Phosphorylation of Protein Kinase Cdelta (PKCdelta ) at Threonine 505 Is Not a Prerequisite for Enzymatic Activity
EXPRESSION OF RAT PKCdelta AND AN ALANINE 505 MUTANT IN BACTERIA IN A FUNCTIONAL FORM*

(Received for publication, September 27, 1996, and in revised form, December 2, 1996)

Luise Stempka Dagger , Andreas Girod Dagger §, Hans-Joachim Müller , Gabriele Rincke , Friedrich Marks , Michael Gschwendt and Dirk Bossemeyer §

From the Divisions of Biochemistry of Tissue-specific Regulation and § Pathochemistry, German Cancer Research Center, D-69120 Heidelberg, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES


ABSTRACT

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 alpha , beta I, and beta II of protein kinase C (PKC) are synthesized as inactive precursors and require phosphorylation by a putative "PKC kinase" for permissive activation. Expression of PKCalpha 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 PKCalpha and PKCbeta II could be identified as Thr497 and Thr500, respectively. We report here that PKCdelta , contrary to PKCalpha , 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 PKCdelta expressed in baculovirus-infected insect cells. By site-directed mutagenesis we were able to show that Thr505, corresponding to Thr497 and Thr500 of PKCalpha and PKCbeta II, respectively, is not essential for obtaining a catalytically competent conformation of PKCdelta . 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 PKCdelta and that at least this PKC isoenzyme differs from the isotypes alpha , beta I, and beta II regarding the permissive activation by a PKC kinase.


INTRODUCTION

PKC1 covers a family of 11 isoenzymes (PKCalpha , beta I, beta II, gamma , delta , epsilon , eta , theta , zeta , lambda /iota , 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. PKCdelta is a member of the so-called novel PKC subgroup consisting of Ca2+-unresponsive diacylglycerol (12-O-tetradecanoylphorbol-13-acetate (TPA))-activated isoenzymes. PKCdelta 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) PKCdelta , and insect cells infected with recombinant baculovirus containing the cDNA coding for human PKCdelta (11). Expression of functional recombinant PKCdelta or any other functional recombinant PKC isoenzyme in bacteria has not been described yet.

PKCdelta is a ubiquitously expressed PKC isoenzyme (12) and exhibits some unique properties. The tyrosine kinase c-Src selectively phosphorylates the type delta  PKC isoenzyme in vitro. The tyrosine phosphorylation induces a modification of PKCdelta activity exhibiting some substrate selectivity (13). Tyrosine phosphorylation of PKCdelta could be demonstrated also in vivo (14, 15). In addition to the substrate specificity acquired by tyrosine phosphorylation, PKCdelta appears to possess an intrinsic substrate specificity, for example, toward the elongation factor 1alpha and an elongation factor 1alpha peptide (16). PKCdelta 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 PKCdelta , some role of this PKC isoenzyme in growth suppression and induction of differentiation has been suggested (18-20). The finding that PKCdelta 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 PKCdelta . Contrary to PKCalpha (22-24), PKCdelta 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 PKCalpha (24) and beta II (25), respectively, is not essential for a permissive activation of PKCdelta .


EXPERIMENTAL PROCEDURES

Materials

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 PKCdelta full-length cDNA clone (3000 base pairs) and the recombinant baculovirus containing the sequence coding for PKCdelta 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 delta  (MNRRGSIKQAKI) was synthesized by Dr. R. Pipkorn (German Cancer Research Center). Other materials were bought from the following companies: bovine brain L-alpha -phosphatidylserine (PS) and histone III-S (lysine-rich fraction from calf thymus) from Sigma; [gamma -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.

Polymerase Chain Reaction Amplification and Cloning of Wild Type and Mutant PKCdelta cDNA

For the construction of a PKCdelta 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 PKCdelta 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 pET28delta 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 PKCdelta 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 PKCdelta cDNA are given, and changed bases are underlined): (T/A)505, 5'-CGG GCC AGC <UNL>GCT</UNL> TTC TGC GGC-3'; (S/A)504, 5'-GAG AAC CGG GCC <UNL>GCC</UNL> ACA TTC TGC GGC ACT-3'; and (S/A)504(T/A)505, 5'-GAG AAC CGG GCC <UNL>GCC:G</UNL>CA TTC TGC GGC ACT-3'. Successful mutation was confirmed by sequencing. The PKCdelta mutant cDNAs were cloned into pET28 as described above. The resulting plasmids were termed pET28delta Ala505, pET28delta Ala504, and pET28delta Ala504/Ala505.

Bacterial Expression of PKCdelta

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-beta -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 PKCdelta . The insoluble fraction was resuspended in SDS sample buffer and boiled for 5 min before application to PAGE.

Partial Purification of Recombinant His-tagged PKCdelta

Partial purification of soluble PKCdelta 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). PKCdelta was detected in the 100 mM imidazole fraction by immunoblotting and assaying PS- and TPA-stimulated kinase activity.

Recombinant PKCdelta from Baculovirus-infected Insect Cells

Sf9 cells were infected with the recombinant baculovirus, and cells were extracted as described previously (27).

Protein Kinase Assay

Phosphorylation reactions were carried out in a total volume of 100 µl containing buffer C (50 mM Tris-HCl, pH 7.5, 10 mM beta -mercaptoethanol), 4 mM MgCl2, 10 µg of PS, 100 nM TPA, 2 µg of pseudosubstrate-related peptide delta  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 [gamma -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 and Tyrosine Phosphorylation of PKCdelta

Autophosphorylation was carried out essentially as described for the protein kinase assay, but 37 µM ATP containing 8 µCi of [gamma -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

Immunoblotting was performed as described previously (28). As the primary antibody a polyclonal PKCdelta -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 beta -mercaptoethanol, 2% SDS, and 63 mM Tris-HCl, pH 6.7, at 60 °C for 1 h.

Determination of Protein Concentration

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.


RESULTS

At transformation with the expression vector pET28delta wt containing the full-length cDNA of rat PKCdelta , although not with the pET28 vector alone, the E. coli cells BL21(DE3)pLysS produced PKCdelta , as demonstrated by immunoblotting of bacterially expressed proteins with a PKCdelta -specific antibody (Fig. 1A). A portion of the ectopically expressed rat PKCdelta 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.


Fig. 1. A, expression of PKCdelta in E. coli BL21(DE3)pLysS cells. Bacteria were transformed with the plasmid pET28 alone or the plasmid pET28delta wt (wt) containing the full-length cDNA of rat PKCdelta (PKCdelta wild type) and were grown as described under "Experimental Procedures." Bacteria were extracted with 1 ml of buffer I and buffer I-insoluble proteins dissolved in 1 ml of sample buffer containing 1% SDS. Soluble (a) and insoluble (b) proteins (3 µl each) were separated by SDS-PAGE. PKCdelta (arrow) was identified by immunoblotting with a PKCdelta -specific antibody and an alkaline phosphatase-conjugated goat anti-rabbit IgG as secondary antibody. Molecular masses were determined from the standard proteins myosin (205 kDa), beta -galactosidase (116 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa), and ovalbumin (45 kDa). B, comparison of the concentration of recombinant PKCdelta in extracts from bacteria and baculovirus-infected insect cells. Extracts from bacteria that were transformed with the plasmids pET28delta wt or pET28delta Ala505 and produced the PKCdelta wild type (wt) or the PKCdelta Ala505 mutant (Ala505) and extracts from baculovirus-infected Sf9 insect cells expressing PKCdelta wild type (Baculo) were diluted as indicated (µg protein). On separation of the extracted proteins by SDS-PAGE the amount of PKCdelta was estimated by immunoblotting (see A).
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PKCdelta could not be detected on staining of bacterial proteins with Coomassie Blue but only by immunoblotting. The concentration of PKCdelta in the bacterial extract was approximately one-third of that in an extract of insect cells that produced PKCdelta on infection with a recombinant baculovirus (Fig. 1B). The bacterial recombinant PKCdelta 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 PKCdelta from bacteria with that from baculovirus-infected insect cells, nearly equal amounts of PKCdelta 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 PKCdelta in both extracts (see Fig. 1B). The activity of PKCdelta produced by the bacteria proved to be comparable to that of PKCdelta expressed in insect cells (Fig. 2). Enzyme activity was measured with the substrates histone III-S (Fig. 2) and pseudosubstrate-related peptide delta  (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 delta  and histone III-S (Table I).


Fig. 2. Kinase activity of bacterial recombinant PKCdelta wild type (wt) and PKCdelta Ala505 mutant (Ala505) compared with that of recombinant PKCdelta expressed in baculovirus-infected insect cells (Baculo). Bacterial extracts (wt and Ala505) were diluted 1:4 and 1:6, respectively, and the insect cell extract was diluted 1:11 to approximately equalize the concentration of PKCdelta in the extracts (see Fig. 1B). The enzyme activity of PKCdelta in 10 µl of the diluted extracts was determined by the kinase assay with histone III-S as substrate, as described under "Experimental Procedures." Values are the means of three determinations (bars, ±S.E.).
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Fig. 6. Kinase activity of bacterial recombinant PKCdelta wild type (wt) and PKCdelta Ala505 (Ala505), PKCdelta Ala504 (Ala504), and PKCdelta Ala504/Ala505 (Ala504/505) mutants. Extracts from bacteria transformed with the respective plasmids (see "Experimental Procedures") were brought to approximately equal final concentrations of PKCdelta , as described in Figs. 2 and 1B (respective immunoblots of Ala504 and Ala504/Ala505 are not shown). The enzyme activity of PKCdelta in the diluted extracts was determined by the kinase assay in the presence or absence of PS and PS and TPA and with the pseudosubstrate-related peptide delta  as substrate, as described under "Experimental Procedures." Values are the means of three determinations (bars, ±S.E.).
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Table I.

Specific activities and Km values of bacterial recombinant PKCdelta wt and PKCdelta Ala505 mutant as well as of recombinant PKCdelta expressed in baculovirus-infected insect cells (Baculo)

Phosphorylation of extracts (20 µg) with [32P]ATP in the presence of PS and TPA and with histone III-S or pseudosubstrate-related peptide delta  as substrates was performed as described under "Experimental Procedures" (protein kinase assay).
Peptide delta
Histone III-S
Km Spec. act.a Km Spec. act.a

µM units/mg µg/ml units/mg
Baculo 13 3.95 70 2.0
wt 5 1.4 125 1.1
Ala505 6 2.0 140 1.5

a Spec. act., specific activity.

The expression of functional PKCdelta in bacteria indicated that PKCdelta , unlike PKCalpha (24) and beta II (25), does not require phosphorylation of Thr505 (corresponding to Thr497 and Thr500 in PKCalpha and beta 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 PKCdelta , we exchanged threonine 505 for alanine by site-directed mutagenesis and transformed bacteria with the expression vector pET28delta Ala505 containing the PKCdelta -mutant cDNA. The PKCdelta mutant (PKCdelta Ala505) was expressed by the bacteria as effectively as the PKCdelta 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 delta  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 delta , and 125 and 140 µM, respectively, for histone III-S) were essentially the same (Table I).

For a more detailed characterization of bacterial recombinant PKCdelta wild type and the mutant PKCdelta 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 PKCdelta Ala505 behaved like the wild type. No autophosphorylated PKCdelta could be detected in extracts of bacteria transformed with the pET28 vector alone.


Fig. 3. Autophosphorylation of bacterial recombinant PKCdelta wild type and PKCdelta Ala505 mutant. 10 µl of the diluted extracts (see Fig. 2) from bacteria transformed with the plasmids pET28, pET28delta wt (wt), or pET28delta Ala505 (Ala505) were phosphorylated with [32P]ATP in vitro in the presence or absence of PS and PS and TPA as described under "Experimental Procedures." Phosphorylated proteins were separated by SDS-PAGE and visualized by autoradiography. The location of PKCdelta , as identified by immunoblotting, is indicated by an arrow.
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Phosphorylation of PKCdelta 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 PKCdelta Ala505, were phosphorylated by Src and exhibited decreased mobility on tyrosine phosphorylation, as shown by immunoblotting with an anti-PKCdelta 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.


Fig. 4. Phosphorylation of bacterial recombinant PKCdelta wild type (wt) and PKCdelta Ala505 mutant (Ala505) with the tyrosine kinase Src. Diluted bacterial extracts (10 µl, see Fig. 2) were phosphorylated with 6 units of c-Src as described under "Experimental Procedures." PKCdelta was identified by immunoblotting with an anti-PKCdelta antibody (A). After stripping of the blot, tyrosine-phosphorylated proteins, including PKCdelta , were identified using an anti-phosphotyrosine antibody (B), as described under "Experimental Procedures." As secondary antibodies goat anti-rabbit and goat anti-mouse peroxidase-conjugated antibodies were used for the first and second blots, respectively. The location of PKCdelta is indicated by arrows (note the shift on tyrosine phosphorylation).
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Finally, we studied the inhibition of both types of recombinant PKCdelta 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 PKCdelta , 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 PKCdelta 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.


Fig. 5. Suppression of the kinase activity of bacterial recombinant PKCdelta wild type (wt) and PKCdelta Ala505 mutant (Ala505) by the staurosporine-derived inhibitors Gö 6976 (A) and Gö 6983 (B). The enzyme activity of PKCdelta in the diluted extracts (10 µl, see Fig. 2) was determined in the absence or presence of the inhibitors (concentrations as indicated) by the kinase assay, as described under "Experimental Procedures." Kinase activity was determined in the presence of PS and TPA and is given as percentage of control (activity in the absence of inhibitor).
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We could not entirely exclude the possibility that in PKCdelta 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 PKCalpha and beta II, respectively. Therefore, we expressed two other mutants of PKCdelta , PKCdelta Ala504 and PKCdelta 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 delta  in a TPA-dependent manner and thus did not differ from the wild type. The same could be demonstrated with autophosphorylation of the PKCdelta mutants (data not shown).

In preliminary experiments bacterial recombinant PKCdelta (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 (PKCdelta Ala505), and 54.2 ± 6.1 units/mg protein (PKCdelta Ala504/Ala505).


DISCUSSION

Fabbro and co-workers (32) provided the first evidence of PKCalpha 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 PKCalpha results in a recombinant protein devoid of kinase activity (22-24). It was suspected that phosphorylation by another protein kinase is necessary for PKCalpha to gain the ability of being activated, and that bacteria lack this putative PKC kinase. According to our results, PKCdelta can be expressed in bacteria in a functional form. Agreeing with previous reports (22-24), we were unable to express enzymatically active PKCalpha in bacteria using the same expression vector and the same conditions as for the expression of PKCdelta (data not shown). The activity of bacterially expressed PKCdelta regarding substrate phosphorylation and TPA dependence as well as its Km values for two substrates are comparable to those of recombinant PKCdelta expressed in baculovirus-infected insect cells. Moreover, partial purification of recombinant PKCdelta 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 PKCdelta 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 PKCdelta 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 PKCalpha (24) and rat PKCbeta 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 PKCalpha (24) or PKCbeta II (25) with a neutral, nonphosphorylatable residue results in kinases that cannot be activated. The corresponding site in rat PKCdelta is Thr505. Bacterial expression of a PKCdelta 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 PKCdelta can be expressed in bacteria, and that Thr505, contrary to Thr497 and Thr500 in PKCalpha and beta II, respectively, is not a critical site for permissive activation of PKCdelta . As Ser504 is another amino acid that can be phosphorylated in this region of PKCdelta , 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 PKCdelta 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 PKCdelta and native PKCdelta from porcine spleen (see above). This proves that neither Thr505 nor Ser504 is essential for gaining a catalytically competent conformation of PKCdelta . As each PKC isoenzyme known so far contains threonine in a position corresponding to positions 497 and 500 of PKCalpha and beta II, respectively, the findings regarding the critical role of this threonine residue for the activation process of PKCalpha and beta II have been thought to be valid for all PKC isoenzymes (24, 40). According to our results, however, at least the isotype delta  is an exception to this apparent rule. PKCdelta 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 PKCalpha (32, 41, 42) and on the in vivo phosphorylation sites of PKCbeta II (40, 43), Newton and co-workers (40, 44) have shown that phosphorylation of threonine 500, putatively by the PKC kinase, enables PKCbeta 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 PKCbeta I phosphorylation of Thr642 (corresponding to Thr641 in PKCbeta 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 PKCalpha (corresponding to Thr642 of PKCbeta I and Thr641 of PKCbeta 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 PKCdelta also requires autophosphorylation of the corresponding site, i.e. Ser643, for stabilization of a catalytically competent conformation. As indicated by our results, however, PKCdelta is able to autophosphorylate without having been prephosphorylated by another kinase. As a consequence, a putative regulation of this PKC kinase would not affect PKCdelta , and thus PKC kinase might be a target for a differential regulation of PKC isoenzymes. Identification of the in vivo phosphorylation sites of PKCdelta should allow an answer to the question of whether any of the three in vivo phosphorylation sites found in PKCbeta II (40, 43) play a role also in PKCdelta .

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 PKCdelta . To get a better idea of the structural situation around Thr505 in PKCdelta , we exchanged the side chains in the crystal structure of the catalytic core of cAPK for those of PKCdelta 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 PKCdelta ; His87 from the small lobe is replaced by Cys391 in PKCdelta , and Thr195 is replaced by Ala503. A cysteine in the position equivalent to His87 of cAPK is also conserved in PKCalpha and PKCbeta 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 PKCdelta . It is likely that Arg165, which precedes the catalytic base, and perhaps Lys189 are conserved in protein kinases and thus also in PKCdelta 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 PKCdelta the dispensable Thr505-P may be functionally substituted. Several possibilities can be discussed to explain the observed Thr505-independent activity of PKCdelta . If an ionic interaction at this site is also needed for PKCdelta , 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 PKCdelta 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 PKCdelta . The fact that Thr505 is dispensable for the permissive activation of PKCdelta 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.


Fig. 7. Model of the region around Thr505 in PKCdelta (lower part) based on the crystal structure of cAPK (Protein Data Bank entry 1CDK; Ref. 47; upper part). In cAPK, the network of ionic and nonionic interactions in the region of the Thr197 phosphoryl group is indicated by dotted lines. The interactions of Arg165 may be significant, because this residue precedes the catalytic base in the kinase reaction. A serine inserted in the position of the bound pseudosubstrate alanine (SerPEP) illustrates the contact of the seryl hydroxyl with the catalytic base Asp166 (dotted line) prior to nucleophilic attack of the gamma -phosphorus (a zigzag symbolizes in-line transfer). Metal ligands of the bound nucleotide are shown as crosses. In the model of PKCdelta , residues that are not conserved between both enzymes are displayed in bold. A phosphoryl group at Thr505 and any possible interactions with Thr505 are omitted. Unless the conserved residues adopt different conformations, from the complex network seen in cAPK, only the hydrogen bonds of the guanidinium group of Arg470 with the carbonyl of Met492 and the hydroxyl of Tyr523 remain possible (dotted lines).
[View Larger Version of this Image (24K GIF file)]



FOOTNOTES

*   This work was supported by Deutsche Forschungsgemeinschaft Grants Ma 381/12-2 and Ki 173/13-2.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.
Dagger    These authors contributed equally to these studies.
   To whom correspondence should be addressed. Tel.: 49-6221-424505; Fax: 49-6221-424554; E-mail: m.gschwendt@dkfz- heidelberg.de.
1   The abbreviations used are: PKC, protein kinase C; cAPK, cAMP-dependent protein kinase; TPA, 12-O-tetradecanoylphorbol-13-acetate; PS, phosphatidyl serine; PAGE, polyacrylamide gel electrophoresis; wt, wild type; MES, 4-morpholineethanesulfonic acid.

Acknowledgment

We thank Dr. Volker Kinzel for fruitful discussions.


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