Requirements of Protein Kinase C
for Catalytic Function
ROLE OF GLUTAMIC ACID 500 AND AUTOPHOSPHORYLATION ON SERINE
643*
Luise
Stempka,
Martina
Schnölzer,
Susanne
Radke,
Gabriele
Rincke,
Friedrich
Marks, and
Michael
Gschwendt
From the German Cancer Research Center,
D-69120 Heidelberg, Germany
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ABSTRACT |
Recently, we reported that, in contrast to
protein kinase C (PKC)
and
II, PKC
does not
require phosphorylation of a specific threonine (Thr505) in
the activation loop for catalytic competence (Stempka et al. (1997) J. Biol. Chem. 272, 6805-6811). Here, we show
that the acidic residue glutamic acid 500 (Glu500) in the
activation loop is important for the catalytic function of PKC
. A
Glu500 to valine mutant shows 76 and 73% reduced kinase
activity toward autophosphorylation and substrate phosphorylation,
respectively. With regard to thermal stability and inhibition by the
inhibitors Gö6976 and Gö6983 the mutant does not differ
from the wild type, indicating that the general conformation of the
molecule is not altered by the site-directed mutagenesis. Thus,
Glu500 in the activation loop of PKC
might take over at
least part of the role of the phosphate groups on Thr497
and Thr500 of PKC
and
II, respectively.
Accordingly, PKC
exhibits kinase activity and is able to
autophosphorylate probably without posttranslational modification.
Autophosphorylation of PKC
in vitro occurs on
Ser643, as demonstrated by matrix-assisted laser desorption
ionization mass spectrometry of tryptic peptides of autophosphorylated
PKC
wild type and mutants. A peptide containing this site is
phosphorylated also in vivo, i.e. in
recombinant PKC
purified from baculovirus-infected insect cells. A
Ser643 to alanine mutation indicates that
autophosphorylation of Ser643 is not essential for the
kinase activity of PKC
. Probably additional (auto)phosphorylation
site(s) exist that have not yet been identified.
 |
INTRODUCTION |
The PKC1 family (for
review, see Refs. 1-3) consists of 11 isoenzymes that, because of
structural and enzymatic differences, can be subdivided into three
groups: the Ca2+-dependent,
diacylglycerol-activated conventional PKCs (
,
1,
2, and
), the Ca2+-independent,
diacylglycerol-activated novel PKCs (
,
,
,
, and µ), and
the Ca2+-independent diacylglycerol-non-responsive atypical
PKCs (
and
/
). PKCµ is an novel PKC, but with some special
structural and enzymatic properties.
PKC
is one of the most thoroughly studied members of the novel PKC
subfamily. After the discovery of the enzyme in 1986 (4), its cloning
in 1987 (5), and its first purification to homogeneity in 1990 (6),
several groups have focused their interest on this PKC isoenzyme and
have reported on its structural and enzymatic properties (7-13),
regulation of expression (14-18), interaction with binding proteins
(19-24), specific substrate phosphorylation (25-27), and cellular
functions (28-36). Recently, the role of phosphorylation of PKC
,
i.e. either autophosphorylation or phosphorylation by an
exogenous protein kinase, for the regulation of its enzymatic activity
could, to some extent, be elucidated and compared with that of other
PKC isoforms. It was shown that, in contrast to PKC
(37) and
PKC
II (38), PKC
does not require phosphorylation of a
specific threonine (Thr505 corresponding to
Thr497 and Thr500 of PKC
and
II, respectively) in the activation loop for catalytic competence (39). PKC
wild type as well as a Thr505 to
alanine mutant were expressed in bacteria in a catalytically competent
form. Specific activities of these enzymes were comparable with that of
native PKC
from porcine spleen (39). Recent studies with a
Ser643 to alanine mutant of PKC
indicated that
phosphorylation of this residue might be required for enzymatic
activity (40). Ser643 was not unequivocally identified as
an autophosphorylation site of PKC
but corresponds to one of the
in vivo autophosphorylation sites of PKC
II,
i.e. Thr641 (41, 42). Finally, it was reported
by several groups that tyrosine phosphorylation affects the kinase
activity of PKC
and also other PKC isoforms in vitro and
in vivo (10, 43-47).
Here we demonstrate that glutamic acid 500 is important for the
catalytic function of PKC
and thus might take over at least part of
the role of the phosphate groups of Thr497 and
Thr500 of PKC
and
II, respectively. By
MALDI mass spectrometry of tryptic PKC
peptides we provide evidence
that Ser643 is an autophosphorylation site of PKC
in vitro and that a PKC
peptide containing
Ser643 is phosphorylated also in vivo.
 |
EXPERIMENTAL PROCEDURES |
Materials--
TPA was supplied by Dr. E. Hecker (German Cancer
Research Center). Gö6976 and Gö6983 were kindly provided by
Goedecke AG (Freiburg, Germany). The rat PKC
full-length
cDNA clone (3000 bp) containing the sequence coding for PKC
was
a gift from Dr. C. Polke (University of Würzburg, Würzburg,
Germany). The vectors pET28
Ala505 and pET28
Ala504/5 were provided
by Dr. D. Bossemeyer and Dr. A. Girod (German Cancer Research Center).
The pseudosubstrate-related peptide
(MNRRGSIKQAKI) was synthesized
by Dr. R. Pipkorn (German Cancer Research Center). Other materials were
bought from the following companies: PS from Sigma;
[
-32P]ATP (specific activity, 5000 Ci/mmol) from
Hartmann Analytic (Braunschweig, Germany); mouse monoclonal anti-PKC
antibody P36520 from Transduction Laboratories (Lexington, KY);
alkaline phosphatase-conjugated goat anti-mouse antibodies from Dianova
(Hamburg, Germany); expression vector pET28 and Escherichia
coli strain BL21(DE3) pLysS from AGS GmbH (Heidelberg, Germany);
T7-Sequencing kit and Mono-Q from Amersham Pharmacia Biotech; Pwo DNA
polymerase from Boehringer Mannheim; modified trypsin, sequencing
grade, from Promega; and nickel-nitrilo-triacetic acid resin from
Qiagen GmbH (Hilden, Germany).
Polymerase chain reaction Amplification and Cloning of Wild Type
and Mutant PKC
-cDNA--
As described previously (39) a PKC
full-length cDNA with an NdeI restriction site at the
initiation signal ATG and an EcoRI restriction site behind
the stop codon TGA was amplified by polymerase chain reaction and
cloned into the expression vector pET28. The plasmid was termed
pET28
wt.
Site-directed mutagenesis was performed using the "overlap
extension" method as described previously (39). A rat PKC
full-length clone of 3000 bp served as a template. The following pairs
of mutagenic oligonucleotides were used (only the sense
oligonucleotides are given, and changed bases are underlined):
(E/V)500, 5'-GAAT ATA TTT GGG GTG AAC CGG GCT*
AGC ACA TTC-3'; (S/A)643, 5'-GAG AAA CCC CAA CTT GCC TTC
AGT GAC AAG AAC C-3'; and (S/A)643(S/A)645, 5'-G AAA CCC CAA CTT
GCC TTC GCT GAC AAG AAC CTC-3' (synthesized by
Dr. W. Weinig, German Cancer Research Center). Successful mutation was
confirmed by sequencing and, in the case of (E/V)500, in addition by
introducing a new NheI restriction site that was created by a silent point mutation (see base with asterix). The mutant cDNAs were cloned into pET28 and were termed pET28
Val500,
pET28
Ala643, and pET28
Ala643/5.
Bacterial Expression and Partial Purification of Recombinant
His-tagged PKC
--
E. coli BL21(DE3)pLysS cells were
used for expression of the recombinant PKC
wt and mutants. Cells
were grown under conditions described previously (39). Washed and
sedimented cells were cracked by the method of freezing and thawing and
resuspended in ice-cold buffer (50 mM sodium phosphate, pH
8.0, 150 mM NaCl, 5 mM imidazole, 1% Triton
X-100, 10% glycerol, and protease inhibitors: phenylmethylsulfonyl
fluoride, aprotinin, leupeptin, and pepstatin). After sonication with a
Branson sonifier and centrifugation at 100,000 × g for
30 min at 4 °C, the supernatant was applied to an
nickel-nitrilo-triacetic acid column following the manufacturer's recommendation. Bound proteins, eluted with 50 mM and 100 mM imidazole, were pooled, diluted 1:5 with buffer (10 mM Tris-HCl, pH 7.5, and protease inhibitors), and further
purified by chromatography on a Mono-Q column. Elution of bound
proteins was achieved with NaCl (steps of 100, 200, 300, and 500 mM). The 200 mM NaCl fraction was selected,
because it contained PKC
with the highest specific activity.
According to Coomassie Blue staining a 75% purity of PKC
was
estimated. Eluted PKC
was stabilized by addition of 10% glycerol
and 10 mM
-mercaptoethanol and stored at
70 °C.
Expression of Recombinant PKC
Containing a C-terminal His Tag
in the Baculovirus-Insect Cell System--
For the cloning of PKC
full-length cDNA into the pBac1 baculovirus transfer plasmid
(Novagen), a PKC
cDNA with an EcoRI restriction site
at the initiation signal ATG and an XhoI restriction site
following the removed stop codon TGA was amplified by polymerase chain
reaction as described for the pET28
wt-plasmid (39). The oligonucleotides 5'-GAC GAA TTC ATG GCA CCG TTC-3' and 5'-GGA CCC TCG
AGT TCC AGG AAT TGC-3' were used as 5' and 3' primers, respectively.
The construction and amplification of recombinant baculovirus were
performed using the Bac Vector 2000 transfection kit (Novagen)
following the manufacturer's recommendation. Sf9 cells were
harvested after 65 h of infection with the recombinant baculovirus, and recombinant PKC
was partially purified as described above for the bacterially expressed enzyme. According to Coomassie Blue
staining, 80% purity of PKC
was estimated.
Protein Kinase Assay and
Autophosphorylation--
Phosphorylation reactions were carried out at
30 °C for 5 min as described previously (39). The
pseudosubstrate-related peptide
was used as a substrate.
32P-labeled phosphoproteins were visualized and quantitated
by measuring the intensity of photo-stimulated luminescence using a
Bio-Imaging Analyzer (Fuji Bas 1500).
Immunoblotting--
Proteins were separated by
SDS-polyacrylamide gel electrophoresis (7.5%). PKC
was detected and
quantitated by immunoblotting using the monoclonal anti-PKC
antibody
P36520 as the first antibody and an alkaline phosphatase-conjugated
second antibody. The blots were scanned using a scanner (MacIntosh),
and the values were expressed as arbitrary units relative to the background.
Enzymatic Digestion and MALDI Mass
Spectrometry--
Autophosphorylation of 10 µg of partially purified
PKC
wild type and mutants was performed as described above, but
[
-32P]ATP was omitted, and the reaction was terminated
by precipitation of the proteins with methanol-chloroform (49).
Proteins were separated by SDS-polyacrylamide gel electrophoresis
(7.5%, 0.75 mm). After staining with Coomassie R250 (Bio-Rad), the
PKC
bands were excised, cut into small pieces (1 × 1 mm),
washed, dehydrated (2 × 30 min with H2O, 3 × 15 min with 50% acetonitrile, and 1 × 15 min with acetonitrile),
and incubated with 2 µg of trypsin in 50 µl of digest buffer (50 mM NH4HCO3, pH 8.0) at 37 °C for 16 h. The digest was sonicated and centrifuged, and the
supernatant was subsequently analyzed by MALDI mass spectrometry using
the thin film preparation technique (50). Aliquots of 0.3 µl of a
saturated solution of
-cyano-4-hydroxycinnamic acid in acetone containing nitrocellulose were deposited onto individual spots on the
target. Subsequently, 1 µl of 10% formic acid and 0.5 µl of the
digest were loaded on top of the thin film spots and allowed to dry
slowly at ambient temperature. The spots were washed with 10% formic
acid and deionized water to remove salts.
MALDI mass spectra were recorded in the positive ion mode on a Reflex
II time-of-flight instrument (Bruker-Franzen, Bremen, Germany) equipped
with a SCOUT multiprobe inlet and a 337-nm nitrogen laser. Ion
acceleration voltage was set to 25 kV, and the reflector voltage was
26.5 kV. When using delayed extraction the first extraction plate was
set to 18.5 kV. Mass spectra were obtained by averaging 20-50
individual laser shots. Calibration of the spectra was performed externally by a two-point linear fit using angiotensin I and oxidized insulin
-chain.
 |
RESULTS AND DISCUSSION |
Phosphorylation of Thr497 and Thr500 in
the activation loop of PKC
(37) and PKC
II (38),
respectively, is known to be required for catalytic competence of the
enzymes. Recently, we demonstrated that PKC
exhibits full enzymatic
activity without phosphorylation of the corresponding
Thr505 (39). As previously discussed, a structural
difference in the activation loops of the isoenzymes could possibly
explain the differential behavior of PKC
to PKC
and
II. In contrast to PKC
and PKC
II,
PKC
contains a glutamic acid in position 500 (Glu500)
that might take over the role of the phosphorylated threonines of
PKC
and
II.
To test this possibility, we mutated Glu500 to the neutral
amino acid valine and determined the enzymatic activity of the
bacterially expressed PKC
Val500 mutant. All
phosphorylation assays were performed in the presence of PS and TPA.
Autophosphorylation of partially purified wild type and
PKC
Val500 mutant is shown in Fig.
1. Approximately equal amounts of the enzymes were applied to the assay. The values determined by
autoradiography were normalized according to the slight difference in
the amount of wild type and mutant enzyme (factor, 1.2) that was
observed by immunoblotting with an anti-PKC
antibody and scanning
the immunoblots (Fig. 1). Using different preparations of the enzymes in two independent experiments, one of which is shown in Fig. 1, a
76 ± 4% reduction in PKC
Val500
autophosphorylation activity, compared with the wild type, was observed. A similar decrease in enzymatic activity after mutation of
Glu500 to Val was seen when substrate phosphorylation by
wild type and mutant were compared. Phosphorylation of the
pseudosubstrate-related peptide
by PKC
Val500 was
reduced by 73 ± 2% compared with that by the wild type (Table I). The low incorporation of phosphate
into the peptide by PKC
Val500 was essentially
attributable to a slower velocity of the phosphorylation reaction.
Vmax of the PKC
Val500 catalyzed
reaction was reduced by 72% compared with Vmax
of the wild type, whereas the Km values for the
peptide substrate did not differ significantly (Table I). The
Km value for ATP, however, was approximately three
times higher with PKC
Val500 (116 µM) than
with the wild type (42 µM; Fig.
2 and Table I). Thus, binding of ATP
appeared to be impeded with mutation of Glu500 to Val. For
comparative purposes, some kinetic data of the partially purified
PKC
Ala505 and PKC
Ala504/505 mutants are
also shown in Table I. The kinase activities of these mutants did not
differ from the wild type, as reported previously (39). Taking the
autophosphorylation and substrate phosphorylation data together,
mutation of Glu500 to Val causes an ~75% reduction in
PKC
kinase activity. These results clearly support the idea that the
negatively charged carboxylate of Glu500 in the activation
loop is important for the kinase activity of PKC
and might function
in a similar way as the phosphate groups of Thr497 and
Thr500 in PKC
and
II, respectively, in
correctly aligning residues involved in catalysis by interaction with
positively charged residues of the catalytic core. However, the
residual activity of PKC
Val500 (~25%) indicates that,
in addition to Glu500, some as yet unknown structural
features of PKC
might be required for its catalytic function.
Several kinases are known that contain acidic residues such as Glu,
instead of phosphorylated residues, in the activation loop (51).
Moreover, catalytic competence of PKC
II could be
attained by mutation of Thr500 to Glu (38). No other PKC
isoenyzme contains a glutamic acid residue in a position corresponding
to position 500 of PKC
; however, aspartic acid is located in this
position in some isoforms (PKC
,
/
, and
). It is not yet
known whether these aspartic acid residues function similarly to the
glutamic acid 500 of PKC
. According to Orr and Newton (38), aspartic
acid may be less suited than glutamic acid for the electrostatic
interactions that cause correct alignment of catalytic residues.

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Fig. 1.
Autophosphorylation of PKC wild type
(wt) and PKC Val500 mutant
(Val500). Bacterial recombinant PKC wild type and
mutant were purified by metal chelate affinity and Mono-Q
chromatography (see "Experimental Procedures"). Subsequently, the
enzymes (0.3 µg of each protein) were phosphorylated at 30 °C for
5 min with [32P]ATP in the presence of PS and TPA. The
proteins were separated by SDS-polyacrylamide gel electrophoresis as
described under "Experimental Procedures." PKC
(arrow) was detected by immunoblotting and quantitated with
a scanner. Values are given as arbitrary units (a.u.).
Radiolabeled PKC was visualized by autoradiography and quantitated
by measuring the intensity of photostimulated luminescence
(PSL) using a Bio-Imaging Analyzer.
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Table I
Kinase activities and Km and Vmax values of PKC
wild type and mutants
Phosphorylation of the pseudosubstrate-related peptide (peptide
) with partially purified PKC wild type (wt) and mutants (0.3 µg each of protein) in the presence of PS and TPA was performed as
described under "Experimental Procedures." ND, not determined.
Vmax of wt (100%), 384 units/mg; kinase activity of
wt (100%), 259,700 cpm. The values of kinase activities are the mean
of two determinations of two independent experiments ± S.E.
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Fig. 2.
Lineweaver-Burk plots for the determination
of the Km and Vmax values
for ATP of PKC wild type and Val500 mutant. The
kinase activities of partially purified PKC wild type (A)
and Val500 mutant (B) were determined as
described under "Experimental Procedures." Five µg of the
pseudosubstrate-related peptide were phosphorylated by PKC wild
type and mutant (0.3 µg each of protein) with [32P]ATP
(concentrations as indicated) in the presence of PS and TPA at 30 °C
for 9 min. The reciprocal values of phosphate incorporation
(1/V) were plotted as a function of the reciprocal ATP
concentrations. The intercepts of the double reciprocal plots with the
x- and y-axis give the
Km and Vmax values,
respectively (see Table I).
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Mutation of Glu500 to Val affected neither the thermal
stability of PKC
nor its inhibition by the specific PKC inhibitors
Gö6983 and Gö6976. The kinase activity of the mutant, like
wild type PKC
, remained relatively stable at room temperature for at
least 40 min (Fig. 3). Inhibition of
mutant and wild type PKC
was almost identical, either by
Gö6983 in the nM range or by Gö6976 in the
µM range (Fig. 4). These
data indicate that site-directed mutagenesis did not alter the general
conformation of the molecule.

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Fig. 3.
Thermal stability of PKC wild type and
PKC Ala643 (Ala643) and PKC
Val500 (Val500) mutants. Partially
purified PKC wild type and mutants were preincubated at 25 °C.
After preincubation for the indicated times (0-40 min) the enzymes
(0.3 µg of each protein) were assayed for kinase activity. Values are
the mean of three determinations ± S.E. and are given as percent
of control (kinase activity without preincubation).
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Fig. 4.
Suppression of the kinase activity of
recombinant PKC wild type (wt) and PKC
Val500 (Val500) mutant by the inhibitors
Gö6983 (A) and Gö6976 (B). The
enzyme activities of partially purified PKC wild type and mutant
were determined in the absence or presence of the inhibitors
(concentrations as indicated) by the kinase assay, as described under
"Experimental Procedures." Kinase activities are given as percent
of control (activity in the absence of inhibitor: 0.5 µg of Val500,
161,300 cpm; 0.15 µg of wt, 139,800 cpm).
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PKC
II autophosphorylates residues Thr641 and
Ser660 (41, 42). Phosphorylation of Thr641
appears to be essential for maintaining catalytic competence of the
enzyme (38). The same holds true for Thr642 of
PKC
I (52). Phosphorylation of the corresponding
Thr638 of PKC
, however, is not required for the
catalytic function of the enzyme, as reported by Bornacin and Parker
(53). To elucidate a putative role of autophosphorylation in the
enzymatic activity of PKC
, as a first step we attempted to identify
autophosphorylation sites of in vitro phosphorylated
PKC
.
Recombinant PKC
partially purified from bacterial extracts was
phosphorylated in the presence of PS and TPA. Phosphorylated and
nonphosphorylated PKC
were applied to SDS-polyacrylamide gel
electrophoresis. The PKC
bands were cut out of the gels, and the gel
slices were washed and incubated with trypsin. Aliquots of the tryptic
digests were analyzed by MALDI mass spectrometry. Expanded views of the
obtained mass spectra from m/z 2760 to m/z 2910 are given in Fig. 5. The upper
panel shows ion signals of tryptic peptides from nonphosphorylated
PKC
wild type, whereas the lower panel shows the
corresponding tryptic peptides from the phosphorylated PKC
wild
type. The signal at m/z 2807 present in all mass spectra
(Figs. 5-7) was observed previously when tryptic digests from gel
slices were applied. Its nature is not known. The ion signal at
m/z 2791 in the mass spectrum of nonphosphorylated PKC
(Fig. 5, upper panel) could be assigned to the PKC
peptide 624SPSDYSNFDPEFLNEKPQLSFSDK647. In the
mass spectrum of phosphorylated PKC
(Fig. 5, lower panel) the ion signal at m/z 2791 had disappeared almost
completely. Instead a signal at m/z 2871 was observed. Thus,
the digest of in vitro phosphorylated PKC
contained
predominantly the phosphorylated peptide 624-647 (m/z 2791 + 80, i.e. the mass of one phosphate group). A signal at
m/z 2871, which was rather weak compared with that at
m/z 2791, could be observed also in the mass spectrum of the
nonphosphorylated PKC
(Fig. 5, upper panel),
indicating that peptide 624-647 of bacterially expressed PKC
was to
some degree phosphorylated in vivo. However, a quantitative
evaluation and comparison of signals in MALDI mass spectra is possible
only to a very limited extent. Precursor ion selection was applied to
confirm that the ion signal at m/z 2871 indeed represented a
phosphopeptide. When the precursor ion selector was set to
m/z 2791, no fragmentation occurred (Fig. 5, upper
panel, inset). However, when the molecular ion at m/z
2871 was selected as precursor ion, one additional signal was observed
at m/z 2783, which is attributable to the loss of
H3PO4 (Fig. 5, lower panel, inset). The signal at m/z 2783 was seen also in the original
spectrum of the phosphorylated PKC
(Fig. 5, lower panel).
This metastable fragmentation is characteristic for peptides containing
phosphoserine and phosphothreonine (46, 54). The ion signal for the
peptide fragment produced by metastable fragmentation did not appear at its correct m/z value because the mass scale was not
calibrated for fragment ions. However, previous studies with
phosphopeptides have shown that under the tuning parameters used in
this experiment the observed mass deviation is in fact characteristic
for the loss of H3PO4 (46).

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Fig. 5.
Expanded view of the positive ion MALDI mass
spectra obtained from the total tryptic digest of nonphosphorylated
(upper panel) and phosphorylated (lower panel)
PKC . The ion signal at m/z 2791 corresponds to the
nonphosphorylated tryptic peptide 624-647 of PKC (upper
panel). The ion signals of the phosphorylated peptide 624-647 are
observed at m/z 2871 in the upper and lower spectra.
Insets, precursor ion selection was performed for the ion
signals at m/z 2791 (upper panel) and 2871 (lower panel). Only the ion signal at m/z 2871 is
accompanied by an additional ion signal at m/z 2783, which
is attributable to the loss of H3PO4
characteristic for phosphorylated serine and threonine peptides. The
mass value of the metastable fragment ion is not corrected (see
text).
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The phosphorylated tryptic peptide
624SPSDYSNFDPEFLNEKPQLSFSDK647 contains 5 serine residues, each of which could have been phosphorylated. However,
Ser643 corresponds to the in vivo
autophosphorylation site Thr641 of PKC
II
(41, 42) and thus could be assumed to be one of the autophosphorylated
residues of PKC
. Furthermore, comparison of phosphopeptide maps of
in vivo phosphorylated PKC
wild type and
Ser643 to alanine mutant indicated that Ser643
might be an in vivo phosphorylation site of PKC
(40).
However, autophosphorylation was not proven, and a phosphopeptide
containing Ser643 was not identified. Therefore, we mutated
Ser643 to alanine (PKC
Ala643) and also
produced the double mutant Ser643/645-Ala
(PKC
Ala643/5) by site-directed mutagenesis. Both mutants
were phosphorylated and then treated as described above for the PKC
wild type. Expanded views of the MALDI mass spectra obtained from the
tryptic peptides of the phosphorylated mutants
PKC
Ala643/5 (upper panel) and
PKC
Ala643 (lower panel) are shown in Fig.
6. The expected mass signals of the
nonphosphorylated peptides corresponding to the peptide m/z
2791 of the PKC
wild type with masses of 2759 (2791
32, i.e. the mass of two oxygens) for PKC
Ala643/5
and 2775 (2791
16, i.e. the mass of one oxygen) for
PKC
Ala643 were found. However, we failed to detect
signals of significant intensity at m/z 2839 (2759 + 80) or
m/z 2855 (2775 + 80), corresponding to the phosphorylated
forms of the mutants (see Fig. 6, arrows). Thus, the mass
spectra of the phosphorylated mutants are distinctly different from the
mass spectrum of the phosphorylated wild type, which almost completely
lacked the signal of the nonphosphorylated peptide and instead showed
the signal of the phosphorylated peptide (compare Fig. 5). This result
clearly demonstrates that Ser643 is one of the in
vitro autophosphorylated residues of PKC
. A small, not clearly
recognizable, signal at m/z 2855 in the mass spectrum of
PKC
Ala643 (Fig. 6, lower panel, arrow) may
indicate a very weak phosphorylation of Ser645, because the
corresponding signal at m/z 2839 is completely missing in
the mass spectrum of the double mutant PKC
Ala643/5 (Fig.
6, upper panel, arrow). The slight phosphorylation of
Ser645 possibly occurs only with mutation of the major
phosphorylation site Ser643. The tryptic peptide 624-647
containing Ser643 was found to be phosphorylated also in
the digest of recombinant PKC
partially purified from
baculovirus-infected insect cells. The MALDI mass spectrum indicated
complete phosphorylation of PKC
at this site, because exclusively
the signal of the phosphorylated peptide at m/z 2871 (m/z 2791 + 80) was observed (Fig.
7). Precursor ion selection (Fig. 7,
inset) was applied to confirm that the ion signal at
m/z 2871 indeed represented a phosphopeptide. As with the
in vitro phosphorylated PKC
(see above, Fig. 5), one additional signal was observed at m/z 2783 which is
attributable to the loss of H3PO4. This signal
was distinct also in the original spectrum. Even though PKC
can be
expected to contain more than one (auto)phosphorylation site (see
below), neither in vitro nor in vivo additional
phosphopeptides could be detected by MALDI mass spectrometry as yet. In
two independent experiments using different preparations of the
enzymes, autophosphorylation of the PKC
Ala643 mutant was
reduced to 54 ± 11% of that of the wild type. One of the two
experiments is shown in Fig. 8. Values
determined by autoradiography were normalized according to the slightly
different amount of PKC
wild type and mutant (see the immunoblot in
Fig. 8), as described above. A similar result was obtained with the PKC
Ala643/5 mutant (data not shown). The impeded
autophosphorylation of the mutants supports the result of the mass
spectrometric study showing that Ser643 is an
autophosphorylation site of PKC
. It also indicates, however, that
additional phosphorylation site(s) exist, because the mutation of
Ser643 did not completely abolish autophosphorylation.
Mutation of Ser643 or Ser643/5 to alanine
affected neither kinase activity, and Km and
Vmax values of PKC
(Table I), nor its thermal
stability (Fig. 3). The lack of requirement of Ser643
autophosphorylation for kinase activity of PKC
is in agreement with
the entirely intact kinase activity of untreated PKC
wild type
expressed in bacteria, which, according to the mass spectrum (Fig. 5),
contains predominantly nonphosphorylated Ser643. It is also
in agreement with the finding of Bornacin and Parker (53) showing that
the corresponding Thr638 of PKC
is not required for the
catalytic function of the enzyme. However, it is in contrast to a
previous report by Li et al. (40) that indicated a reduced
kinase activity after Ser643 to alanine mutation of murine
PKC
expressed in myeloid 32D cells. One possible explanation for
these contradictory results might be the different sources and purities
of PKC
used in the two in vitro studies. For example, it
is conceivable that the Ser643 to alanine mutation causes
an increased sensitivity of the enzyme to proteolysis or
dephosphorylation and that the PKC
Ala643 mutant from
myeloid 32D cells is partially inactivated because of the action of
contaminating proteases and/or phosphatases.

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Fig. 6.
Expanded view of the positive ion MALDI mass
spectra obtained from the total tryptic digests of the phosphorylated
PKC Ala643/5 mutant (upper panel) and of the
phosphorylated PKC Ala643 mutant (lower
panel). The observed ion signals at m/z 2759 and
2775 correspond to the nonphosphorylated tryptic peptides 624-647
containing the Ser643/645 to Ala643/645 and
Ser643 to Ala643 mutations, respectively. No
ion signal (upper panel) or possibly a very weak ion signal
(lower panel) could be detected for the phosphorylated forms
of those peptides at m/z +80, as indicated by
arrows.
|
|

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|
Fig. 7.
Expanded view of the positive ion MALDI mass
spectrum obtained from the total tryptic digest of recombinant PKC
from baculovirus-infected insect cells. The ion signal at
m/z 2871 corresponds to the phosphorylated tryptic peptide
624-647 of PKC . The signal of the nonphosphorylated peptide is
barely visible (m/z 2791). Inset,
precursor ion selection was performed for the ion signal at
m/z 2871. An additional ion signal at m/z 2783 occurs, which is attributable to the loss of
H3PO4 characteristic for phosphorylated serine
and threonine peptides (see Fig. 5).
|
|

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|
Fig. 8.
Autophosphorylation of PKC wild type
(wt) and PKC Ala643 mutant
(Ala643). Partially purified PKC wild type and
mutant (0.3 µg of each protein) were phosphorylated at 30 °C for 5 min and quantitated with a scanner after immunoblotting
(a.u., arbitrary units) as described in Fig. 1 and under
"Experimental Procedures." Radiolabeled PKC was visualized by
autoradiography and quantitated using a phosphoimager (PSL,
photostimulated luminescence; see Fig. 1).
|
|
Taken together, we have shown that the catalytic function of PKC
depends in part on the acidic residue Glu500 in the
activation loop and, as reported earlier (39), not on phosphorylated
Thr505. Thus, PKC
exhibits kinase activity and is able
to autophosphorylate without the posttranslational modification that is
required for catalytic competence of PKC
and
II.
Autophosphorylation in vitro, and most likely also in
vivo, occurs on Ser643, which corresponds to the
autophosphorylation site Thr641 of PKC
II.
Probably other (auto)phosphorylation site(s) of PKC
exist that have
not yet been identified.
 |
FOOTNOTES |
*
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: German Cancer Research
Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Tel.:
49-6221-424505; Fax: 49-6221-424554; E-mail: m.gschwendt{at}dkfz-heidelberg.de.
 |
ABBREVIATIONS |
The abbreviations used are:
PKC, protein kinase
C;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
PS, phosphatidyl serine;
MALDI, matrix-assisted laser desorption
ionization.
 |
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