Activation Loop Sequences Confer Substrate Specificity to Phosphoinositide 3-Kinase alpha  (PI3Kalpha )

FUNCTIONS OF LIPID KINASE-DEFICIENT PI3Kalpha IN SIGNALING*

Luciano PirolaDagger §, Marketa J. Zvelebil, Genevieve Bulgarelli-LevaDagger , Emmanuel Van Obberghen||, Michael D. Waterfield**, and Matthias P. WymannDagger DaggerDagger

From the Dagger  Institute of Biochemistry, University of Fribourg, CH-1700 Fribourg, Switzerland,  Ludwig Institute for Cancer Research, London W1W 7BS, United Kingdom, || INSERM U-145, IFR 50, Faculté de Medecine, 06107 Nice Cedex 2, France, and ** Department of Biochemistry and Molecular Biology, University College London, London WCE 6BT, United Kingdom

Received for publication, December 15, 2000, and in revised form, February 9, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phosphoinositide 3-kinases (PI3Ks) are dual specificity lipid and protein kinases. While the lipid-dependent PI3K downstream signaling is well characterized, little is known about PI3K protein kinase signaling and structural determinants of lipid substrate specificity across the various PI3K classes. Here we show that sequences C-terminal to the PI3K ATP-binding site determine the lipid substrate specificity of the class IA PI3Kalpha (p85/p110alpha ). Transfer of such activation loop sequences from class II PI3Ks, class III PI3Ks, and a related mammalian target of rapamycin (FRAP) into p110alpha turns the lipid substrate specificity of the resulting hybrid protein into that of the donor protein, while leaving the protein kinase activity unaffected. All resulting hybrids lacked the ability to produce phosphatidylinositol 3,4,5-trisphosphate in intact cells. Amino acid substitutions and structure modeling showed that two conserved positively charged (Lys and Arg) residues in the activation loop are crucial for the functionality of class I PI3Ks as phosphatidylinositol 4,5-bisphosphate kinases. By transient transfecion of 293 cells, we show that p110alpha hybrids, although unable to support lipid-dependent PI3K signaling, such as activation of protein kinase B/Akt and p70S6k, retain the capability to associate with and phosphorylate insulin receptor substrate-1, with the same specificity and higher efficacy than wild type PI3Kalpha . Our data lay the basis for the understanding of the class I PI3K substrate selectivity and for the use of PI3Kalpha hybrids to dissect PI3Kalpha function as lipid and protein kinase.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phosphoinositide 3-kinase lipid products play a central role in the regulation of a number of cellular processes; the monophosphorylated PtdIns 3-P1 is a key lipid regulator of vesicle trafficking in all eukaryotic organisms, including yeast (1). The polyphosphoinositides PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are transiently produced in multicellular organisms in response to receptor activation by extracellular stimuli and, by acting as second messengers, orchestrate a large array of mitogenic, metabolic, and antiapoptotic responses (reviewed in Refs. 2-4).

Production of 3-phosphorylated phosphoinositides is dependent on the activity of phosphoinositide 3-kinases (PI3Ks) (5). According to their substrate specificity and mode of activation, PI3Ks are divided into three classes (6). Class I PI3Ks phosphorylate, in vitro, PtdIns, PtdIns 4-P, and PtdIns(4,5)P2 and are placed into two subclasses depending on their coupling to cell surface receptors. Class IA heterodimeric PI3Ks, to which the prototypic PI3Kalpha (p85alpha /p110alpha ) belongs, are composed of an adaptor subunit of 50-85 kDa containing two SH2 domains and a tightly bound 110-kDa catalytic subunit of either the p110alpha , p110beta , or p110delta Type. Upon receptor tyrosine kinase activation, the SH2 domains of the adaptor bring the heterodimer to the cell surface, thereby allowing the p110 catalytic subunit to phosphorylate its lipid substrate(s). The class IB PI3Kgamma is associated to a p101 adapter, which was proposed to mediate activation via Gbeta gamma subunits of trimeric G proteins (7). The most recently described class II PI3Ks are monomeric enzymes (~170 kDa in size) containing a C-terminal C2 domain. To date, three mammalian isoforms have been identified: the ubiquitously expressed PI3K-C2alpha (also referred as m-Cpk, p170, or Dm_68D, (8, 9)), PI3K-C2beta (10), and the liver-specific isoform PI3K-C2gamma (11). Class II PI3Ks preferentially phosphorylate PtdIns and PtdIns 4-P, while class III PI3K only produces PtdIns 3-P, which is central to vesicle trafficking (12). Besides, several PI3K-related enzymes exist that do not have demonstrated lipid kinase activity but are functional as protein kinases. These include enzymes involved in DNA repair such as the target of rapamycin (mTOR/FRAP), the catalytic subunit of the DNA-dependent protein kinase (DNAPK), and others (for a review, see Ref. 13).

In addition to their function as lipid kinases, PI3Ks have an intrinsic protein kinase activity, of which the physiological significance is less well characterized. Initial studies showed that p110alpha -mediated phosphorylation on the p85alpha adapter reduces the lipid kinase activity of the heterodimer (14). Likewise, a shut-off of activity has been observed in p110delta upon phosphorylation on serine 1039 (15). Recently, it has also been shown that the protein kinase activity of the class IB PI3Kgamma is sufficient to activate the mitogen-activated protein kinase Erk-2, thus overcoming the requirement for 3'-phosphorylated lipids in this pathway (16). An involvement for protein autophosphorylation in the activation of the class III PI3K has also been described, via an intrinsic serine/threonine kinase activity of Vps34 (17). The function of the different PI3Ks is thus determined by their class-specific lipid substrate specificity and protein kinase activities.

The lipid substrate specificity of PI3Ks, as well as of other members of the phosphoinositide kinases superfamily, appears to be dependent on activation loop sequences as determined by the following approaches: (a) sequence swapping on PI3Kgamma (16), (b) activation loop swapping between type 1 and type 2 phosphatidylinositol phosphate kinases (18), and (c) molecular modeling studies on the x-ray-solved structure of PI3Kgamma (19). The activation loop of phosphoinositide kinases is located C-terminally to the conserved DFG motif of the catalytic domain and can be viewed as the topological counterpart of the activation loop of protein kinases (20).

In this study, we show that swapping of foreign sequences into the activation loop of p110alpha permits us to switch its lipid substrate specificity into that of class II or class III PI3Ks or even to fully abolish its lipid kinase activity, while leaving the protein kinase functionality unaltered. We then use the manipulated p110alpha to dissect the functionalities of the PI3Kalpha heterodimer as a lipid kinase and as a protein kinase. Further, we identify by site-directed mutagenesis key residues of class I PI3K required for phosphorylation of PtdIns(4, 5)P2. Our biochemical data provide a better understanding of the PI3K substrate specificity, and the chimeric construct described here should provide a powerful tool to distinguish lipid kinase versus protein kinase functions of PI3K in signaling.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression Plasmids and Site-directed Mutagenesis-- To generate hybrid PI3Ks, silent PflMI and KpnI restriction sites were introduced by the unique site elimination method (21) into wild type p110alpha (22) within codons for GHFLD and RVP, respectively (see Fig. 1A). Oligonucleotide cassettes coding for selected amino acid sequences of class I, II, and III PI3Ks and FRAP as depicted in Fig. 1 were then ligated into the PflMI/KpnI-digested plasmids. All insertions were confirmed by DNA sequencing (Microsynth, Balgach, Switzerland) and transferred as PstI-HindIII fragments to cytomegalovirus promoter-driven expression vectors pSCT1 (23) and pSTC-tkGST (24) carrying the p110alpha gene. Mutated regions were equally inserted into a p110alpha with an N-terminal Myc tag and an extension for a C-terminal isoprenylation sequence from Ha-Ras (Myc-p110alpha -CAAX in pSG5; see Ref. 25). The pMT2-based expression vector for the p85alpha regulatory subunit was described before (23, 26). Point mutations in the previously described GST-PI3Kgamma wild type and cII hybrid (16, 24) were obtained by inserting appropriate nucleotide cassettes into the AvrII and KpnI silent sites and confirmed by sequencing. The expression plasmids Myc-p70S6k in pRK5 and Myc-iSH2 in pCMV6 were previously described (27, 28). Mouse insulin receptor substrate-1 (IRS-1) (29) was subcloned into pcDNA3 as a HindIII-HindIII fragment.

Protein Expression and Transient Transfections-- Human embryonic kidney (HEK) 293 and COS-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS; Life Technologies, Inc.). HEK 293 cells were seeded (2 × 106/10-cm dish) 24 h prior to transfection by the calcium phosphate method (typically with 1 µg of p85alpha and 10 µg of p110alpha expression vectors or 10 µg of GST-PI3Kgamma expression vector). A medium change 12 h later was followed by cell lysis at 48 h (23). COS-7 cells (3 × 105/6-cm dish) were seeded 24 h before transfection by the DEAE-dextran method. One microgram of expression vector was added to 10 µl of 20 mg/ml DEAE-dextran (Amersham Pharmacia Biotech), and the volume was brought to 600 µl with PBS. Cells were incubated with the resulting mixture for 30 min, and then 3 ml of DMEM supplemented with 171 µM chloroquine was added. After 3 h, the medium was exchanged with DMEM supplemented with 10% FCS (v/v). Cells were lysed or metabolically labeled 48-60 h later.

Heterodimers of p85alpha /p110alpha were immunoprecipitated from cell lysates with anti-p85alpha antibodies and immobilized on protein A-Sepharose (CL-4B; Amersham Pharmacia Biotech). Alternatively, glutathione-Sepharose (4B; Amersham Pharmacia Biotech) was used to isolate GST-p110alpha ·p85alpha complexes and GST-PI3Kgamma s. Sepharose beads were washed twice with lysis buffer (20 mM Tris·HCl, 138 mM NaCl, 2.7 mM KCl, pH 8.0, supplemented with 5% glycerol, 1 mM MgCl2, 1 mM CaCl2, 1 mM sodium-o-vanadate, 20 µM leupeptin, 18 µM pepstatin, 1% Nonidet P-40, 5 mM EDTA, and 20 mM NaF), twice with 0.1 M Tris-HCl plus 0.5 M LiCl (pH 7.4), and twice with the respective reaction buffers (see below).

Antibodies and Immunoblotting-- Rabbit polyclonal antibodies directed against wortmannin, p110alpha , and p85alpha were used as previously described (23). Monoclonal anti-Myc antibodies (9E10) were from Babco, rabbit anti-IRS-1 antibodies were from Upstate Biotechnology, Inc., and rabbit anti-phospho-PKB Ser473 antibodies were from New England Biolabs. Monoclonal anti-phosphotyrosine antibodies were raised from hybridoma cells (clone PY72). Proteins were separated on standard SDS-PAGE and transferred to polyvinylidene difluoride (Millipore Corp.) according to Ref. 30. Primary antibodies were detected with species-specific goat anti-rabbit or anti-mouse peroxidase-conjugated secondary antibodies (Sigma) and enhanced chemiluminescence (ECL; Pierce).

Lipid and Protein Kinase Assays-- Lipid kinase assays were basically carried out as previously described (31). Immobilized proteins were suspended in a presonicated mixture of 10 µg of phosphatidylserine and 10 µg of PtdIns, PtdIns 4-P, or PtdIns(4,5)P2 in 40 µl of reaction buffer (20 mM HEPES, 5 mM MgCl2, pH 7.4). Sodium cholate (2.3%, w/v) was included in mixtures containing PtdIns(4,5)P2. The reaction was initiated by the addition of 10 µl of 50 µM ATP supplemented with 10 µCi of [gamma -32P]ATP (3000 Ci/mmol; Hartmann Analytic) and kept at 30 °C for 10 min. Reactions were stopped by adding 50 µl of 1 M HCl and 200 µl of chloroform/methanol (1:1, v/v). Extracted lipids were separated on oxalate-treated silica 60 TLC plates (Merck) with chloroform/acetone/methanol/acetic acid/water (80:30:26:24:14, v/v/v/v/v). Phosphorylated lipids were detected by autoradiography on Kodak X-Omat films or phosphorimaging (GS-525 from Bio-Rad). The identity of lipid spots was verified by deacylation and HPLC analysis on a Partisphere SAX column as described by Arcaro and Wymann (32).

Protein serine kinase activity of PI3K was assayed as in Ref. 14 with modifications given in Ref. 23. The activities of Myc-PKB and Myc-p70S6k were determined by using 30 µM Crosstide peptide as substrate as described (33).

Cellular PI3K Activity-- Transiently transfected COS-7 were starved overnight in DMEM without FCS and then rinsed with phosphate-free DMEM (Life Technologies, Inc). Subsequently, cells were incubated with 250 µCi of [32P]orthophosphate (Hartmann Analytic) in 1.8 ml of phosphate-free DMEM per 6-cm dish for 3 h at 37 °C, 5% CO2. Total lipids were extracted according to Ref. 34 and deacylated for HPLC analysis (32).

Wortmannin Labeling of PI3K and Substrate Competition-- Glutathione-Sepharose with immobilized GST-p110alpha ·p85 complexes was washed twice with PBS supplemented with 0.5% Triton X-100 and suspended in PBS plus 0.1% Triton X-100. Wortmannin (1000× stock in Me2SO) was added to a final concentration of 200 nM and incubated for 15 min on ice. For substrate competition experiments, immobilized PI3Ks were incubated with 1 mM ATP or 0.1 mg/ml presonicated PtdIns(4,5)P2 for 15 min at 37 °C before the sample was put on ice and exposed to wortmannin. Excess inhibitor was removed by two PBS plus 0.5% Triton X-100 washes. SDS-PAGE and immunodetection of covalently bound wortmannin was carried out as in Ref. 23. Wortmannin sensitivity of PI3K hybrids was assayed by measuring lipid or protein serine kinase activity of glutathione Sepharose-bound GST-p110alpha ·p85 after a 15-min incubation with the indicated concentrations of wortmannin at 37 °C.

Two-dimensional Phosphopeptide Mapping and Phosphoamino Acid Analysis-- PI3Kalpha ·IRS-1 immune complexes subjected to a PI3K protein kinase assay were separated on a 7.5% SDS-PAGE. After separation, the gel was thoroughly rinsed with water and exposed overnight to a Kodak X-Omat film. The portion of the gel containing the radioactive IRS-1 band was excised and digested for 36 h with trypsin as described (35). Tryptic digests (>3000 cpm) were spotted on a cellulose plate and the two-dimensional separation of the phosphopeptides consisted of a high voltage electrophoresis (900 V, 2.5 h in 30% formic acid) followed by a TLC separation with water/2-butanol/acetic acid/pyridine (48:60:12:40, v/v/v/v).

For phosphoaminoacid analysis, the radioactively labeled IRS-1 was separated as above; HCl hydrolysis and phosphoaminoacid separation were performed as described (36).

PI3K Structure Modeling-- The model of p110alpha was generated using QUANTATM with the x-ray structure of PI3Kgamma (19) as the template. The location and orientation of ATP was directly transferred from the PI3Kgamma structure to the model of p110alpha . The activation loop (not resolved in the PI3Kgamma structure) was modeled by searching through a proprietary high resolution data base of fragments. The loop was then energetically minimized locally to alleviate steric hindrances. This was followed by Steepest Descent global minimization until the energy leveled out. PtdIns(4,5)P2 was modeled and docked manually using the SURFACE and GAPS programs of Laskowski (75). Each orientation was examined subsequently for putative hydrogen bonds, salt bridges, and bad contacts. Once a satisfactory initial orientation was obtained, the orientation of the activation loop was modeled to allow for contacts to be formed with the PtdIns(4,5)P2. This required only minor changes in Phi  and Psi  angles and an adjustment of the Arg949 side chain. The new model was then subjected to a steepest descent minimization.

Differences in accessibility were calculated using the program NACCESS (76) and where calculated for all residues within 15 Å from the PtdIns(4,5)P2 ligand accessed with a 1.4-Å probe.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction and Expression of p110alpha Hybrids-- While the ATP-binding site of PI3Kalpha was biochemically localized by the identification of Lys802 as the residue taking part in the phosphate transfer reaction (23) and most recently confirmed by crystallographic studies (19), little is known about the productive interaction of PI3Ks with phosphoinositides. We have shown previously that wortmannin covalently binds to Lys802 in PI3Kalpha and that this binding can be prevented by preincubation of PI3Ks with PtdIns(4,5)P2. This suggests a spatial overlap of the wortmannin and the PtdIns(4,5)P2-binding site. Putative PI-interacting residues were found C-terminal to the highly conserved DFG motif within homology region 1. In p110alpha , this region contains a polybasic stretch, 941KKKKFGYKRER951, characterized by two basic boxes, 941KKKK and 947KRER; class II PI3Ks retain a KRDR box, similar to the 947KRER of p110alpha , but do not display homology to the 941KKKK box; class III members and PI3K-related proteins (FRAP/mTOR, DNAPK, ATM, and RAD 3 (13)) possess, in the same region, sequences unrelated to the sequences observed in the PI3K classes I and II; and, inside the PI3K-related protein group, these sequences are also unrelated to each other.

To establish whether the DFG C-terminal region plays a role in directing the substrate specificity of the various PI3K classes, we produced p110alpha hybrids in which the p110alpha amino acid sequence comprised between 933DFGHF and the conserved Leu956 was exchanged with corresponding sequences from class II p170/Cpk (8, 9), class III human Vps34 (37), and the PI3K-related protein kinase FRAP (38). We hereby refer to these p110alpha hybrids as cII, cIII, and cIV upon insertion of p170/Cpk, Vps34, and FRAP sequences, respectively (Fig. 1). Since the alignment between p110alpha and Vps34 presents a 5-amino acid gap and the Vps34 region is rich in prolines, which may have disrupting effects on the secondary structure of the protein, several cIII hybrids were constructed; the entire region between 933DFGHFL and Leu956 was replaced by the sequence from Vps34 (cIII.2) or shorter pieces (cIII.1 and cIII.3), and arbitrary amino acids (AVAAV or AV; cIII.4 and cIII.5, respectively) were added to fill the 5-amino acid gap of the sequence alignment. Due to the absence of significant gaps among p110alpha , p170/Cpk, and FRAP and from the information gained in preliminary experiments with the cIII hybrids, a single cII and cIV hybrid was constructed (Fig. 1B). The p110alpha hybrids were transiently expressed in 293 cells as PI3Kalpha heterodimers; p110alpha was either expressed untagged or as a GST fusion. The integrity of the heterodimer was not affected by the insertion of sequences from other PI3K classes, and the intact heterodimer was recovered either by immunoprecipitation with anti-p85alpha antibodies (see Fig. 5A) or glutathione beads fishing of GST-p110alpha (data not shown).


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 1.   PI3K classes and p110alpha hybrid constructs. A, schematic alignments of PI3Ks and related enzymes. The homology regions (HR1-HR4) are shown in gray, and the binding sites for Ras (on class I PI3K only), wortmannin (Wm), and ATP are indicated by triangles. B, amino acid sequences of members of classes I-III and related members (IV, e.g. FRAP) are aligned with hybrid constructs obtained by manipulation of the nonconserved region following the DFG motif of the ATP-binding site of p110alpha . Class IV included e.g. mTOR and other PI3K-related protein kinases. The triangle shows the conserved Leu956.

Lipid Kinase Activity of the p110alpha Hybrids-- We first used PtdIns as a substrate to evaluate the lipid kinase activity of the p110alpha hybrids, since it is an in vitro substrate common to the three PI3K classes (5, 39). The lipid kinase activity of cIII.1, cIII.4, and cIII.5 p110alpha hybrids was 40% with respect to the control p110alpha WT, suggesting a minor affinity for the substrate of the Vps34 sequences inserted. Insertion of the whole Vps34 region comprised in the 933DFGHFL/Leu956 stretch, although corresponding to the best matched alignment, gave rise to a protein almost unable to support PtdIns phosphorylation, equal to for the hybrid carrying a 2-amino acid deletion (cIII.2 and cIII.3, respectively). The higher activities were thus observed in the hybrids maintaining the p110alpha VPFV sequence N-terminal to Leu956, as in the cIII.1 hybrid, regardless of further insertion of the AVAAV or AV sequences (cIII.4 and cIII.5 hybrids). Based on these results and on the lost specificity toward PtdIns 4-P and PtdIns(4,5)P2 phosphorylation (see below), it appears that the minimal Vps34 amino acid sequence required to transform p110alpha into a class III-like lipid kinase is the 8-amino acid stretch GRDPKPLP (Fig. 1B). The cII hybrid was more active toward PtdIns phosphorylation (143 ± 38% of the PtdIns (Fig. 2A), and the cIV hybrid was lipid kinase-inactive. Insertion of the short region from FRAP (a PI3K-related protein with no lipid kinase activity assigned thus far), rendering p110alpha unable to behave as a lipid kinase, confirms the idea that the swapped region is involved in lipid substrate recognition and phosphorylation (Fig. 2).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 2.   PI3K activities of p110alpha hybrid constructs. The GST or GST fusion proteins produced from constructs shown in Fig. 1 by transfection of 293 cells are indicated at the bottom. The numbers correspond to various constructs of cIII hybrids. A, GST-p110alpha ·p85 complexes were immobilized on glutathione beads and subjected to a PI3K assay using PtdIns as a substrate (see "Experimental Procedures"; mean ± S.E., n = 3). B, a typical experiment of PtdIns 3-P formation by the hybrid constructs. C, expression pattern of GST-p110alpha hybrids probed with anti-p110alpha antiserum.

To assess whether the insertion of sequences from different PI3K classes affects the substrate specificity of the p110alpha hybrids, in vitro lipid kinase assays were performed using the three substrates accepted by class I PI3Kalpha . As previously shown (22), PI3Kalpha WT phosphorylated PtdIns, PtdIns 4-P, and PtdIns(4,5)P2. The cII hybrid phosphorylated PtdIns and PtdIns 4-P but not PtdIns(4,5)P2, as described for p170/Cpk and other class II PI3Ks (8, 9, 40). The lipid kinase-active cIII hybrids (cIII.1 (Fig. 3) and cIII.4 and cIII.5 (data not shown)) acquired the substrate specificity of class III Vps34 (17, 37), only phosphorylating PtdIns. The cIV hybrid and the cIII.2,3 hybrids, which do not phosphorylate PtdIns (Fig. 2A), were also unable to support PtdIns 4-P and PtdIns(4,5)P2 phosphorylation (Fig. 3 and data not shown). The identity of 32P-labeled 3'-phosphorylated products of Fig. 3 was confirmed by HPLC analysis of the deacylated radioactive spots. Notably, the two slowly migrating radioactive products obtained when using PtdIns as substrate both represent a 3'-monophosphorylated phosphoinositide (lyso-PtdIns 3-P and not PtdIns(3,4)P2 or PtdIns(3,4,5)P3), since deacylation and HPLC separation of each radioactive spot give a single peak eluting at the same retention time of the upper PtdIns 3-P. Likewise, the higher Rf signal produced by the cII hybrid upon presentation of PtdIns 4-P as substrate is PtdIns 3-P. This probably arises from a PtdIns contamination in the PtdIns 4-P substrate, and it is produced only by the cII hybrid and only marginally by p110alpha WT notwithstanding the higher activity of the former. The nature of the radioactive spots thus analyzed has been established by comparison with standards produced by phosphorylation of the lipid substrates with baculovirus-produced PI3Kalpha WT (data not shown).


View larger version (56K):
[in this window]
[in a new window]
 
Fig. 3.   Top, altered PI substrate specificity of p110alpha hybrid constructs. GST fusion proteins of the type indicated at the bottom were subjected to PI3K assays using PtdIns, PtdIns 4-P, or PtdIns(4,5)P2 as substrates. The arrows on the right indicate the migration distance of PtdIns 3-P, PtdIns(3,4)P2, or PtdIns(3,4,5)P3 standards. The origin is indicated. The asterisk represents an ATP degradation product separating on an oxalate-treated TLC plate. Bottom, quantitation of the relative activity of the hybrids compared with the WT for the three different substrates. (Data for PtdIns as substrate are as in Fig. 2; data for PtdIns 4-P and PtdIns(4,5)P2 as substrates are from the shown TLC and typical of at least three independent experiments).

To investigate their in vivo behavior, p110alpha hybrids were constitutively activated by recloning into a Myc-tagged p110alpha bearing a carboxyl-terminal farnesylation signal from Ha-Ras that localizes the protein to the plasma membrane (Myc-p110alpha -CAAX (25, 41, 42)). COS-7 cells were transiently transfected with CAAX-boxed p110alpha hybrids (Fig. 4B) and serum-starved overnight to suppress the activity of endogenous PI3Ks. Following [32P]orthophosphate metabolic labeling, TLC analysis of extracted lipids from cells transfected with wild type p110alpha -CAAX showed production of PtdIns(3,4,5)P3, while, consistent with the in vitro data, any transfected p110alpha -CAAX hybrid failed to produce it (Fig. 4A); subsequent HPLC analysis of deacylated lipids also demonstrated accumulation of PtdIns(3,4)P2 in WT p110alpha -CAAX-transfected cells but not in cells transfected with any of the hybrids (Fig. 4C). By HPLC analysis, we never detected any increase in PtdIns 3-P levels (data not shown), and this is in agreement with the accepted idea that PtdIns is not an in vivo substrate for class I PI3Ks (see "Discussion").


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 4.   PI production in COS-7 cells. COS-7 cells were transfected with the indicated Myc-tagged p110alpha constructs (M, mock transfection; KR, Lys802 to Arg; for chimera, see Fig. 1B) with C-terminal isoprenylation sequences (CAAX box). A, cells were metabolically labeled with 32Pi, and lipids were extracted and analyzed by TLC. The arrows indicate the location of standards. PI, phosphatidylinositol; PIP, phosphatidylinositol phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3 phosphatidylinositol 3,4,5-trisphosphate. B, in parallel, cells were lysed and probed with anti-Myc antibodies to evaluate Myc-p110alpha -CAAX expression. C, cells treated as for A were extracted, and lipids were deacylated and subjected to HPLC separation. Traces from cells transfected with wild type p110alpha -CAAX are displayed as a solid line, and traces from cells transfected with a cII-CAAX construct are shown as a broken line.

Modification of Lipid Kinase Activity Does Not Affect Other Properties of the p85/p110alpha Hybrid Heterodimers-- To prove that the alteration in lipid substrate specificity of the p110alpha hybrids is due to a specific effect of the exchanged activation loops and not to major structural alterations of the polypeptide caused by the manipulations performed, we analyzed the autokinase activity of the hybrids (14, 43) and interaction with the PI3K-specific inhibitor wortmannin (32). The cII and cIII hybrids efficiently supported p85alpha serine 608 phosphorylation; a lower, but still significant as compared with the kinase-dead K802R mutant (23), p85 phosphorylation was mediated by the cIV hybrid (Fig. 5). Serine phosphorylation on p85 leads to a feedback negative control on the lipid kinase activity of the heterodimer (14, 43). Here we observed the same extent of feedback inhibition on the wild type heterodimer and p85-p110alpha heterodimers bearing cII or cIII.1 hybrid catalytic subunits (Fig. 6).


View larger version (52K):
[in this window]
[in a new window]
 
Fig. 5.   Protein kinase activity of p110alpha hybrids. PI3Kalpha was immunoprecipitated with anti-p85 antibodies from mock-transfected cells (M) or cells transfected with p85alpha and kinase-dead (KR, Lys802 to Arg mutation), WT, or hybrid p110alpha (as indicated at the bottom). A, expression of the p85alpha /p110alpha complex was detected by Coomassie Blue staining. B, immobilized PI3Kalpha was subjected to a protein kinase assay (see "Experimental Procedures"), and incorporated 32P was analyzed after reducing SDS-PAGE by autoradiography.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 6.   Feedback control of PI3Kalpha lipid kinase activity by p85 phosphorylation. A, PI3Kalpha heterodimers were immunoprecipitated from transfected 293 cells with anti-p85 antiserum and incubated in protein kinase buffer in the presence (+) or absence (-) of 1 mM ATP. Samples were subsequently split for a kinase assay in the presence of [gamma -32P]ATP (B) or a lipid kinase assay (mean ± S.E., n = 2) (C). The type of p110alpha cotransfected with p85 is indicated at the bottom (cIII represents cIII.1).

GST-p110alpha hybrids were subsequently analyzed for their interaction with wortmannin (Fig. 7A). The cII and cIII hybrids were covalently modified by wortmannin. Unexpectedly, the cIV hybrid was not covalently attacked by wortmannin, both under standard assay conditions (200 nM wortmannin in PBS plus 0.1% Triton X-100; Fig. 7A) and even in the absence of detergent in the incubation mixture (data not shown). Once we demonstrated that wortmannin binding still takes place in the cII and cIII hybrids, we further investigated the inhibitor-protein interaction by performing inhibitor-substrate competition experiments. Preincubation of p85·GST-p110alpha WT and the cII and cIII.1 hybrids with either ATP or PtdIns(4,5)P2 prior to wortmannin treatment totally prevented the covalent binding of wortmannin to the catalytic subunit (Fig. 7C). Effective ATP competition for wortmannin binding, associated with the retained catalytic activity of the heterodimer, both as a lipid and protein kinase, demonstrates that the cII and cIII hybrids were not altered in the ATP binding site. The observation that PtdIns(4,5)P2 inhibits covalent wortmannin binding to the cII and cIII hybrids was less expected, these hybrids being unable to phosphorylate this substrate, and demonstrates that PtdIns(4,5)P2 binding to the cII and cIII hybrids still takes place on other regions of p110alpha also required for wortmannin binding. This is in keeping with structural data obtained on the PI3Kgamma isoform in which the lipid-binding surface comprises, in addition to the activation loop, the crevice between the C- and N-terminal lobes of the catalytic domain (19). Thus, PtdIns(4,5)P2 efficiently competes for wortmannin binding in the cII and cIII hybrids despite not being phosphorylated to PtdIns(3,4,5)P3 due to the exchanges made in the activation loop. Wortmannin binding experiments, although informative on the specificity of the protein-inhibitor interaction, do not allow us to test the inhibitory effect of wortmannin on the enzymatic activity of the protein. To address this point, the lipid kinase activity of cII and cIII hybrids and autophosphorylation of the cIV hybrid were measured after preincubation with increasing wortmannin concentrations; the inhibition curves for cII and cIII.1 lipid kinase activities and cIV autokinase activity were identical to the inhibition curve obtained for the WT protein with an IC50 in the low nanomolar range (Fig. 7D).


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 7.   Interaction of hybrid PI3Ks with inhibitor and substrate. PI3K and mutants were isolated as p85alpha ·GST-p110alpha complexes and immobilized on glutathione-Sepharose. A, immobilized PI3Kalpha preparations (type indicated below panel B) were incubated with 200 nM wortmannin (+ Wm) or Me2SO (-) and analyzed after SDS-PAGE with anti-wortmannin antiserum on immunoblots (alpha Wm). B, expression of p110alpha was verified by reprobing the membranes with anti-p110alpha antisera (alpha p110). C, competition of PI3K substrates with wortmannin. ATP (1 mM; left) or presonicated PtdIns(4,5)P2 (0.1 mg/ml; right) was added to p85alpha ·GST-p110alpha before the complexes were incubated with 200 nM wortmannin. Detection of covalently bound wortmannin was carried out as in A. D, PtdIns 3-P formation in the presence of the given wortmannin concentrations was tested for wild type (closed circles), cII (closed squares), and cIII.1 (closed triangles) PI3Ks (left panel). Phosphorylation of immunoprecipitated p85 by coexpressed wild type or cIV PI3K is shown to the right.

Site-directed Mutagenesis Defines Key Residues of the p110 Catalytic Subunit as Determinants of Lipid Substrate Specificity-- Once we defined the DFG C-terminal region as the determinant for lipid substrate specificity in p110alpha , we sought to determine which are the basic residues necessary for discriminating the lipid substrate by inserting single/double amino acid substitutions. To this end, we employed PI3Kgamma instead of p110alpha because it has a less positively charged 972YKSF box, which aligns with the 941KKKK box of p110alpha (Figs. 1A and 8A). The conservation of the second lysine throughout the class I PI3Ks suggests that this is the key positive residue in this box. Lys973 of PI3Kgamma was therefore replaced by an aspartic acid, and Lys980 (located in the 979NKER sequence, which aligns to the 948KRER of p110alpha ) was also replaced by an aspartic acid. Finally, a lysine-serine motif was reinserted in the PI3Kgamma -cII hybrid (see Fig. 8A and Ref. 16), thus restoring the positively charged character of the 972YKSF box in a cII background. The three mutants (PI3Kgamma K973Q (equivalent to p110alpha K942), PI3Kgamma K980Q (equivalent to p110alpha R949), and cIIQM/KS; Fig. 8A) were transiently expressed as GST fusions in 293 cells. The expression level of the mutants was comparable with that of wild-type GST-PI3Kgamma , and the mutants could undergo autophosphorylation (Fig. 8B). When tested for lipid kinase activity, the three mutants were effective in phosphorylating PtdIns and (albeit to a lesser extent) PtdIns 4-P, as expected for wild-type p110 and the cII hybrid in both alpha  and gamma  isoforms (Fig. 3). Modifications in the lipid kinase behavior were instead observed when PtdIns(4,5)P2 was presented as substrate; both the K973Q and K980Q mutations yielded a protein unable to produce PtdIns(3,4,5)P3, while restoration of the positively charged character of the first basic box lost in the cII background (cIIQM/KS) restored the ability of the protein to phosphorylate PtdIns(4,5)P2 (Fig. 9). Such discrimination of the mutants toward PtdIns(4, 5)P2 phosphorylation was also observed in reactions were a mixture of PI lipids was used as substrate (data not shown).


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 8.   Construction, expression, and protein kinase activity of GST-PI3Kgamma point mutants. A, sequence alignment highlighting 1) the basic residues in the activation loop conserved among class I PI3Ks (gray shading) and 2) the single (or double) amino acid mutation produced (boldface letters). The GST-PI3Kgamma cII hybrid is from Ref. 16. B, GST-PI3Kgamma variants were immobilized on glutathione-Sepharose and subjected to an autophosphorylation assay. The autophosphorylation pattern of the mutants is shown at the top (32P), and the Coomassie stained protein expression pattern is shown at the bottom (St.).


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 9.   Altered PI substrate specificity of GST-PI3Kgamma point mutants. The mutants of the type indicated at the top were subjected to PI3K assays using PtdIns, PtdIns 4-P, or PtdIns(4,5)P2 as substrates as indicated. The arrows on the left indicate the migration distance of PtdIns 3-P, PtdIns(3,4)P2, or PtdIns(3,4,5)P3 standards. A, quantification of lipid kinase activity (mean ± S.E., n = 3). B, typical TLC showing the production of 3-phosphorylated phosphoinositides.

The importance of the positive charges provided by Lys973 and Lys980 (Lys942 and Arg949 in p110alpha , respectively) for PtdIns(4,5)P2 turnover was further supported by model calculations on a structure of p110alpha (see "Experimental Procedures") derived from the recently published crystal structure of the PI3Kgamma catalytic subunit (19). PtdIns(4,5)P2 could be easily docked into the catalytic groove of p110alpha to mediate a close contact of the 3-OH group of the inositol head group and the gamma -phosphate group of ATP. The unresolved activation loop (19) could be rearranged to make contact with the PI head group; K942p110alpha ended up interacting with the 5-phosphate group, and R949p110alpha interacted with the 4-phosphate group. Energy minimization did not disrupt this arrangement. The assembled ATP·PtdIns(4,5)P2·p110alpha complex tightly incorporated the two substrates (Fig. 10). This structural complex was then used to estimate the solvent accessibility of various residues in the presence and absence of ATP and PtdIns(4,5)P2. Calculations were carried out using a 1.4-Å probe (see "Experimental Procedures"). As positively charged, PtdIns(4,5)P2-interacting residues Lys776 p110alpha (19.9% contribution), Lys941 (10.4%), Lys942 (30.5%), and Arg949 (13.4) were identified (Table I). Lys776 is conserved in PI3Kgamma but substituted in p110beta and p110delta by a Met. This Met is, however, embedded between two Lys residues, so that these might compensate for the missing positive charge. A positively charged patch at this position is not present in class II and III PI3Ks. Within the activation loop, only the positive charges Lys942 and Arg949 (Lys973 and Lys980 in PI3Kgamma ) are present throughout the class I PI3Ks. Together with the mutational analysis, this clearly illustrates that these residues provide an important charge compensation upon PtdIns(4,5)P2 binding.


View larger version (64K):
[in this window]
[in a new window]
 
Fig. 10.   Model of the p110alpha substrate complex. A, space-filling model of PtdIns(4,5)P2 (PIP2) and ATP docked into the catalytic pocket of p110alpha , which is shown as a sliced surface plot (red). Lys942 (K942) and Arg949 (R949) are shown as space-filling residues and interact with the 5- and 4-phosphate groups (numbered) of PtdIns(4,5)P2, respectively. B, PtdIns(4,5)P2, ATP, Lys942, and Arg949 are represented as sticks embedded in a ribbon diagram of p110alpha (green). The activation loop is highlighted in red, and the sequence of the activation loop is given in the same color (C). Green, carbon; blue, nitrogen; orange, phosphate; red, oxygen; pink, hydrogen.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Percentage accessibility of the catalytic pocket of p110alpha
Residues within the catalytic site of p110alpha that become accessible to a 1.4-Å probe when either PtdIns(4,5)P2 (PIP2) or PIP2 and ATP are removed from a p110alpha substrate complex are shown. The columns give the percentage accessibility for the indicated side chains. The PIP2/ATP column gives the accessibility for the pocket when both PIP2 and ATP are present. The ATP column shows the accessibility when PIP2 has been removed, while "No ligand" represents values in the absence of both ATP and PIP2. The columns marked Delta PIP2 and Delta ATP indicate inferred changes by removal of the respective ligand (Delta PIP2 = ATP - PIP2/ATP; Delta ATP = No ligand - ATP). Increasing values in Delta PIP2 thus characterize PtdIns(4,5)P2-interacting residues.

p110alpha Hybrids Lose Lipid Kinase but Retain Protein Kinase Signaling Capabilities-- To study the effect of p110alpha hybrids on downstream signaling, we co-transfected COS-7 cells with a Myc-tagged PKB and p110alpha -CAAX wild type/hybrids, and PKB activities were assayed in anti-Myc immunoprecipitates 36-60 h post-transfection. We observed that Myc-PKB kinase activity was elevated only in cells co-transfected with p110alpha -CAAX WT and not by the cII and cIII hybrids. Accordingly, Western blot with anti-active PKB Ser473 antibodies showed a strong activation of Myc-PKB as well as of the endogenous PKB (Fig. 11A). Likewise, we co-transfected 293 cells with Myc-p70S6k, p110alpha wild type/hybrids, and a Myc-tagged inter-SH2 (iSH2) domain of p85, which constitutively activates the p110alpha catalytic subunit (44). Immunoprecipitated Myc-p70S6k from starved cells was assayed for kinase activity using crosstide peptide as substrate (33). In this experiment, iSH2-p110alpha wild type induced a 6-fold activation of p70S6k, while any other transfected form of PI3K was unable to activate p70S6k (Fig. 11B). These data, associated with the previously described in vivo lipid measurements (Fig. 4), confirm that production of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 is an absolute requirement for PI3K-mediated activation of PKB/Akt and the downstream p70S6k, and loss of lipid kinase activity by the p110alpha hybrids renders them unable to support PKB/Akt and p70S6k activation.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 11.   Signaling properties of PI3K hybrids. A, COS-7 cells were co-transfected with the indicated Myc-tagged p110alpha -CAAX constructs and Myc-PKB. PKB activity was assayed on anti-Myc immunoprecipitates from overnight starved cells and is expressed as -fold increase relative to cells transfected with kinase-dead p110alpha -CAAX-KR (mean ± S.E., n = 3). In parallel, PKB activation was monitored by anti-active PKB Ser473 Western blot on cell lysates. B, catalytic p110alpha WT, kinase-dead p110alpha , and chimeras (as indicated) were co-transfected with Myc-p70S6k and Myc-iSH2 in 293 cells. Protein kinase activity of Myc-p70S6k was assayed in anti-Myc immunoprecipitates of starving cells 24-36 h post-transfection and is displayed relative to p70S6k activity in cells not transfected with p110alpha ·iSH2 complex (mean ± S.E., n = 3, top). Expression levels of Myc-p70S6k and p110alpha were evaluated by Western blotting on cell lysates and anti-Myc-iSH2 immunoprecipitates by anti-Myc and anti-p110alpha antibody, respectively (bottom). C, 293 cells were cotransfected with p85alpha , p110alpha s, and IR in an IRS-1 background as indicated, and lysates were immunoprecipitated with alpha -p85 antiserum. p85 immunoprecipitates were subjected to an in vitro protein kinase assay. IRS-1 phosphorylation was quantified after SDS-PAGE separation (32P, middle) by phosphor imaging (mean ± S.E., n = 3, bottom). Expression levels of immunoprecipitated p85 (Coomassie stain, St.) and associated IRS-1 (alpha -IRS-1) and tyrosine phosphorylation of IRS-1 (alpha -pY) are shown at the top.

To determine whether p110alpha hybrids retain protein kinase-associated signaling capabilities, we co-transfected 293 cells with the insulin receptor, IRS-1, and various PI3Kalpha s as described in the legend to Fig. 11C. In cells transfected with the insulin receptor, p85 immunoprecipitates contained associated IRS-1 in a tyrosine-phosphorylated form, while no IRS-1 association to p85 (nor IRS-1 tyrosine phosphorylation) was detected in cells not transfected with the insulin receptor (Fig. 11C) or transfected with a catalytically inactive form of the receptor (data not shown). In vitro PI3K protein kinase assay on p85 immunoprecipitates showed that the hybrids cII and cIII.1 can phosphorylate IRS-1, as it was previously known for the PI3Kalpha (45). The tested hybrids were even more effective than the wild type in phosphorylating IRS-1. We exclude an occurrence of IRS-1 phosphorylation by other protein kinases such as mitogen-activated protein kinase (46), GSK-3 (47), or PKB (48) for two reasons: first, immunoprecipitates from cells transfected with the catalytically inactive p110alpha K802R yield a lower IRS-1 phosphorylation; second, IRS-1 phosphorylation was inhibited by 100 nM wortmannin preincubation (data not shown). To evaluate whether the increase in IRS-1 phosphorylation observed in cells transfected with the cII and cIII.1 hybrid reflects a higher phosphorylating activity of the hybrid PI3K toward IRS-1 or the utilization of random phosphorylation sites, we performed phosphoamino acid analysis and two-dimensional tryptic phosphopeptide analysis on phosphorylated IRS-1. Phosphoamino acid analysis indicated that p110alpha wild type as well as cII (Fig. 12B) or cIII.1 phosphorylate IRS-1 exclusively on serine and threonine residues. The phosphopeptide pattern observed for IRS-1 phosphorylated by PI3Kalpha wild type, cII (Fig. 12A) and cIII.1 (not shown), is superimposable, indicating that the more extensive phosphorylation reflects a higher activity of the cII and cIII.1 hybrids. A difference in the intensity of the signal is, in fact, observed, thus indicating that certain serine/threonine residues on IRS-1 are more effectively phosphorylated by the cII and cIII.1 hybrids.


View larger version (105K):
[in this window]
[in a new window]
 
Fig. 12.   A, tryptic phosphopeptide mapping of IRS-1 in vitro phosphorylated by p110alpha WT (top) and cII hybrid (bottom). Electrophoretic separation is on the horizontal axis, and TLC is shown on the vertical axis. The origin is marked by a white spot. B, phospho-amino acid analysis of phosphorylated IRS-1 showing the presence of both Ser(P) and Thr(P) residues.

Our experiments in 293 cells lead us to conclude that p110alpha hybrids, although unable to signal via 3'-phosphorylated lipid products, may retain signaling capabilities as protein kinases with respect to IRS-1 serine/threonine phosphorylation.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

While the catalytic region of PI3Ks is located in the conserved kinase domain, members of different PI3K classes display a typical substrate specificity. Indeed, in vitro, class IA p85/p110alpha , -beta , and -delta and class IB p101·PI3Kgamma heterodimers phosphorylate PtdIns, PtdIns 4-P, and PtdIns(4,5)P2 (22, 49-51); C2 domain-containing class II PI3Ks are commonly considered to phosphorylate PtdIns and PtdIns 4-P but not PtdIns(4,5)P2 (8, 9, 11, 40, 52), although a report exists indicating that PtdIns(4,5)P2 phosphorylation occurs if the substrate is presented in PtdSer-containing micelles (53), and class III PI3Ks of the Vps34 type only phosphorylate PtdIns (17, 37). Moreover, protein kinases belonging to the PI3K superfamily (yeast and mammalian TOR/FRAP, the ATM gene product, and DNA-dependent protein kinase among them (see Ref. 54 for a review) have not been shown to possess any lipid kinase activity (13). The aim of our investigation was to understand the basis for such different substrate specificity among the members of the PI3K superfamily and to determine whether signaling capabilities can be retained if the PI3K p110alpha catalytic subunit is manipulated in such a way that its substrate specificity is altered.

We previously demonstrated that wortmannin inhibits class I PI3Ks p85/p110alpha and p101·PI3Kgamma at low nanomolar concentration by binding covalently to a lysine residue required for the phosphate transfer reaction to the lipid substrate (Lys802 in p110alpha and Lys833 in PI3Kgamma ; (23, 24)). Although this lysine is conserved among PI3Ks, some PI3Ks are less sensitive to wortmannin inhibition (e.g. IC50 for yeast Vps34 is 3 µM), indicating that noncovalent wortmannin-protein interactions directing the inhibitor to the conserved lysine residue occur and determine the effectiveness of nucleophilic attack on the lysine epsilon -amino group. Moreover, wortmannin binding on p110alpha and PI3Kgamma is effectively competed for by preincubation of the enzyme with its substrates PtdIns(4,5)P2 and ATP, indicating that wortmannin and the substrates interact within the same region of the PI3K catalytic subunit, namely the kinase domain. We therefore hypothesized that the affinity for a specific subset of phosphoinositides as substrates observed in the various PI3K classes is driven by specific substrate-recognizing motifs in the kinase domain.

The primary sequence of p110alpha displays, C-terminally to the conserved 933DFG motif, a 941KKKKFGYKRER951 positively charged stretch resembling a KXnKXKK (n = 3-7) motif that was proposed to be the PtdIns(4,5)P2 binding site of gelsolin and phosphoinositide-specific phospholipases C (55, 56). This region may therefore be involved in recognizing the polar head group of the phosphoinositide. Sequence alignment of the C-terminal region to the DFG motif of PI3Ks shows a basic profile for the class I members, represented in p110alpha by the sequences 941KKKK and 948KRER and in PI3Kgamma by the sequences 972YKSF and 979NKER (Figs. 1B and 8A). Class II members retain the homology with the KRER sequence but no relationship to the KKKK sequence, and class III members do not display any similarity in this region and do not have basic amino acid stretches (Fig. 1B). Insertion of DFG C-terminal sequences from class II and III PI3Ks into p110alpha conferred to p110alpha the substrate selectivity of the cII and cIII hybrids (see "Results" and Fig. 3). Phosphorylation of PtdIns and PtdIns 4-P by the cII hybrid was increased, and phosphorylation of PtdIns by the cIII.1, cIII.4, and cIII.5 hybrids lowered, as compared with p110alpha WT. We speculate that the swapped activation loops can be responsible for the variation in the lipid kinase activity of the hybrids. Consistently, the reported specific activity of the mammalian class II PI3K p170 toward PtdIns is 3 nmol min-1 mg-1 (9) as compared with 2 nmol min-1 mg-1 for p110alpha (40). Also, in a direct comparative study, the PtdIns phosphorylating activity of the human class III PI3K Vps34 has been shown to be lower than that of PI3Kalpha (37). The complete loss of lipid kinase activity of the cIV hybrid further demonstrated that the region investigated is specifically required for recognition and phosphorylation of the lipid substrate (Fig. 2). Based on the results obtained from the in vitro lipid kinase assays, we speculate that the 941KKKK and 948KRER boxes are both required for the productive binding of the 5- and 4-phosphate groups of PtdIns(4,5)P2. Therefore, elimination of the 941KKKK p110alpha positively charged box in the cII hybrid accounts for its inability to phosphorylate PtdIns(4,5)P2 and is in agreement with results obtained in a similar mutant of PI3Kgamma (16). The subsequent production of the two point mutants, PI3Kgamma K973Q and PI3Kgamma K980Q, confirmed the need for two positively charged amino acids in specific positions within these boxes in order to achieve PtdIns(4,5)P2 turnover. In keeping with this, the restoring of a positive charge in the cII background (cIIQM/KS mutant) yielded a cII variant with reacquired functionality toward PtdIns(4,5)P2 phosphorylation. Our biochemical data are in agreement with structure modeling performed on the basis of a p110alpha substrate complex generated with the recently reported x-ray structure of a class IB PI3Kgamma (19) as a template. In the PI3Kgamma structure, the activation loop sequence is not ordered. Together with the mutational analysis, our modeling data suggest that K942p110alpha interacts with the 5-phosphate and R949p110alpha with the 4-phosphate group of PtdIns(4,5)P2. A simulation of solvent accessibility further supports the importance of K942p110alpha and R949p110alpha . Within the activation loop, these two positively charged residues seem to be crucial and sufficient to mediate PtdIns(3,4,5)P3 production. It can therefore be speculated that the activation loop becomes ordered upon PtdIns(4,5)P2 binding and that solvent molecules are removed from the 5- and 4-phosphate groups when these interact with K942p110alpha and R949p110alpha . By this process, the activation loop seals the catalytic pocket off from solvent access, which is a prerequisite for a successful transfer of the gamma -phosphate group of ATP to the 3-OH group of PtdIns(4,5)P2.

The above finding and the fact that manipulations of the activation loop switch p110alpha substrate specificity indicate that PI kinase activation loops function in general as dynamic structures to limit access to the correct substrate among the PIs present in the cell's membranes. Accordingly, Kunz et al. (18), by reciprocally exchanging the activation loops of type 1 and type 2 phosphatidylinositol phosphate kinases, where able to invert their lipid substrate specificities. In a different manner, selective polyphosphoinositide head group discrimination is observed for the PH domains of Grp1, Btk, and PDK-1, which exclusively bind PtdIns(3,4,5)P3, versus the PH domains of DAPP1 and PKB, which bind to both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 (70), and in the FYVE domains of yeast Vps27p and human early endosome autoantigen 1, which exclusively recognize PtdIns 3-P and not PtdIns 5-P or PtdIns (71, 72).

Swapping of foreign sequences into p110alpha could, in principle, alter the structure of the hybrid protein, and the observed variations in substrate specificity could therefore be due to nonspecific structural changes. To exclude this possibility, we sought to establish whether other properties of the enzyme, namely its intrinsic protein kinase activity (14, 43) and interaction with the inhibitor wortmannin (23), are unaffected in the p110alpha hybrids. In vitro p110alpha -mediated p85alpha serine 608 phosphorylation was unaffected in the cII and cIII hybrids; cIV showed a reduced protein kinase activity but still significantly higher than the negative control provided by the p110alpha K802R kinase-dead catalytic subunit (23). Serine phosphorylation on the p85alpha subunit is functionally relevant, since, by decreasing the lipid kinase activity of the heterodimer, it provides a feedback control mechanism on the activity of the protein (14, 43). Here we show that both the hybrids cII and cIII.1 did not display any variation in this kind of feedback control with respect to the wild type heterodimer. Wortmannin binding was also unaffected in the cII and cIII hybrids. Our biochemical observations relative to wortmannin binding can be rationalized with respect to the reported structure of PI3Kgamma bound to wortmannin (60). In this recent study, it is shown that wortmannin binds in the ATP binding site of the enzyme with one face directed toward the N-terminal lobe of the ATP binding site and the other face packed against the C-terminal lobe, where an interaction with residues 950, 953, 961, 963, and 964 is occurring. Interestingly, these residues are placed immediately before the DFG motif, and it is shown in the same study that mutagenesis of residues N-terminal to the DFG motif modulates the sensitivity of the enzyme to wortmannin. On the contrary, our manipulations on the C-terminal side of the DFG motif did not affect wortmannin binding to the hybrids cII and cIII. Although the cII and cIII hybrids retained wortmannin binding, the cIV hybrid was unable to covalently bind wortmannin, even in the absence of detergent in the incubation mixture, which provides a less stringent assay condition. This may be due to the lower wortmannin sensitivity of FRAP (61). In addition, wortmannin-binding competition experiments showed that 1) the ATP binding site remains intact after construction of the hybrid proteins, since ATP effectively competes for wortmannin binding, and 2) the exchanged regions, although responsible for the differential phosphorylation of the phosphoinositide polar head group, do not preclude the binding of phosphoinositides to the surface of the enzyme, as demonstrated by the competition of PtdIns(4,5)P2 toward wortmannin binding in the hybrids cII and cIII.1. This is in accord with structural data on PI3Kgamma in which the C- and N-terminal lobes of the catalytic domain are described as taking part in the binding of both lipid substrate and wortmannin (19, 60). To complement the in vitro experiments, we next explored the in vivo behavior of the p110alpha hybrids. To eliminate interference from endogenous PI3Ks, we took advantage of an agonist-independent constitutively active form of p110alpha (Myc-p110alpha -CAAX; Ref. 25); WT and K802R kinase-dead as well as cII, cIII.1, cIII.2, and cIV Myc-p110alpha -CAAX were tested for the in vivo production of 3'-phosphorylated phosphoinositides in serum-starved COS-7 cells. Cells transfected with WT Myc-p110alpha -CAAX accumulated PtdIns(3,4)P2 and PtdIns(3,4,5)P3, while these lipids were not produced in cells transfected with any of the p110alpha hybrids. In our 32P labeling experiments, we never observed changes in PtdIns 3-P levels, irrespective of the transfected p110alpha -CAAX. Thus, class I PI3Ks do not behave, in vivo, as PtdIns kinases, although PI3K activities are often measured in vitro by presenting PtdIns as substrate. The fact that the cII hybrid was unable to produce PtdIns(3,4)P2 implies that also PtdIns 4-P may be not an in vivo substrate for class I PI3Ks, and the appearance of PtdIns(3,4)P2 upon p110alpha -CAAX WT overexpression may be due to SHIP-dependent 5' dephosphorylation of PtdIns(3,4,5)P3 (73) rather than phosphorylation of PtdIns 4-P. Accordingly, several lines of evidence place PtdIns(4,5)P2 as the unique in vivo substrate for class I PI3Ks (7, 27, 63-65), and our data from metabolic labeling experiments are consistent with this idea, since none of the hybrids could produce PtdIns(3,4,5)P3 or the less phosphorylated PtdIns(3,4)P2. Nevertheless, the reason why class I PI3Ks can phosphorylate PtdIns, PtdIns 4-P, and PtdIns(4,5)P2 in vitro but exclusively PtdIns(4,5)P2 in vivo is not yet understood. A report indicates that this may be due to the fact that in vivo substrate presentation to PI3Ks is carried out by a phosphatidylinositol transfer protein, which may thus regulate the in vivo preferential utilization of PtdIns(4,5)P2 (74).

The fact that the cII hybrid, opposed to its in vitro behavior, was unable to produce PtdIns(3,4)P2 in vivo could not permit us to investigate the different roles played in signaling cascades by PtdIns(3,4)P2 and PtdIns(3,4,5)P3, yet the cII and cIII hybrids were exploited as tools to investigate the contribution that PI3K plays in signaling either as a lipid kinase or as a protein kinase.

The requirement for PtdIns(3,4)P2 and PtdIns(3,4,5)P3 is definitely established for the regulation of the PI3K-PDK-PKB/Akt-p70S6k mitogenic signaling pathway (66, 67); PtdIns(3,4,5)P3 binds to and activates phosphoinositidedependent kinases, which in turn phosphorylate and activate PKB/Akt (68-70) and p70S6k (28). In addition, PKB itself, upon binding to PtdIns(3,4)P2 via its PH domain (27, 70, 71) provides further activation signals to p70S6k (72). We studied here the effect of constitutively activated wild-type p110alpha and p110alpha hybrids on PKB and p70S6k activation, in transiently transfected COS-7 and 293 cells, respectively, and found that none of the hybrids tested could activate p70S6k, in keeping with the above.

The requirement for 3'-phosphoinositides in some PI3K-mediated signaling pathways does not rule out an involvement of this kinase as a protein kinase in others. In fact, PI3Kalpha protein kinase activity has been implicated in IRS-1 serine phosphorylation in insulin-treated adipocytes (45) and in STAT3 and IRS-1 phosphorylation upon activation of the type 1 IFN receptor by IFN-alpha (73, 74). In addition, previous work from our laboratory demonstrated that PI3Kgamma -mediated activation of the mitogen-activated protein kinase Erk-2 relies solely on PI3Kgamma protein kinase action (16). Now, we show here that p110alpha hybrids, although unable to activate PKB and p70S6k, still retain the capability to associate and in vitro phosphorylate the insulin receptor substrate IRS-1 to an even bigger extent than the p85/p110alpha wild type. By two-dimensional phosphopeptide mapping, we also show that the increased IRS-1 phosphorylation supported by the cII and cIII.1 hybrid as compared with p110alpha wild type reflects a higher activity of the hybrids rather than a utilization of nonspecific phosphorylation sites. Finally, by phosphoamino acid analysis, we show that phosphorylation takes place on serine and threonine residues (Fig. 12B).

The data described here clearly demonstrate that the two activities of PI3Ks (i.e. the lipid- and protein-phosphorylating activities) are generated by different structural kinase motifs. Moreover, the p110alpha hybrids that we have designed will allow further investigation of whether PI3Kalpha protein kinase activity is per se sufficient to sustain known PI3K signaling pathways and open the possibly of uncovering new PI3K-dependent signaling events not requiring production of 3'-phosphorylated phosphoinositides.

    ACKNOWLEDGEMENTS

We thank J. Downward for the Myc-p110alpha -CAAX construct; T. Franke, B. Hemmings, and G. Thomas for PKB/Akt and p70S6k expression plasmids; S. Keller for the IRS-1 cDNA; and B. Hemmings for crosstide peptide and help with the PKB/Akt assay. We thank S. Bonnafous and C. Filloux for help with two-dimensional map experiments and Piers Gaffney and Roger Williams for advice during the modeling of the PtdIns(4,5)P2·PI3Kgamma complex.

    FOOTNOTES

* This work was supported by Swiss Cancer League Grant SKL 780-2-1999 and a Human Frontier Science Program grant (to M. P. W.).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.

§ Recipient of "Swiss National Science Foundation" predoctoral fellowship (3100-50506.97; to M. P. W.) and postdoctoral fellowship. Present address: INSERM U-145, Faculté de Médecine, Av. de Valmbrose, 06107 Nice Cedex 2, France.

Dagger Dagger To whom correspondence should be addressed: Inst. of Biochemistry, Rue du Musée 5, CH-1700 Fribourg, Switzerland. Tel.: 41 26 300 86 55; Fax: 41 26 300 9735; E-mail: MatthiasPaul.Wymann@UniFR.ch.

Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M011330200

    ABBREVIATIONS

The abbreviations used are: PtdIns 3-P, phosphatidylinositol 3-phosphate; PtdIns(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; PtdIns(3, 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns 4-P, phosphatidylinositol 4-phosphate; PtdIns(4, 5)P2 or PIP2, phosphatidylinositol 4,5-bisphosphate; PI3K, phosphoinositide 3-kinase; SH2, Src homology 2; iSH2, inter-SH2; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; IRS, insulin receptor substrate; PKB, protein kinase B; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PI, phosphatidylinositol; HPLC, high pressure liquid chromatography; S6k, S6 kinase; cI to cIV, class I to IV, respectively.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Wurmser, A. E., Gary, J. D., and Emr, S. D. (1999) J. Biol. Chem. 274, 9129-9132[Free Full Text]
2. Leevers, S. J., Vanhaesebroeck, B., and Waterfield, M. D. (1999) Curr. Opin. Cell Biol. 11, 219-225[CrossRef][Medline] [Order article via Infotrieve]
3. Vanhaesebroeck, B., and Waterfield, M. D. (1999) Exp. Cell Res. 253, 239-254[CrossRef][Medline] [Order article via Infotrieve]
4. Fruman, D. A., Meyers, R. E., and Cantley, L. C. (1998) Annu. Rev. Biochem. 67, 481-507[CrossRef][Medline] [Order article via Infotrieve]
5. Wymann, M. P., and Pirola, L. (1998) Biochim. Biophys. Acta 1436, 127-150[Medline] [Order article via Infotrieve]
6. Domin, J., and Waterfield, M. D. (1997) FEBS Lett. 410, 91-95[CrossRef][Medline] [Order article via Infotrieve]
7. Stephens, L. R., Eguinoa, A., Erdjument-Bromage, H., Lui, M., Cooke, F., Coadwell, J., Smrcka, A. S., Thelen, M., Cadwallader, K., Tempst, P., and Hawkins, P. T. (1997) Cell 89, 105-114[Medline] [Order article via Infotrieve]
8. Molz, L., Chen, Y. W., Hirano, M., and Williams, L. T. (1996) J. Biol. Chem. 271, 13892-13899[Abstract/Free Full Text]
9. Virbasius, J. V., Guilherme, A., and Czech, M. P. (1996) J. Biol. Chem. 271, 13304-13307[Abstract/Free Full Text]
10. Arcaro, A., Volinia, S., Zvelebil, M. J., Stein, R., Watton, S. J., Layton, M. J., Gout, I., Ahmadi, K., Downward, J., and Waterfield, M. D. (1998) Mol. Cell. Biol. 273, 33082-33090
11. Misawa, H., Ohtsubo, M., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., and Yoshimura, A. (1998) Biochem. Biophys. Res. Commun. 244, 531-539[CrossRef][Medline] [Order article via Infotrieve]
12. De Camilli, P., Emr, S. D., McPherson, P. S., and Novick, P. (1996) Science 271, 1533-1539[Abstract]
13. Hunter, T. (1995) Cell 83, 1-4[Medline] [Order article via Infotrieve]
14. Dhand, R., Hiles, I., Panayotou, G., Roche, S., Fry, M. J., Gout, I., Totty, N. F., Truong, O., Vicendo, P., Yonezawa, K., Kasuga, M., Courtneidge, S. A., and Waterfield, M. D. (1994) EMBO J. 13, 522-533[Abstract]
15. Vanhaesebroeck, B., Higashi, K., Raven, C., Welham, M., Anderson, S., Brennan, P., Ward, S. G., and Waterfield, M. D. (1999) EMBO J. 18, 1292-1302[Abstract/Free Full Text]
16. Bondeva, T., Pirola, L., Bulgarelli-Leva, G., Rubio, I., Wetzker, R., and Wymann, M. P. (1998) Science 282, 293-296[Abstract/Free Full Text]
17. Stack, J. H., and Emr, S. D. (1994) J. Biol. Chem. 269, 31552-31562[Abstract/Free Full Text]
18. Kunz, J., Wilson, M. P., Kisseleva, M., Hurley, J. H., Majerus, P. W., and Anderson, R. A. (2000) Mol. Cell 5, 1-11[Medline] [Order article via Infotrieve]
19. Walker, E. H., Perisic, O., Ried, C., Stephens, L., and Williams, R. L. (1999) Nature 402, 313-320[CrossRef][Medline] [Order article via Infotrieve]
20. Rao, V. D., Misra, S., Boronenkov, I. V., Anderson, R. A., and Hurley, J. H. (1998) Cell 94, 829-839[Medline] [Order article via Infotrieve]
21. Deng, W. P., and Nickoloff, J. A. (1992) Anal. Biochem. 200, 81-88[Medline] [Order article via Infotrieve]
22. Hiles, I. D., Otsu, M., Volinia, S., Fry, M. J., Gout, I., Dhand, R., Panayotou, G., Ruiz-Larrea, F., Thompson, A., Totty, N. F., Hsuan, J. J., Courtneidge, S. A., Parker, P. J., and Waterfield, M. D. (1992) Cell 70, 419-429[Medline] [Order article via Infotrieve]
23. Wymann, M. P., Bulgarelli-Leva, G., Zvelebil, M. J., Pirola, L., Vanhaesebroeck, B., Waterfield, M. D., and Panayotou, G. (1996) Mol. Cell. Biol. 16, 1722-1733[Abstract]
24. Stoyanova, S., Bulgarelli-Leva, G., Kirsch, C., Hanck, T., Klinger, R., Wetzker, R., and Wymann, M. P. (1997) Biochem. J. 324, 489-495[Medline] [Order article via Infotrieve]
25. Khwaja, A., Rodriguez-Viciana, P., Wennstrom, S., Warne, P. H., and Downward, J. (1997) EMBO J. 16, 2783-2793[Abstract/Free Full Text]
26. Kaufman, R. J., Davies, M. V., Pathak, V. K., and Hershey, J. W. (1989) Mol. Cell. Biol. 9, 946-958[Medline] [Order article via Infotrieve]
27. Franke, T. F., Kaplan, D. R., Cantley, L. C., and Toker, A. (1997) Science 275, 665-668[Abstract/Free Full Text]
28. Pullen, N., Dennis, P. B., Andjelkovic, M., Dufner, A., Kozma, S. C., Hemmings, B. A., and Thomas, G. (1998) Science 279, 707-710[Abstract/Free Full Text]
29. Keller, S. R., Aebersold, R., Garner, C. W., and Lienhard, G. E. (1993) Biochim. Biophys. Acta 1172, 323-326[Medline] [Order article via Infotrieve]
30. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract]
31. Kaplan, D. R., Whitman, M., Schaffhausen, B., Pallas, D. C., White, M., Cantley, L., and Roberts, T. M. (1987) Cell 50, 1021-1029[Medline] [Order article via Infotrieve]
32. Arcaro, A., and Wymann, M. P. (1993) Biochem. J. 296, 297-301[Medline] [Order article via Infotrieve]
33. Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M., and Hemmings, B. A. (1995) Nature 378, 785-789[CrossRef][Medline] [Order article via Infotrieve]
34. Traynor-Kaplan, A. E., Thompson, B. L., Harris, A. L., Taylor, P., Omann, G. M., and Sklar, L. A. (1989) J. Biol. Chem. 264, 15668-15673[Abstract/Free Full Text]
35. Gual, P., Baron, V., Lequoy, V., and Van Obberghen, E. (1998) Endocrinology 139, 884-893[Abstract/Free Full Text]
36. Nguyen, T. T., Scimeca, J. C., Filloux, C., Peraldi, P., Carpentier, J. L., and Van Obberghen, E. (1993) J. Biol. Chem. 268, 9803-9810[Abstract/Free Full Text]
37. Volinia, S., Dhand, R., Vanhaesebroeck, B., MacDougall, L. K., Stein, R., Zvelebil, M. J., Domin, J., Panaretou, C., and Waterfield, M. D. (1995) EMBO J. 14, 3339-3348[Abstract]
38. Brown, E. J., Albers, M. W., Shin, T. B., Ichikawa, K., Keith, C. T., Lane, W. S., and Schreiber, S. L. (1994) Nature 369, 756-758[CrossRef][Medline] [Order article via Infotrieve]
39. Toker, A., and Cantley, L. C. (1997) Nature 387, 673-676[CrossRef][Medline] [Order article via Infotrieve]
40. MacDougall, L. K., Domin, J., and Waterfield, M. D. (1995) Curr. Biol. 5, 1404-1415[Medline] [Order article via Infotrieve]
41. Klippel, A., Reinhard, C., Kavanaugh, W. M., Apell, G., Escobedo, M. A., and Williams, L. T. (1996) Mol. Cell. Biol. 16, 4117-4127[Abstract]
42. Leevers, S. J., Paterson, H. F., and Marshall, C. J. (1994) Nature 369, 411-414[CrossRef][Medline] [Order article via Infotrieve]
43. Carpenter, C. L., Auger, K. R., Duckworth, B. C., Hou, W. M., Schaffhausen, B., and Cantley, L. C. (1993) Mol. Cell. Biol. 13, 1657-1665[Abstract]
44. Hu, Q., Klippel, A., Muslin, A. J., Fantl, W. J., and Williams, L. T. (1995) Science 268, 100-102[Medline] [Order article via Infotrieve]
45. Lam, K., Carpenter, C. L., Ruderman, N. B., Friel, J. C., and Kelly, K. L. (1994) J. Biol. Chem. 269, 20648-20652[Abstract/Free Full Text]
46. De Fea, K., and Roth, R. A. (1997) J. Biol. Chem. 272, 31400-31406[Abstract/Free Full Text]
47. Eldar-Finkelman, H., and Krebs, E. G. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9660-9664[Abstract/Free Full Text]
48. Paz, K., Liu, Y. F., Shorer, H., Hemi, R., LeRoith, D., Quan, M., Kanety, H., Seger, R., and Zick, Y. (1999) J. Biol. Chem. 274, 28816-28822[Abstract/Free Full Text]
49. Hu, P., Mondino, A., Skolnik, E. Y., and Schlessinger, J. (1993) Mol. Cell. Biol. 13, 7677-7688[Abstract]
50. Stoyanov, B., Volinia, S., Hanck, T., Rubio, I., Loubtchenkov, M., Malek, D., Stoyanova, S., Vanhaesebroeck, B., Dhand, R., Nurnberg, B., Gierschik, P., Seedorf, K., Hsuan, J. J., Waterfield, M. D., and Wetzker, R. (1995) Science 269, 690-693[Medline] [Order article via Infotrieve]
51. Vanhaesebroeck, B., Welham, M. J., Kotani, K., Stein, R., Warne, P. H., Zvelebil, M. J., Higashi, K., Volinia, S., Downward, J., and Waterfield, M. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4330-4335[Abstract/Free Full Text]
52. Brown, R. A., Ho, L. K., Weber-Hall, S. J., Shipley, J. M., and Fry, M. J. (1997) Biochem. Biophys. Res. Commun. 233, 537-544[CrossRef][Medline] [Order article via Infotrieve]
53. Domin, J., Pages, F., Volinia, S., Rittenhouse, S. E., Zvelebil, M. J., Stein, R. C., and Waterfield, M. D. (1997) Biochem. J. 326, 139-147[Medline] [Order article via Infotrieve]
54. Zakian, V. A. (1995) Cell 82, 685-687[Medline] [Order article via Infotrieve]
55. Simoes, A. P., Reed, J., Schnabel, P., Camps, M., and Gierschik, P. (1995) Biochemistry 34, 5113-5119[Medline] [Order article via Infotrieve]
56. Yu, F. X., Sun, H. Q., Janmey, P. A., and Yin, H. L. (1992) J. Biol. Chem. 267, 14616-14621[Abstract/Free Full Text]
57. Walker, E. H., Pacold, M. E., Perisic, O., Stephens, L., Hawkins, P. T., Wymann, M. P., and Williams, R. L. (2000) Mol. Cell 6, 909-919[Medline] [Order article via Infotrieve]
58. Brunn, G. J., Williams, J., Sabers, C., Wiederrecht, G., Lawrence, J. C. J., and Abraham, R. T. (1996) EMBO J. 15, 5256-5267[Abstract]
59. Kucera, G. L., and Rittenhouse, S. E. (1990) J. Biol. Chem. 265, 5345-5348[Abstract/Free Full Text]
60. Nolan, R. D., and Lapetina, E. G. (1991) Biochem. Biophys. Res. Commun. 174, 524-528[Medline] [Order article via Infotrieve]
61. Stephens, L. R., Hughes, K. T., and Irvine, R. F. (1991) Nature 351, 33-39[CrossRef][Medline] [Order article via Infotrieve]
62. Downward, J. (1998) Science 279, 673-674[Free Full Text]
63. Alessi, D. R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R., Reese, C. B., and Cohen, P. (1997) Curr. Biol. 7, 261-269[Medline] [Order article via Infotrieve]
64. Stephens, L., Anderson, K., Stokoe, D., Erdjument-Bromage, H., Painter, G. F., Holmes, A. B., Gaffney, P. R., Reese, C. B., McCormick, F., Tempst, P., Coadwell, J., and Hawkins, P. T. (1998) Science 279, 710-714[Abstract/Free Full Text]
65. Stokoe, D., Stephens, L. R., Copeland, T., Gaffney, P. R., Reese, C. B., Painter, G. F., Holmes, A. B., McCormick, F., and Hawkins, P. T. (1997) Science 277, 567-570[Abstract/Free Full Text]
66. Klippel, A., Kavanaugh, W. M., Pot, D., and Williams, L. T. (1997) Mol. Cell. Biol. 17, 338-344[Abstract]
67. Burgering, B. M., and Coffer, P. J. (1995) Nature 376, 599-602[CrossRef][Medline] [Order article via Infotrieve]
68. Pfeffer, L. M., Mullersman, J. E., Pfeffer, S. R., Murti, A., Shi, W., and Yang, C. H. (1997) Science 276, 1418-1420[Abstract/Free Full Text]
69. Uddin, S., Fish, E. N., Sher, D. A., Gardziola, C., White, M. F., and Platanias, L. C. (1997) J. Immunol. 158, 2390-2397[Abstract]
70. Ferguson, K. M., Kavran, J. M., Sankaran, V. G., Fournier, E., Isakoff, S. J., Skolnik, E. Y., and Lemmon, M. A. (2000) Mol. Cell 6, 373-384[Medline] [Order article via Infotrieve]
71. Kutateladze, T. G., Ogburn, K. D., Watson, W. T., de Beer, T., Emr, S. G., Burd, C. G., and Overduin, M. (1999) Mol. Cell 3, 805-811[CrossRef][Medline] [Order article via Infotrieve]
72. Misra, S., and Hurley, J. H. (1999) Cell 97, 657-666[Medline] [Order article via Infotrieve]
73. Damen, J. E., Liu, L., Rosten, P., Humphries, R. K., Jefferson, A. B., Majerus, P. W., and Krystal, G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1689-1693[Abstract/Free Full Text]
74. Kular, G., Loubtchenkov, M., Swigart, P., Whatmore, J., Ball, A., Cockcroft, S., and Wetzker, R. (1997) Biochem. J. 325, 299-301[Medline] [Order article via Infotrieve]
75. Laskowski, R. A. (1995) J. Mol. Graph. 13, 323-330[CrossRef][Medline] [Order article via Infotrieve]
76. Hubbard, S. J., Campbell, S. F., and Thornton, J. M. (1991) J. Mol. Biol. 220, 507-530[Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.