect an intrinsic
property of the protein, because both unphosphorylated wild-type
protein and the T641AAA mutant are soluble when expressed in a
cell-free translation system. Rather, the insolubility appears to
result from a specific interaction with a detergent-insoluble cellular component.
One possibility to account for the detergent insolubility is that the
phosphorylation site mutants are retained in the endoplasmic reticulum;
for example, the detergent insolubility of methionyl-tRNA synthetase
arises from its tight association with the endoplasmic reticulum (31).
However, confocal images of COS-7 cells stained for T641AAA or S660A
and for the endoplasmic reticulum marker, calreticulin, suggest that
protein kinase C mutants are not retained in this subcellular
compartment. This suggests that the detergent-insoluble component is
cytoskeletal. One obvious candidate could be the actin-based
cytoskeleton; however, confocal microscopy indicated that protein
kinase C does not appear to co-distribute with actin. Consistent with
this, disruption of the actin-based cytoskeleton does not alter the
subcellular distribution of protein kinase C Phorbol Esters Do Not Release Cytoskeletal-bound Protein Kinase C
but Do Cause Soluble Protein Kinase C to Redistribute to the
Cytoskeleton--
As has been extensively documented over the past 15 years, phorbol ester treatment of cells causes a dramatic
redistribution of protein kinase C from the cytosol to the membrane
(32-34). This translocation arises because phorbol esters act as
molecular glue, altering the membrane affinity of protein kinase C by
many orders of magnitude (for example, 1 mol % PMA increases membrane
affinity of protein kinase C by 4 orders of magnitude (35)). The
structural basis for this effect results from phorbol esters capping a
hydrophilic ligand-binding pocket in the C1 domain, thus altering the
surface hydrophobicity of the domain such that its top third now
constitutes a continuous hydrophobic surface (36).
In marked contrast to its effects on wild-type protein kinase C
The phorbol ester-dependent relocation to the
detergent-insoluble fraction noted for S660A was also observed, to a
lesser extent, for wild-type enzyme. In this case, however,
redistribution to the cytoskeleton was accompanied by dephosphorylation
of the protein as assessed by its increased electrophoretic mobility. The latter observation is consistent with a report by Parker and co-workers (37) showing that phorbol ester treatment promotes the
dephosphorylation of protein kinase C
Phorbol esters did, however, affect the nature of the phosphorylation
site mutants' interaction with the cytoskeleton. Specifically, confocal microscopy studies revealed that phorbol esters caused the
staining of T641AAA and S660A to become more punctate. This suggests
that interaction of the protein with the detergent-insoluble fraction
is altered, without actually releasing the proteins from this compartment.
Phorbol ester treatment caused marked changes in the morphology of
untransfected COS-7 cells or ones transfected with functional protein
kinase C Conclusions--
Phosphorylation provides two key switches
in regulating protein kinase C: 1) transphosphorylation at the
activation loop, which likely structures the active site for catalysis,
and 2) autophosphorylation at the carboxyl terminus which locks the
enzyme in a catalytically competent conformation and dictates its
subcellular localization. The activation loop switch has been well
characterized in many kinases (5, 38). Now, the growing number of
reports of kinases phosphorylated on their carboxyl terminus suggests that the mechanism described for protein kinase C may also provide a
general regulatory switch for the kinase superfamily. For example, S6
kinase and protein kinase B (Akt kinase) both require carboxyl-terminal phosphorylation as part of their activation (39-41). Most striking, mutagenesis of Ser-371 to Ala (or Asp) in S6 kinase, corresponding to
Thr-641 in protein kinase C II. Mutation of this residue to Ala
results in compensating phosphorylations at adjacent sites, so that a
triple Ala mutant was required to address the function of phosphate at
Thr-641. Biochemical and immunolocalization analyses of phosphorylation
site mutants reveal that negative charge at this position is required
for the following: 1) to process catalytically competent protein kinase
C; 2) to allow autophosphorylation of Ser-660; 3) for cytosolic
localization of protein kinase C; and 4) to permit phorbol
ester-dependent membrane translocation. Thus, phosphorylation of Thr-641 in protein kinase C
II is essential for
both the catalytic function and correct subcellular localization of
protein kinase C. The conservation of this residue in every protein
kinase C isozyme, as well as other members of the kinase superfamily
such as protein kinase A, suggests that carboxyl-terminal phosphorylation serves as a key molecular switch for defining kinase function.
INTRODUCTION
Top
Abstract
Introduction
References
with
Ala, or of protein kinase C
II with Val, resulted in inactive enzyme (6, 7), whereas replacement with Glu in protein kinase C
II resulted
in activable enzyme (7).
and
II are phosphorylated at three positions in vivo (8):
in protein kinase C
II these correspond to Thr-500 on the activation
loop and Thr-641 and Ser-660 on the enzyme's carboxyl terminus (8, 9)
(see Fig. 1). These residues are conserved among all protein kinase C
isozymes, with the exception of two isozymes (protein kinase C
and
) which have a Glu at the position corresponding to Ser-660 (8).
Although phosphorylation on the activation loop is required to process
mature protein kinase C, phosphatase treatment of the mature enzyme
revealed that phosphate on Thr-500 is no longer required for activity
once Thr-641 has been phosphorylated. Specifically, kinase activity is
retained upon dephosphorylation of Thr-500 and Ser-660 but not Thr-641. In contrast, additional dephosphorylation of Thr-641 results in complete loss of activity (8, 10).
II, that of
Thr-500, is mediated by another protein kinase (8, 11), with recent
studies showing that the phosphoinositide-dependent protein
kinase, PDK-1, can mediate this phosphorylation both for conventional
(12) and atypical (13, 14) protein
kinase Cs. This phosphorylation is followed by rapid phosphorylations
at the two carboxyl-terminal positions, with in vivo data
suggesting that Thr-641 precedes phosphorylation of Ser-660 (8).
Mutagenesis of Ser-660 to Glu or Ala recently revealed that phosphate
at this position is not critical for function; rather, it structures
determinants in both the active site and regulatory region thereby
allowing tighter binding of both substrates and cofactors (15). Similar mutagenesis of Ser-657 in protein kinase C
showed that, in addition to increasing the stability of the protein, negative charge at this
position increases the phosphatase resistance of protein kinase C (16).
This residue is part of an unusual hydrophobic phosphorylation motif
(FXXF(S/T)(F/Y)) that is found in a number of other kinases
such as S6 kinase and protein kinase B (Akt kinase) (17, 18).
I suggested that phosphorylation here is essential for activity (19), whereas similar studies with protein kinase C
concluded that this residue is not essential for activity (20). The possibility of compensating phosphorylations was not taken
into account in these studies.
II is
Thr-634, which neighbors the in vivo site, Thr-641. It is
noteworthy that neither the in vivo nor in vitro
autophosphorylation sites conform to "consensus" phosphorylation
motifs for protein kinase C (23); for intramolecular reactions, the
high local concentration of substrate may ablate the necessity for
optimal substrate binding.
II. Analysis of phosphorylation site
mutants reveals that lack of negative charge at this position 1)
results in expression of inactive enzyme, 2) prevents
autophosphorylation at the second carboxyl-terminal site, 3) causes
association of the protein with the detergent-insoluble cell fraction,
and 4) prevents the enzyme from translocating to the plasma membrane in
response to phorbol esters. As part of this work, we discovered that
the mutation of both Thr-634 and Thr-641 to Ala results in a
compensating phosphorylation at a neighboring residue that mimics the
effect of phospho-Thr-641, underscoring the importance of negative
charge on the carboxyl terminus for the function of protein kinase C. Thus, this residue serves as a key molecular switch in regulating
protein kinase C. Its conservation in a number of other kinases
suggests that protein kinases are regulated by two phosphorylation
switch mechanisms: the well characterized phosphorylation of the
activation loop, and phosphorylation events at the carboxyl terminus
described in this report.
MATERIALS AND METHODS
-phosphatidylserine and
sn-1,2-dioleoylglycerol were purchased from Avanti Polar
Lipids, Inc. Dithiothreitol (DTT),1 HEPES, PDBu, PMA, and
ATP were from Sigma. [
-32P]ATP (3000 Ci
mmol
1) and [35S]methionine (1175 Ci
mmol
1) were from NEN Life Science Products, and calcium
chloride (analytical grade) was from J. T. Baker, Inc.
Peroxidase-conjugated goat anti-rabbit antibodies and bovine serum
albumin were obtained from Boehringer Mannheim. Antibodies to protein
kinase C were purchased from Santa Cruz Biotechnologies (
II
carboxyl-terminal antibodies) and Transduction Laboratories (
-hinge
antibodies; recognize protein kinase C
and
II).
Chemiluminescence SuperSignal substrates were from Pierce. Fluorescein
isothiocyanate- or Texas Red-conjugated antibodies were from Molecular
Probes. Lipofectin reagent was purchased from Life Technologies, Inc.,
and BaculoGold DNA was from PharMingen. In vitro
transcription/translation kit (TNT) was purchased from Promega. A
protein kinase C-selective peptide
(Ac-FKKSFKL-NH2 (24)) was synthesized by Dr.
Elizabeth Komives, University of California, San Diego. All other
chemicals were reagent grade.
II with mutation of Ser-660 to Ala or
Glu were made as described previously (15). The T634A/T641A double
mutant in pBluescript and in baculovirus was a generous gift of Drs.
A. J. Flint and D. E. Koshland, Jr. The T641E and
T634A/T641A/S654A mutants were made by polymerase chain reaction using
wild-type protein kinase C
II or the Thr-634/Thr-641 mutant,
respectively, in the pBlueScript vector (pBluePKC) as the template.
The sense primer used for both mutants was GGAGCATGCATTTTTCCG
and contained an NsiI restriction site. Antisense
primers corresponding to the sequence around the codon for Thr-641 or
Ser-654 and containing the necessary nucleic acid changes to encode the
desired mutation were ATCCTTCGAATTCTGCTTGGTCAATATTCCTG
(S654A mutation) and
ATCCTTCGAATTCTGATTGGTCAATATTCCTGATGACTTCCTGGTCAGGAGGTTCTAGGACTGGTGG (T641E mutation). These antisense primers contained a
BstBI restriction site. The polymerase chain reaction
product and the pBluePKC template were digested with NsiI
and BstBI (unique sites in pBluePKC), and the products were
gel-purified and ligated together. The mutant protein kinase C gene was
then subcloned into pcDNA3 (for mammalian expression) using
XhoI/XbaI or into the pVL1393 baculovirus
transfer vector (Invitrogen) as described (15). The sequence of all
mutants was verified by DNA sequencing.
II or its mutants was introduced into COS-7 cells
by calcium-phosphate transfection. Cells were lysed by sonication in 50 mM HEPES (pH 7.4), 1 mM EDTA, 1 mM
EGTA, 1 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride, 85 µM leupeptin, 2 mM benzamidine,
and 100 nM microcystin (lysis buffer). A portion of the
lysate was retained, and the remainder was centrifuged at 100,000 × g for 20 min at 4 °C. The supernatant (cytosol) was
saved, and the pellet was resuspended in lysis buffer containing 1%
Triton X-100. The resuspended pellet was centrifuged (100,000 × g, 20 min, 4 °C), and the supernatant (membrane) and
pellet (detergent-insoluble) fractions were saved. All fractions were
diluted 2-fold in glycerol and stored at
20 °C. For some
experiments, cells were treated with 200 nM PDBu for 20 min
prior to cell lysis.
II or its mutants, and the
detergent-soluble supernatants were analyzed for protein kinase C
activity as described (15).
II or the phosphorylation site mutants were
analyzed by SDS-polyacrylamide gel electrophoresis (7%
polyacrylamide). The proteins were then transferred to polyvinylidene difluoride (Immobilon-P, Millipore) and probed with antibodies to the
carboxyl terminus of protein kinase C
II and peroxidase-conjugated secondary antibodies. Labeling was detected using chemiluminescence.
II (50 ng) or the T641AAA mutant (1 µg) in pcDNA3
were incubated with 20 µl of the cell-free transcription/translation
lysate provided by Promega, in the presence of 0.4 µM
[35S]methionine (1175 Ci mmol
1) following
the manufacturer's protocol. After 90 min incubation at 30 °C,
lysates were centrifuged at 100,000 × g for 20 min at 4 °C, and the resulting supernatant and pellet were analyzed by SDS-polyacrylamide gel electrophoresis followed by detection of radioactivity using a Bio-Rad Imager or autoradiography.
-32P]ATP (0.1 µCi nmol
1), 5 mM MgCl2, and either 0.5 mM
Ca2+ and lipid (sonicated dispersion of brain
phosphatidylserine (140 µM) and diacylglycerol (3.8 µM), prepared as described (21)) or 0.5 mM
EGTA. Samples were incubated at 30 °C for 4-6 min and quenched by
the addition of 25 µl of a solution containing 0.1 M ATP
and 0.1 M EDTA (pH 8-9). Aliquots (85 µl) were spotted
on P81 ion-exchange chromatography paper and washed 4 times with 0.4%
(v/v) phosphoric acid, followed by a 95% ethanol rinse, and 32P incorporation was detected by liquid scintillation
counting in 5 ml of scintillation fluid (Biosafe II, Research Products International Corp.).
II plasmid cDNA expression constructs using the
calcium phosphate precipitation method. Cells were incubated with the
DNA for 16 h at 37 °C under 5% CO2, washed with
phosphate-buffered saline (PBS), and incubated for an additional
48 h in fresh growth medium (Dulbecco's modified Eagle's medium,
10% fetal calf serum, 1% penicillin/streptomycin). In some cases,
cells were incubated with 100 nM PMA for 15 min at 37 °C
under 5% CO2. Cells were then washed twice with PBS, fixed
for 10 min in PBS containing 3.7% formaldehyde, washed with PBS, and
permeabilized for 1 min in ice-cold acetone. Cells were washed with PBS
and blocked for 30 min in PBS, 0.2% bovine serum albumin. Cells were
incubated for 1 h with primary antibody (1:500 dilution of mouse
anti-protein kinase C
/
, Transduction Laboratories; 1:100
dilution of rabbit anti-calreticulin, Affinity Bioreagents, Inc.) and
washed three times with PBS, 0.2% bovine serum albumin. The cells were
then incubated for 1 h with fluorescent secondary antibodies
(donkey anti-mouse Texas Red or donkey anti-rabbit Cy5, Jackson
Immunoresearch; fluorescein isothiocyanate-phalloidin, Molecular
Probes), followed by washing with PBS, 0.2% bovine serum albumin. The
coverslips were then mounted on glass slides using the Prolong system
(Molecular Probes), and indirect immunofluorescent staining was
detected in successive focal planes using a UV-visible laser-scanning
confocal microscope system (Bio-Rad).
RESULTS
II, Thr-641 and Ser-660, were constructed in order to assess how phosphorylation at these positions modulates the function of protein kinase C. Hydroxyl-containing residues were replaced with either Glu as a mimic
of phosphate (26) or the neutral, non-phosphorylatable residue, Ala.
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Fig. 1.
Schematic of protein kinase C showing
conserved carboxyl-terminal phosphorylation sites. Top,
cartoon representation of the primary structure of protein kinase C
showing ligand binding domains (C1 and C2) in
regulatory moiety, and ATP and substrate-binding moieties
(C3 and C4) in kinase core (reviewed in Ref. 2).
The three in vivo phosphorylation sites are indicated as
follows: Thr-500 in the activation loop and Thr-641 and Ser-660 in the
carboxyl terminus (protein kinase C II numbering) (8).
Bottom, alignment of carboxyl termini of protein kinase C
(PKC)
II,
, and
I, S6 kinase (S6K), and
protein kinase A (PKA) showing conserved phosphorylation
sites (black rectangles). Asterisks indicate
potential compensating phosphorylation sites in protein kinase C
II;
underlined residue with asterisk is in
vitro autophosphorylation site of protein kinase C
II (22).
Sequences shown are for rat protein kinase C
I and
II (43),
bovine
(44), rat S6 kinase (45), and mouse protein kinase A
(46).
). We have
previously established that the slower migrating form is quantitatively
phosphorylated at Thr-641 and Ser-660,2 and the
fastest migrating species is not phosphorylated at the carboxyl
terminus (8). A minor intermediate species was also visible (lane
3, *); previous analyses showed that this intermediate species is
phosphorylated at Thr-641 but not Ser-660 (8). Under the expression
conditions in Fig. 1, approximately 10% of the total protein kinase C
II migrated as the dephosphorylated form partitioning in the
detergent-insoluble fraction (
); however, this percentage depended on
protein expression levels, with increased expression typically
resulting in increased representation of the dephosphorylated
species.
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Fig. 2.
Negative charge at position 641 or 660 alters
electrophoretic mobility and subcellular partitioning of protein kinase
C II. Western blot of whole cell lysate
(L), detergent-soluble supernatant (S), and
detergent-insoluble pellet (P) from COS-7 cells expressing
wild-type protein kinase C
II (wt
II), T641E mutant,
T634A/T641A/S654A (T641AAA) mutant, S660A mutant, or S660E
mutant; blot was probed with a polyclonal antibody against protein
kinase C
II's carboxyl terminus that recognizes phosphorylated and
dephosphorylated protein kinase C with equal affinity. Lanes
1-6 and 13-15 contained sample from approximately
2 × 104 cells; lanes 7-12 contained
sample from approximately 4 × 104 cells. Double
asterisk indicates the position of protein kinase C with negative
charge at both carboxyl-terminal positions (Thr-641 and Ser-660);
single asterisk denotes the position of protein kinase C
with a single negative charge on the carboxyl-terminal phosphorylation
sites, and the dash indicates the position of protein kinase
C with no phosphates on the carboxyl terminus, as determined previously
by mass spectrometry (8). Note that the faint slower migrating band in
lane 7 is not protein kinase C
II and is not labeled by
other protein kinase C-specific antibodies; it is a protein endogenous
to COS cells whose cross-reactivity is apparent when higher amounts of
cell extract are loaded on gels (see text).
); this mutant partitioned equally in the detergent-soluble (lane 8) and
detergent-insoluble (lane 9) fractions. The lack of bands
co-migrating with partially phosphorylated species of protein kinase C
indicated that the mutant was not phosphorylated on Ser-660 nor at any
other position on the carboxyl terminus that could alter the
electrophoretic mobility of the protein. Note that a minor slower
migrating band is visible in the lysate (lane 7) and, to a
lesser extent, in the pellet (lane 9). This band is not
protein kinase C
II; first, it is present in control COS cells
transfected with pcDNA3 vector alone, and second, it is not labeled
with a different antibody that labels the kinase domain in a
phosphorylation-independent manner (data not shown). Because the Ala
mutants expressed less well than wild-type or Glu mutants, twice as
much cell extract of the former is loaded on gels.
) and a minor
band co-migrating with wild-type enzyme modified at only one
carboxyl-terminal position (*). Both species were present in the
detergent-soluble (lane 11) and detergent-insoluble
(lane 12) fractions. The S660E mutant also migrated as two
bands in the lysate (lane 13). However, the mobilities of
the bands were shifted by approximately 2 kDa compared with the Ala
mutant, so that the fastest migrating band co-migrated with wild-type
enzyme with one carboxyl-terminal phosphate (*) and the slowest
migrating band co-migrated with wild-type enzyme with two
carboxyl-terminal phosphates (**). Thus, Ala at position 660 mimicked
protein with no phosphate at that position, whereas Glu at that
position mimicked protein with phosphate at that position, as reported
previously based on baculovirus expression studies (15). In addition,
the ratios and subcellular fractionation patterns of the two species were comparable to that of wild-type enzyme; the upper band was the
major species and partitioned in the detergent-soluble fraction (lane 14) and lower band was the minor species and localized
to the detergent-insoluble fraction (lane 15).
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Fig. 3.
Precursor protein kinase C and T641AAA mutant
are soluble proteins when expressed in cell-free translation
system. Autoradiogram showing 35S-labeled wild-type
protein kinase C II and T641AAA mutant in the supernatant
(S; lanes 1 and 3) and pellet
(P; lanes 2 and 4) fractions following
high speed centrifugation of in vitro
transcription/translation reaction mixtures. Details are presented
under "Materials and Methods."
was subtracted
from the total activity; it accounted for 50% of the activity observed
when wild-type protein kinase C
II was transfected. When assayed in
the presence of saturating cofactor concentrations for wild-type enzyme
(see legend to Fig. 4), no significant activity of the T641AAA mutant
was detected. Similarly, no activity of the T641AAA mutant was detected when expressed in insect cells, where there is no endogenous protein kinase C, even in the presence of ATP and peptide concentrations 20-fold higher than the Km for these substrates
(data not shown). In insect cells, the lipid-dependent
activity of the T641E mutant was 55 ± 9% that of wild-type
enzyme; this reduced activity likely arose from thermal instability of
the enzyme, because addition of glycerol increased the activity of the
mutant to levels similar to those of wild-type enzyme. Glycerol,
however, was unable to promote the activity of the T641AAA mutant.
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Fig. 4.
Negative charge at position 641 is required
for catalytic activity. Protein kinase C activity in the
detergent-soluble supernatant from COS-7 cells expressing wild-type
protein kinase C II or T641AAA was measured in the presence of
Ca2+ (500 µM) and sonicated dispersions of
phosphatidylserine (140 µM) and diacylglycerol (3.8 µM). ATP and peptide concentrations were 0.1 mM and 50 µM, respectively. Data are
expressed as protein kinase C
II activity (total activity minus
endogenous protein kinase C
activity) divided by the amount of
protein kinase C
II based on Western blot analysis with protein
kinase C
II-specific antibodies (mean ± S.E. for one
representative experiment performed in triplicate).
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Fig. 5.
Negative charge at the carboxyl terminus
modulates the phorbol ester-dependent redistribution of
protein kinase C II. Western blot of
cytosol (S), membrane (M), and
detergent-insoluble pellet (P) from COS-7 cells expressing
wild-type protein kinase C
II, the T641AAA mutant, or the S660A
mutant; blot was probed as described in the legend to Fig. 2. Cells
were treated without (
) or with (+) 200 nM PDBu for 20 min at 37 °C prior to cell lysis, as described under "Materials
and Methods." Lanes 1-6 contained sample from
approximately 2 × 104 cells; lanes 7-18
contained sample from approximately 4 × 104 cells.
The barely visible slower migrating band in lanes 9 and
12 is not protein kinase C, as discussed in the legend to
Fig. 3.
II. Therefore, the intracellular locations of these mutants were analyzed immunocytochemically. COS-7
cells were transiently transfected with wild-type protein kinase C
II or phosphorylation site mutants, and the subcellular distribution
of the proteins was monitored immunocytochemically using a monoclonal
antibody that recognizes protein kinase C
and
II independently
of phosphorylation state.
II displayed diffuse cytoplasmic distribution and
staining of the plasma membrane, consistent with results of
fractionation studies. This staining was distinct from that observed
with the actin (Fig. 6C) or endoplasmic reticulum (not
shown) markers, and composite analysis revealed that protein kinase C
did not co-distribute with either the endoplasmic reticulum or the
actin-based cytoskeleton (Fig. 6C).
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Fig. 6.
Immunocytochemical analysis of wild-type
protein kinase C II and carboxyl-terminal
phosphorylation mutants. Confocal microscopy of COS-7 cells
transfected with wild-type protein kinase C
II (A-C),
S660A mutant (D-F), or T641AAA mutant
(G-I). Cells were treated without (A, C, D, F,
G, and I) or with (B, E, and H)
100 nM PDBu for 15 min prior to fixation, as described
under "Materials and Methods." Transfected cells were stained
immunocytochemically to visualize protein kinase C in all panels and
also with phalloidin to visualize F-actin (C, F, and
I). Fluorescent staining was imaged by laser scanning
confocal microscopy. The images shown are representative focal planes.
Co-staining of protein kinase C
II (red) and F-actin
(green) are shown as composite images in C, F,
and I. The data shown are representative from at least three
separate transfection experiments for each construct.
DISCUSSION
II is essential for both the catalytic function and correct
subcellular location of protein kinase C. Specifically, lack of
negative charge at this position results in expression of inactive
enzyme, incapable of phosphorylating at Ser-660, that displays a
different subcellular distribution and phorbol ester responsiveness
compared with wild-type enzyme.
, corresponding to Thr-641 in protein kinase C
II, to Ala and
reported that the protein retained significant activity. Based on our
results with protein kinase C
II, this activity could have resulted
from a compensating phosphorylation that partially mimicked the effect
of phosphate on Thr-638. Both the compensating phosphorylation sites
found in protein kinase C
II, Thr-634, and Ser-654, are conserved in
protein kinase C
. Consistent with this, the migration of the T638A
mutant on SDS-polyacrylamide gel electrophoresis did not appear to
differ detectably from that of fully phosphorylated wild-type protein kinase C
(20), suggesting the same number of carboxyl-terminal phosphorylations as present on wild-type enzyme.
I, mutation of Thr-642 to Ala, corresponding to
Thr-641 in protein kinase C
II, results in expression of inactive
protein that associates with the detergent-insoluble fraction (19),
consistent with the results reported here for the T641AAA mutant. For
the
I isozyme, the proposed compensating phosphorylation site,
Ser-654, is absent.
II (Fig. 1). Mutagenesis
studies established that negative charge at position 338 is required
for protein kinase A stability, presumably by strengthening the
interaction of the carboxyl terminus with the small lobe (29).
Molecular modeling suggests that phosphorylation of Thr-641 on protein
kinase C
II could also tether the carboxyl terminus away from the
active site, thus stabilizing the kinase core (30). In support of an
electrostatic contact mediated by phosphate at position 641, Glu, with
a single negative charge, is much less effective at allowing
activation. Thus, a weaker potential electrostatic contact formed by
Glu at position 641 compared with phospho-Thr, may not structure the
core adequately for maximal catalysis. This would account for the
decreased thermal stability of Glu mutations at this position both in
protein kinase C
II (above) and protein kinase C
(20).
II in COS-7
cells.4 Despite eliminating potential sites for
sequestration of the kinase, the nature and mechanism of the
cytoskeletal tether for non- or partially phosphorylated protein kinase
C remain to be determined.
II,
fractionation studies revealed that phorbol esters do not cause the
carboxyl-terminal phosphorylation site mutants, T641AAA and S660A, to
associate with the membrane. In unstimulated cells, the T641AAA mutant
is primarily associated with the detergent-insoluble fraction and
phorbol ester treatment has no significant effect on this fractionation
pattern. Interestingly, a significant fraction of the S660A mutant is
soluble (present both in cytosolic and membrane fraction), yet phorbol
ester treatment promotes the association of this soluble protein with
the detergent-insoluble pellet. Thus, at the time point measured,
phorbol ester treatment did not recruit the phosphorylation site
mutants to the membrane. Rather, for the mutant that displayed some
solubility, the effect of phorbol esters was to target the protein to
the detergent-insoluble fraction.
. Our results indicate that
this dephosphorylation is accompanied by targeting of the kinase to the
detergent-insoluble fraction. Thus, dephosphorylation of the carboxyl
terminus may regulate the binding of protein kinase C to a cytoskeletal
component. Wild-type enzyme appears to be first targeted to the
membrane in response to phorbol esters, followed by dephosphorylation
and association with the detergent-insoluble fraction. The S660A mutant
may also first translocate to the membrane; however, increased
phosphatase sensitivity (16) could have resulted in a much more rapid
redistribution to the detergent-insoluble fraction such that membrane
translocation was not detected in our experiments.
II. Curiously, these changes were not apparent in cells
transfected with the inactive phosphorylation site mutants (e.g. T641AAA), suggesting that these inactive protein
kinase Cs may be acting as dominant negatives in preventing protein
kinase C-mediated cell morphological changes.
II, abolishes activity in this kinase
also (42). In addition, the archetypal kinase, protein kinase A
requires carboxyl-terminal phosphorylation to stabilize the kinase
(29). Thus, carboxyl-terminal phosphorylation may provide an
electrostatic anchor that structures the kinase and, in addition,
alters the surface of the kinase to promote or disrupt protein-protein interactions.
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ACKNOWLEDGEMENTS |
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We thank Andrew Flint and Daniel E. Koshland, Jr., for the construct of the T634A/T641A mutant; Erica Dutil for helpful discussions; and Lorene Langeberg for assistance with confocal microscopy and imaging.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM 43154 (to A. C. N.) and GM 48231 (to J. D. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 619-534-4527; Fax: 619-534-6020; E-mail: anewton{at}ucsd.edu.
3 A. Behn-Krappa and A. C. Newton, unpublished data.
4 E. M. Dutil and A. C. Newton, manuscript in preparation.
2 This species is approximately 60% phosphorylated on Thr-500; negative charge at this position does not alter the protein's electrophoretic mobility (8).
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ABBREVIATIONS |
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The abbreviations used are: DTT, dithiothreitol; PDBu, phorbol dibutyrate; PMA, phorbol myristate acetate; PBS, phosphate-buffered saline.
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REFERENCES |
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