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J. Biol. Chem., Vol. 277, Issue 48, 45866-45873, November 29, 2002

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Lack of Constitutive Activity of the Free Kinase Domain of Protein Kinase C

DEPENDENCE ON TRANSPHOSPHORYLATION OF THE ACTIVATION LOOP^*

Lucinda Smith and Jeffrey B. Smith

From the Department of Pharmacology and Toxicology, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294

Received for publication, June 28, 2002, and in revised form, September 5, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Following the induction of apoptosis in mammalian cells, protein kinase C  (PKC ) is processed between the regulatory and catalytic domains by caspases, which increases its kinase activity. The catalytic domain fragments of PKC isoforms are considered to be constitutively active, because they lack the autoinhibitory amino-terminal regulatory domain, which includes a pseudosubstrate segment that plugs the active site. Phosphorylation of the activation loop at Thr⁴¹⁰ is known to be sufficient to activate the kinase function of full-length PKC , apparently by inducing a conformational change, which displaces the amino-terminal pseudosubstrate segment from the active site. Amino acid substitutions for Thr⁴¹⁰ of the catalytic domain of PKC  (CAT ) essentially abolished the kinase function of ectopically expressed CAT  in mammalian cells. Similarly, substitution of Ala for a Phe of the docking motif for phosphoinositide-dependent kinase-1 prevented activation loop phosphorylation and abolished the kinase activity of CAT . Treatment of purified CAT  with the catalytic subunit of protein phosphatase 1 decreased activation loop phosphorylation and kinase activity. Recombinant CAT  from bacteria lacked detectable kinase activity. Phosphoinositide-dependent kinase-1 phosphorylated the activation loop and activated recombinant CAT  from bacteria. Treatment of HeLa cells with fetal bovine serum markedly increased the phosphothreonine 410 content of CAT  and stimulated its kinase activity. These findings indicate that the catalytic domain of PKC  is intrinsically inactive and dependent on the transphosphorylation of the activation loop.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The protein kinase C (PKC)¹ family comprises a dozen or more structurally related phospholipid-dependent serine/threonine kinases, which consist of a carboxyl-terminal catalytic domain and an amino-terminal regulatory domain. The regulatory domain includes an inhibitory pseudosubstrate segment and allosteric sites for phosphatidylserine and, depending on the isoform, also for calcium and/or diacylglycerol (1-3). Two family members,  and /, are atypical because they have only half of the C1 domain and hence are not activated by C1 ligands, such as diacylglycerol, phorbol ester tumor promoters, or bryostatins (1-4). Phosphorylation of threonine 410 of PKC  by 3-phosphoinositide-dependent kinase-1 appears to be necessary and sufficient to activate its kinase function (5, 6), although PtdIns 3,4-bisphosphate and PtdIns 3,4,5-P₃ stimulate PKC  activity (7-9). PDK-1 has a pleckstrin homology domain, which binds PtdIns 3,4,5-P₃, a product of the phosphoinositide 3-kinase pathway (10). Phosphorylation by PDK-1 regulates a wide variety of AGC family members in addition to conventional and atypical PKCs (11). Consequently, PDK-1 is a pivotal effector enzyme of the phosphatidylinositol 3-kinase pathway, which transduces prosurvival and mitogenic signals in mammalian cells (12). Interestingly, PKC  also transduces growth and survival signals. The lack of PKC  in embryonic fibroblasts impairs NF-B transcriptional activity (13). Activation of NF-B can explain at least part of the prosurvival function of PKC , because NF-B is known to induce genes that negate apoptotic signals, such as those for inhibitor of apoptosis proteins (14-18).

Although there is little understanding of the precise functions of either of the atypical PKCs in apoptosis, they both seem to promote cell survival, albeit by somewhat different mechanisms (13, 19, 20). PKC /, but not PKC , protected human K562 leukemia cells from apoptosis (20). Following the induction of apoptosis, the , but not the /, isoform of PKC undergoes processing by caspases, a family of highly specific proteases that implement a cell-autonomous death pathway (20-23). Processing of PKC  occurs chiefly at two aspartates (Asp²¹⁰ and Asp²³⁹), which separates the regulatory and catalytic domains, and increases PKC  activity (22). Caspase blockade prevented the production of the two catalytic domain fragments and the increase in kinase activity following the induction of apoptosis in either nontransfected parotid C5 cells or in HeLa cells transfected with epitope-tagged PKC  (22).

Caspase processing may activate PKC  by separating the catalytic domain from the autoinhibitory pseudosubstrate motif, and the free catalytic domain may or may not depend on transphosphorylation for activity. For example, PKC  is highly active without transphosphorylation (24). An acidic residue (Glu⁵⁰⁰ of PKC ) that immediately precedes the PDK-1 substrate motif is critical for the inherent catalytic activity of bacterial expressed nontransphosphorylated PKC  and partially fulfills the role of activation loop phosphorylation of other PKC isoforms (25). The only other PKC isoforms with an acidic residue immediately preceding the PDK-1 substrate motif are PKC /, , and , all of which have an aspartyl residue. In contrast to other isoforms, PKC  has a glutamate at the conserved hydrophobic autophosphorylation site (Glu⁵⁷⁹), which might contribute to constitutive kinase activity. Like PKC , the , , and µ PKC isoforms are efficiently processed by caspases to catalytic domain fragments (26-28). Because they are divorced from the inhibitory regulatory domain, the kinase domain fragments are considered to be constitutively active (1-3). For example, ectopic expression of the kinase domain of PKC  is the method of choice for increasing PKC  activity in mammalian cells (29-31). Additionally, the kinase domain polypeptide of PKC  (also called PKM ) is endogenously expressed in rat brain and considered to be constitutively active (32). We show here, however, that the free catalytic domain of PKC  is inherently inactive and dependent on transphosphorylation by PDK-1 for activation, in contrast to the kinase domain of PKC .

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture, Plasmid Constructs, and Transfection-- HeLa and transformed human embryonic kidney (HEK) 293 cells (ATCC number CRL-1573) were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 units/ml penicillin G, and 0.1 mg/ml streptomycin. Transformed human embryonic retinoblast (HER) 911 cells were grown in a 1:1 mixture of Ham's F-12 and Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 0.1 mg/ml streptomycin (33). Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO₂ and 95% air. HEK 293 and HER 911 cells were transfected by a modified calcium phosphate method (34). HeLa cells were transfected with the previously described pcDNA3.1/GS constructs, human full-length PKC  (amino acids 1-592), or CAT  (amino acids 1-15 and 240-592), with carboxyl-terminal V5 and His₆ tags, using X-tremeGENE Q2 reagent as recommended by the manufacturer (Roche Molecular Biochemicals) (22). Transfection efficiencies were optimized with pcDNA3.1LacZ plasmid (Invitrogen) and histochemical staining with 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal), a -galactosidase substrate (35).

Amino acid substitution mutants were produced by the QuikChange method as described by the manufacturer (Stratagene). Wild type and the T410A mutant of CAT  (amino acids 238-592) flanked by KpnI and EcoRI were produced by PCR with Taq DNA polymerase, TA cloned into pCR2.1 (Invitrogen), and subcloned into the KpnI/EcoRI sites of pcDNA4C to produce CAT  with amino-terminal His₆ and Xpress tags.

PDK-1 and the kinase-dead K111N PDK-1 mutant were cloned into pcDNA4HisMax plasmid (Invitrogen) in order to produce PDK-1 and K/N PDK-1 with amino-terminal His₆ and Xpress epitope tags to facilitate affinity purification from transfected mammalian cells. PDK-1 and K/N PDK-1 cDNAs were produced by PCR with Pfu DNA polymerase using PDK-1 and K/N PDK-1 pcDNA3 plasmids as templates. The cDNAs were simultaneously digested with the restriction enzymes and purified by electrophoresis in a low melt agarose gel. The purified cDNAs were ligated into the KpnI/EcoRI sites of pcDNAHisMax4C. Each of the cDNA constructions was validated by sequencing using dye terminator chemistry and an ABI Prism 377 automated sequencer.

Bacterial Expression and Purification of CAT  and CAT -- CAT  (amino acids 240-592) and human CAT  (amino acids 330-676) cDNAs with flanking 5' HindIII and 3' NotI restriction sites were produced by PCR using Pfu Turbo DNA polymerase (Stratagene) using the above described PKC  pcDNA3.1GS or pBlueBac/PKC  (ATCC number 80048) as template, cloned by the zero blunt method (Novagen), and subcloned into the HindIII/NotI sites of the ProTet.E133 6×HN vector (CLONTECH). Production of CAT  and CAT  proteins with an amino-terminal (His-Asn)₆ tag was induced by treatment of a liter of the BL21PRO strain of Escherichia coli (A₆₀₀ = 0.7) with 100 ng/ml anhydrotetracycline for 24 h at 18 °C. Bacteria were harvested by centrifugation and frozen in a 80 °C freezer. The bacteria were thawed with 10 ml of ice-cold lysis buffer, which contained 1% Triton X-100, 50 mM sodium phosphate, pH 8.0, 0.3 M NaCl, 25 µM acetyl-Leu-Leu-norleucinal, 1 mM Pefabloc SC and PSC protector (Pierce), 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM benzamidine. The cells were disrupted by sonication on ice with a Branson Cell Disrupter 350 at number 5 output (four cycles of 10 s with a 20-s rest between cycles, pulse mode at 50% duty cycle), and the lysate was clarified by centrifugation for 30 min at 4 °C at 80,000 × g. CAT  and  proteins were purified at room temperature with TALON metal affinity resin (CLONTECH). The supernatant (5 ml) was incubated for 1.5 h with 0.5 ml of packed resin, which had been equilibrated with lysis buffer. The resin was collected by centrifugation, suspended with 5 ml of wash buffer containing 50 mM sodium phosphate, pH 8.0, and 0.3 M NaCl, and incubated for 10 min before transferring the suspension to a 2-ml gravity flow column. The resin was washed again with 5 ml of wash buffer and finally with wash buffer containing 5 mM imidazole. Proteins were eluted with wash buffer containing 0.2 M imidazole, and 0.5-ml fractions were collected. EDTA, EGTA, and DTT were added to the fractions to final concentrations of 0.1, 0.1, and 1 mM, glycerol was added to 20%, and the fractions were frozen in liquid N₂ and stored at 80 °C. Purified proteins were fractionated by SDS-PAGE (10% gel), subjected to Western blot analysis, and immunostained with polyclonal antibodies to the extreme carboxyl-terminal segment of CAT  (C-20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or CAT  (C-20; Santa Cruz Biotechnology). The eluate fraction with the highest concentration of CAT  or  (usually the second fraction) was used directly for treatment with PDK-1 and assaying kinase activity.

Affinity Purification of PKC , CAT , and PDK-1 from Mammalian Cells-- Enzymatically active forms of these three proteins were affinity-purified following transient expression in mammalian cell lines. Two days after transfection, the cells were rinsed three times with ice-cold phosphate-buffered saline, detached by scraping, collected by centrifugation, and suspended with lysis buffer, which contained 25 mM Tris-HCl, pH 7.7, 150 mM NaCl, 20 mM -glycerophosphate, 10 mM imidazole, 0.1% Nonidet P-40, 0.5 mM DTT, 0.1 mM sodium orthovanadate, 1 mM Pefabloc SC and PSC protector, 25 mM acetyl-Leu-Leu-norleucinal, and 2 µg/ml leupeptin. The cells were homogenized with a 26-gauge needle, and the homogenate was centrifuged for 20 min at 16,000 × g at 4 °C. The supernatant was incubated for 30 min with 0.05 ml of packed nickel-nitrilotriacetic acid resin (Qiagen) that had been equilibrated with ice cold lysis buffer. The resin was washed at 4 °C by centrifugation as follows: once with lysis buffer lacking acetyl-Leu-Leu-norleucinal, Pefabloc SC, and PSC protector; three times with wash buffer (50 mM sodium phosphate, pH 8.0, containing 20 mM imidazole, and 300 mM NaCl); and three times with wash buffer lacking NaCl. Proteins were eluted with two 0.1-ml volumes of elution buffer that contained 50 mM sodium phosphate buffer, pH 8.0, 250 mM imidazole, and 20% glycerol. EDTA, EGTA, and DTT were added to 1 mM, and the fractions were frozen in liquid N₂ and stored at 80 °C. Purified proteins were fractionated by SDS-PAGE (10% gel) and subjected to Western blot analysis. Purified wild type and mutant forms of PKC , CAT , and PDK-1 were prepared by this method. Transfected HER 911 cells were the source of PDK-1 and amino-terminal His₆ Xpress-tagged CAT  and T410A CAT . Transfected HEK 293 cells were the source of carboxyl-terminal V5 His₆ tagged wild type and mutant PKC  and CAT .

Assay of Kinase Activity and Treatment of CAT  with PDK-1 or the Catalytic Subunit of Protein Phosphatase 1 (CS1)-- Immunoprecipitation of epitope-tagged PKC  or CAT  with a monoclonal antibody to the V5 epitope tag (Invitrogen) and assay of immune complex kinase activity using MBP as phosphoacceptor were done as previously described (22). Each assay (30 µl) contained 25 mM Hepes-Tris, pH 7.4, 20 mM -glycerophosphate, 10 mM MgCl₂, 2 mM DTT, 0.1 mM sodium orthovanadate, 20 µM ATP, and an immune complex or affinity-purified PKC , CAT , and/or PDK-1. For the treatment of affinity-purified CAT  or F578A CAT  with PDK-1 or K110N PDK-1, the reactions contained 10 µM sn-1,2, dipalmitoyl PtdIns 3,4,5-P₃ (Calbiochem), and 100 µM each of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (Avanti Polar Lipids) for 1 h at 30 °C (30 µl/assay). Kinase assays were started by adding (7.5 µl/assay) 5 µCi of [-³²P]ATP and either 5 µg of MBP or 40 µM  peptide (ERMRPRKRQGSVRRRV) as substrate. After 30 min at 30 °C, the reactions were stopped by pipetting 25 µl onto a 2.1-cm phosphocellulose P81 disc (Whatman) and rinsing the discs with 5% acetic acid. The amount of ³²P-labeled peptide bound to the disc was determined by scintillation counting.

Immunoprecipitated CAT  was incubated at 22 °C for 1 h with 2 µg of human recombinant CS1 ( isoform, 3 units/mg; Calbiochem) in a buffer (30 µl total) that contained 20 mM Hepes-Tris, pH 7.5, 1 mM DTT, 0.2 mM MnCl₂, 0.5 mM CaCl₂, and 0.04 mM EDTA. The reaction was stopped by rinsing the immunoprecipitate three times with kinase buffer containing 1 µM calyculin A. Kinase activity was assayed with MBP as substrate as described above. Affinity-purified CAT  (2 µl) was incubated with 0.4 µg of CS1 in the buffer described above with 0.1% bovine serum albumin and 1 mM MnCl₂. Reactions were stopped by the addition of calyculin A to 1 µM, and kinase activity was assayed with MBP as substrate.

Western Blot Analyses-- A horseradish peroxidase-conjugated anti-V5 antibody (Invitrogen) was used for Western analysis of immunoprecipitates from HeLa cells transfected with epitope-tagged PKC  or CAT . Affinity-purified PDK-1 or K/N PDK-1 were quantified by Western blot analysis with anti-Xpress antibody (Invitrogen). Western analysis was done essentially as previously described (22, 36). Briefly, proteins were fractionated by SDS-PAGE (10% gel), transferred electrophoretically to a polyvinylidene difluoride membrane (Millipore Corp.). The membrane was blocked for 1 h at room temperature with 5% (w/v) nonfat dry milk in Tris-buffered saline (TBS), which contained 8 g/liter NaCl, 0.2 g/liter KCl, and 3 g/liter Tris and was adjusted to pH 7.4 with HCl and with 0.05% (w/v) Tween 20 in the case of TTBS. Following the incubation with horseradish peroxidase-conjugated first or second antibody, the membrane was rinsed with TTBS and incubated with a chemiluminescent substrate (36). Some membranes were immunostained with a rabbit polyclonal antibody (catalog no. 2291; Cell Signaling Technology) that specifically recognizes the phosphothreonine-FCGT motif.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Caspase Processing between the Regulatory and Catalytic Domains Activates Wild Type, but Not Threonine 410 Mutants of PKC -- It is generally assumed that the free kinase domain of various PKC isoforms is constitutively active because it lacks the inhibitory regulatory domain that includes a pseudosubstrate motif (2, 3). To determine whether or not the free kinase domain of PKC  depended on threonine 410 for activity, HeLa cells were transfected with wild type or threonine 410 mutants of PKC . A brief treatment of HeLa cells with TNF plus CHX, which activates caspase-3, evoked the processing of most full-length PKC  to two catalytic domain fragments and increased immune complex kinase activity (Fig. 1), as previously reported (22). The TNF/CHX treatment induced caspase processing of the T410E mutant similarly to wild type PKC ; however, the carboxyl-terminal fragment of the T410E mutant had low but significant kinase activity (Fig. 1). This result suggests that threonine 410 has a marked impact on the kinase function of the free catalytic domain of PKC .

View larger version (63K): [in this window] [in a new window]   Fig. 1.   Caspase processing of PKC  to the free catalytic domain activates the wild type but not threonine 410 mutants following induction of apoptosis in HeLa cells. Wild type or the T410A or T410E mutants of PKC  or CAT  were expressed in HeLa cells. The indicated cultures were treated with 50 ng/ml TNF plus 10 µg of CHX for 4 h, the cells were lysed, and PKC  and CAT  were immunoprecipitated with antibody to the V5 epitope. Immunoprecipitates were assayed for kinase activity with MBP as substrate and subjected to Western analysis with horseradish peroxidase-conjugated antibody to the V5 epitope. The data are representative of five experiments.

Although the TNF/CHX treatment evoked efficient processing of the T410A PKC  mutant, relatively little of the carboxyl-terminal fragments accumulated (Fig. 1). Hence, HeLa cells were transfected with wild type or threonine 410 mutants of CAT . The CAT  constructs migrated slightly more slowly than the major fragment produced by caspase processing of full-length PKC , because they have 15 additional amino acids (residues 1-15 of PKC ). Fig. 1 shows that the T410A CAT  immune complex lacked detectable kinase activity and that the T410E CAT  immune complex had low but significant kinase activity. These findings agree with the results with affinity-purified T410 CAT  mutants (Fig. 2A) and suggest that threonine 410 modulates the kinase function of the carboxyl-terminal fragment of PKC .

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Free kinase domain of PKC  depends on the phosphorylation of threonine 410 for catalytic activity in transfected cells. A, carboxyl-terminal V5 His6-tagged wild type full-length PKC  and CAT  and the T410A and T410E/HER mutants were affinity-purified from HEK 911 cells, assayed for kinase activity with MBP as substrate, and subjected to Western analysis with an antibody that specifically recognized phosphothreonine 410 or the V5 epitope. B, carboxyl-terminal V5 His6-tagged wild type CAT  and the T410A and F578A CAT  mutants were affinity-purified from HEK 293 cells, assayed for kinase activity with MBP as substrate, and subjected to Western analysis with an antibody that specifically recognized phosphothreonine 410 or the V5 epitope. C, amino-terminal His6 Xpress-tagged CAT  or the T410A mutant were affinity-purified from HER 911 cells, assayed for kinase activity with MBP as substrate, and subjected to Western analysis with antibody to the Xpress epitope. The data are representative of at least four experiments.

The carboxyl-terminal fragments of the T410A CAT  appear to be less stable than the wild type or T410E fragments following their production by the TNF/CHX treatment. CAT  is a relatively short lived protein and, like full-length PKC , is ubiquitinated and degraded by the proteasome (22). The half-life of CAT  was 1.6 ± 0.4 h (n = 4) (mean ± S.E.), as determined by Western blot analysis following the treatment of the HeLa cells with CHX for 1-4 h.

Dependence of CAT  on Thr⁴¹⁰ Phosphorylation and the PDK-1 Docking Motif for Activity-- The deficiency in the kinase activity of the T410A and T410E CAT  immune complexes from transfected HeLa cells was confirmed following metal affinity purification from transfected HER 911 cells (Fig. 2A). Wild type CAT  was active and positively immunostained with an antibody that specifically recognized the phosphorylated PDK-1 substrate motif (phospho-Thr-Phe-Cys-Gly-Thr) (Fig. 2A). The T410A CAT  mutant lacked detectable kinase activity and was not recognized by the phospho-Thr⁴¹⁰ antibody (Fig. 2A). The mutant with the negatively charged glutamate substituted for Thr⁴¹⁰ (T410E CAT ) to partially mimic phosphothreonine had significant kinase activity, although it was much less active than wild type CAT  (Fig. 2A).

PDK-1 was recently shown to dock to a hydrophobic motif in the carboxyl-terminal tail of PKC  (37). Substitution of Ala for Phe⁵⁷⁸ of this motif essentially abolished Thr⁴¹⁰ phosphorylation and the kinase activity of immune complexes from 293 cells transfected with amino-terminal epitope-tagged full-length PKC  (37). We have confirmed this result and extended it to the free catalytic domain. In contrast to wild type CAT , F578A CAT  had no detectable kinase activity and was not immunostained by the phospho-Thr⁴¹⁰ antibody following affinity purification from transfected HEK 293 cells (Fig. 2B). The F578A mutant of full-length PKC  was weakly immunostained by the phospho-Thr⁴¹⁰ antibody and exhibited weak but significant kinase activity (Fig. 2B). The regulatory domain apparently influences Thr⁴¹⁰ phosphorylation in 293 cells, because F578A CAT  lacked detectable phospho-Thr⁴¹⁰, whereas F578A PKC  was partially active and phosphorylated at Thr⁴¹⁰.

To address the possibility that the carboxyl-terminal V5 and His₆ tags or the inclusion of the first 15 amino acids of PKC  was responsible for the requirement for phospho-Thr⁴¹⁰ for kinase activity, amino-terminal His₆ Xpress-tagged CAT  (amino acids 238-592 of PKC ) was affinity-purified from transfected HER 911 cells. Wild type His₆-Xpress-tagged CAT  had similar kinase activity as the carboxyl-terminal tagged CAT , whereas the T410A mutant of amino-terminal tagged CAT  had no detectable activity (Fig. 2C). Additionally, the wild type amino-terminal tagged CAT  immunostained positively for phospho-Thr⁴¹⁰ similarly to the carboxyl-tagged CAT .² Thus, the kinase function of CAT  depended on phospho-Thr⁴¹⁰ whether the epitope tags were on the amino- or the carboxyl terminus and whether or not the CAT  included the first 15 amino acids of PKC .

Dephosphorylation of CAT  by CS1 Decreased Phospho-Thr⁴¹⁰ and Kinase Activity-- Treatment of CAT  with CS1, the catalytic subunit of protein phosphatase 1, markedly decreased the immune complex kinase activity of CAT  immunoprecipitated from transfected HeLa cells (Fig. 3A). The presence of the phosphatase inhibitor, calyculin A, during the incubation with CS1 prevented the decrease in the kinase activity of the CAT  immune complex (Fig. 3A). In order to determine the effect of CS1 on phospho-Thr⁴¹⁰, CAT  was affinity-purified from HER 911 cells and treated with CS1 in the presence or absence of calyculin A (Fig. 3B). CS1 treatment for 2 or 18 h markedly decreased immunostaining of phospho-Thr⁴¹⁰ in the absence but not in the presence of the phosphatase inhibitor (Fig. 3B). As expected, the kinase activity of CAT  paralleled the decrease in phospho-Thr⁴¹⁰ produced by the CS1 treatment (Fig. 3B).

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Treatment of immunoprecipitated or affinity-purified CAT  with the catalytic subunit of protein phosphatase 1 decreased its catalytic activity. A, carboxyl-terminal V5 His6-tagged CAT  was immunoprecipitated from transfected HeLa cells and treated for 1 h with 2 µg of CS1 in the presence or absence of 1 µM calyculin A as indicated. Immunoprecipitates were rinsed with kinase buffer, assayed for kinase activity with MBP as substrate, and subjected to Western analysis with horseradish peroxidase-conjugated antibody to the V5 epitope. B, carboxyl-terminal V5 His6-tagged wild type CAT  was affinity-purified from HER 911 cells, treated for 2 or 18 h with 0.4 µg of CS1 as indicated in the presence or absence of 1 µM calyculin A, assayed for kinase activity with MBP as substrate, and subjected to Western analysis with an antibody that specifically recognized phosphothreonine 410 or the V5 epitope. The data are representative of three experiments.

Activation of Recombinant CAT  by PDK-1 in Vitro-- CAT  (Fig. 3), like full-length PKC  (5, 37), is phosphorylated at Thr⁴¹⁰ following expression in mammalian cells. To obtain CAT  that was not phosphorylated at Thr⁴¹⁰, recombinant human CAT  (amino acids 240-592) with an amino-terminal (His-Asn)₆ tag was affinity-purified from bacteria. Bacterial expressed CAT  lacked detectable phospho-Thr⁴¹⁰ and kinase activity, which was assayed with a peptide substrate based on the sequence of the pseudosubstrate sequence of PKC  (Fig. 4A). Recombinant purified CAT  was treated for 1 h with PDK-1 or a kinase-dead K/N PDK-1 mutant and assayed for phospho-Thr⁴¹⁰ by Western analysis and for kinase activity. Treatment with PDK-1, but not the K/N PDK-1 mutant, phosphorylated Thr⁴¹⁰ and activated bacterial expressed CAT  (Fig. 4). The activity of the bacterial expressed, PDK-1-activated CAT  was essentially the same as that of a similar amount of affinity-purified CAT  from HEK 293 cells (Fig. 4A), which was based on immunostaining with the anti-V5 epitope tag (Fig. 4B). In contrast to CAT , treatment of bacterial expressed F578A CAT  with PDK-1 failed to phosphorylate Thr⁴¹⁰ or to activate its kinase function (Fig. 4). For these experiments, the PDK-1 treatment was done in the presence of PtdIns 3,4,5-P₃, phosphatidylserine, and phosphatidylcholine, because PDK-1 binds PtdIns 3,4,5-P₃ via a pleckstrin homology domain, which may influence its interaction with substrates (10). We carried out similar experiments in the absence of PtdIns 3,4,5-P₃ or in the absence of all three lipids. These experiments showed that treatment with PDK-1 produced the same extent of CAT  activation in the presence or absence of added lipids.² These findings indicate that phosphorylation of Thr⁴¹⁰ and activation of bacterial expressed CAT  in vitro required the hydrophobic PDK-1 docking motif and was independent of added phospholipids.

View larger version (62K): [in this window] [in a new window]   Fig. 4.   Recombinant CAT  from bacteria depends on Thr410 phosphorylation for kinase activity. Amino-terminal HN6-tagged wild type and F578A CAT  were affinity-purified from E. coli and treated with affinity-purified PDK-1 or the kinase-dead K/N PDK-1 as indicated. Kinase activity was assayed with the  peptide as substrate (A). Values are mean ± S.D. (n = 3-5). Following the treatment with or without PDK-1 or K/N PDK-1, wild type and F578A CAT  were subjected to Western analysis with an antibody that specifically recognizes phosphothreonine 410 or the extreme carboxyl-terminal segment of PKC  (C-20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Note that bacterial expressed ProTet CAT  is predicted to be 2.5 kDa smaller than pcDNA3 CAT  (45.8 kDa) from HEK 293 cells, which accounts for the mobility difference by SDS-PAGE.

In contrast to CAT , the kinase domain of PKC  (CAT ) was highly active following expression in bacteria, and treatment with PDK-1 had no effect on its activity.² CAT  had no detectable immunostaining for phosphothreonine of the activation loop.²

Activation of CAT  by Treatment of HeLa Cells with FBS-- HeLa cells were transfected with CAT  or empty vector and serum-starved for 48 h. The cultures were incubated for 1 or 2 h with or without 10% FBS in the presence of 2 µg/ml actinomycin D. In the absence of actinomycin D, which blocks transcription, the FBS treatment increased the level of CAT  protein, which complicated the analysis of the effect of FBS on phosphothreonine 410 (data not shown). In the presence of actinomycin D, treatment of the cells with FBS for 2 h markedly increased the phosphothreonine 410 content of CAT  without affecting the level of CAT  as determined by immunostaining with antibody to the V5 epitope (Fig. 5A). No CAT  or phosphothreonine 410 were observed by Western blot analysis of cells transfected with empty vector instead of CAT  (Fig. 5A). FBS treatment increased phosphothreonine 410 by 2.9 ± 0.4-fold at 1 h and by 2.4 ± 0.4-fold at 2 h (n = 6) (Fig. 5B). The FBS treatment similarly increased the kinase activity of CAT , which was immunoprecipitated from the cells and assayed. The immune complex kinase activity increased by 2.75 ± 0.4-fold following a 2-h treatment of the cells with FBS (Fig. 5C). Immune complexes from cells transfected with empty vector had no detectable CAT  kinase activity. These results show that FBS markedly increased the phosphothreonine 410 content and kinase activity of CAT  in serum-starved HeLa cells.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Treatment of serum-starved HeLa cells with FBS stimulated CAT  kinase activity and its phosphothreonine 410 content. HeLa cells were transfected with CAT  or empty vector (pcDNA3.1 GS) and serum-starved for 48 h. The serum-free culture medium was replaced 24 h before the addition of actinomycin D to each culture to 2 µg/ml. FBS was added to some cultures to 10% as indicated. The cultures were rinsed and lysed with Nonidet P-40-containing solution (22) after a 1- or 2-h interval. The lysates were subjected to Western blot analysis for CAT  with antibody to the V5 epitope or to phosphothreonine 410 (A). The X indicates an unknown protein immunostained by the antibody that recognizes phosphothreonine 410. Western blots were scanned and analyzed by volume densitometry (GS-670; Bio-Rad). Values (B) are mean ± S.E. (n = 6). Samples (0.1 mg) were subjected to immunoprecipitation with antibody to the V5 epitope, and the kinase activity of the immune complex was assayed with the PKC  peptide as substrate (C). Values are mean ± S.E. (n = 8). Statistical analyses were done by Student's t test, and the asterisk indicates significant differences (p < 0.01).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The free kinase domain of protein kinase C  and other PKC isoforms is considered to be "constitutively active" because it lacks the amino-terminal regulatory domain, which includes a pseudosubstrate segment that plugs the phosphoacceptor site (2, 3, 19, 30, 31). However, we found that CAT , the free catalytic domain of PKC  depends on phosphorylation of Thr⁴¹⁰ for activity. Indeed, CAT  lacked detectable catalytic activity following expression in bacteria (Fig. 4A). Phosphorylation of Thr⁴¹⁰ by PDK-1 was necessary and sufficient to activate the kinase function of recombinant CAT  (Fig. 4). Threonine 410 mutants either lacked (T410A) or had little (T410E) kinase activity following expression in mammalian cells (Figs. 1 and 2). Treatment of CAT  with a phosphatase decreased its kinase activity and the phospho-Thr⁴¹⁰ content (Fig. 3). FBS treatment strongly stimulated CAT  kinase activity and its phosphothreonine 410 content in serum-starved HeLa cells (Fig. 5). These findings indicate that the catalytic domain of PKC  is intrinsically inactive and subject to regulation by transphosphorylation like full-length PKC . Hence, it is misleading to refer to the catalytic domain of PKC  as "constitutively active," because whether PDK-1 is active and colocalized with the PKC  depends on cellular phenotype and environmental stimuli, as discussed below (5, 37-39). Interestingly, transphosphorylation of PKC  was recently shown to be regulated rather than constitutive, in contrast to conventional PKC isoforms (, , and ) (40). It remains to be determined whether other PKC isoforms, all of which have the PDK-1 substrate motif, are subject to intrinsic kinase domain regulation by transphosphorylation.

The intrinsic regulation of the catalytic domain of PKC  is expected to have physiological significance because caspase processing generates kinase domain fragments, which lack the regulatory domain (22, 23). Three other PKC isoforms, , µ, and , like PKC , are prominent caspase substrates (26-28). In contrast to PKC , the , µ, and  isoforms have proapoptotic functions. Interestingly, the catalytic domain of PKC  (amino acids 330-676), which is produced by caspase processing, appears to be constitutively active in contrast to CAT . Thus, the recombinant catalytic domain of PKC  was highly active after purification from bacteria and lacked detectable phospho-Thr⁵⁰⁵ in the PDK-1 substrate motif.² Similarly to the bacterially expressed kinase domain, full-length PKC  is highly active following expression in bacteria (24). Whereas an acidic residue (Glu⁵⁰⁰) at least partially fulfills the role of activation loop phosphorylation of PKC  (25), the aspartate of the activation loop of CAT  is not sufficient to activate its kinase function (Fig. 4).

Phosphorylation of Thr⁴¹⁰ of CAT  by PDK-1 presumably induces a conformational change in the activation loop, which activates the kinase function by repositioning catalytic groups involved in interactions with substrates and phosphate transfer. Additionally, the conformational change may relieve an intrinsically autoinhibited state of CAT . For example, a conformational change evoked by the binding of cyclin A to CDK2 realigns active site residues and relieves the blockade of the catalytic cleft (41, 42). The activation loop of this and other AGC kinases, including PKC , have conserved structural and functional features (42, 43). In the case of full-length PKC , phosphorylation of Thr⁴¹⁰ would be expected to evoke a conformational change in the amino-terminal regulatory domain that removes the pseudosubstrate segment (amino acids 106-143) from the substrate binding pocket. The precise conformational changes that are elicited by phosphorylation of the activation loop of PKC  and other isoforms remain to be determined.

PDK-1 seems to be the major upstream effector kinase of PKC  and CAT  in mammalian cells, because amino acid substitutions at the PDK-1 docking site markedly depressed the activity of full-length PKC  and essentially abolished the activity of CAT  in transfected mammalian cells (Fig. 2B and Ref. 37). Generally full-length PKC  is active in proliferating cells and depressed in growth factor-deprived cell cultures (5, 37-39). Insulin, for example, has been shown to stimulate PKC  in adipocytes by increasing transphosphorylation by PDK-1 and by a phosphorylation-independent relief of autoinhibition, which appeared to be caused by PtdIns 3,4,5-P₃ binding to the regulatory domain of PKC  (9). PDK-1 is subject to regulation by growth factors and other stimuli that affect phosphatidylinositol 3-kinase activity or by kinases such as JNK and tyrosine kinases that directly phosphorylate PDK-1 (44). Phosphorylation of Ser²⁴¹ of PDK-1 turns on its kinase function, and bacterial expressed PDK-1 is active due to autophosphorylation of Ser²⁴¹ (45). However, PDK-1 is probably not constitutively active in mammalian cells, because it is autoinhibited (46, 47). In addition to relieving the autoinhibition, PtdIns 3,4,5-P₃ and PtdIns 3,4-bisphosphate recruit PDK-1 and its substrates to cell membranes. For example, PtdIns 3,4,5-P₃ binding colocalizes PDK-1 and PKB and influences its efficiency as a PDK-1 substrate (46, 48). PtdIns 3,4,5-P₃ and PtdIns 3,4-bisphosphate also appear to modulate the phosphorylation of PKC , because inhibition of phosphatidylinositol 3-kinase markedly decreased phosphorylation of PKC  in 293 cells (6).

The primary determinants that influence the interaction of PDK-1 with PKC  or PKC II lie in the carboxyl terminus of protein kinase C and include a hydrophobic motif near the carboxyl terminus (37, 49). A recent study by Newton and co-workers (49) suggested that PDK-1 influences the maturation of conventional isoforms of protein kinase C by PDK-1 in two ways. First, PDK-1 associates with newly synthesized unphosphorylated protein kinase C and transfers phosphate to the activation loop threonine. Second, PDK-1 docking to the hydrophobic segment in the carboxyl terminus impedes the autophosphorylation of conventional PKC isoforms at two carboxyl-terminal sites and hence maturation to competency. Autophosphorylation occurs at the "turn motif" (Thr⁵⁶⁰ of PKC ), so called because it corresponds to phosphorylation site in protein kinase A located at the apex of a turn and, with the exception of the atypical PKCs, at the "hydrophobic motif," which consists of a Ser or Thr residue flanked by bulky hydrophobic residues. Alessi and co-workers (37) demonstrated that atypical PKC  (or /) docks with PDK-1 via a hydrophobic motif as do PKC-related kinase isoforms (PRKs). The C-terminal Phe-Xaa-Xaa-Phe-Asp/Glu Phe/Tyr hydrophobic motif of PKC  or /, like that PRKs, has an acidic residue flanked by bulky hydrophobic residues instead of a Ser or Thr as in the case of other PKC isoforms (37). Newton and co-workers (39) have suggested that the release of PDK-1 is rate-limiting and hence dictates the rate of autophosphorylation and maturation of conventional PKC isoforms. It is unclear to what extent autophosphorylation contributes to the regulation of atypical PKC . By docking with PDK-1, full-length PKC  or its free catalytic domain may accelerate release of PDK-1 from conventional PKC isoforms and other AGC kinases and thereby modulate their functions. Further work is needed to examine the effectiveness of full-length PKC  compared with the free kinase domain as a putative PDK-1-releasing factor.

	ACKNOWLEDGEMENTS

We thank Anning Lin for the HeLa cells and Alex Toker for the PDK-1 plasmids that were used as PCR templates.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM60383.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294-0019. Tel.: 205-934-7434; Fax: 205-975-5841; E-mail: jeff.smith@ccc.uab.edu.

Published, JBC Papers in Press, September 19, 2002, DOI 10.1074/jbc.M206420200

² L. Smith, and J. B. Smith, unpublished data.

	ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; CAT  and , catalytic domain of PKC  and , respectively; CLA, calyculin A; CS1, catalytic subunit of protein phosphatase 1; CHX, cycloheximide; DTT, dithiothreitol; FBS, fetal bovine serum; HEK, human embryonic kidney; HER, human embryonic retinoblast; MBP, myelin basic protein; PDK-1, phosphoinositide-dependent kinase 1; PRK, protein kinase C-related kinase; PtdIns, phosphatidylinositol; PtdIns 3, 4,5-P₃, phosphatidylinositol 3,4,5-trisphosphate; TBS, Tris-buffered saline; TNF, tumor necrosis factor .

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES