Identification and Characterization of the Autophosphorylation Sites of Phosphoinositide 3-Kinase Isoforms beta  and gamma *

Cornelia CzupallaDagger §, Miran Culo§, Eva-Christina Müller||, Carsten BrockDagger §**, H. Peter ReuschDagger Dagger , Karsten Spicher§, Eberhard Krause§§, and Bernd NürnbergDagger §¶¶

From the Dagger  Institut für Biochemie und Molekularbiologie II, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany, § Institut für Pharmakologie, ** Institut für Biochemie, and Dagger Dagger  Institut für Klinische Pharmakologie und Toxikologie, Freie Universität Berlin, 14195 Berlin, Germany, || Max-Delbrück-Zentrum für Molekulare Medizin, Charité, Humboldt Universität zu Berlin, 13092 Berlin, Germany, and §§ Forschungsinstitut für Molekulare Pharmakologie, 13125 Berlin, Germany

Received for publication, October 9, 2002, and in revised form, December 18, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Class I phosphoinositide 3-kinases (PI3Ks) are bifunctional enzymes possessing lipid kinase activity and the capacity to phosphorylate their catalytic and/or regulatory subunits. In this study, in vitro autophosphorylation of the G protein-sensitive p85-coupled class IA PI3Kbeta and p101-coupled class IB PI3Kgamma was examined. Autophosphorylation sites of both PI3K isoforms were mapped to C-terminal serine residues of the catalytic p110 subunit (i.e. serine 1070 of p110beta and serine 1101 of p110gamma ). Like other class IA PI3K isoforms, autophosphorylation of p110beta resulted in down-regulated PI3Kbeta lipid kinase activity. However, no inhibitory effect of p110gamma autophosphorylation on PI3Kgamma lipid kinase activity was observed. Moreover, PI3Kbeta and PI3Kgamma differed in the regulation of their autophosphorylation. Whereas p110beta autophosphorylation was stimulated neither by Gbeta gamma complexes nor by a phosphotyrosyl peptide derived from the platelet-derived growth factor receptor, autophosphorylation of p110gamma was significantly enhanced by Gbeta gamma in a time- and concentration-dependent manner. In summary, we show that autophosphorylation of both PI3Kbeta and PI3Kgamma occurs in a C-terminal region of the catalytic p110 subunit but differs in its regulation and possible functional consequences, suggesting distinct roles of autophosphorylation of PI3Kbeta and PI3Kgamma .

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Class I phosphoinositide 3-kinases (PI3Ks)1 are lipid kinases that are activated in response to a variety of extracellular stimuli including hormones, neurotransmitters, and growth factors, which act via G protein-coupled receptors or receptor tyrosine kinases. These lipid kinases phosphorylate the D3 position of the inositol ring of phosphoinositides, thus generating intracellular lipid second messengers (1, 2). PtdIns, PtdIns-4-P, and PtdIns-4,5-P2 are in vitro substrates of class I PI3Ks, although these enzymes predominantly produce PtdIns-3,4,5-P3 in vivo. Class I PI3K lipid products transmit signals by recruiting intracellular effector molecules to the membrane, which contain particular pleckstrin homology domain modules. Effectors include serine/threonine kinases like Akt/protein kinase B, Tec family tyrosine kinases, and guanine nucleotide exchange factors for monomeric GTP-binding proteins like Grp1 and Vav (3). Consequently, class I PI3Ks are involved in the regulation of a wide variety of cellular functions such as differentiation, proliferation, survival, migration, and metabolism (4, 5).

All class I PI3Ks are heterodimers consisting of a p110 catalytic and a p85 or p101 type regulatory subunit. According to the type of their associated regulatory subunit, class I PI3Ks can be further distinguished. The class IA catalytic p110alpha , -beta , and -delta subunits complex with adaptor molecules containing two Src homology 2 domains, of which p85 is the prototype. Through interaction of the adaptor Src homology 2 domains with phosphotyrosines, class IA PI3Ks are activated by receptor tyrosine kinases. In contrast, the only class IB member, p110gamma , associates with a p101 regulatory subunit and is not recruited to tyrosine-phosphorylated receptor tyrosine kinases. PI3Kgamma is regulated by G protein-coupled receptors through direct interaction with Gbeta gamma complexes of heterotrimeric G proteins (6-9). Furthermore, class IA PI3Kbeta is also sensitive to Gbeta gamma and thus may function as a coincidence detector integrating tyrosine kinase- and G protein-dependent signals (10-12).

In addition to their lipid kinase activity, all class I PI3Ks possess an intrinsic protein kinase activity in vitro (13). This enzymatic quality was first characterized as autophosphorylation of catalytic and regulatory subunits. PI3Kalpha phosphorylates its p85 adaptor subunit at serine 608, whereas autophosphorylation of PI3Kdelta occurs at serine 1039 of the catalytic p110delta subunit. Both phosphorylations result in down-regulation of the lipid kinase activity (14-16). Moreover, in Jurkat T cells, p110delta phosphorylation was stimulated by CD28 in vivo (15). There is some in vitro evidence that class IA PI3Ks can phosphorylate other substrates such as the insulin receptor substrate-1 adaptor protein and PDE3B, but the physiological significance of these phosphorylations remains unknown (17-21). Recently it was reported that a "protein kinase-only" mutant of the G-protein-regulated PI3Kgamma still activated mitogen-activated protein kinase pathways in cells, whereas no activation of Akt/protein kinase B by this mutant occurred (22).

Autophosphorylation of both catalytic and regulatory subunits of PI3Kbeta and PI3Kgamma has been proposed, but many questions regarding the autophosphorylation sites and the functional relevance of these autophosphorylation events remain unanswered. Therefore, in the present study, we examined the in vitro autophosphorylation of the G protein-sensitive class I PI3Kbeta and PI3Kgamma . Recombinant heterodimeric PI3Ks were purified and analyzed for their autophosphorylation. Phosphorylated amino acids were identified, and the effect of autophosphorylation on PI3K lipid kinase activity was studied. Interestingly, we observed significant differences in the regulation and functional consequences of the autophosphorylation of PI3Kbeta and PI3Kgamma .

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Recombinant PI3Ks-- Construction and characterization of recombinant baculoviruses for expression of GST-p110alpha , p110gamma , GST-p110gamma , GST-p110gamma K833R, GST-p101, p101, and p85alpha were described previously (6-8). A pFastBacHTa baculovirus transfer vector (Invitrogen) was used to generate His-p110beta and His-p110gamma full-length constructs using NcoI/SalI and NcoI/BamHI cloning sites, respectively. Point mutations were introduced using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).

Oligonucleotides for generation of p110beta S1070 mutants (mutated residues are underlined) were as follows: 5'-GGA AAG ACT ACA GAG CTT AAG CTG CAG TCG-3' and 5'-CGA CTG CAG CTT AAGCTC TGT AGT CTT TCC-3' (for S1070A), 5'-GGA AAG ACT ACA GAG ATT AAG CTG CAG TCG-3' and 5'-CGA CTG CAG CTT AAT CTC TGT AGT CTT TCC-3' (for S1070D), and 5'-GGA AAG ACT ACA GAG AGT AAG CTG CAG TCG-3' and 5'-CGA CTG CAG CTT ACT CTC TGT AGT CTT TCC-3' (for S1070E).

Oligonucleotides used to create the p110gamma S1101 mutants were as follows: 5'-GGC ATC AAA CAA GGA GAG AAA CAT GCA GCC TAA TAC TTT AGG CTA GAA TC-3' and 5'-GAT TCT AGC CTA AAG TAT TAG GCT GCA TGT TTC TCT CCT TGT TTG ATG CC-3' (for S1101A), 5'-GGC ATC AAA CAA GGA GAG AAA CAT GAC GCC TAA TAC TTT AGG CTA GAA TC-3' and 5'-GAT TCT AGC CTA AAG TAT TAG GCG TCA TGT TTC TCT CCT TGT TTG ATG CC-3' (for S1101D), and 5'-GGC ATC AAA CAA GGA GAG AAA CAT GAA GCC TAA TAC TTT AGG CTA GAA TC-3' and 5'-GAT TCT AGC CTA AAG TAT TAG GCT TCA TGT TTC TCT CCT TGT TTG ATG CC-3' (for S1101E).

Recombinant viruses expressing hexahistidine-tagged p110beta , p110gamma , and mutants thereof were generated using the Bac-to-Bac Expression System (Invitrogen) following the manufacturer's instructions. Expression and purification of PI3K isoforms were carried out according to published protocols (11) with the exception that the partially purified proteins were subjected to an additional chromatographic step on a 1-ml Resource Q fast protein liquid chromatography column (Amersham Biosciences). For that purpose, proteins were diluted in buffer A (20 mM Tris/HCl, pH 8.0, 10 mM beta -mercaptoethanol) and loaded onto the column. The column was subsequently washed with buffer A, and proteins were eluted with a linear gradient of 0-500 mM NaCl in buffer A.

Gbeta gamma Complexes and Peptides-- Expression and purification of recombinant Gbeta 1gamma 2-His complexes was carried out as published (11). Purified proteins were quantified by Coomassie Blue staining following SDS-PAGE with bovine serum albumin as the standard (23). The tyrosine-phosphorylated peptide used in this study, CGGpYMDMSKDESVDpYVPMLDM (where pY represents phosphotyrosine), was derived from the human platelet-derived growth factor receptor (24) and kindly donated by Dr. Andreas Steinmeyer (Schering AG, Berlin, Germany). A nonphosphorylated peptide served as a control and had no effect on PI3K enzymatic activity. The peptides derived from the C termini of p110beta and p110gamma (WMAHTVRKDYRS and WFLHLVLGIKQGEKHSA, respectively) were kindly provided by Dr. Michael Beyermann (Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany).

Cell Culture, Transfection, and Preparation of Cell Lysates-- HEK293 cells (American Type Culture Collection, Manassas, VA) were grown in minimal essential medium with Earle's salts supplemented with 10% fetal calf serum and antibiotics. Subconfluent cells were transfected in 3-cm dishes with pcDNA3-fMLP receptor (0.2 µg), pcDNA3-p101 (0.4 µg), and pcDNA3-p110gamma (0.4 µg) variants, using the FuGene 6 transfection reagent (Roche Molecular Biochemicals) following the manufacturer's instructions. For preparation of whole cell lysates, cells were directly lysed in sample buffer according to Laemmli (39).

Gel Electrophoresis, Immunoblotting, and Antibodies-- Characterization of the monoclonal antibody against p110gamma has been described elsewhere (7). The polyclonal anti-extracellular signal-regulated kinase and anti-phospho-Akt antibodies were purchased from New England Biolabs. Whole cell lysates were fractionated by SDS-PAGE and blotted onto nitrocellulose membranes (Amersham Biosciences). Visualization of specific antisera was performed using the ECL chemiluminescence system (Amersham Biosciences) according to the manufacturer's instructions.

Lipid Kinase Assay-- In vitro lipid kinase activity was determined basically as described previously (11). In brief, assays were conducted in a final volume of 50 µl, containing 0.1% bovine serum albumin, 1 mM EGTA, 0.2 mM EDTA, 7 mM MgCl2, 100 mM NaCl, 40 mM HEPES, pH 7.4, 1 mM dithiothreitol, 1 mM beta -glycerophosphate (vesicle buffer) with some modifications. Lipid vesicles (30 µl containing 320 µM phosphatidylethanolamine, 300 µM phosphatidylserine, 140 µM phosphatidylcholine, 30 µM sphingomyelin, and 40 µM PtdIns-4,5-P2 in vesicle buffer) were mixed with stimuli as indicated and incubated on ice for 10 min. It should be noted that we ensured that the effects of Gbeta gamma on PI3K activity were not affected by their detergent-containing vehicles. Thereafter, the enzyme (20-100 ng of PI3Kbeta or 2-10 ng of PI3Kgamma ) was added, and the mixture was incubated for further 10 min at 4 °C in a final volume of 40 µl. The assay was started by adding 40 µM ATP (1 µCi of [gamma -32P]ATP; PerkinElmer Life Sciences) in 10 µl of vesicle buffer. After an incubation period of 15 min (unless otherwise stated) at 36 °C, the reaction was stopped by adding 150 µl of ice-cold N HCl, and lipids were extracted with 450 µl of chloroform/methanol (1:1). Following centrifugation, the organic phase was washed twice with 150 µl of 1 N HCl. Subsequently, 40 µl of the organic phase were resolved on potassium oxalate-pretreated TLC plates (Whatman, Maidstone, UK) using a mixture of 35 ml of 2 N acetic acid and 65 ml of n-propyl alcohol as the mobile phase. Dried TLC plates were exposed to Fuji imaging plates, and autoradiographic signals were quantitated with a BAS 1500 Fuji-Imager (Raytest, Straubenhardt, Germany).

Protein Kinase Assay-- In vitro protein kinase activity was determined as described for the lipid kinase activity with some modifications. The assay volume was 25 µl (2 µCi of [gamma -32P]ATP/tube), the vesicle buffer contained 0-10 mM MgCl2 and/or 0-10 mM MnCl2 as indicated, and lipid vesicles lacked PtdIns-4,5-P2. The reaction was stopped after an incubation period of 30 min (unless otherwise stated) at 36 °C by adding 10 µl of 4× sample buffer according to Laemmli (39). Following separation on SDS-polyacrylamide gels, proteins were transferred to nitrocellulose membranes. Dried membranes were exposed to Fuji imaging plates, and autoradiographic signals were quantitated. For the determination of PI3K autophosphorylation sites, 20-50 µg of PI3Kbeta or PI3Kgamma were phosphorylated in a final volume of 1,500 µl. Samples were subjected to SDS-PAGE, and gels were stained with Coomassie Blue. Calculation of the stoichiometry of p110 autophosphorylation was based on the specific activity of [gamma -32P]ATP incorporated into p110, and counts were determined by Cerenkov counting. The amount of p110 was estimated from the Coomassie-stained gel by comparison with stained bovine serum albumin standards.

Phosphoamino Acid Analysis-- Autophosphorylated PI3Kgamma was subjected to SDS-PAGE and blotted onto a polyvinylidene difluoride membrane (Millipore Corp.), and the phosphorylated p110gamma band was excised. The protein was hydrolyzed in 6 N HCl for 1 h at 110 °C. The sample was vacuum-dried, and amino acids were resuspended in 5 µl of pH 1.9 buffer (0.078% (v/v) acetic acid, 0.025% (v/v) formic acid) containing 2 µg each of phosphoserine, phosphothreonine, and phosphotyrosine as internal standards. The sample was applied to a cellulose thin layer plate, and electrophoresis in the first dimension was carried out in pH 1.9 buffer at 550 V for 1 h. After drying the plate, an electrophoretic separation in the second dimension was carried out in pH 3.5 buffer (0.05% (v/v) acetic acid, 0.005% (v/v) pyridine, 0.5 mM EDTA) at 500 V for 50 min. The unlabeled phosphoamino acids were visualized by spraying the plate with 0.2% (w/v) ninhydrin in acetone, and the radiolabeled phosphoamino acids were detected by autoradiography.

Determination of p110beta and p110gamma Autophosphorylation Sites-- Gel-excised autophosphorylated p110 spots (100-150 pmol) were washed with 50% (v/v) acetonitrile in 25 mM ammonium bicarbonate, shrunk by dehydration in acetonitrile, and dried in a vacuum centrifuge. Disulfide bonds were reduced by incubation with 10 mM dithiothreitol in 100 mM ammonium bicarbonate for 45 min at 55 °C. Alkylation was performed by replacing the dithiothreitol solution with 55 mM iodoacetamide in 100 mM ammonium bicarbonate. Following a 20-min incubation period at 25 °C in the dark, the gel pieces were washed with 50% (v/v) acetonitrile in 25 mM ammonium bicarbonate, shrunk by dehydration in acetonitrile, and dried in a vacuum centrifuge. The gel pieces were incubated overnight at 37 °C in 5 mM ammonium bicarbonate, containing 1 µg of chymotrypsin (sequencing grade; Roche Molecular Biochemicals), for p110gamma or at room temperature in 50% (v/v) trifluoroacetic acid, containing 10 mg/ml cyanogen bromide, for p110beta . To extract the peptides, 0.5% (v/v) trifluoroacetic acid in acetonitrile was added, and the separated liquid was dried under vacuum, redissolved in 5 µl of buffer B (0.1% (v/v) formic acid), and loaded onto a Vydac C18 column (150 × 1 mm, 5 µm, type 218 TP 5115) for micro-liquid chromatography separation. Elution was performed using a linear gradient of 5-80% buffer C in 60 min at an eluent flow rate of 30 µl/min. Buffer C was 0.1% (v/v) formic acid in acetonitrile/water (8:2, v/v), containing 0.1% (v/v) formic acid. Fractions were collected, their radioactivity was determined by Cerenkov counting, and phosphopeptides were identified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). MALDI-MS measurements were performed on a Voyager-DE STR BioSpectrometry work station MALDI-TOF mass spectrometer (Perseptive Biosystems, Inc., Framingham, MA) using alpha -cyano-4-hydroxycinnamic acid as the matrix. The program FindMod (available on the World Wide Web at expasy.ch/tools/findmod) was used to interpret the MS spectra of protein digests. Amino acid sequences of the phosphopeptides were determined by nanoelectrospray tandem mass spectrometry (nanoESI-MS/MS). The liquid chromatography fractions were lyophilized and redissolved in 5 µl of 1% (v/v) formic acid in methanol/water (1:1, v/v). The MS/MS measurements were performed with a nanoelectrospray hybrid quadrupole mass spectrometer Q-TOF (Micromass, Manchester, UK). The collision gas was argon at a pressure of 6.0 × 10-5 millibar in the collision cell.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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In Vitro Autophosphorylation of Class IA PI3Ks-- Class IA PI3Kalpha and PI3Kdelta isoforms phosphorylate either their p85 adaptor subunit and/or the catalytic p110 subunit itself (14, 15). Autophosphorylation of p110delta occurs on serine 1039 within the C terminus. Alignment of the C termini of class IA catalytic subunits shows that p110alpha , which does not autophosphorylate, contains no C-terminal serine, whereas p110beta does have one (serine 1070) (Fig. 1A). Recent reports have described autophosphorylation of both subunits of PI3Kbeta (i.e. p85 and p110beta ) (25-27). In order to analyze autophosphorylation of PI3K isoforms in vitro, we expressed recombinant heterodimeric PI3Kalpha and PI3Kbeta in insect cells and measured their protein kinase activities (Fig. 1B). As anticipated, the p85 adaptor of PI3Kalpha was phosphorylated in the presence of Mn2+ only (see Fig. 1B, left panel). In contrast to p110alpha , p110beta autophosphorylated its catalytic subunit. This autophosphorylation of p110beta was also largely Mn2+-dependent, since in vitro phosphorylation levels in the presence of Mg2+ reached a maximum of only 5-10% compared with the level observed in the presence of Mn2+ (see Fig. 1B, right panel). Furthermore, in the presence of Mn2+, a small but significant phosphorylation of the p85 subunit of PI3Kbeta was evident. These data indicate that both subunits are phosphorylated, with p110beta being the main substrate of PI3Kbeta autophosphorylation.


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Fig. 1.   Autophosphorylation of class IA PI3Ks. A, alignment of the C-terminal amino acid sequences of class IA PI3K catalytic subunits. The arrowhead indicates the p110delta autophosphorylation site. The equivalent serine residue in p110beta is marked in boldface type. B, heterodimeric recombinant PI3Kalpha (GST-p110alpha /p85) and PI3Kbeta (His-p110beta /p85) were purified from Sf9 cells, and the proteins were separated by SDS-PAGE and analyzed by Coomassie staining. PI3Kalpha and PI3Kbeta were assayed for the incorporation of 32P into the catalytic and regulatory subunits in the presence of either Mn2+ (2 mM) or Mg2+ (7 mM) as indicated. Shown are representative autoradiographs and the corresponding Coomassie-stained gels as loading controls.

p110beta Autophosphorylates a C-terminal Serine Residue-- Speculating that p110beta autophosphorylates its C terminus, the in vitro phosphorylated and [32P]phosphate-labeled protein (Fig. 2A) was cleaved with cyanogen bromide in order to generate a C-terminal peptide, which was then analyzed by mass spectrometry. The resulting peptides were separated by reversed-phase HPLC, and the radioactivity of each fraction was determined. The main radioactive fraction was examined by MALDI-MS, and a peptide corresponding to the phosphorylated C terminus of p110beta (m/z 1312.65) could be identified (Fig. 2B). Sequencing of this phosphopeptide by nanoESI-MS/MS revealed the 1061AHTVRKDYRpS1070 (where pS represents phosphoserine) sequence of p110beta and serine 1070 as the site of autophosphorylation (Table I). In order to verify this finding, a p110beta mutant in which serine 1070 was changed to alanine was created, and autophosphorylation of this mutant was compared with the wild-type enzyme in the presence of Mn2+. Fig. 2C shows that no significant phosphate incorporation into the mutant p110beta S1070A of PI3Kbeta took place while this mutant was still catalytically active as a lipid kinase (see below). Hence, results obtained by mass spectrometric and mutagenic analysis demonstrate that serine 1070 represents in fact the main site of autophosphorylation in p110beta .


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Fig. 2.   p110beta autophosphorylation at a C-terminal serine residue. A, heterodimeric recombinant PI3Kbeta purified from Sf9 cells was subjected to SDS-PAGE and visualized by Coomassie staining. Apparent molecular masses (kDa) of marker proteins are indicated. B, peptide mass fingerprint analysis of p110beta . Autophosphorylated p110beta was digested in gel using cyanogen bromide, the resulting peptides were separated by reversed-phase HPLC, and the fractions were analyzed by MALDI-MS. The peak with m/z 1312.65 (calculated m/z 1312.62) corresponds to the phosphorylated sequence 1061AHTVRKDYRpS1070. C, p110beta in which serine 1070 was mutated to alanine shows a loss of autophosphorylating activity. Heterodimeric PI3Kbeta either containing wild-type p110beta (WT) or a p110beta mutant (S1070A) was subjected to a protein kinase assay in the presence of Mn2+. Shown are one representative autoradiograph and the corresponding Coomassie-stained protein bands as loading control. D, lipid kinase activity of mutant p110beta . Equal amounts of purified heterodimeric PI3Kbeta (His-p110beta /p85) either containing wild-type p110beta (WT) or a p110beta mutant (S1070D, S1070E, or S1070A) were tested for their enzymatic activity in a lipid kinase assay in the absence (-) and presence (+) of 120 nM purified Gbeta 1gamma 2-His, 100 nM tyrosine-phosphorylated peptide, and both stimuli. Experiments were carried out in the presence of Mg2+. Indicated are mean values ± S.D. of three independent experiments. E, Mn2+-dependent protein kinase activity of PI3Kbeta in the presence of increasing amounts of a synthetic peptide derived from the C terminus of p110beta . One representative autoradiograph of three independent experiments is shown.


                              
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Table I
MS/MS fragment ions of the p110beta C-terminal phosphopeptide
The triple-charged precursor ion with m/z 438.22 of the phosphopeptide 1061AHTVRKDYRpS1070 was sequenced by nanoESI-MS/MS. The detected y"- and b-type fragment ions confirm the amino acid sequence of the C terminus of p110beta . Since all C-terminal y" ions show corresponding peaks with loss of 98 mass units, whereas only the N-terminal b10 ion shows the neutral loss of H3PO4, the site of phosphorylation could be clearly assigned to the C-terminal amino acid serine 1070.

Both phosphorylation of the p85 adaptor by p110alpha and autophosphorylation of p110delta down-regulate the enzymes' lipid kinase activities (14-16, 20). p110beta , like p110delta , autophosphorylates a serine residue at the extreme C terminus, which may also affect the catalytic activity of PI3Kbeta . In order to test this assumption, we measured lipid kinase activities of p110beta variants in which serine 1070 was mutated. Since the lipid kinase activity of PI3Kbeta can be synergistically stimulated by Gbeta gamma complexes and a tyrosine-phosphorylated peptide derived from the platelet-derived growth factor receptor, we measured formation of PtdIns-3,4,5-P3 under basal conditions and after stimulation of PI3Kbeta variants with either stimuli in the presence of Mg2+ (11). Wild-type p110beta and the nonphosphorylating p110beta S1070A mutant exhibited the same enzymatic activity under basal conditions and after stimulation with Gbeta gamma , phosphotyrosyl peptide, or both stimuli (Fig. 2D). This finding indicates that serine 1070 is not essential for the catalytic activity of p110beta . However, purified mutant PI3Kbeta heterodimers are less stable than the wild-type enzyme (data not shown). In order to mimic the effects of p110beta autophosphorylation, mutants of p110beta containing the negatively charged aspartic and glutamic acid instead of serine 1070 were employed. These p110beta S1070D/E mutants no longer autophosphorylated (data not shown). Moreover, as shown in Fig. 2D, the lipid kinase activity of either p110beta S1070D/E mutant was reduced by 4-7-fold under basal conditions and following stimulation with Gbeta gamma and phosphotyrosyl peptides. This implies a regulatory function of the p110beta autophosphorylation.

Next we created a peptide corresponding to the C terminus of p110beta (WMAHTVRKDYRS) as a pseudosubstrate in order to test whether the protein kinase activity of the autophosphorylated p110beta was still intact. Applying mass spectrometry, no phosphorylation of this peptide by wild-type PI3Kbeta was observed (data not shown). Moreover, as indicated in Fig. 2E, increased amounts of the C-terminal peptide did not influence the autophosphorylation of p110beta by competition. Therefore, we assume that protein phosphorylation by PI3Kbeta requires highly specific protein-protein interactions, and due to the lack of appropriate substrates the effect of p110beta autophosphorylation on the protein kinase activity of the enzyme remains unknown so far.

Regulation of p110beta Autophosphorylation-- The finding that autophosphorylation of p110beta on serine 1070 results in down-regulation of the lipid kinase activity of PI3Kbeta (see Fig. 2D) suggests a regulatory function of this autophosphorylation. Therefore, one may suppose that the protein kinase activity like the lipid kinase activity of PI3Kbeta is controlled by cell surface receptors. In order to address this hypothesis, we compared both kinase activities after incubation of PI3Kbeta with increasing concentrations of Gbeta gamma complexes and the tyrosine-phosphorylated peptide. As indicated, Fig. 3, A and B, shows that the lipid kinase activity of PI3Kbeta was stimulated in a concentration-dependent manner by Gbeta gamma (EC50 = 20 nM) or phosphotyrosyl peptide (EC50 = 5 nM). In contrast, neither Gbeta gamma nor phosphotyrosyl peptide stimulated p110beta autophosphorylation (see Fig. 3, A and B). Moreover, a combination of both stimuli led to a remarkable synergistic activation of PI3Kbeta lipid kinase activity (see Fig. 2B) (11). However, even under these conditions, autophosphorylation of p110beta was not enhanced regardless of whether Mg2+, Mn2+, or mixtures thereof were present (data not shown).


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Fig. 3.   Autophosphorylation of p110beta is not stimulated by Gbeta gamma or a tyrosine-phosphorylated peptide derived from the platelet-derived growth factor receptor. A and B, lipid kinase (closed circles) and protein kinase (open circles) activities of purified PI3Kbeta were examined in response to increasing concentrations of purified Gbeta 1gamma 2-His (A) and a tyrosine-phosphorylated peptide (Tyr-P-peptide) (B). [32P]phosphate-labeled PtdIns-3,4,5-P3 and p110beta were isolated and quantified as described under "Experimental Procedures." Representative autoradiographs are shown at the top, whereas mean values ± S.D. of three independent experiments are shown at the bottom. Lipid and protein kinase activities of PI3Kbeta were illustrated as -fold stimulation of basal activities. C, time course of p110beta autophosphorylation. Purified PI3Kbeta was incubated with phospholipid vesicles and [gamma -32P]ATP either in the absence of stimuli (open circles) or in the presence of 120 nM purified Gbeta 1gamma 2-His (closed circles), 100 nM tyrosine-phosphorylated peptide (closed squares), or both stimuli (closed triangles). At equilibrium, the stoichiometry of p110beta autophosphorylation was ~0.4-0.6 mol of phosphate/mol of p110beta (n = 3). Note that lipid kinase assays were carried out in the presence of Mg2+, whereas protein kinase activity was determined in the presence of Mn2+. Shown is the time course of one representative experiment of three.

These observations suggest a high level of basal p110beta autophosphorylation. Nonetheless, we found that in the presence of Mn2+, the stoichiometry of phosphorylation was maximally 0.5 mol of phosphate/mol of p110beta (Fig. 3C), arguing against a high basal autophosphorylation as the reason for the missing regulation of PI3Kbeta protein kinase activity in vitro. Moreover, no differences in the time course of p110beta autophosphorylation occurred, regardless of whether Gbeta gamma or tyrosine-phosphorylated peptide were present. It is interesting that autophosphorylation peaked after more than 30 min under in vitro conditions. Taken together, the presented data do not exclude the possibility that p110beta autophosphorylation may be involved in receptor-independent regulation of PI3Kbeta enzymatic activity.

Autophosphorylation of p110gamma Is Stimulated by Gbeta gamma -- Major characteristics of PI3Kbeta autophosphorylation such as Mn2+ dependence and inhibition of lipid kinase activity resemble those of class IA PI3Kalpha and -delta autophosphorylation. However, in contrast to class IA kinases, a significant autophosphorylation of p110gamma occurs in the presence of Mg2+. Furthermore, autophosphorylation does not change the lipid kinase activity of PI3Kgamma (11, 28). These findings suggest a role for PI3Kgamma autophosphorylation different from the class IA PI3K isoforms. Our observation that Gbeta gamma stimulates p110gamma autophosphorylation further supports this assumption (11, 12). Since others have reported an inhibitory effect of Gbeta gamma on PI3Kgamma protein kinase activity (29), we reexamined Gbeta gamma -induced p110gamma autophosphorylation using recombinant purified protein (Fig. 4). In the absence of lipid vesicles, Gbeta gamma did not increase autophosphorylation of p110gamma . In contrast, the addition of lipid vesicles led to a significant Gbeta gamma -dependent stimulation of p110gamma autophosphorylation regardless of whether the lipid vesicles contained PtdIns-4,5-P2 (see Fig. 4A). These data may indicate that the orientation of proteins on the lipid bilayer surface facilitates the interaction of Gbeta gamma with PI3Kgamma . Interestingly, we observed phosphorylation of p101, which could not be stimulated by Gbeta gamma even in the presence of lipid vesicles. Since in these experiments, a GST-p101/p110gamma heterodimer was analyzed (see Fig. 4B), we also used a His-p110gamma /p101 heterodimer (Fig. 5A) in order to exclude the possibility that a bulky GST tag may influence the phosphorylation of PI3Kgamma subunits. As indicated in the upper panel of Fig. 5B, phosphorylation of a p101 variant without a GST tag was not visible. Moreover, both Gbeta gamma -stimulated p110gamma autophosphorylation of the His-p110gamma /p101 heterodimer (EC50 = 30 nM) and lipid kinase activity (EC50 = 10 nM) were comparable with the data obtained with the GST-p101/p110gamma heterodimer (see Fig. 5B) (11, 12). Taken together, these results clearly demonstrate that, in contrast to class IA PI3Kbeta , autophosphorylation of PI3Kgamma is sensitive to Gbeta gamma . Whereas under basal conditions autophosphorylation of p110gamma increased linearly for a period of more than 90 min, the presence of Gbeta gamma significantly accelerated this phosphorylation (Fig. 5C). In particular, autophosphorylation of p110gamma reached its maximum after 20 min with a stoichiometry of about 0.95 mol of incorporated phosphate/mol of p110gamma . The latter observation suggests the presence of one autophosphorylation site in p110gamma .


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Fig. 4.   Phospholipid vesicles are required for Gbeta gamma stimulation of PI3Kgamma autophosphorylation. A, heterodimeric recombinant PI3Kgamma (GST-p101/p110gamma ) purified from Sf9 cells was assayed for autophosphorylation in response to increasing concentrations of recombinant Gbeta 1gamma 2-His in the absence (-) and presence (+) of phospholipid vesicles lacking PtdIns-4,5-P2. Shown are autoradiographs of one typical experiment (upper panel) and graph bars with mean values ± S.D. of three independent experiments (lower panel). B, heterodimeric recombinant PI3Kgamma (GST-p101/p110gamma ) was purified from Sf9 cells, subjected to SDS-PAGE, and analyzed by Coomassie staining. Apparent molecular masses of marker proteins are indicated.


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Fig. 5.   Gbeta gamma stimulates p110gamma autophosphorylation. A, heterodimeric recombinant PI3Kgamma (His-p110gamma /p101) was purified from Sf9 cells, subjected to SDS-PAGE, and analyzed by Coomassie staining. Apparent molecular masses of marker proteins are indicated. B, purified PI3Kgamma (His-p110gamma /p101) was assayed for lipid kinase activity (closed circles) and protein kinase activity (open circles) in the presence of increasing concentrations of recombinant Gbeta 1gamma 2-His. [32P]phosphate-labeled PtdIns-3,4,5-P3 and p110gamma were isolated and quantified as detailed under "Experimental Procedures." Representative autoradiographs are depicted at the top, whereas graph bars with mean values ± S.D. of three independent experiments are shown at the bottom. Lipid and protein kinase activities of PI3Kgamma are illustrated as -fold stimulation of basal activities. C, autophosphorylation of p110gamma is accelerated in the presence of Gbeta gamma subunits. Purified His-p110gamma /p101 was incubated with phospholipid vesicles and [gamma -32P]ATP in the absence (open circles) or presence (closed circles) of 120 nM purified Gbeta 1gamma 2-His for the indicated periods of time. At equilibrium, the stoichiometry of Gbeta gamma -stimulated p110gamma autophosphorylation was ~0.95 mol of phosphate/mol of p110gamma . The time course of one representative experiment out of three is shown.

Autophosphorylation of p110gamma Does Not Inhibit PI3Kgamma Lipid Kinase Activity-- In order to examine the effect of p110gamma autophosphorylation on PI3Kgamma lipid kinase activity, p110gamma was phosphorylated in the presence of Gbeta gamma . The catalytic activity of this autophosphorylated PI3Kgamma was compared with the nonphosphorylated counterpart using PtdIns-4,5-P2 as the substrate (Fig. 6). No differences in the production of PtdIns-3,4,5-P3 were detected, regardless of whether PI3Kgamma was autophosphorylated. Therefore, in contrast to the C-terminal autophosphorylation of p110beta and p110delta , which both down-regulate lipid kinase activity of PI3K, autophosphorylation of p110gamma has no obvious inhibitory effects on PI3Kgamma enzymatic activity. Hence, we assumed that autophosphorylation of p110gamma occurs at a site different from a serine residue at its C terminus.


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Fig. 6.   Autophosphorylation of p110gamma does not affect its lipid kinase activity. Heterodimeric recombinant PI3Kgamma was subjected to a protein kinase assay either with (closed circles) or without (open circles) ATP for 30 min at 37 °C in the presence of 120 nM purified Gbeta 1gamma 2-His and phospholipid vesicles lacking PtdIns-4,5-P2. Thereafter, PtdIns-4,5-P2-containing phospholipid vesicles were added to the reaction mixture, the ATP concentration was adjusted, and a lipid kinase assay was performed as described under "Experimental Procedures." Reactions were stopped at the time points indicated, and incorporation of [32P]phosphate was determined (mean values ± S.D. of three independent experiments).

Identification of the p110gamma Autophosphorylation Site-- Phosphoamino acid analysis revealed that p110gamma autophosphorylates serine but not threonine or tyrosine residues (Fig. 7A). In order to identify the phosphorylated serine residue, in vitro [32P]phosphate-labeled p110gamma protein was cleaved using different proteases. The resulting peptides were separated by reversed-phase HPLC, and the radioactivity of each fraction was determined. After digestion with chymotrypsin, a phosphopeptide corresponding to the C terminus of p110gamma was identified by MALDI-MS (m/z 1134.56) and nanoESI-MS (doubly charged ion with m/z 567.77), as shown in Fig. 7B. The site of modification within the C-terminal sequence 1093-1102 was determined by nanoESI-MS/MS (see Fig. 7B, lower panel). In particular, the C-terminal y" fragment ion series and the loss of neutral H3PO4 (98 mass units) confirmed the sequence and identified phosphorylation of serine 1101. Thus, the MS data demonstrate that serine 1101 of p110gamma is the site of autophosphorylation. To confirm this finding, a p110gamma mutant in which serine 1101 was changed to alanine was examined for its autophosphorylation. As indicated in Fig. 7C, no significant Gbeta gamma -stimulated phosphate incorporation into the p110gamma S1101A mutant took place. Hence, both class IB p110gamma as well as class IA p110beta and p110delta isoforms autophosphorylate on a serine residue at the extreme C terminus.


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Fig. 7.   Mapping of the p110gamma autophosphorylation site. A, phosphoamino acid analysis. Purified His-p110gamma /p101 was phosphorylated in the presence of recombinant Gbeta 1gamma 2-His (400 nM). After protein separation by SDS-PAGE and transfer to a polyvinylidene difluoride membrane, the 32P-labeled p110gamma subunit was subjected to phosphoamino acid analysis as described under "Experimental Procedures." The positions of ninhydrin-stained phosphoamino acid standards are indicated by dashed circles. B, MS (upper panel) and MS/MS (lower panel) spectra of the chymotryptic fragment 1093GIKQGEKHpSA1102 of p110gamma isolated by reversed-phase HPLC. The peak with a m/z ratio of 567.74 (upper panel, calculated m/z 567.77) corresponds to the double-charged ion of the phosphorylated sequence. The loss of 98 Da corresponding to neutral H3PO4 from this precursor ion gave rise to the peak with an m/z ratio of 518.77 (lower panel). Relevant ions are labeled according to the nomenclature proposed in Ref. 30. The y" ions in the spectrum that contain the phosphoserine are produced by consecutive fragmentation reactions breaking the amino bond and losing the H3PO4, or vice versa. C, p110gamma with a serine 1101 to alanine mutation does not show any Gbeta gamma -stimulated autophosphorylation. Heterodimeric purified PI3Kgamma (His-p110gamma /p101) either containing wild-type p110gamma (WT) or a p110gamma mutant (S1101A) was subjected to a protein kinase assay in the absence (-) and presence (+) of 120 nM purified Gbeta 1gamma 2-His. One typical autoradiograph and the corresponding Coomassie-stained gel as loading control are shown.

Lipid Kinase Activity of Mutant PI3Kgamma -- Next we examined the in vitro lipid kinase activity of heterodimeric PI3Kgamma variants containing either wild-type p110gamma or a mutant p110gamma in which serine 1101 was replaced by either alanine (see above) or the negatively charged aspartic and glutamic acid (Fig. 8A). No differences in the production of PtdIns-3,4,5-P3 by these PI3Kgamma variants were observed under both basal conditions and followed by stimulation with Gbeta gamma . These data underline that autophosphorylation of p110gamma does not inhibit PI3Kgamma lipid kinase activity. Moreover, HEK293 cells were transiently transfected with wild-type or mutant PI3Kgamma as well as with the G protein-coupled fMLP receptor, and Akt phosphorylation was subsequently determined. In the absence of PI3Kgamma , no fMLP-induced phosphorylation of Akt was observed (data not shown), whereas in the presence of any PI3Kgamma variants (i.e. the wild-type enzyme or the alanine, aspartate, or glutamate mutants), Akt phosphorylation was significantly stimulated by fMLP to a comparable extent (Fig. 8B). Hence, results obtained both in an in vitro assay using recombinant proteins and in a cell-based assay suggest that autophosphorylation of p110gamma does not influence PI3Kgamma lipid kinase activity and thus clearly differs from the autophosphorylation of class IA PI3K isoforms.


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Fig. 8.   Lipid kinase activity of mutant p110gamma . A, in vitro lipid kinase activity of mutant p110gamma . Equal amounts of heterodimeric purified PI3Kgamma (His-p110gamma /p101) containing either wild-type p110gamma (WT) or a p110gamma mutant (S1101D, S1101E, or S1101A) were tested for their enzymatic activity in a lipid kinase assay in the absence (-) and presence (+) of 120 nM Gbeta 1gamma 2-His. Basal PtdIns-3,4,5-P3 formation of all PI3Kgamma variants was similar (0.25 mol/min/mol enzyme). Shown are mean values ± S.D. of three independent experiments. B, stimulation of Akt phosphorylation by mutant p110gamma . HEK293 cells were transiently transfected with plasmids for the fMLP receptor, p101, and wild-type (WT) or mutant (S1101D, S1101E, or S1101A) p110gamma . Serum-starved cells were treated with either vehicle (-) or 1 µM fMLP. Equal amounts of whole cell lysates were subjected to SDS-PAGE followed by immunoblotting with anti-phospho-Akt, anti-p110gamma , and anti-Erk antibodies as loading control.

Mechanism of p110gamma Autophosphorylation-- In order to examine the mechanism of p110gamma autophosphorylation, we used a peptide corresponding to the C terminus of p110gamma (WFLHLVLGIKQGEKHSA). However, this C-terminal peptide was not a substrate for PI3Kgamma protein kinase activity, since a phosphorylation of this peptide by wild-type PI3Kgamma was not detected using mass spectrometry (data not shown). Furthermore, the peptide neither influenced p110gamma autophosphorylation under basal conditions nor in the presence of Gbeta gamma (Fig. 9A). Moreover, a kinase-defective p110gamma K833R mutant did not autophosphorylate, emphasizing that phosphorylation of purified PI3Kgamma preparations was not due to the presence of a contaminant kinase activity copurifying with the lipid kinase (Fig. 9B). Last, co-incubation of p110gamma K833R with enzymatically active wild-type heterodimeric PI3Kgamma did not result in phosphorylation of the mutant p110gamma K833R, whereas the wild-type enzyme autophosphorylated. Hence, a transphosphorylation mechanism can be excluded.


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Fig. 9.   Mechanism of p110gamma autophosphorylation. A, protein kinase activity of purified PI3Kgamma in the presence of increasing amounts of a synthetic peptide derived from the C terminus of p110gamma . Autophosphorylation of p110gamma was monitored in the absence or presence of 120 nM purified Gbeta 1gamma 2-His. Shown is one representative autoradiograph out of three independent experiments. B, the autophosphorylation of p110gamma is not mediated by transphosphorylation. Recombinant purified His-p110gamma /p101 and GST-p110gamma K833R were obtained from Sf9 cells, and proteins were subjected to SDS-PAGE and analyzed by Coomassie staining (left panel). The His-p110gamma /p101 complex was incubated alone or in the presence of kinase-inactive GST-p110gamma K833R with [gamma -32P]ATP and 120 nM Gbeta 1gamma 2-His. Phosphorylated proteins were separated by SDS-PAGE and visualized by autoradiography. Autophosphorylated GST-p110gamma served as a control to indicate the size of a phosphorylated GST-p110gamma K833R. One representative autoradiograph is shown (right panel).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present study describes the autophosphorylation sites of the G protein-sensitive class I PI3Kbeta and -gamma isoforms. The experimental approaches include mass spectrometric analysis of the posttranslationally modified proteins and site-directed mutagenesis, which are independent and complementary methods. With these strategies, we identified the C-terminal residues serine 1070 and serine 1101 of the catalytic p110beta and p110gamma subunits, respectively, as the modified amino acids. Previously, the C-terminal serine 1039 was detected as the site of p110delta autophosphorylation (15). Hence, class I PI3Kbeta , -gamma , and -delta isoforms share the extreme C terminus of the catalytic subunit as a common site of autophosphorylation, whereas p110alpha is not significantly modified, probably due to the lack of a serine residue in this region.

Recently, Williams and co-workers published the crystal structure of p110gamma for a fragment comprising amino acid residues 144-1102 (31). Although serine 1101 was not resolved in this structure it is clear from the data that it should be just beyond the C-terminal helix kalpha 12, which lines the PtdIns-4,5-P2 binding pocket. Therefore, from a sterical point of view, a preferential phosphorylation of this serine appears reasonable. Moreover, lipid and protein kinase activities may compete with each other (32, 33). This assumption is supported by recent data from Yart et al. (27) demonstrating that a "protein kinase-only" (PKO) mutant of p110beta exhibited an even higher protein kinase activity than the wild-type enzyme. Our own studies did not indicate any difference in the autophosphorylation activity of p110gamma , regardless of whether lipid substrates such as PtdIns-4,5-P2 were present (data not shown). Other data are more complex. Bondeva et al. (22) reported that only those PKO p110gamma variants showed an increased autophosphorylation activity that contained a class II or class III donor activation loop but not the class IV counterpart. In contrast, wild-type p110alpha and all PKO p110alpha variants phosphorylated p85 equally well, whereas PKO p110alpha variants containing a class II or class III donor activation loop exhibited autophosphorylation of the p110alpha subunit (20). The latter finding was unexpected, since p110alpha lacks a C-terminal serine. In this context, our observation of a residual phosphorylation of the p110beta S1070A mutant may be of interest (see Fig. 2C). The fact that purified kinase-defective mutants of p110beta were not detectably phosphorylated by a contaminating kinase activity rather indicates the presence of a second, quantitatively less important phosphorylation site in p110beta , presumably at threonine 1063 (see Fig. 1A). Support for this assumption comes from MALDI-post-source decay data, which revealed a double phosphorylated C-terminal peptide; unfortunately, the signal was too weak for sequencing by nanoESI/MS-MS. A corresponding C-terminal threonine of p110alpha (see Fig. 1A) may be a candidate target for significant autophosphorylation by class II and III PKO p110alpha variants (20).

In contrast to the predominant autophosphorylation of p110beta observed in this and previous studies (27), others have reported p85 phosphorylation as the major target of PI3Kbeta autophosphorylation (25, 26). In order to explain the apparent discrepancies, one must consider the experimental conditions used in these studies. Roche et al. (25) added purified p85 to immunoprecipitated p110beta and detected a phosphorylated p85 band, whereas the p110beta band was not shown. More important, PI3Kbeta autophosphorylation was examined with enzyme preparations immobilized to beads in those studies (25, 26). Interestingly, this may affect autophosphorylation, since we noticed an increased p85 phosphorylation when we examined PI3Kbeta bound to Ni2+-nitrilotriacetic acid beads.2 Hence, experimental conditions significantly influence in vitro autophosphorylation of PI3Kbeta . Likewise, in addition to p110gamma autophosphorylation (11, 12, 28), p101 phosphorylation by PI3Kgamma has been reported (29). In fact, using a GST-p101 construct, we also observed in initial experiments a phosphorylation of p101, which, in contrast to p110gamma autophosphorylation, was not stimulated by Gbeta gamma . However, p101 phosphorylation was not detectable when a purified hexahistidine-tagged PI3Kgamma heterodimer was used. Therefore, we cannot exclude the possibility that the artificial bulky GST tag may have facilitated phosphorylation of p101.

The data presented in this study as well as in other reports provide evidence that the C terminus of p110 is a common site of autophosphorylation for three out of four class I PI3K isoforms. Despite this conformity, PI3Kbeta and -gamma differ in all other biochemical characteristics of autophosphorylation. For instance, PI3Kbeta , like the two other class IA isoforms, PI3Kalpha and -delta , autophosphorylates preferentially in the presence of Mn2+ (see Fig. 1B and Refs. 14-16). In contrast, PI3Kgamma exhibits a significantly stimulated autophosphorylation activity in the presence of Mg2+ as shown previously (11, 28) and in this study. Interestingly, in vitro most serine/threonine kinases are Mg2+-dependent, whereas many tyrosine kinases show a greater activity in the presence of Mn2+ (34). Conversely, for phosphorylase kinase, a metal ion-dependent dual kinase specificity was reported (35). The presence of Mg2+ causes serine phosphorylation of phosphorylase b, and Mn2+ activates tyrosine phosphorylation of angiotensin II (35). The basis for these properties are still unclear. In this context, it should be remembered that autophosphorylation, per se, is not a good indicator of protein kinase activity, since many ATP-binding proteins that are not protein kinases are known to autophosphorylate in vitro (32).

We also addressed the control of autophosphorylation by upstream regulators. Under basal conditions, PI3Kgamma slowly autophosphorylated (i.e. 0.1 mol of phosphate was incorporated into 1 mol of p110gamma within 30 min). The addition of Gbeta gamma accelerated phosphate incorporation by 8-10-fold, resulting in an almost stoichiometric phosphorylation (see Fig. 5, B and C). Interestingly, this effect was only seen in the presence of lipid vesicles, which is in contrast to results obtained by Bondev and co-workers (29). Since the EC50 values for the stimulation of lipid and protein kinase activities of PI3Kgamma were concordant, we hypothesized that the molecular mechanisms of stimulation of these kinase activities are similar. In contrast, neither Gbeta gamma nor phosphotyrosyl peptide stimulated autophosphorylation of PI3Kbeta (see Fig. 3) even under low basal autophosphorylation conditions (i.e. in the presence of Mg2+). Unfortunately, in vitro data addressing a possible stimulation of autophosphorylation of PI3Kalpha and -delta by upstream regulators are missing. However, Vanhaesebroeck and co-workers (15) reported a CD28-mediated stimulation of C-terminal p110delta phosphorylation under in vivo conditions.

Data obtained from aspartate and glutamate mutants of p110beta suggest that a phosphorylated PI3Kbeta displays a hampered lipid kinase activity (see Fig. 2D). Unfortunately, a more direct experimental approach (e.g. assaying the effect of autophosphorylation on lipid kinase activity) was inconclusive. In particular, we were unable to completely remove Mn2+, which is necessary for autophosphorylation but disturbed PI3Kbeta lipid kinase activity, without the use of immobilizing agents before carrying out the lipid kinase assay. Nevertheless, our results with the p110beta 1070D/E mutants are consistent with data obtained from the other class IA kinases. Autophosphorylation of PI3Kalpha and -delta and exchange of serine 1039 of p110delta to aspartate or glutamate inhibited lipid kinase activity (14-16, 20). Possible explanations for this effect include an induction of structural/conformational changes of the phosphorylated enzyme or an impact on the phospho-transfer reaction or on the ATP/PtdIns-4,5-P2 interaction (15). Furthermore, based on the crystal structure of p110gamma , Williams and associates (31) have speculated that a phosphorylated C terminus may be a sterical impediment for PtdIns-4,5-P2 substrate binding. Surprisingly, here we provide experimental evidence that autophosphorylation of the C terminus of p110gamma does not inhibit lipid kinase activity as shown under in vitro conditions with a prephosphorylated wild-type enzyme and p110gamma 1101D/E mutants (see Figs. 6 and 8A). These mutants showed full activity on cellular effectors in HEK293 cells in vivo (see Fig. 8B). Hence, we assume that autophosphorylation of p110gamma , which is primarily regulated by Gbeta gamma , has functions distinct from regulating its lipid kinase activity. One possibility may be the existence of PI3Kgamma binding partners that specifically interact with the autophosphorylated form of PI3Kgamma . In fact, recent evidence suggests that PI3Kgamma interacts not only with its principal regulators (i.e. Gbeta gamma and Ras) but also with additional components of signaling cascades such as the beta -adrenergic receptor kinase 1 (36).

We found that PI3Kbeta and -gamma did not phosphorylate peptides derived from their respective C terminus, and vice versa the peptides did not affect the autophosphorylation capacity of the enzyme (see Figs. 2E and 9A). Similar observations were reported for PI3Kalpha and -delta (15), whereas Beeton et al. (26) described that PI3Kalpha and -beta phosphorylated a p85-derived peptide containing serine 608. Mechanistically, PI3Kgamma did not transphosphorylate (see Fig. 9B), which may indicate a high degree of substrate specificity of the protein kinase activity. Notably, auto- but not transphosphorylation has also been reported for the p110gamma monomer and a phosphatidylinositol 4-kinase beta  (37, 38). Surprisingly, while we were searching for in vivo substrates of PI3K protein kinase activity, we failed to detect p110gamma autophosphorylation in HL-60 and Sf9 cells so far, which emphasizes the need for further investigations into the regulation, activity, and targets of PI3Ks in vivo.

    ACKNOWLEDGEMENTS

We thank H. Lerch and J. Malkewitz for excellent technical assistance. We thank Drs. Bart Vanhaesebroeck and Michael Waterfield for providing baculoviruses and Dr. Reinhard Wetzker for the monoclonal anti-p110gamma antibody. We also thank Drs. Michael Beyermann and Andreas Steinmeyer for providing peptides. Valuable discussions with Drs. Reinhard Wetzker, Len Stephens, Phil Hawkins, Lewis Cantley, and Roland Piekorz are appreciated.

    FOOTNOTES

* This work was supported by grants from the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie (DFG Nu53-6/1; SFB518).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.

This work is in partial fulfillment of Ph.D. requirements at Friedrich-Schiller-Universität Jena. Present address: Biotec der TU Dresden, c/o Max-Planck-Institut für Molekulare Zellbiologie und Genetik, Dresden.

¶¶ To whom correspondence should be addressed: Institut für Physiologische Chemie II, Klinikum der Heinrich-Heine-Universität, Universitätsstr. 1, Gebäude 22.03, 40 225 Düsseldorf, Germany. Tel.: 49-211-811-2724; Fax: 49-211-811-2726; E-mail: bernd.nuernberg@uni-duesseldorf.de.

Published, JBC Papers in Press, December 26, 2002, DOI 10.1074/jbc.M210351200

2 C. Czupalla and B. Nürnberg, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: PI3K, phosphoinositide 3-kinase; p110, catalytic subunit of PI3Ks; p101, subunit associated with p110gamma ; p85, regulatory subunit of class IA PI3Ks; PtdIns, phosphatidylinositol; PtdIns-4-P, phosphatidylinositol 4-phosphate; PtdIns-4, 5-P2, phosphatidylinositol 4,5-bisphosphate; PtdIns-3, 4,5-P3, phosphatidylinositol 3,4,5-trisphosphate; GST, glutathione S-transferase; His, hexahistidine tag; MS, mass spectrometry; MALDI-MS, matrix-assisted laser desorption/ionization MS; nanoESI-MS/MS, nanoelectrospray ionization tandem MS; HPLC, high pressure liquid chromatography; fMLP, formylmethionylleucylphenylalanine; PKO, protein kinase-only; TOF, time of flight.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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