(Received for publication, September 27, 1996, and in revised form, December 2, 1996)
From the Divisions of Biochemistry of Tissue-specific Regulation and § Pathochemistry, German Cancer Research Center, D-69120 Heidelberg, Germany
A structural feature shared by many protein
kinases is the requirement for phosphorylation of threonine or tyrosine
in the so-called activation loop for full enzyme activity. Previous
studies by several groups have indicated that the isotypes ,
I, and
II of protein kinase C (PKC) are
synthesized as inactive precursors and require phosphorylation by a
putative "PKC kinase" for permissive activation. Expression of
PKC
in bacteria resulted in a nonfunctional enzyme, apparently due
to lack of this kinase. The phosphorylation sites for the PKC kinase in
the activation loop of PKC
and PKC
II could be
identified as Thr497 and Thr500, respectively.
We report here that PKC
, contrary to PKC
, can be expressed in
bacteria in a functional form. The activity of the recombinant enzyme
regarding substrate phosphorylation, autophosphorylation, and
dependence on activation by
12-O-tetradecanoylphorbol-13-acetate as well as the
Km values for two substrates are comparable to
those of recombinant PKC
expressed in baculovirus-infected insect
cells. By site-directed mutagenesis we were able to show that
Thr505, corresponding to Thr497 and
Thr500 of PKC
and PKC
II, respectively, is
not essential for obtaining a catalytically competent conformation of
PKC
. The mutant Ala505 can be activated and does not
differ from the wild type regarding activity and several other
features. Ser504 can not take over the role of
Thr505 and is not prerequisite for the kinase to become
activated, as proven by the unaffected enzyme activity of respective
mutants (Ala504 and Ala504/Ala505).
These results indicate that phosphorylation of Thr505 is
not required for the formation of functional PKC
and that at least
this PKC isoenzyme differs from the isotypes
,
I, and
II regarding the permissive activation by a PKC
kinase.
PKC1 covers a family of 11 isoenzymes
(PKC,
I,
II,
,
,
,
,
,
,
/
, and µ) known so far to possess
phospholipid-dependent serine and threonine kinase activity
(1-6). These kinases play a key role in signal transduction and are
involved in the regulation of numerous cellular processes. PKC
is a
member of the so-called novel PKC subgroup consisting of
Ca2+-unresponsive diacylglycerol
(12-O-tetradecanoylphorbol-13-acetate (TPA))-activated
isoenzymes. PKC
was purified to homogeneity or partially purified
from various sources, such as porcine spleen (7), rat brain (8), COS1
cells transfected with cDNA coding for rat (9) or mouse (10)
PKC
, and insect cells infected with recombinant baculovirus
containing the cDNA coding for human PKC
(11). Expression of
functional recombinant PKC
or any other functional recombinant PKC
isoenzyme in bacteria has not been described yet.
PKC is a ubiquitously expressed PKC isoenzyme (12) and exhibits some
unique properties. The tyrosine kinase c-Src selectively phosphorylates
the type
PKC isoenzyme in vitro. The tyrosine phosphorylation induces a modification of PKC
activity exhibiting some substrate selectivity (13). Tyrosine phosphorylation of PKC
could be demonstrated also in vivo (14, 15). In addition to
the substrate specificity acquired by tyrosine phosphorylation, PKC
appears to possess an intrinsic substrate specificity, for example,
toward the elongation factor 1
and an elongation factor 1
peptide
(16). PKC
is autophosphorylated to a much higher degree than the
other isoenzymes, and in contrast to other kinases, including PKC
isoenzymes, it is able to accept GTP as a phosphate donor for
autophosphorylation (17). Based on results obtained with cells
overexpressing PKC
, some role of this PKC isoenzyme in growth
suppression and induction of differentiation has been suggested
(18-20). The finding that PKC
is lost from immortalized human
keratinocytes after stable transfection with a c-Ha-ras oncogene also points to this possible function (21).
Here we report on another, possibly unique, feature of PKC. Contrary
to PKC
(22-24), PKC
could be expressed in bacteria in a
functional form. Moreover, we were able to demonstrate by site-directed
mutagenesis that phosphorylation by a putative "PKC kinase" of
Thr505, unlike that of the corresponding Thr497
and Thr500 in PKC
(24) and
II (25),
respectively, is not essential for a permissive activation of
PKC
.
TPA was supplied by Dr. E. Hecker (German Cancer
Research Center). Gö 6976 and Gö 6983 were kindly
provided by Goedecke A.G. (Freiburg, Germany). The rat PKC
full-length cDNA clone (3000 base pairs) and the recombinant
baculovirus containing the sequence coding for PKC
were generously
given by Dr. C. Polke (University of Würzburg, Würzburg,
Germany) and Dr. S. Stabel (Max-Delbrück-Laboratorium, Cologne,
Germany). The pseudosubstrate-related peptide
(MNRRGSIKQAKI)
was synthesized by Dr. R. Pipkorn (German Cancer Research
Center). Other materials were bought from the following companies:
bovine brain L-
-phosphatidylserine (PS) and histone
III-S (lysine-rich fraction from calf thymus) from Sigma; [
-32P]ATP (specific activity,
5000 Ci/mmol) from Hartmann Analytic (Braunschweig, Germany),
recombinant human protein kinase c-Src from Upstate Biotechnology Inc.
(Lake Placid, NY), mouse monoclonal anti-phosphotyrosine antibodies
(PY20) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), peroxidase-
and alkaline phosphatase-conjugated goat anti-rabbit and anti-mouse
antibodies from Dianova (Hamburg, Germany), and ECL reagents from
Amersham Corp. Expression vector pET28 and Escherichia coli
strain BL21(DE3)pLysS were from AGS GmbH (Heidelberg, Germany),
nickel-nitrilo-triacetic acid resin from Quiagen GmbH (Hilden,
Germany), T7-SequencingTM kit from Pharmacia Biotech Inc.,
and Pwo DNA polymerase from Boehringer Mannheim.
For the construction of 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, the following oligonucleotide primers were used:
5
-AAA GGA TCC CAT ATG GCA CCG TTC CTG CGC-3
as 5
-primer and 5
-TCT
GGG AAT TCA CTA CTA TTC CAG GAA TTG CTC-3
as 3
-primer (synthesized by
W. Weinig, German Cancer Research Center). For polymerase chain
reaction amplification (cycle profile: 94 °C/5 min; 10 × 94 °C/15 s, 56 °C/30 s, 72 °C/2 min; 15 × 94 °C/15 s,
56 °C/30 s, 72 °C/2 min, plus cycle elongation of 20 s for each cycle; 72 °C/7 min), a rat PKC
full-length cDNA clone of 3000 base pairs was used as template. The resulting cDNA of 2048 base pairs was cut with NdeI and EcoRI and cloned
into the NdeI-EcoRI-cut expression vector pET28.
The resulting plasmid was termed pET28
wt and used for transformation
of BL21(DE3)pLysS cells.
Polymerase chain reaction-mediated site-directed mutagenesis was
performed using the "overlap extension" method according to Ho
et al. (26). A rat PKC full-length clone of 3000 base pairs served as a template. The following pairs of mutagenic
oligonucleotides were used (only the oligonucleotides that are sense
with respect to the PKC
cDNA are given, and changed bases are
underlined): (T/A)505, 5
-CGG GCC AGC
TTC TGC GGC-3
;
(S/A)504, 5
-GAG AAC CGG GCC
ACA TTC TGC GGC ACT-3
;
and (S/A)504(T/A)505, 5
-GAG AAC CGG GCC
CA TTC TGC
GGC ACT-3
. Successful mutation was confirmed by sequencing. The PKC
mutant cDNAs were cloned into pET28 as described above. The
resulting plasmids were termed pET28
Ala505,
pET28
Ala504, and
pET28
Ala504/Ala505.
E. coli
BL21(DE3)pLysS cells were transformed with the various plasmids
indicated above, grown at 37 °C for 12 h, diluted 1:100 in 1 liter of fresh LB medium supplemented with 50 µg/ml kanamycin and 30 µg/ml chloramphenicol, and incubated at 24 °C until the absorbance
(600 nm) reached 0.5-0.7. Induction was performed with 0.5 mM isopropyl-1-thio--D-galactopyranoside for
16 h at 24 °C. Cells were sedimented and resuspended with
ice-cold buffer I (30 mM MES, pH 6.5, 50 mM
KCl, 1 mM EDTA, 1 mM dithiothreitol) or for
further purification on nickel-nitrilo-triacetic acid resin with buffer
II (50 mM sodium phosphate, pH 8.0, 500 mM
NaCl, 10 mM imidazole). Resuspension in buffer resulted in
an absorbance (600 nm) of approximately 40. After sonication with a
Branson sonifier and centrifugation at 80,000 × g for
45 min at 4 °C, the supernatant (termed bacterial extract) was used
as a source of bacterial recombinant His-tagged PKC
. The insoluble
fraction was resuspended in SDS sample buffer and boiled for 5 min
before application to PAGE.
Partial purification of soluble PKC was achieved by
metal chelate affinity chromatography of the bacterial extract
under native conditions using nickel-nitrilo-triacetic acid resin
and following the manufacturer's recommendation. Bound proteins were eluted with imidazole (100, 150, 200, 250, and 500 mM).
PKC
was detected in the 100 mM imidazole fraction by
immunoblotting and assaying PS- and TPA-stimulated kinase activity.
Sf9 cells were infected with the recombinant baculovirus, and cells were extracted as described previously (27).
Protein Kinase AssayPhosphorylation reactions were carried
out in a total volume of 100 µl containing buffer C (50 mM Tris-HCl, pH 7.5, 10 mM -mercaptoethanol), 4 mM MgCl2, 10 µg of
PS, 100 nM TPA, 2 µg of pseudosubstrate-related peptide
or 30 µg of histone III-S as substrate, 10 µl of bacterial
extracts diluted as indicated in figure legends, and 37 µM ATP containing 1 µCi [
-32P]ATP. In
some experiments, PS and TPA were omitted, and in some others, Gö
6976 or Gö 6983 at concentrations indicated in figure legends
were added. After incubation for 7 min at 30 °C, the reaction was
terminated by transferring 50 µl of the assay mixture onto a 20-mm
square piece of phosphocellulose paper (Whatman p81). After washing the
paper three times in deionized water and twice in acetone,
radioactivity was determined by liquid scintillation counting. 1 unit
of kinase activity equals 1 nmol of phosphate incorporated into
substrate/min.
Autophosphorylation was carried out essentially as
described for the protein kinase assay, but 37 µM ATP
containing 8 µCi of [-32P]ATP was added and
substrate was omitted. The reaction was terminated by addition of 10%
trichloroacetic acid. Precipitated proteins were redissolved in sample
buffer, separated by SDS-PAGE (7.5%), and visualized by immunoblotting
and autoradiography with an exposure time of 16 h. Tyrosine
phosphorylation was performed essentially as autophosphorylation, but
37 µM ATP (without [32P]ATP), 250 µM MnSO4, and 6 units c-Src were added, and
incubation at 30 °C was performed for 15 min.
Immunoblotting was performed as described
previously (28). As the primary antibody a polyclonal PKC-specific
antibody (7) or a monoclonal anti-phosphotyrosine antibody and as the
secondary antibody alkaline phosphatase- or horseradish
peroxidase-conjugated goat anti-rabbit IgG (see figure legends) were
used. Immunoblots were stripped by incubation with 100 mM
-mercaptoethanol, 2% SDS, and 63 mM Tris-HCl, pH 6.7, at 60 °C for 1 h.
Protein concentration was determined with the protein dye reagent concentrate (according to the method of Bradford; Ref. 53) from Bio-Rad, using bovine serum albumin as standard.
At transformation with the expression vector pET28wt containing
the full-length cDNA of rat PKC
, although not with the pET28 vector alone, the E. coli cells BL21(DE3)pLysS produced
PKC
, as demonstrated by immunoblotting of bacterially expressed
proteins with a PKC
-specific antibody (Fig.
1A). A portion of the ectopically expressed
rat PKC
was soluble in a buffer without detergent (herein termed
bacterial extract). The insoluble fraction, present in the bacteria
probably in the form of inclusion bodies, was dissolved in sample
buffer containing 1% SDS. Slow growth of the bacteria at 24 °C was
expected to result in an increased portion of active recombinant
enzyme.
PKC could not be detected on staining of bacterial proteins with
Coomassie Blue but only by immunoblotting. The concentration of PKC
in the bacterial extract was approximately one-third of that in an
extract of insect cells that produced PKC
on infection with a
recombinant baculovirus (Fig. 1B). The bacterial recombinant PKC
was found to be enzymatically active. As shown in Fig.
2, histone III-S was phosphorylated by the recombinant
enzyme in the bacterial extract in a TPA-dependent manner.
Only weak incorporation of phosphate occurred in the absence of PS and
TPA or in the presence of PS alone. An extract of bacteria transformed
with the pET28 vector alone served as a control and did not show any
TPA-inducible kinase activity. To be able to compare the kinase
activity of recombinant PKC
from bacteria with that from
baculovirus-infected insect cells, nearly equal amounts of PKC
had
to be applied to the kinase assay. This was achieved by diluting the
insect cell extract 1:11 (final concentration of protein in the assay,
0.08 mg/ml) and the bacterial extract 1:4 (final concentration of
protein in the assay, 0.2 mg/ml), according to the data on the
concentration of PKC
in both extracts (see Fig. 1B). The
activity of PKC
produced by the bacteria proved to be comparable to
that of PKC
expressed in insect cells (Fig. 2). Enzyme activity was
measured with the substrates histone III-S (Fig. 2) and
pseudosubstrate-related peptide
(data not shown, but see Fig. 6).
Moreover, no significant difference between both enzymes could be
detected regarding either activation by TPA (Fig. 2) or specific
activities and Km values for the
pseudosubstrate-related peptide
and histone III-S (Table
I).
|
The expression of functional PKC in bacteria indicated that PKC
,
unlike PKC
(24) and
II (25), does not require
phosphorylation of Thr505 (corresponding to
Thr497 and Thr500 in PKC
and
II, respectively) as a prerequisite of a catalytically competent form of the enzyme. This could be assumed, since bacteria are
thought to lack the as yet unidentified protein kinase, which is
responsible for phosphorylation of this site (22-24). To prove that
Thr505 is not essential for the activation of PKC
, we
exchanged threonine 505 for alanine by site-directed mutagenesis and
transformed bacteria with the expression vector
pET28
Ala505 containing the PKC
-mutant cDNA. The
PKC
mutant (PKC
Ala505) was expressed by the bacteria
as effectively as the PKC
wild type (Fig. 1B) and did not
significantly differ from the wild type regarding its TPA-stimulated
kinase activity (Fig. 2). Equal amounts of wild type and mutant were
applied to the kinase assay, as determined by immunoblotting (see above
and Fig. 1B). Specific activities of wild type and mutant
(1.4 and 2.0 units/mg, respectively, with peptide
as substrate, and
1.1 and 1.5 units/mg, respectively, with histone III-S as substrate) as
well as the Km values (5 and 6 µM,
respectively, for peptide
, and 125 and 140 µM, respectively, for histone III-S) were essentially the same (Table I).
For a more detailed characterization of bacterial recombinant PKC
wild type and the mutant PKC
Ala505 we compared both
enzymes with respect to several features, such as autophosphorylation,
tyrosine phosphorylation by Src, and inhibition by
staurosporine-related inhibitors.
The wild type as well as mutant in the respective bacterial extracts
could be autophosphorylated in the presence of PS and TPA, as shown in
Fig. 3. In the absence of PS and TPA or in the presence
of PS alone, no or just weak autophosphorylation was observed.
Regarding intensity and TPA dependence of autophosphorylation, the
mutant PKCAla505 behaved like the wild type. No
autophosphorylated PKC
could be detected in extracts of bacteria
transformed with the pET28 vector alone.
Phosphorylation of PKC at tyrosine residue(s) in vitro by
the tyrosine kinase Src, a characteristic property of this PKC isoenzyme, results in decreased mobility of the protein in SDS-PAGE (13). Both recombinant enzymes, wild type and PKC
Ala505,
were phosphorylated by Src and exhibited decreased mobility on tyrosine
phosphorylation, as shown by immunoblotting with an anti-PKC
antibody (Fig. 4A). Immunoblotting with an
anti-phosphotyrosine antibody proved that phosphorylation did indeed
occur at tyrosine residue(s) (Fig. 4B). Again, no
significant difference between wild type and mutant could be seen.
Finally, we studied the inhibition of both types of recombinant PKC
by the staurosporine-related protein kinase inhibitors Gö 6976 and Gö 6983, which are specific for PKC. The two inhibitors are
known to differ significantly in their capacity to suppress PKC
,
with IC50 values of 1 µM and larger for
Gö 6976 and 10-100 nM for Gö 6983 (29-31). A
similar difference in inhibitory potency was observed with the
bacterial recombinant PKC
wild type and mutant (Fig.
5). Suppression by Gö 6976 occurred, with
IC50 values in the range of 10-30 µM and by
Gö 6983 in the range of 25 nM.
We could not entirely exclude the possibility that in PKC
Ser504 might be able to take over the role of
Thr505 and might serve as an essential phosphorylation
site, such as Thr497 and Thr500 of PKC
and
II, respectively. Therefore, we expressed two other mutants of PKC
, PKC
Ala504 and
PKC
Ala504/Ala505, in the bacteria, in which
serine 504 alone or serine 504 and threonine 505 were exchanged for
alanine. As shown in Fig. 6, both mutants actively
phosphorylated the pseudosubstrate-related peptide
in a
TPA-dependent manner and thus did not differ from the wild
type. The same could be demonstrated with autophosphorylation of the
PKC
mutants (data not shown).
In preliminary experiments bacterial recombinant PKC (wild type and
mutants) containing a short His tag could be partially purified by
affinity chromatography on a nickel-nitrilo-triacetic acid resin. On
elution from the column with 100 mM imidazole the purity of
the enzymes was around 20%, as estimated from Coomassie Blue-stained
SDS-polyacrylamide gels. The specific activities (the mean of two
experiments), as determined with histone III-S as substrate, were
46.2 ± 6.2 units/mg protein (wild type), 59.5 ± 13.3 units/mg protein (PKC
Ala505), and 54.2 ± 6.1 units/mg protein (PKC
Ala504/Ala505).
Fabbro and co-workers (32) provided the first evidence of PKC
being synthesized as an inactive nonphosphorylated precursor, which is
at first converted to a transient and finally to a "mature" phospho
form. Further studies by this and another group showed that bacterial
expression of PKC
results in a recombinant protein devoid of kinase
activity (22-24). It was suspected that phosphorylation by another
protein kinase is necessary for PKC
to gain the ability of being
activated, and that bacteria lack this putative PKC kinase. According
to our results, PKC
can be expressed in bacteria in a functional
form. Agreeing with previous reports (22-24), we were unable to
express enzymatically active PKC
in bacteria using the same
expression vector and the same conditions as for the expression of
PKC
(data not shown). The activity of bacterially expressed PKC
regarding substrate phosphorylation and TPA dependence as well as its
Km values for two substrates are comparable to those
of recombinant PKC
expressed in baculovirus-infected insect cells.
Moreover, partial purification of recombinant PKC
from bacterial
extracts by one-step affinity chromatography yields an enzyme with a
specific activity (46.2 units/mg protein) comparable to that of
partially purified native PKC
from porcine spleen, which previously
was found to be 15 units/mg protein on the second purification step
(phenyl-Sepharose) and 54 units/mg protein on the third purification
step (protamine-agarose; see Ref. 7). As the partially purified
bacterial enzyme is around 20% pure, its specific activity is also in
good agreement with the specific activity of the native enzyme purified
to homogeneity from porcine spleen (304.2 units/mg protein; Ref. 7).
Provided that the bacteria strain used in our studies indeed lacks the
PKC kinase, these results indicate that PKC
does not require
phosphorylation by this kinase to become a functional enzyme. Some
protein kinases are able to autoactivate during bacterial expression by
a presumably cotranslational intermolecular phosphorylation (33).
The sites phosphorylated by another kinase were identified in bovine
PKC (24) and rat PKC
II (25) as Thr497 and
Thr500, respectively. These threonines are located in an
activation loop that is also crucial for the regulation of other
protein kinases (34-39). Replacement of these critical residues in
PKC
(24) or PKC
II (25) with a neutral,
nonphosphorylatable residue results in kinases that cannot be
activated. The corresponding site in rat PKC
is Thr505.
Bacterial expression of a PKC
mutant containing alanine in position
505 yields a fully functional kinase. The mutant does not differ from
the wild type in many respects, such as effectiveness of expression in
bacteria, TPA-stimulated kinase activity, Km values
for substrates, autophosphorylation, tyrosine phosphorylation by Src,
and inhibition by staurosporine-related inhibitors. This clearly
demonstrates that functional PKC
can be expressed in bacteria, and
that Thr505, contrary to Thr497 and
Thr500 in PKC
and
II, respectively, is
not a critical site for permissive activation of PKC
. As
Ser504 is another amino acid that can be phosphorylated in
this region of PKC
, we wished to exclude the possibility that this
residue might be able to replace Thr505 as a
phosphorylation site for the putative PKC kinase and thus as the
critical site for permissive activation. Exchange of Ser504
alone or both Ser504 and Thr505 for alanine
does not result in any loss of kinase activity. Partial purification of
the bacterial PKC
mutants Ala505 and
Ala504/Ala505 by affinity chromatography yields
enzymes with specific activities of 59.5 and 54.2 units/mg protein,
respectively, which are comparable to those of partially purified
bacterial wild type PKC
and native PKC
from porcine spleen (see
above). This proves that neither Thr505 nor
Ser504 is essential for gaining a catalytically competent
conformation of PKC
. As each PKC isoenzyme known so far contains
threonine in a position corresponding to positions 497 and 500 of
PKC
and
II, respectively, the findings regarding the
critical role of this threonine residue for the activation process of
PKC
and
II have been thought to be valid for all PKC
isoenzymes (24, 40). According to our results, however, at least the
isotype
is an exception to this apparent rule. PKC
can be
activated even when containing a neutral, nonphosphorylatable amino
acid instead of this threonine residue.
In accordance with reports on a stepwise phosphorylation of PKC (32,
41, 42) and on the in vivo phosphorylation sites of
PKC
II (40, 43), Newton and co-workers (40, 44) have shown that phosphorylation of threonine 500, putatively by the PKC
kinase, enables PKC
II to autophosphorylate at
Thr641 and Ser660. Phosphorylation at
Thr641 replaces the requirement for phosphate on
Thr500 and stabilizes the functional form of the enzyme. A
report by Zhang et al. (45) indicates that in
PKC
I phosphorylation of Thr642
(corresponding to Thr641 in PKC
II) is an
early event in the processing of the newly synthesized enzyme and is
required for enzymatic functioning. Very recently, Bornancin and Parker
(46) reported that phosphorylation of Thr638 of PKC
(corresponding to Thr642 of PKC
I and
Thr641 of PKC
II) is not required for the
catalytic function of the enzyme per se, but serves to
control the duration of activation by regulating the rate of
dephosphorylation and inactivation of the protein. This is achieved
through the cooperative interaction between Thr638 and the
catalytic core site, Thr497. It is conceivable that PKC
also requires autophosphorylation of the corresponding site,
i.e. Ser643, for stabilization of a
catalytically competent conformation. As indicated by our results,
however, PKC
is able to autophosphorylate without having been
prephosphorylated by another kinase. As a consequence, a putative
regulation of this PKC kinase would not affect PKC
, and thus PKC
kinase might be a target for a differential regulation of PKC
isoenzymes. Identification of the in vivo phosphorylation sites of PKC
should allow an answer to the question of whether any
of the three in vivo phosphorylation sites found in
PKC
II (40, 43) play a role also in PKC
.
In the crystal structure of cAPK (47, 48) the dianionic phosphoryl
group of Thr197-P in the activation loop neutralizes a
cluster of positively charged residues from several regions of the
protein. Thr197 of cAPK aligns with Thr505 of
PKC. To get a better idea of the structural situation around Thr505 in PKC
, we exchanged the side chains in the
crystal structure of the catalytic core of cAPK for those of PKC
according to the alignment of Hanks and Quinn (49) while keeping the
backbone unchanged (Fig. 7). Two of the side chain
interactions with Thr197-P in cAPK are not possible in
PKC
; His87 from the small lobe is replaced by
Cys391 in PKC
, and Thr195 is replaced by
Ala503. A cysteine in the position equivalent to
His87 of cAPK is also conserved in PKC
and
PKC
II, which both require phosphorylation at
Thr497 and Thr500, respectively, for activity.
The basic residues Arg470 and Lys494,
corresponding to Arg165 and Lys189 of cAPK, are
both conserved in PKC
. It is likely that Arg165, which
precedes the catalytic base, and perhaps Lys189 are
conserved in protein kinases and thus also in PKC
for similar functions. Both residues form salt bridges to Thr197-P in
cAPK. The contact to Arg165 may directly promote the
correct assembly of the active site by controlling the orientation of
the catalytic base Asp166 via its peptide backbone, whereas
the contact to Lys189 may help in correctly positioning the
metal binding loop, i.e. essentially Asp184.
However, from this model it is not apparent how in PKC
the dispensable Thr505-P may be functionally substituted.
Several possibilities can be discussed to explain the observed
Thr505-independent activity of PKC
. If an ionic
interaction at this site is also needed for PKC
, it could be
provided by a bound ion, similarly as in casein kinase 1 (50, 51). On
the other hand, a negatively charged residue from outside the catalytic core might reach into the activation site. Another possibility is that
the PKC
activation loop, which contains the three-residue insert
Gly499-Glu500-Asn501, folds back to
orient the Glu500 carboxylate in a position where it can
interact with Arg470 and Lys494. Finally,
nonionic interactions with Arg470, similarly to mammalian
casein kinase 1 (51), are conceivable. The question remains why residue
505 is conserved as a threonine in PKC
. The fact that
Thr505 is dispensable for the permissive activation of
PKC
does not exclude its phosphorylation for other purposes, such as
protein-protein interaction, as indicated in the interaction of cAPK
catalytic and regulatory subunits (52), or enzyme inactivation.
We thank Dr. Volker Kinzel for fruitful discussions.