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(PKC-) ATP Binding Mutant
ENZYMATIC ACTIVITY (*)
(Received for publication, December 16, 1994; and in revised form, January 13, 1995)
From the
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
To investigate the function of protein kinase C (PKC)-, we
mutated its ATP binding site by converting the invariant lysine in the
catalytic domain (amino acid 376) to an arginine. Expression vectors
containing wild type and mutant PKC- cDNAs were generated either
with or without an influenza virus hemagglutinin epitope tag. After
expression in 32D cells by transfection, the PKC- ATP binding
mutant (PKC-K376R) was not able to phosphorylate itself or the
PKC- pseudosubstrate region-derived substrate, indicating that
PKC-K376R was an inactive enzyme. PKC activity was inhibited by
67% in 32D cells coexpressing both PKC- wild type (PKC-WT)
and PKC-K376R when compared to 32D cells expressing only
PKC-WT. Mixture of PKC-WT and PKC-K376R kinase sources in vitro also reduced the enzymatic activity of PKC-WT.
These results suggest that PKC-K376R competes with PKC-WT and
inhibits PKC-WT phosphorylation of its in vitro substrate. While PKC-WT overexpressed in 32D cells
demonstrated 12-O-tetradecanoylphorbol-13-acetate
(TPA)-dependent translocation from the cytosolic to the membrane
fraction, PKC-K376R was exclusively localized in the membrane
fraction even prior to TPA stimulation. Unlike PKC-WT which was
phosphorylated on tyrosine residue(s) only after TPA treatment,
PKC-K376R was constitutively phosphorylated on tyrosine
residue(s). Although exposure of PKC-WT transfectants to TPA
induced 32D monocytic differentiation, the 32D/PKC-K376R
transfectants were resistant to TPA-induced differentiation. Thus,
expression of active PKC- is required to mediate 32D monocytic
differentiation in response to TPA stimulation.
Protein kinase C (PKC)- (
)is a serine/threonine
kinase which belongs to a novel subgroup of the PKC isoenzyme family
comprised of PKC-, PKC-
, PKC-
, and PKC-
(1, 2) . The lack of C2 domains in their regulatory
region distinguishes this subgroup from conventional PKCs that are
dependent on Ca
for enzymatic activation. We recently
demonstrated that PKC- was one of two PKC isoenzymes whose
expression could mediate 12-O-tetradecanoylphorbol-13-acetate
(TPA)-induced monocytic differentiation of 32D cells(3) . The
32D line is an interleukin-3-dependent myeloid progenitor line which
can differentiate to macrophages or neutrophils when appropriate
differentiation signaling pathways are
activated(4, 5, 6) . The 32D line
endogenously expresses low levels of PKC-, -, and
-
(3) . When different PKCs (, 
, , ,
, and 
) were individually transfected into 32D cells, their
overexpression did not abrogate factor dependence. However, PKC-
and PKC- transfectants underwent monocytic differentiation when
exposed to TPA, whereas the other transfectants and parental 32D cells
did not(3) . These results indicate that monocytic
differentiation of these myeloid progenitor cells is positively
regulated by the activation of PKC- and PKC-.
Recently, we
cotransfected PKC- and platelet-derived growth factor-
receptor (PDGF-R) into 32D cells in order to investigate the
possible involvement of PKC- in the PDGF-R signaling pathway (7) . When 32D cells coexpressing PDGF-R and PKC-
were stimulated with PDGF-BB, PKC- was translocated from the
cytosol to the membrane and activated. Overnight incubation of these
cells with PDGF-BB induced monocytic differentiation, whereas 32D cells
expressing only PDGF-R did not undergo readily detectable
differentiation. Interestingly, PKC- was phosphorylated on
tyrosine in vivo in response to TPA or PDGF stimulation of the
appropriate 32D transfectants(7) . Tyrosine-phosphorylated
PKC- could be detected only in the membrane fraction, where
PKC- enzymatic activity was increased in response to agonist
stimulation(7) . When baculovirus-derived PKC- was
phosphorylated on tyrosine by different tyrosine kinases, its serine
kinase activity was also increased(8) . Therefore, tyrosine
phosphorylation of PKC- positively correlated with its activation (7, 8) . In the present study, we have mutated the
putative ATP binding site of PKC- at amino acid 376 by converting
lysine to arginine and analyzed the effects of expressing this mutant
in 32D cells.
, cDNA Expression Vectors, and Cell Lines was changed to arginine. The mutation was confirmed by
DNA sequencing. The mutant cDNA, designated PKC-K376R, was cloned
into the pLTR (3) and pCEV27 (9) expression vectors,
containing the gpt and neo selection markers,
respectively. Wild type mouse PKC- (PKC-WT) cDNA had been
previously cloned into pLTR and metallothionine expression vectors (3) . The metallothionine vector contains a neo selection marker.Utilizing the same mutagenesis method
mentioned above, the original stop codons in both PKC-WT and
PKC-K376R were replaced with an EcoRI restriction site
using the following mutation primer: 5`-TTCCTGGACATTAGAATTCTTAAGCTC-3`.
The mutation was confirmed by restriction enzyme analysis. The 5` ends
of PKC-WT and PKC-K376R were inserted with polylinker
sequence containing SalI restriction site
(5`-GATCCCTCGAGAAGCTTGTCGACA-3`). To establish pCEV-HA vector, an
111-base pair oligonucleotides containing three repeats of influenza
virus hemagglutinin epitope HA 1 sequence (3 
YPYDVPDYA) was
synthesized as described previously(10) . The 111-base pair DNA
was amplified by polymerase chain reaction using primers containing
restriction enzyme sites, EcoRI at the 5` end and SphI at the 3` end. The sequences of the primers are as
follows: 5`-CAGAATTCGCGGCCGCATCTTTTACCCATAC-3` and 5`-
AGCATGCGGCCGCACTGAGCAGCGTAATCTGGA-3`. The polymerase chain
reaction-amplified fragment was digested with EcoRI and cloned
into EcoRI and AscI sites of pCEV29 which is a
pCEV27-based vector containing additional cloning sites. (
)Afterwards, the triple stop codon was introduced into the
3` end of HA epitope sequence by inserting the oligonucleotides at the BstEII site of the vector, resulting in the HA epitope-tagged
expression vector designated pCEV-HA. The oligonucleotides carrying
triple stop codons (boldfaced) are as follows:
5`-GTGACGGCGCGCCTTGAATCGTAGATACTGAG-3` and
5`-GTCACCTCAGTATCTACGATTCAAGGCGCGCC-3`. The PKC-WT and
PKC-K376R were cloned into pCEV-HA by SalI at the 5` end
and EcoRI at the 3` end linked to the HA epitope in the
correct reading frame, generating pCEV-PKC-WT-HA and
pCEV-PKC-K376R-HA. The 32D cells were transfected with different
cDNA expression vectors using the electroporation procedure described
previously(3) . 32D cells and transfectants were cultured in
RPMI 1640 medium supplemented with 10% fetal calf serum and 5% WEHI-3B
conditioned medium as a source of murine interleukin-3(6) .
peptide serum (4 µl/sample,
Calbiochemical Inc.) together with 40 µl of protein G-coupled
Sepharose (Pharmacia Biotech Inc.), 25 µg of anti-phosphotyrosine
(anti-pTyr) monoclonal antibody (mAb) covalently linked to agarose
beads (UBI) or anti-HA antibody (6 µl of ascites/sample, 12CA5,
BAbCo) together with protein A-Sepharose beads (25 µl, Pharmacia).
Anti-pTyr (2 µg/ml, UBI), the affinity purified anti-PKC-
serum (1 µg/ml, Life Technologies, Inc./BRL), and anti-HA (1:1000)
were used for immunoblot analysis. 
I-Protein A was used
for autoradiography unless otherwise indicated. In some cases, the
picoBlue immunoscreening kit from Stratagene utilizing the alkaline
phosphatase detection system was used to visualize the bands. The
method for subcellular fractionation was described before(7) .
The intensities of the bands on the Immobilon membranes (I-protein A processed) were quantitated by using a
PhosphorImager analysis (Molecular Dynamics).
Autophosphorylation Assay and PKC
Activity Assay
autophosphorylation assay was performed by following a previously
described protocol with some minor changes(11) . Briefly, the
cell lysates were immunoprecipitated with anti-PKC- serum or
anti-HA mAb. The washed immunoprecipitates were incubated on ice for 30
min with 50 µl of autophosphorylation buffer which contained 20
mM Tris-HCl, pH 7.5, 5 mM magnesium acetate, 50
µg/ml phosphatidylserine, 100 ng/ml TPA, 10 µg/ml leupeptin, 1
mM Na
VO
, 1 µM cold ATP,
and 5 µCi of [
-
P]ATP (3000 Ci/mmol,
Amersham). The reaction was stopped by washing twice with Triton X-100
containing lysis buffer(7) , and denatured proteins were
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). The procedures for enrichment of PKC- from cell
lysates by DE52 ion exchange chromatography (Bio-Rad) and the
subsequent PKC activity assay using a PKC- pseudosubstrate
region-derived peptide (MNRRGSIKQAKI; Peptide Technologies Corp.) as
the substrate have been reported(3, 7, 8) .
Briefly, cell lysates containing equal amounts of proteins were first
enriched for PKC by one-step DE52 ion exchange chromatography according
to the protocol supplied by the company (Life Technologies, Inc./BRL).
Then, 10 µl of DE52 column eluates were added on ice to 40 µl
of assay mixture which contained 10 µM PKC- substrate
peptide, 20 mM Tris-HCl, pH 7.5, 1 mM CaCl, 10 mM magnesium acetate, 1 µM TPA, 50 µg/ml phosphatidylserine, 0.1 mM cold ATP,
and 1 µCi of [-P]ATP. The reactions
were incubated at 30 °C for 30 min, and subsequently 25 µl of
each reaction was spotted onto phosphocellulose disk sheets (Life
Technologies, Inc./BRL). The sheets were washed twice with 1%
phosphoric acid, twice with distilled water, and samples were analyzed
by liquid scintillation. The nonspecific catalytic activity was
measured as above except that TPA and phosphatidylserine were omitted
from the reaction. The specific PKC activity was calculated by
subtracting the nonspecific catalytic activity from the total catalytic
activity and expressed either as counts/min 10/mg
protein/min or as direct counts/min in the mixture experiment described
below. In the mixture experiment, 5 µl of DE52 eluates from either
32D cells or the 32D/K376R1 transfectant were mixed with 5 µl
of eluates from the 32D/WT2 transfectant to demonstrate the
possible inhibitory effect of PKC-K376R on PKC-WT activity.
RII,
or isotype matched controls as described
previously(3, 7) . Cells incubated with anti-Mac-2 for
30 min were washed and incubated with phycoerytherin-conjugated goat
anti-rat IgG. The cells were subjected to flow cytometry using a
Becton-Dickinson FACScan.
and Expression of this Mutant in 32D Cells functions in 32D monocytic
differentiation, an ATP binding mutant of PKC- was constructed by
introducing a point mutation resulting in the conversion of a lysine to
an arginine at amino acid 376 in the catalytic domain of the enzyme.
The mutant cDNA, designated PKC-K376R, was inserted into two
LTR-driven expression vectors, pLTR (3) and pCEV27(9) .
Cells transfected with PKC-K376R inserted into the pLTR vector
were designated 32D/K376R1, and those in the pCEV27 vector were
termed 32D/K376R2. LTR- and metallothionine-driven expression
vectors containing PKC-WT cDNA were previously introduced into 32D
cells and termed 32D/WT1 and 32D/WT2,
respectively(3) . The 32D/WT2 line was transfected with
PKC-K376R in the pLTR vector, generating a cotransfectant
designated 32D/K376R1+WT2. The levels of PKC-WT and
PKC-K376R expressed in the various transfectants were determined
by immunoblot analysis utilizing anti-PKC- serum (Fig. 1).
The 82-kDa PKC--specific protein in 32D/K376R1 and
32D/K376R2 transfectants was increased 4.0- and 1.8-fold,
respectively, when compared to endogenous PKC- present in 32D
cells (Fig. 1). The 32D/WT1 and 32D/WT2 lines
expressed 19.6- and 6.5-fold more PKC-, respectively, than the
parental line (Fig. 1). 32D/K376R1+WT2 expressed
a combined 11.0-fold increase in PKC- protein levels compared to
the parental line (Fig. 1). The 32D cells were also transfected
with pCEV-PKC-WT-HA and pCEV-PKC-K376R-HA, resulting in
32D/WT-HA and 32D/K376R-HA transfectants, respectively.
Expression of HA epitope-tagged PKC-WT and PKC-K376R from
32D/WT-HA and 32D/K376R-HA transfectants were also detected
by using anti-PKC- immunoprecipitation followed by anti-HA
immunoblot analysis (see Fig. 3B).
Figure 1:
The PKC-K376R protein is
expressed in 32D transfectants. 32D cells and transfectants were
untreated or stimulated with 100 ng/ml TPA and lysed. Equal amounts of
cell lysates (150 µg/lane) were denatured and proteins were
resolved by SDS-PAGE, and transferred proteins were immunoblotted (Blot) with anti-PKC- serum as described under
``Experimental Procedures.'' Markers are shown in
kDa.
Figure 3:
The
PKC-K376R protein is exclusively localized in the membrane
fraction in the 32D transfectants. 32D cells or transfectants were
untreated or stimulated with TPA. The membrane fraction was separated
from the cytosolic fraction according a previously established
method(7) . A, equal amounts (100 µg/lane) of
membrane (P100) and cytosolic (S100) proteins were resolved by SDS-PAGE
and transferred proteins were immunoblotted (Blot) with
anti-PKC- serum. B, equal amounts (2 mg/lane) of membrane
(P100) and cytosolic (S100) proteins were immunoprecipitated (IP) with anti-PKC- serum. The transferred proteins were
immunoblotted with anti-HA mAb using the alkaline phosphatase detection
system. The mature PKC-WT or PKC-K376R is indicated by a bracket. Markers are shown in kDa.
K376R Is Abolished in
Vitro.
As shown in Fig. 2A, the level of PKC-
autophosphorylation observed in the anti-PKC- immunoprecipitates
from 32D/K376R1 lysates was the same as that detected in parental
cells, suggesting that PKC-K376R had lost its autophosphorylation
capacity. Similarly, the 32D/K376R1+WT2 cotransfectant
had the same level of autophosphorylation as that of the 32D/WT2
transfectant, further implying that autophosphorylation by the mutant
was abolished. Phosphoproteins of 160, 115, 73, 69, 50, 44, and 32 kDa
were also detected after in vitro autophosphorylation of
immunoprecipitates from the 32D/WT2 transfectant (Fig. 2A). Some, but not all, of these proteins were
detected in immunoprecipitates from 32D/K376R1+WT2
cotransfectant but not from 32D/K376R1 transfectant or 32D cells.
Figure 2:
In
vitro autophosphorylation by PKC-K376R is abolished. A,
cell lysates (4 mg/lane) were immunoprecipitated (IP) with
anti-PKC- serum and the immunoprecipitates were subjected to an in vitro autophosphorylation assay (see ``Experimental
Procedures''). Radiolabeled proteins were resolved by SDS-PAGE and
autoradiographed. B, cell lysates (6 mg/lane) were
immunoprecipitated with anti-HA mAb and subjected to an in vitro autophosphorylation assay. Radiolabeled proteins were resolved by
SDS-PAGE and autoradiographed. Markers are shown in
kDa.
We cloned both PKC-WT and PKC-K376 into pCEV-HA, an
expression vector containing 3 HA repeats which serve as an antigenic
epitope for anti-HA antibody recognition (12) to further
confirm that PKC-K376R had lost autophosphorylation capacity. The
use of the HA-tagged constructs also allowed us to exclude the
possibilities that PKC-K376R was transphosphorylated by endogenous
or overexpressed PKC- or that PKC-K376R could coprecipitate a
serine/threonine or tyrosine kinase, leading to the
transphosphorylation of PKC-K376R in the autophosphorylation assay
described above (Fig. 2A). Therefore, the
32D/WT-HA and 32D/K376R-HA transfectants were subjected to an in vitro autophosphorylation assay utilizing the anti-HA mAb
for immunoprecipitation. As shown in Fig. 2B, the
anti-HA immunoprecipitates from 32D/WT-HA lysates were strongly
phosphorylated, resulting in the detection of a 90-kDa protein which
corresponded to the expected size of PKC-WT-HA. Phosphorylation of
endogenous PKC- from 32D cell immunoprecipitates was not observed
because untagged endogenous PKC- was not recognized by the anti-HA
mAb. Importantly, no phosphoproteins were detected in anti-HA
immunoprecipitates from 32D/K376R-HA lysates, although the
expression of PKC-K376R-HA was easily detected by anti-HA
immunoblot analysis (data not shown) or by anti-PKC-
immunoprecipitation followed by anti-HA immunoblot analysis (see Fig. 3B). Interestingly, other phosphoproteins in the
size range of 190, 125, 102, 73, 55, 50, and 38 kDa were specifically
detected from 32D/WT-HA immunoprecipitates but not from
32D/K376R-HA immunoprecipitates. Some proteins with similar
mobilities were also detected from the 32D/WT2 transfectant in the
autophosphorylation assay (see Fig. 2A). The expression
levels of these additional proteins were too low to be detected by
Coomassie Blue staining (data not shown), so we were unable to
determine if these proteins were also coprecipitated with the
PKC-K376R. However, since two different antibodies detect
phosphoproteins of similar size only from the PKC-WT transfectants
but not from the PKC-K376R transfectants, this suggests that they
may be substrates of PKC-. Taken together, the results clearly
demonstrate that PKC-K376R has lost its autophosphorylation
capacity. If, indeed, the other phosphoproteins detected in the
autophosphorylation assay are substrates of PKC-, this indicates
that the PKC-K376R has also lost transphosphorylation ability.
K376R Is Enzymatically Inactive but
Competitively Inhibits PKC-WT Activity in VitroK376R was inactive and to determine if
expression of PKC-K376R would affect PKC-WT enzymatic
activity, we performed an in vitro PKC activity assay
utilizing the PKC- pseudosubstrate region-derived peptide as the
substrate. As shown in Table 1, PKC-enriched eluates from 32D
cells possessed low levels of activity presumably resulting from the
presence of endogenous PKCs, such as PKC-, -, and -
which are weakly expressed in 32D cells (see Fig. 1, (3) ). PKC activity was increased 6.0-fold in the eluates from
the 32D/WT2 transfectant. This increased activity correlated well
with the increased levels of PKC- protein expressed in this
transfected line (see Fig. 1). In contrast, PKC-enriched eluates
from 32D/K376R1 transfectant possessed slightly lower activity
when compared to 32D eluates, despite a 4.0-fold higher level of
PKC-K376R protein in the transfectant (see Fig. 1). This
result indicates that PKC-K376R is inactive in its ability to
transphosphorylate an exogenous substrate in vitro.
Interestingly, the enzymatic activity detected from the
32D/K376R1+WT2 cotransfectant accounted for only 33% of
that observed from the 32D/WT2 transfectant, although the
cotransfectant expressed roughly twice the amount of PKC-
expressed in 32D/WT2 and 32D/K376R1 single transfectants (see Fig. 1). Moreover, it is probable that the PKC-WT levels
expressed in 32D/K376R1+WT2 cotransfectant were
identical to those in 32D/WT2, since the
32D/K376R1+WT2 cotransfectant was generated by
transfecting 32D/WT2 cells with K376R1. This is also
suggested by the same level of autophosphorylation of PKC-WT in
these two transfectants (see Fig. 2A).
To further
investigate the possible competitive effect of PKC-K376R on
PKC-WT enzymatic activity, PKC-enriched eluates from both
32D/K376R1 and 32D/WT2 transfectants were mixed and measured
for PKC activity. As shown in Table 2, PKC-K376R1 possessed
lower activity than that detected from the eluates of 32D cells and the
activity detected from 32D/WT2 cells was 6.9-fold higher than that
from 32D cells, confirming results obtained in Table 1. The PKC
activity from a mixture of eluates from 32D/PKC-K376R1 and
32D/PKC-WT2 transfectants was reduced by 19% when compared with
the activity from 32D/PKC-WT2 transfectant. Coincubation of
eluates from 32D with those from 32D/PKC-WT2 did not inhibit
PKC-WT2 activity. The mixture experiments were performed several
times and inhibition of PKC-WT activity by PKC-K376R was
consistently observed, ranging from 19 to 25% (data not shown). Taken
together, these results strongly suggest that the PKC-K376R mutant
is not only an inactive enzyme, but also competitively inhibits
PKC-WT to phosphorylate its in vitro substrate.
K376R Is Exclusively Localized in the Membrane
FractionWT
was required for translocation from the cytosolic fraction (S100) to
the membrane fraction (P100), the subcellular location of
PKC-K376R was analyzed before and after TPA stimulation.
Surprisingly, immunoblot analysis with anti-PKC- serum revealed
that the PKC-K376R protein in the 32D/K376R1 transfectant was
detectable only in the membrane fraction even prior to TPA stimulation (Fig. 3A, lane 3 of P100 fraction). The
remaining PKC- detected in the cytosolic fraction (Fig. 3A, lane 3 of S100 fraction) represents
endogenous PKC- since equal amounts of protein were also detected
in this fraction from the parental line (Fig. 3A,
compare lanes 1 and 3 of the S100 fraction).
Similarly, the slight increase in membrane-bound PKC- in
32D/K376R1 transfectant after TPA stimulation reflects the
translocation of endogenous PKC- from the cytosol to the membrane (Fig. 3A, lane 4 of P100 fraction). The
exclusive membrane localization of PKC-K376R was also observed in
32D/K376R2 derived by transfection with a different expression
vector (Fig. 3A, lanes 9 and 10). In contrast,
more than 50% of the PKC-WT expressed in the two 32D/WT
transfectants remained localized in the cytosolic fraction even after
TPA stimulation (Fig. 3A, lanes 5-8).To
confirm the exclusive localization of PKC-K376R in the membrane
fraction, we also analyzed the HA epitope-tagged transfectants. As
demonstrated in Fig. 3B, PKC--specific proteins in
the 88-90 kDa size range were detectable in the membrane fraction
from both 32D/WT-HA and 32D/K376R-HA transfectants but not
from 32D parental cells. The PKC-WT-HA protein level was greatly
increased in the membrane fraction in response to TPA stimulation,
reflecting translocation of PKC-WT-HA from the cytosol to the
membrane (compare lanes 3 and 4 and lanes 9 and 10 in Fig. 3B). However, the
PKC-WT-HA protein (88 kDa) was also clearly detected in the
cytosolic fraction even after TPA stimulation (Fig. 3B, lane
10). In contrast, no PKC-K376R-HA protein was detectable in
the cytosolic fraction, supporting the original observation utilizing
the non-epitope-tagged expression vectors as shown in Fig. 3A. Therefore, the levels of PKC-K376R in the
membrane fraction detected from 32D/K376R-HA lysates in both
non-stimulated and TPA-stimulated lanes were similar to each other (Fig. 3B, lanes 5 and 6). Taken together,
these results clearly demonstrate that the 1 amino acid substitution in
PKC-K376R dramatically affects the subcellular localization of
this protein.
K376R Is Constitutively Phosphorylated on
Tyrosine in the Membrane Fraction was dependent on PKC-
activation and whether membrane localization of PKC- would always
coincide with tyrosine phosphorylation, tyrosine phosphorylation of
PKC- in the various transfectants was investigated. Lysates from
untreated and TPA-stimulated cells were immunoprecipitated with
anti-pTyr and subsequently immunoblotted with the same antibody. As
shown in Fig. 4A, an 82-kDa protein was phosphorylated
on tyrosine only after TPA treatment in both the 32D and 32D/WT2
transfectants, whereas the 82-kDa protein derived from the
32D/K376R1 transfectant was clearly phosphorylated on tyrosine
residue(s) independent of TPA stimulation. The identification of the
82-kDa proteins as PKC- or PKC-K376R was confirmed by
anti-pTyr immunoprecipitation followed by anti-PKC- immunoblot
analysis (Fig. 4B). Constitutive tyrosine
phosphorylation in untreated cells and increased tyrosine
phosphorylation in response to TPA were both observed in the
32D/K376R1+WT2 cotransfectant (Fig. 4, A and B). Constitutive tyrosine phosphorylation of
PKC-K376R compared to ligand-dependent tyrosine phosphorylation of
PKC-WT was also clearly observed when both 32D/WT-HA and
32D/K376R-HA transfectants were analyzed (Fig. 4C). Interestingly, additional proteins of 44 and
150 kDa were tyrosine phosphorylated in the parental cells after TPA
stimulation and their phosphorylation was greatly increased in the
PKC-WT but not in the PKC-K376R transfectant (Fig. 4A). Whether these proteins are tyrosine kinase
substrates whose phosphorylation is dependent on PKC- activation
remains to be determined.
Figure 4:
The PKC-K376R protein expressed in
32D cells is constitutively phosphorylated on tyrosine residue(s).
Cells were untreated or stimulated with TPA. A, equal amounts
of the proteins (4 mg/lane) were immunoprecipitated (IP) with
anti-pTyr and transferred proteins were immunoblotted (Blot)
with the same antibody. B, equal amounts of the proteins (4.5
mg/lane) were immunoprecipitated with anti-pTyr and transferred
proteins were immunoblotted with anti-PKC- serum. C, cell
lysates (4 mg/lane) were immunoprecipitated with anti-HA mAb.
Transferred proteins were immunoblotted with anti-pTyr mAb. The
alkaline phosphatase color reaction was used to visualize the bands. D, cells were fractionated according to a previously
established method(7) . Equal amounts (1 mg/lane) of membrane
or cytosolic (data not shown) proteins were immunoprecipitated with
anti-PKC- serum and transferred proteins were immunoblotted with
anti-pTyr. Markers are shown in kDa.
Subcellular fractionation followed by
immunoprecipitation with anti-PKC- and subsequent immunoblot
analysis with anti-pTyr revealed that tyrosine-phosphorylated
PKC-K376R and PKC-WT were exclusively detected in the
membrane fraction (Fig. 4D). The cytosolic fraction
contained no detectable tyrosine-phosphorylated PKC-WT or
PKC-K376R protein (data not shown). After normalizing for the
amounts of PKC- translocated to the membrane, the tyrosine
phosphorylation content of PKC-K376R1 was calculated to be
51.8-fold higher in untreated samples and 16.0-fold higher in
TPA-stimulated samples than that observed for PKC-WT1. Analysis of
PKC-K376R2 also revealed 51.3- and 12.1-fold higher tyrosine
phosphorylation content than that of PKC-WT1 in untreated and
stimulated cells, respectively.
K376R Transfectants Do Not Undergo Monocytic
Differentiation allowed TPA to mediate monocytic
differentiation of 32D cells(3, 7) , we analyzed the
ability of the PKC-K376R transfectant and cotransfectant to do so.
Parental and transfected cells were either untreated or exposed to TPA
overnight, and subjected to flow cytometric analysis to detect cell
surface markers indicative of monocytic differentiation. Overnight TPA
stimulation led to obvious increases in Mac-1 (Fig. 5A), Mac-2 (Fig. 5B), and
FcRII (Fig. 5C) expression on the surface of
32D/WT1 and 32D/WT2cells, whereas no increases were observed
in 32D/K376R1, 32D/K376R2 transfectants, and 32D cells. The
32D/K376R1+WT2 cells differentiated equally as well as
PKC-WT transfectants after TPA treatment. The 32D/WT-HA
transfectant also underwent TPA-dependent monocytic differentiation,
whereas the 32D/K376R-HA transfectant was resistant to TPA
induction (data not shown). These results indicate that expression of
sufficient levels of an active PKC- molecule is required to induce
32D monocytic differentiation.
Figure 5:
PKC-K376R expressed in 32D cells is
not able to mediate TPA-induced monocytic differentiation. Cells were
untreated (-) or exposed to TPA (
)
overnight and subjected to flow cytometry after incubation with
anti-Mac-1 (A), anti-Mac-2 (B), or anti-FcRII (C). The x axis represents the mean fluorescence
intensity (FL1 represents fluorescence of fluorescein
isothiocyanate, and FL2 represents fluorescence of
phycoerytherin) and y axis represents relative cell
number.
PKC has been found to be involved in many signaling pathways
which affect different cell functions(1, 2) . ATP
binding mutants of PKC-, PKC-, and PKC- have been
generated(13, 14, 15, 16) . The
PKC- mutant generated by Ohno et al.(13) was
found to be down-regulation insensitive, while the one established by
Pears and Parker (14) was down-regulation sensitive.
Down-regulation of PKC-K376R was similar to that of PKC-WT. ()The PKC- ATP binding mutant was shown to partially
inhibit NF-
B activity induced by the wild type enzyme, to inhibit
mitogenesis of fibroblasts, and to block oocyte
maturation(16) . In the present study, we report that
PKC-K376R lacked autophosphorylation capacity and was unable to
phosphorylate an exogenous substrate in vitro. Our data also
suggest that PKC- K376R partially inhibited PKC-WT enzymatic
activity in vitro. This mutant was found to be exclusively
localized in the membrane fraction. It was also constitutively
phosphorylated on tyrosine. PKC-K76R was not able to mediate 32D
cell monocytic differentiation when it was ectopically expressed in
these cells. Furthermore, the utilization of expression vectors
containing HA epitope-tagged PKC-WT and PKC-K376R cDNAs
conclusively confirmed that the mutant enzyme was inactive and
possessed properties described above.
It is thought that PKC is
translocated to the plasma membrane to form a quaternary structure with
diacylglycerol, phospholipid, and calcium and that formation of this
complex will activate the enzyme to phosphorylate its substrates and
transduce downstream signals(17, 18) . However, the
mechanism which mediates this phenomenon is still not clear. Recently,
an interesting model for PKC-II maturation and localization was
proposed by Newton and her colleagues (19) based on many in
vitro and in vivo studies. They suggest that PKC-II
is synthesized as an inactive precursor that is membrane-bound. The
precursor is then recognized and phosphorylated by a putative PKC
kinase which phosphorylates a threonine residue in the activation loop
residing in the C4 domain of the PKC-II molecule (20) .
This transphosphorylation activates PKC which then autophosphorylates a
threonine residue within its COOH terminus. This autophosphorylation
would presumably stimulate a phosphatase activity which would then
dephosphorylate PKC. This would decrease the enzyme's membrane
affinity so that it would be partitioned to the cytosol. This model has
been supported by two studies in which the autophosphorylation site
within the COOH terminus of PKC-I was mutated from a threonine to
an alanine or the transphosphorylation site of PKC-II within the
activation loop was mutated from a threonine to a glutamic acid,
valine, or an aspartic acid. In each case, the majority of the inactive
molecules was constitutively localized in the membrane
fraction(20, 21) .
Our data demonstrate that
PKC-K376R also does not reside in the cytosolic fraction (Fig. 3). Without autophosphorylation together with consequent
dephosphorylation, the previously described model would suggest that
the inactive molecule not receive the signal to partition to the
cytosol. In contrast to the mutant, the PKC-WT precursor molecule
would receive both phosphorylation and dephosphorylation signals so
that mature PKC-WT molecules would reside in the cytosolic
fraction (Fig. 3). When the cells would then be stimulated
exogenously by agents such as TPA or mitogens leading to increased
diacylglycerol production, mature PKC-WT would be recruited to the
membrane where phosphorylation and activation of PKC-WT would take
place. Thus, classical cytosol to membrane translocation of PKC-WT
would be observed. However, the activation of PKC- cannot be
permanently sustained. Therefore, a reoccurrance of the
dephosphorylation signal would relocalize the enzyme to the cytosol.
This idea is supported by the faster migration of PKC-WT in the
cytosolic fraction (88 kDa) compared to that in the membrane fraction
(90 kDa) in Fig. 3B (also Fig. 3A and (7) , Fig. 3). This would indicate that immediately
after dephosphorylation of PKC-WT in the membrane, the molecule
would repartition to the cytosol after fulfilling its function in the
membrane. Currently, we are generating a PKC- mutant where a
putative serine autophosphorylation site in the COOH terminus will be
replaced with an alanine to further investigate this model. We also
speculate that all inactive PKC mutants established up to date will be
localized exclusively in the membrane fraction as long as they lack
autophosphorylation capacity.
We recently reported that PKC-
became phosphorylated on tyrosine residue(s) in response to TPA or PDGF
stimulation in vivo(7) . Since PKC- activity was
increased after PKC- became tyrosine phosphorylated by several
different tyrosine kinases in vitro(8) , we predicted
that tyrosine phosphorylation may positively affect PKC- activity.
The results presented here demonstrate that PKC- tyrosine
phosphorylation is not dependent on PKC- activity, excluding the
possibility that the tyrosine kinase which phosphorylates PKC-
lies downstream of PKC- activation. The amount of
tyrosine-phosphorylated versus total protein was much greater
for PKC-K376R than PKC-WT, indicating that conformational
changes in PKC-K376R either unmask additional tyrosine
phosphorylation sites or allow greater access of the previously
observed tyrosine phosphorylation site(s) to an intracellular tyrosine
kinase. The exclusive localization of PKC-K376R in the membrane
fraction compared to the TPA-dependent translocation of only some
PKC-WT to the membrane would support the latter possibility
because only the membrane-bound PKC- is phosphorylated on
tyrosine. We are currently mapping the tyrosine phosphorylation site(s)
on both PKC-K376R and PKC-WT to determine the influence of
tyrosine phosphorylation on the enzymatic activity of PKC-WT and
on the translocation of both PKC-K376R and PKC-WT.
Nevertheless, since tyrosine-phosphorylated PKC-K376R was detected
only in the membrane fraction, this result further supports our
previous finding that tyrosine phosphorylation can be used as an
indication of translocation or membrane localization of PKC- .
In this report, we demonstrate that PKC-K376R can partially
inhibit enzymatic activity of PKC-WT based on the comparison of
PKC activity in the 32D/WT2 and 32D/K376R1+WT2
transfectants and on mixture experiments ( Table 1and Table 2). However, coexpression of PKC-K376R and PKC-WT
did not block PKC-WT-mediated monocytic differentiation in
vivo in response to TPA stimulation. It is possible that
PKC-K376R could exert its inhibitory effect by competitively
binding to substrates that normally associate with and are
phosphorylated by PKC-WT. Since it is likely that PKC-WT
expression was as high or higher than that of PKC-K376R in the
cotransfectant, this would probably allow the PKC-WT to
effectively compete for substrates in vivo. Moreover, not all
the PKC-WT activity in the in vitro assay was blocked by
PKC-K376R. Interestingly, just prior to submission of this
manuscript, another ATP binding mutant of PKC- (K376A) was
established by Hirai et al.(22) and demonstrated to
inhibit wild type PKC--induced AP-1 activity. Thus, both studies
provide biochemical evidence that an ATP binding mutant of PKC-
may act in a dominant negative fashion to block wild type enzyme
function. Future studies should determine whether PKC-K376R can
affect other biochemical and biological events mediated by PKC-WT
and whether expression of this mutant will have biological consequences
in other model systems. Further characterization of PKC-K376R
should also help elucidate the mechanisms of PKC- translocation
and tyrosine phosphorylation.
)R, platelet-derived growth factor- receptor; anti-pTyr,
anti-phosphotyrosine; mAb, monoclonal antibody; PAGE, polyacrylamide
gel electrophoresis; HA, hemagglutinin; LTR, long terminal repeat.))
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