Requirements of Protein Kinase Cdelta 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 GschwendtDagger

From the German Cancer Research Center, D-69120 Heidelberg, Germany

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Recently, we reported that, in contrast to protein kinase C (PKC)alpha and beta II, PKCdelta 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 PKCdelta . 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 PKCdelta might take over at least part of the role of the phosphate groups on Thr497 and Thr500 of PKCalpha and beta II, respectively. Accordingly, PKCdelta exhibits kinase activity and is able to autophosphorylate probably without posttranslational modification. Autophosphorylation of PKCdelta in vitro occurs on Ser643, as demonstrated by matrix-assisted laser desorption ionization mass spectrometry of tryptic peptides of autophosphorylated PKCdelta wild type and mutants. A peptide containing this site is phosphorylated also in vivo, i.e. in recombinant PKCdelta purified from baculovirus-infected insect cells. A Ser643 to alanine mutation indicates that autophosphorylation of Ser643 is not essential for the kinase activity of PKCdelta . Probably additional (auto)phosphorylation site(s) exist that have not yet been identified.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 (alpha , beta 1, beta 2, and gamma ), the Ca2+-independent, diacylglycerol-activated novel PKCs (delta , epsilon , eta , theta , and µ), and the Ca2+-independent diacylglycerol-non-responsive atypical PKCs (zeta  and lambda /iota ). PKCµ is an novel PKC, but with some special structural and enzymatic properties.

PKCdelta 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 PKCdelta , 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 PKCalpha (37) and PKCbeta II (38), PKCdelta does not require phosphorylation of a specific threonine (Thr505 corresponding to Thr497 and Thr500 of PKCalpha and beta II, respectively) in the activation loop for catalytic competence (39). PKCdelta 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 PKCdelta from porcine spleen (39). Recent studies with a Ser643 to alanine mutant of PKCdelta indicated that phosphorylation of this residue might be required for enzymatic activity (40). Ser643 was not unequivocally identified as an autophosphorylation site of PKCdelta but corresponds to one of the in vivo autophosphorylation sites of PKCbeta II, i.e. Thr641 (41, 42). Finally, it was reported by several groups that tyrosine phosphorylation affects the kinase activity of PKCdelta 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 PKCdelta and thus might take over at least part of the role of the phosphate groups of Thr497 and Thr500 of PKCalpha and beta II, respectively. By MALDI mass spectrometry of tryptic PKCdelta peptides we provide evidence that Ser643 is an autophosphorylation site of PKCdelta in vitro and that a PKCdelta peptide containing Ser643 is phosphorylated also in vivo.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

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 PKCdelta full-length cDNA clone (3000 bp) containing the sequence coding for PKCdelta was a gift from Dr. C. Polke (University of Würzburg, Würzburg, Germany). The vectors pET28delta Ala505 and pET28delta Ala504/5 were provided by Dr. D. Bossemeyer and Dr. A. Girod (German Cancer Research Center). The pseudosubstrate-related peptide delta  (MNRRGSIKQAKI) was synthesized by Dr. R. Pipkorn (German Cancer Research Center). Other materials were bought from the following companies: PS from Sigma; [gamma -32P]ATP (specific activity, 5000 Ci/mmol) from Hartmann Analytic (Braunschweig, Germany); mouse monoclonal anti-PKCdelta 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 PKCdelta -cDNA-- As described previously (39) 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 was amplified by polymerase chain reaction and cloned into the expression vector pET28. The plasmid was termed pET28delta wt.

Site-directed mutagenesis was performed using the "overlap extension" method as described previously (39). A rat PKCdelta 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 pET28delta Val500, pET28delta Ala643, and pET28delta Ala643/5.

Bacterial Expression and Partial Purification of Recombinant His-tagged PKCdelta -- E. coli BL21(DE3)pLysS cells were used for expression of the recombinant PKCdelta 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 PKCdelta with the highest specific activity. According to Coomassie Blue staining a 75% purity of PKCdelta was estimated. Eluted PKCdelta was stabilized by addition of 10% glycerol and 10 mM beta -mercaptoethanol and stored at -70 °C.

Expression of Recombinant PKCdelta Containing a C-terminal His Tag in the Baculovirus-Insect Cell System-- For the cloning of PKCdelta full-length cDNA into the pBac1 baculovirus transfer plasmid (Novagen), a PKCdelta 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 pET28delta 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 PKCdelta was partially purified as described above for the bacterially expressed enzyme. According to Coomassie Blue staining, 80% purity of PKCdelta 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 delta  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%). PKCdelta was detected and quantitated by immunoblotting using the monoclonal anti-PKCdelta 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 PKCdelta wild type and mutants was performed as described above, but [gamma -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 PKCdelta 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 alpha -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 beta -chain.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Phosphorylation of Thr497 and Thr500 in the activation loop of PKCalpha (37) and PKCbeta II (38), respectively, is known to be required for catalytic competence of the enzymes. Recently, we demonstrated that PKCdelta 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 PKCdelta to PKCalpha and beta II. In contrast to PKCalpha and PKCbeta II, PKCdelta contains a glutamic acid in position 500 (Glu500) that might take over the role of the phosphorylated threonines of PKCalpha and beta II.

To test this possibility, we mutated Glu500 to the neutral amino acid valine and determined the enzymatic activity of the bacterially expressed PKCdelta Val500 mutant. All phosphorylation assays were performed in the presence of PS and TPA. Autophosphorylation of partially purified wild type and PKCdelta 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-PKCdelta 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 PKCdelta 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 delta  by PKCdelta Val500 was reduced by 73 ± 2% compared with that by the wild type (Table I). The low incorporation of phosphate into the peptide by PKCdelta Val500 was essentially attributable to a slower velocity of the phosphorylation reaction. Vmax of the PKCdelta 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 PKCdelta 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 PKCdelta Ala505 and PKCdelta 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 PKCdelta 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 PKCdelta and might function in a similar way as the phosphate groups of Thr497 and Thr500 in PKCalpha and beta II, respectively, in correctly aligning residues involved in catalysis by interaction with positively charged residues of the catalytic core. However, the residual activity of PKCdelta Val500 (~25%) indicates that, in addition to Glu500, some as yet unknown structural features of PKCdelta 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 PKCbeta 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 PKCdelta ; however, aspartic acid is located in this position in some isoforms (PKCzeta , iota /lambda , and theta ). It is not yet known whether these aspartic acid residues function similarly to the glutamic acid 500 of PKCdelta . 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 PKCdelta wild type (wt) and PKCdelta Val500 mutant (Val500). Bacterial recombinant PKCdelta 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." PKCdelta (arrow) was detected by immunoblotting and quantitated with a scanner. Values are given as arbitrary units (a.u.). Radiolabeled PKCdelta 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 PKCdelta wild type and mutants
Phosphorylation of the pseudosubstrate-related peptide delta  (peptide delta ) with partially purified PKCdelta 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 PKCdelta wild type and Val500 mutant. The kinase activities of partially purified PKCdelta wild type (A) and Val500 mutant (B) were determined as described under "Experimental Procedures." Five µg of the pseudosubstrate-related peptide delta  were phosphorylated by PKCdelta 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).

Mutation of Glu500 to Val affected neither the thermal stability of PKCdelta nor its inhibition by the specific PKC inhibitors Gö6983 and Gö6976. The kinase activity of the mutant, like wild type PKCdelta , remained relatively stable at room temperature for at least 40 min (Fig. 3). Inhibition of mutant and wild type PKCdelta 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 PKCdelta wild type and PKCdelta Ala643 (Ala643) and PKCdelta Val500 (Val500) mutants. Partially purified PKCdelta 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 PKCdelta wild type (wt) and PKCdelta Val500 (Val500) mutant by the inhibitors Gö6983 (A) and Gö6976 (B). The enzyme activities of partially purified PKCdelta 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).

PKCbeta 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 PKCbeta I (52). Phosphorylation of the corresponding Thr638 of PKCalpha , 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 PKCdelta , as a first step we attempted to identify autophosphorylation sites of in vitro phosphorylated PKCdelta .

Recombinant PKCdelta partially purified from bacterial extracts was phosphorylated in the presence of PS and TPA. Phosphorylated and nonphosphorylated PKCdelta were applied to SDS-polyacrylamide gel electrophoresis. The PKCdelta 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 PKCdelta wild type, whereas the lower panel shows the corresponding tryptic peptides from the phosphorylated PKCdelta 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 PKCdelta (Fig. 5, upper panel) could be assigned to the PKCdelta peptide 624SPSDYSNFDPEFLNEKPQLSFSDK647. In the mass spectrum of phosphorylated PKCdelta (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 PKCdelta 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 PKCdelta (Fig. 5, upper panel), indicating that peptide 624-647 of bacterially expressed PKCdelta 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 PKCdelta (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) PKCdelta . The ion signal at m/z 2791 corresponds to the nonphosphorylated tryptic peptide 624-647 of PKCdelta (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).

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 PKCbeta II (41, 42) and thus could be assumed to be one of the autophosphorylated residues of PKCdelta . Furthermore, comparison of phosphopeptide maps of in vivo phosphorylated PKCdelta wild type and Ser643 to alanine mutant indicated that Ser643 might be an in vivo phosphorylation site of PKCdelta (40). However, autophosphorylation was not proven, and a phosphopeptide containing Ser643 was not identified. Therefore, we mutated Ser643 to alanine (PKCdelta Ala643) and also produced the double mutant Ser643/645-Ala (PKCdelta Ala643/5) by site-directed mutagenesis. Both mutants were phosphorylated and then treated as described above for the PKCdelta wild type. Expanded views of the MALDI mass spectra obtained from the tryptic peptides of the phosphorylated mutants PKCdelta Ala643/5 (upper panel) and PKCdelta 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 PKCdelta wild type with masses of 2759 (2791 - 32, i.e. the mass of two oxygens) for PKCdelta Ala643/5 and 2775 (2791 - 16, i.e. the mass of one oxygen) for PKCdelta 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 PKCdelta . A small, not clearly recognizable, signal at m/z 2855 in the mass spectrum of PKCdelta 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 PKCdelta 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 PKCdelta partially purified from baculovirus-infected insect cells. The MALDI mass spectrum indicated complete phosphorylation of PKCdelta 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 PKCdelta (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 PKCdelta 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 PKCdelta 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 PKCdelta wild type and mutant (see the immunoblot in Fig. 8), as described above. A similar result was obtained with the PKCdelta 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 PKCdelta . 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 PKCdelta (Table I), nor its thermal stability (Fig. 3). The lack of requirement of Ser643 autophosphorylation for kinase activity of PKCdelta is in agreement with the entirely intact kinase activity of untreated PKCdelta 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 PKCalpha 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 PKCdelta expressed in myeloid 32D cells. One possible explanation for these contradictory results might be the different sources and purities of PKCdelta 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 PKCdelta 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 PKCdelta Ala643/5 mutant (upper panel) and of the phosphorylated PKCdelta 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 PKCdelta from baculovirus-infected insect cells. The ion signal at m/z 2871 corresponds to the phosphorylated tryptic peptide 624-647 of PKCdelta . 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 PKCdelta wild type (wt) and PKCdelta Ala643 mutant (Ala643). Partially purified PKCdelta 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 PKCdelta 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 PKCdelta depends in part on the acidic residue Glu500 in the activation loop and, as reported earlier (39), not on phosphorylated Thr505. Thus, PKCdelta exhibits kinase activity and is able to autophosphorylate without the posttranslational modification that is required for catalytic competence of PKCalpha and beta II. Autophosphorylation in vitro, and most likely also in vivo, occurs on Ser643, which corresponds to the autophosphorylation site Thr641 of PKCbeta II. Probably other (auto)phosphorylation site(s) of PKCdelta 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.

Dagger 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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