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Originally published In Press as doi:10.1074/jbc.M205893200 on August 23, 2002

J. Biol. Chem., Vol. 277, Issue 44, 41556-41562, November 1, 2002
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The p85 Regulatory Subunit Controls Sequential Activation of Phosphoinositide 3-Kinase by Tyr Kinases and Ras*

Concepción Jiménez, Carmen Hernández, Belén Pimentel, and Ana C. CarreraDagger

From the Department of Immunology and Oncology, Centro Nacional de Biotecnología, Universidad Autónoma de Madrid, Cantoblanco, Madrid E-28049, Spain

Received for publication, June 13, 2002, and in revised form, July 25, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Class IA phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit that regulates a variety of cell responses, including cell division and survival. PI3K is activated following Tyr kinase stimulation and by Ras. We found that the C-terminal region of p85, including the C-Src homology 2 (C-SH2) domain and part of the inter-SH2 region, protects the p110 catalytic subunit from Ras-induced activation. Although the p110 activity associated with a C-terminal p85 deletion mutant increased significantly in the presence of an active form of Ras, purified wild type p85-p110 was only slightly stimulated by active Ras. Nonetheless, incubation of purified p85-p110 with Tyr-phosphorylated peptides, which mimic the activated platelet-derived growth factor receptor, restored Ras-induced p85-p110 activation. In conclusion, p85 inhibits p110 activation by Ras; this blockage is released by Tyr kinase stimulation, showing that the classical mechanism of class IA PI3K stimulation mediated by Tyr kinases also regulates Ras-induced PI3K activation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phosphoinositide 3-kinases (PI3K)1 are enzymes that transfer phosphate to position 3 of the phosphoinositide ring, regulating a variety of cell responses including survival, division, and transformation. PI3Ks are divided into three subclasses based on their primary structure and substrate specificity, but only the class I enzymes generate phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (3-poly-PtdIns) products in vivo. Basal levels of these lipids are very low in quiescent cells but increase rapidly and transiently following growth factor receptor (GFR) stimulation (for a review, see Refs. 1-4). 3-Poly-PtdIns recruits pleckstrin homology domain-containing proteins such as phosphoinositide-dependent kinase-1 and protein kinase B (PKB), which mediate PI3K signal propagation (5-8).

Class IA PI3K is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit, of which there are several isoforms (1, 2, 9-14). The prototypic p85 regulatory subunit has an SH3 domain, a BcR homology domain (BH) flanked by proline-rich sequences, and two SH2 domains separated by the so-called inter-SH2 domain, to which p110 binds (15-17). The p110 catalytic subunit contains a p85-interacting region, a Ras-binding domain, a region homologous to PI4K and the PI3K catalytic domain (4, 15).

The p85 subunit protects p110 from degradation and inhibits its enzymatic activity in quiescent cells (18). When cells are stimulated by receptors with intrinsic Tyr kinase activity, such as platelet-derived growth factor receptor (PDGFR), p85 mediates p110 translocation to the cell membrane, where PI3K substrates are found (11). In addition to mediating PI3K translocation, p85 appears to transmit an activating conformational change to p110 (19-21), because binding to p85 of Tyr-phosphorylated peptides representing the activated PDGFR increases p110 enzymatic activity (16, 22). Other receptors activate PI3K by stimulating cytosolic Tyr kinases of the Src family; this would be the case for cytokine receptors, such as the interleukin-2 receptor (IL-2R) (23). In this case, the receptor associates to PI3K constitutively (24, 25), and p110 is activated following IL-2R-induced Lck stimulation (27). In addition, phosphorylation of p85 on Tyr-688 by Src kinases increases p85-p110 kinase activity (28). p110 can also associate with and be activated by the GTP-binding protein Ras (29, 30). Finally, p85 not only mediates p110 activation; p85 binding to 3-poly-PtdIns or to SHP-1 phosphatase appears to control PI3K signal down-regulation (30, 31).

We described previously (32) the cloning and characterization of a natural oncogenic mutant of the p85alpha regulatory subunit, p65PI3K, isolated from a murine thymic lymphoma. Similar mutations have been found in a number of human tumors, increasing the interest in understanding how p65PI3K-like mutations control p110 activation (33-35). p65PI3K lacks the C-SH2 domain and part of the inter-SH2 region but still binds to p110 and enhances PI3K activity. Here we studied the mechanisms underlying p65PI3K-induced PI3K/PKB activation using cells stimulated via the PDGFR or IL-2R. p65PI3K expression increased both basal and receptor-induced PI3K activation (32) but also reduced the rate of PI3K signal down-regulation. To examine the cause of the enhanced p110 activation by p65PI3K, we compared p85-p110 and p65PI3K-p110 activation in vitro. The results indicate that in addition to controlling PI3K signal termination, the C-terminal region of p85 regulates sequential PI3K activation by Tyr kinases and by Ras.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Transfections-- CTLL2 cells were cultured in RPMI medium containing 10% fetal calf serum and 20 units/ml IL-2. CTLL2 cells were transfected by electroporation using 50 µg of each cDNA as described (36) and collected 24 h later. Stable CTLL2 cell lines (32) were cultured with 2 µg/ml puromycin (Sigma). NIH3T3 cells were cultured in DMEM containing 10% calf serum (Invitrogen) and were transiently transfected using LipofectAMINE reagent (Invitrogen) according to the manufacturer's recommendations. Stably transfected NIH3T3 cells (37) were cultured in DMEM with 10% calf serum and 1 mg/ml G418 (Calbiochem).

cDNAs, Antibodies, and Reagents-- The following cDNAs were used for transfections: PefBos-P505-Lck (36), pSG5 empty vector, pSG5-HAp65PI3K, pCG-HAp85, pSG5-V12Ras, and pSG5-HA-PKB (32). SHP1 cDNA was amplified from poly(A)-mRNA of Jurkat cells and subcloned in pRK5. For in vitro transcription and translation, the following cDNAs were used: pBSSKp50alpha (38), pSG5-V12-Ras, pSG5p110, pSG5p65, and pSG5p85 (32). Anti-HA (12CA5), -PKB, and -pPKB antibodies were purchased from Babco, Santa Cruz Biotechnology, and New England Biolabs, respectively. Anti-human SH3 PI3-kinase, -NT-Lck, -SHP1, -PDGFR and -rat PI3K antisera were from Upstate Biotechnology, Inc., and anti-Ras (Ab3) monoclonal antibody was from Oncogene Science. Recombinant IL-2 was from Roche Molecular Biochemicals; PDGF was from Calbiochem.

Immunoprecipitation and Western Blot-- Cells were lysed in Triton X-100 lysis buffer (50 mM Hepes, pH 8.0, 150 mM NaCl, 1% Triton X-100) containing protease and phosphatase inhibitors (1 mM Na3VO4, 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 10 µg/ml leupeptin). For the association of Ras with PI3K, cells were lysed in Brij 96 lysis buffer (20 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1% in Brij 96) containing phosphatase and protease inhibitors (as above). Cell lysates were cleared by centrifugation. Total protein concentration was measured with the Micro BCA kit (Pierce). Immunoprecipitation was performed by incubating lysates (4 °C, 2 h) with the appropriate antibody (2 µl of polyclonal Ab or 1 µg of purified monoclonal Ab), followed by incubation with 30 µl of 50% protein A-Sepharose slurry (Amersham Biosciences) for 1 h. Immunoprecipitates were washed once with Triton X-100 lysis buffer, twice with 50 mM Tris-HCl, pH 7.5, 0.5 M LiCl, and once with 50 mM Tris-HCl, pH 7.5. Immunoprecipitated proteins were resolved by SDS-PAGE and then transferred to nitrocellulose for Western blot analysis (32). Other gels were dried, and radiolabeled proteins were visualized by autoradiography.

Ras Peptide Columns-- A peptide encompassing H-Ras residues 17-42 (SALTIQLIQNHFVDEYDPTIEDSYRK) was coupled to Actigel ALD superflow beads using ALD coupling solutions (Sterogene Bioseparations). Extracts (500 µg) of p65PI3K- and p85alpha -expressing CTLL2 cells were incubated with the peptide-specific or a control column (1 h, 4 °C). Beads were washed once with Triton X-100 lysis buffer, twice with 50 mM Tris-HCl, pH 7.5, 0.5 M LiCl, and once with 50 mM Tris-HCl, pH 7.5. Control and Ras peptide-coupled beads were boiled in sample buffer, and the retained proteins were resolved by SDS-PAGE and examined in a Western blot using anti-p85 antibody.

In Vitro Transcribed/Translated Proteins-- The cDNAs for in vitro transcription/translation were purified by phenol/chloroform (1:1 v/v) extraction followed by EtOH precipitation. DNA (2 µg) was used to develop the TNT-coupled Reticulocyte Lysate kit (Promega), as described by the manufacturer. Briefly, DNA was mixed with 75 µl of TNT reticulocyte lysate, 6 µl of TNT reaction buffer, 3 µl of an amino acid mixture without methionine, 1 µl of RNasin (40 units/µl), 3 µl of RNA polymerase T7, and 12 µl of L-[35S]methionine (1000 Ci/mmol, Amersham Biosciences) was added. The reaction was allowed to proceed at 30 °C for 2 h. Subsequently, 1 µl of RNase (10 mg/ml) was added (30 min, 37 °C). The reaction was analyzed by immunoprecipitating 10% of this reaction mixture with anti-p85 or anti-Ras antibody. Precipitates were resolved in SDS-PAGE, and the radioactive products were visualized by autoradiography.

PKB and PI3K Assays-- PKB kinase activity was determined in a Western blot using anti-phospho-Ser-473 PKB Ab or by in vitro kinase assay. Endogenous PKB or recombinant HA-PKB were immunoprecipitated as above. Immunopurified PKB was resuspended in 25 µl of kinase buffer (50 mM Tris, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol) and mixed with 2.5 µg of histone H2B, 1 µM protein kinase inhibitor P0300 (Sigma), 50 µM ATP, and 10 µCi of [gamma -32P]ATP (3000 Ci/mmol, Amersham Biosciences). Reactions were incubated (20 min, room temperature) and resolved by SDS-PAGE. H2B phosphorylation was detected by autoradiography.

The PI3K assay was reported previously (32). For assays with in vitro translated PI3K complexes, translation mixtures were purified using anti-SH3 p85 Ab. Similar results were obtained using the anti-rat PI3K antiserum. Immunopurified PI3K was resuspended in 20 µl of 50 mM Hepes containing phosphoinositide (brain extract) micelles (0.5 mg/ml; Sigma) as substrate. The kinase reaction was initiated by adding 5 µl of the kinase buffer containing 10 µCi of [32P]ATP, 100 mM MgCl2, and 200 µM unlabeled ATP (25 °C, 5 min). The reaction was terminated by adding 100 µl of 1 M HCl and 200 µl of a methanol/chloroform mixture (1:1 v/v). The extracted phospholipids were resolved by thin layer chromatography (Silica Gel 60; Merck) on plates coated with 1% potassium oxalate and developed in glacial acetic acid/H2O/n-propyl alcohol (4:31:70 v/v/v). The radioactive products were visualized by autoradiography and quantified with the NIH image program. For peptide stimulation (16, 22), the phosphopeptide including Tyr-740 and Tyr-751 of PDGFR (GESDGGpYMDMSKDESVDpYVPMLTM, where pY is phosphotyrosine) was diluted in 10 mM Hepes and added (50 µM) simultaneously with the lipid substrate to the kinase reaction. In the case of V12-Ras activation of PI3K, the V12-Ras translation reaction was combined with the PI3K translation reaction, and PI3K complexes were immunopurified from this mixture as above.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Prolonged PI3K/PKB Activation in p65PI3K-expressing Cells-- To determine the consequences of p65PI3K expression on p110 activity, we followed the activation of the PI3K effector PKB in CTLL2 cells expressing p65PI3K or p85. PKB activation can be determined by analyzing the phosphorylation status of Ser-473 (39). Phospho-Ser-473-PKB content was examined prior to and following stimulation with IL-2; results were confirmed by in vitro kinase assays. The two cell lines expressed similar amounts of hemagglutinin (HA)-tagged regulatory subunits (Fig. 1A), which were within the range of that of the endogenous p85alpha molecule (not shown). Although IL-2 addition leads to transient PKB activation in control and HA-p85-expressing cells, HA-p65PI3K-expressing cells showed higher basal PKB phosphorylation, greater phosphorylation increases after IL-2 addition, and slower PKB down-regulation kinetics at later time points (Fig. 1B). Similar results were obtained in NIH3T3 cells expressing p85 or p65PI3K and activated with PDGF (Fig. 2, A and B), although in this system the higher activation peak at early time points was less evident. This difference between PDGFR and IL-2R may reflect the distinct PI3K recruitment mechanisms by the two receptors, with IL-2R showing constitutive PI3K association (24, 25) and PDGFR requiring both SH2 domains for efficient PI3K recruitment to the cell membrane (9). In conclusion, in addition to mediating higher basal PI3K activity in quiescent cells, p65PI3K expression enhances PI3K activation in response to GFR activation (more clearly in IL-2R) and results in prolonged PI3K/PKB activation kinetics.


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  		Fig. 1.   Prolonged PI3K/PKB activation in CTLL2 cells expressing p65PI3K. A, control, HA-p85- and HA-p65PI3K-expressing CTLL2 cells were lysed; 30 µg of total cell extracts were resolved by SDS-PAGE and analyzed in Western blot using anti-HA. B, control, HA-p85-, and HA-p65PI3K-expressing CTLL2 cells stimulated with 500 units/ml IL-2 for the times indicated were lysed; 30 µg of total cell extracts were resolved by SDS-PAGE and analyzed in Western blot using anti-phospho-Ser-473-PKB or anti-PKB antibody (indicated). The intensity of the phospho-PKB signal was quantitated using NIH image software. The figure illustrates a representative assay of three with similar results.


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  		Fig. 2.   Prolonged PI3K/PKB activation in p65PI3K-expressing CTLL2 cells. A, p85- and p65PI3K-expressing NIH3T3 cells were lysed; 30 µg of total cell extracts were resolved by SDS-PAGE and analyzed in Western blot using anti-p85. B, p85- and p65PI3K-expressing NIH3T3 cell lines incubated with 50 ng/ml PDGF for the periods indicated were lysed; 30 µg of total cell extracts were resolved by SDS-PAGE and analyzed in Western blot using anti-phospho-Ser-473-PKB or anti-PKB antibody (indicated). The intensity of the phospho-PKB signal was quantitated using NIH image software and calculated relative to the signal at time 0. The figure illustrates one representative assay of three with similar results.

Previous studies (31) indicated that p85 binds Tyr-phosphorylated SHP-1 phosphatase via the C-SH2 p85 domain and that SHP-1 binding mediates down-regulation of both Tyr phosphorylation and PI3K activation. Other authors (40) nonetheless showed a contribution by the N-SH2 domain in p85-SHP-1 association. To examine whether the absence of the C-SH2 domain in p65PI3K impaired association to SHP-1, we compared the ability of p65PI3K and p85 to bind to this phosphatase. COS-7 cells were transfected with cDNAs encoding SHP-1 and HA-tagged versions of both regulatory subunits. Transfections were performed in the absence or presence of cDNA encoding a constitutive active version of the Src kinase family member Lck (P505-Lck) (36). The distinct proteins were expressed to a similar extent in the p65PI3K and p85 samples (Fig. 3). Moreover, when p65PI3K and p85 regulatory subunits were immunopurified with anti-HA Ab, both subunits recruited similar amounts of SHP-1 (Fig. 3). This suggests that p85 binding to SHP-1 is not mediated exclusively by the C-SH2 domain. Minor changes in p65PI3K and p85 association to SHP-1 were observed in the absence or presence of P505-Lck, consistent with the moderate effect on SHP-1-p85 complex formation when Jurkat cells were examined prior to and following T cell receptor ligation, which triggers Lck activation (31). Considering that p65PI3K binds SHP-1 to a similar extent as p85, the slower PI3K/PKB down-regulation kinetics observed in p65PI3K-expressing cells is not related to defective p65PI3K association to SHP-1.


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  		Fig. 3.   p65PI3K is more stably associated to PDGFR than p85. COS-7 cells were cotransfected with a vector encoding SHP-1 and a vector encoding either HA-p65PI3K or HA-p85. cDNA for P505-Lck was included in some transfections. Lysates (30 µg) were resolved by SDS-PAGE and analyzed in Western blot using anti-Lck, anti-HA Ab, or anti-SHP1 Ab (indicated). Lysates (500 µg) were also immunoprecipitated with anti-HA Ab and resolved in SDS-PAGE, and gels were examined by Western blot using anti-SHP1 antibody. The figures illustrate a representative assay of three with similar results.

We also examined whether the lack of the C-SH2 domain in p65PI3K could affect the kinetics of binding and detachment of PI3K to the activated PDGFR (9, 30). The amount of p65PI3K bound to the receptor at any time point was lower than that of p85 (not shown), confirming that both SH2 domains contribute to mediating PI3K association to PDGFR (9). In addition, whereas the amount of PDGFR associated to p85 returned to basal levels at 3 h post-stimulation, a small fraction of p65PI3K remained PDGFR-associated (not shown). This small difference may not account for the significantly more stable activation of PI3K/PKB in cells expressing p65PI3K. Despite the open questions, the distinct PI3K/PKB down-regulation kinetics of p85- and p65PI3K-expressing cells demonstrates that the p85 C-terminal region contributes to control PI3K signal termination.

Activated Ras Enhances p65PI3K-p110 Activation in Vivo-- p85-p110 class IA PI3K is activated following stimulation of Tyr kinases and by active forms of Ras (1-4, 29). To evaluate which pathway acts more efficiently in p65PI3K- compared with p85-expressing cells following GFR stimulation, we deprived cells of GF and examined the consequences of expressing constitutive active forms of Ras or Lck (29, 36). V12-Ras and P505-Lck expression were confirmed by Western blot (not shown). V12-Ras and P505-Lck increased phospho-PKB content in the three CTLL2 lines analyzed (control and p85- and p65PI3K-expressing cells; Fig. 4A). V12-Ras nonetheless consistently induced more efficient PI3K/PKB activation in p65PI3K-expressing than in control cells and reduced PKB activation in p85-expressing compared with control cells (Fig. 4A). Comparable results were obtained when PKB activation was examined by in vitro kinase assay (not shown). p65PI3K and p85 NIH3T3 stable cell lines (32) were also examined. Cells were co-transfected with PKB and either V12-Ras or P505-Lck. P505-Lck mediated PKB activation in both p85- and p65PI3K-expressing cells (Fig. 4B). However, V12-Ras induced strong PKB activation in p65PI3K-expressing cells and very modest PKB stimulation in p85-expressing cells (Fig. 4B). These observations suggest that the p85alpha C-terminal region (residues 572-724), absent in p65PI3K, impairs Ras-induced p110 activation.


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  		Fig. 4.   Activated Ras enhances p65PI3K-p110 activation in vivo. A, control, p85- and p65PI3K-expressing CTLL2 lines were transfected with control cDNA or cDNAs encoding P505-Lck or V12-Ras. Cells were incubated 12 h in complete medium and then 16 h in medium without serum or IL-2. Cells were lysed, and normalized lysates (30 µg) were resolved by SDS-PAGE and examined in Western blot using anti-phospho-Ser-473-PKB or anti-PKB Ab (indicated). B, p85- and p65PI3K-expressing NIH3T3 lines were transfected with a vector encoding HA-PKB alone or in combination with cDNAs coding for P505-Lck or V12-Ras (indicated). Cells were incubated 12 h in complete medium and then for 16 h in serum-free medium. Cells were lysed and normalized lysates (300 µg) immunoprecipitated using anti-HA Ab. HA-PKB kinase activity was tested in vitro using histone 2B as substrate. Lysates (30 µg) were also resolved by SDS-PAGE and examined in Western blot using anti-HA Ab (indicated). The figure illustrates a representative experiment of three with similar results. Wt, wild type.

An N-terminal Ras Peptide Binds p65PI3K-p110 and p85-p110 with Similar Affinity-- To examine the differential effect of V12-Ras on p65PI3K-p110 and p85alpha -p110 complexes, we first analyzed the affinity of these complexes for Ras. Rodriguez-Viciana et al. (29) showed that the p85-p110 complex binds Ras-GTP and that this interaction can be displaced with an H-Ras peptide encompassing residues 17-42. If this peptide displaces H-Ras association to p85-p110, we inferred that this peptide could bind directly p85-p110. We prepared beads covalently linked to the peptide; extracts of p65PI3K- and p85alpha -expressing CTLL2 cells were prepared and incubated with the Ras peptide-specific column or a control column. p65PI3K and p85 were both retained in the Ras column, with slightly greater retention of p85 than of p65PI3K (Fig. 5A). We also examined the interaction of p65PI3K-p110 and of p85alpha -p110 with Ras in vivo. Exponentially growing COS-7 cells expressing HA-p65PI3K-p110 and HA-p85alpha -p110 were lysed in Brij 96-containing buffer. Cell extracts were immunoprecipitated using anti-Ras Ab, and the interaction of Ras with PI3K was examined by Western blot using anti-HA monoclonal Ab. As shown in Fig. 5B, HA-p65PI3K and HA-p85 associated with Ras to a similar extent. Formation of this complex was confirmed in COS-7 cells deprived of GF and expressing V12-Ras and either p65PI3K-p110 or p85alpha -p110, which yielded similar results (not shown). These observations confirm that p65PI3K-p110 does not show higher affinity for Ras binding than p85-p110.


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  		Fig. 5.   Ras binds p65PI3K-p110 and p85-p110 with similar affinity. A, extracts (500 µg) of p65PI3K- and p85alpha -expressing CTLL2 cells were incubated with the control column or a Ras-peptide column. After washing, retained material was recovered by boiling beads in SDS sample buffer, resolved by SDS-PAGE, and examined in Western blot using anti-p85 Ab. Total lysates (30 µg) were examined in parallel. B, COS-7 cells were cotransfected with a vector encoding wt-p110 and a vector encoding either HA-p65PI3K or HA-p85. Lysates obtained from exponentially growing cells (30 µg) were resolved by SDS-PAGE and analyzed in Western blot using anti-HA Ab or anti-Ras Ab (indicated). Lysates (250 µg) were also immunoprecipitated (IP) with anti-Ras Ab (indicated), resolved in SDS-PAGE, and gels examined by Western blot using anti-HA antibody. The figure illustrates a representative experiment of three with similar results.

The p85 C-SH2 Domain Regulates Ras-induced p110 Activation-- To test whether the p85alpha C-terminal region (residues 572-724) impairs Ras-induced p110 activation, we compared the action of V12-Ras on p65PI3K-p110 and p85-p110 complex activities using purified in vitro translated proteins. p85alpha -p110 and p65PI3K-p110 complexes were transcribed and translated with similar efficiency (Fig. 6A) and showed similar basal in vitro kinase activity (Fig. 6B), suggesting that p65PI3K does not activate p110 constitutively. We next evaluated whether incubation with a Tyr-phosphorylated peptide mimicking the activated PDGFR (22) would have a distinct effect on p65PI3K-p110 and p85alpha -p110 complexes. Phosphopeptide addition significantly increased p85-p110 complex activity and had a lower activating effect on the p65PI3K-p110 complex (Fig. 6B). These observations confirmed that a mechanism other than Tyr kinase activation may be responsible for enhanced p110 activation by p65PI3K.


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  		Fig. 6.   The p85 C-SH2 domain regulates Ras-induced p110 activation. A, a control empty vector or vectors encoding p85 or p65PI3K were combined with a cDNA encoding wild type p110, in vitro transcribed and translated, and then analyzed by SDS-PAGE. B, purified p85-p110 and p65PI3K-p110 complex activities were assayed in vitro alone or in the presence of a PDGFR phosphopeptide (indicated). PtdIns-3-P spot intensity was quantitated using NIH image software and calculated relative to the signal in the control vector lane (translation with empty vector). C, cDNAs encoding p85, p65PI3K, or p50alpha were combined with a cDNA encoding wild type p110, in vitro transcribed and translated, and then analyzed by SDS-PAGE. An empty vector and a cDNA encoding V12-Ras were processed similarly (indicated). D, control, p85-p110, p65PI3K-p110, or p50alpha -p110 complexes were incubated alone or in combination with V12-Ras, and their lipid kinase activities were assayed in vitro. PtdIns-3-P spot intensity was quantitated and calculated as in B. The figures illustrate a representative assay of three with similar results.

GTP-Ras has been shown to trigger PI3K activation in vitro (29). We next compared the ability of V12-Ras to activate p65PI3K-p110 and p85-p110 in vitro. Both V12-Ras and PI3K molecules were in vitro transcribed and translated (Fig. 6C). V12-Ras moderately enhanced p85-p110 kinase activity but had a significantly higher activating effect on p65PI3K-p110 (Fig. 6D). In different assays, p85-p110 induction by V12-Ras ranged from moderate to none, although p65PI3K-p110 was reproducibly highly responsive to V12-Ras addition. The results show that the p85 C terminus, absent in p65PI3K, protects p110 from V12-Ras-induced activation. To confirm the role of the p85 C-terminal domain in the protection of p110 from Ras action, we examined the consequences of adding V12-Ras on the activity of the p50alpha -p110 complex. p50alpha is a natural alternative splicing form of p85alpha that lacks the N-terminal SH3 and BcR homology domains but retains the p85alpha SH2 domains and the inter-SH2 domain (38). Defective V12-Ras-induced p50alpha -p110 activation (Fig. 6D) confirming that the C-terminal domain impairs Ras action on p110.

Tyr Phosphorylation Releases p110 from the p85 Inhibitory Effect, Allowing V12-Ras to Activate p110-- The observations reported here suggest that Ras-induced p110 activation is controlled by the C-terminal region of p85. As class IA PI3K activation is selectively induced by receptors that trigger Tyr kinase activation (1-4), it was therefore possible that Tyr kinase stimulation is a prerequisite for V12-Ras-induced p110 activation. To test this hypothesis, we examined whether PDGFR phosphopeptide addition rendered p85-p110 sensitive to V12-Ras-induced activation. Fig. 7A shows the in vitro translated V12-Ras and PI3K complexes. Consistent with the results described above (Fig. 6), V12-Ras alone activated p85-p110 moderately and p65PI3K-p110 more efficiently (Fig. 7B), whereas the PDGFR phosphopeptide alone activated p85-p110 more efficiently than p65PI3K-p110 (Fig. 7B). Nonetheless, the combination of V12-Ras and the Tyr-phosphorylated peptide activated p85-p110 synergistically, whereas activation of p65PI3K-p110 was similar to that observed with V12-Ras alone (Fig. 7B). In conclusion, Tyr-phosphorylated peptide binding to p85 releases the inhibition exerted by the C-terminal region, rendering p110 sensitive to V12-Ras action. We propose that Ras action depends on prior activation of Tyr kinases.


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  		Fig. 7.   Tyr phosphorylation releases p110 from p85 inhibition, allowing V12-Ras to activate p110. A, cDNAs encoding p85 or p65PI3K were combined with a cDNA encoding wild type p110, in vitro transcribed and translated, and then analyzed by SDS-PAGE. cDNA encoding V12-Ras was processed similarly (indicated). B, p85-p110 or p65PI3K-p110 complexes were incubated alone, with V12-Ras, the PDGFR phosphopeptide, or both (indicated); lipid kinase activity was then assayed in vitro. PtdIns-3-P spot intensity was quantitated and calculated as in Fig. 6. The figure illustrates a representative assay of three with similar results.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The observations presented here extend our knowledge of the functional role of the p85 regulatory subunit in the control of PI3K activity, showing that the p85 C-terminal region blocks Ras-induced p110 activation. The data illustrate that Tyr kinase stimulation releases p110 from p85 inhibition, triggering partial p110 activation and sensitizing p110 for further activity increases induced by active forms of Ras. Such a sequential mechanism explains why only receptors that stimulate Tyr kinases trigger class IA PI3K activation (1-4), as active Ras operates only after Tyr kinase induction. This study also clarifies the mechanism by which p65PI3K, a mutation found in human cancer (33-35), contributes to PI3K deregulation and induction of cell transformation. In contrast to p85-p110, p65PI3K-p110 complexes are susceptible to Ras action in the absence of Tyr kinase activation. In addition, p65PI3K-expressing cells show prolonged PI3K/PKB activation kinetics in vivo. The C-terminal region encompassing residues 572-724, which include the final part of the inter-SH2 domain and the C-SH2 domain, is thus an essential regulatory region of class IA PI3K that enables p85 to control PI3K activation as well as its down-regulation kinetics in vivo.

The existence of an inhibitory activity in the p85 C-terminal region was first postulated in view of the higher PI3K activity detected in p65PI3K-expressing cells (32). Here we characterized the kinetics of this activation in greater detail, and we explored the mechanism responsible for p65PI3K-induced p110 activation. We show that p65PI3K expression affects PI3K activity in the following three ways: increasing basal PI3K activity in quiescent cells, enhancing PI3K activation following GFR stimulation, and delaying p110 down-regulation. Both IL-2R stimulation of CTLL2 cells (Fig. 1) and PDGFR stimulation of NIH3T3 cells (Fig. 2) yielded similar PI3K activation profiles after p65PI3K expression, although the initial PI3K activation peak following GFR ligation was more pronounced in CTLL2 cells. This difference reflects the distinct PI3K recruitment mechanisms used by the two receptors, with IL-2R showing constitutive PI3K association (24, 25) and PDGFR requiring both SH2 domains for efficient PI3K recruitment to the cell membrane (9).

The basal 3-poly-PtdIns content of quiescent p65PI3K-expressing cells is probably related to the fact that a small proportion of p65PI3K is constitutively located at the membrane fraction (32). With regard to the slower PI3K/PKB down-regulation kinetics observed in p65PI3K-expressing cells, we first considered the possibility that p65PI3K could bind the SHP-1 phosphatase to a lesser extent than does p85. Cuevas et al. (31) reported that SHP-1 phosphatase binds p85 via the C-SH2 domain and contributes to the down-regulation of Tyr kinase and PI3K activities. Other authors report (40) that the N-SH2 domain also mediates binding to SHP-1. We found that p65PI3K and p85 bind SHP-1 to a similar extent, supporting the idea that the N-SH2 domain mediates this interaction. In conclusion, a defect in SHP-1 binding does not appear to be the cause of prolonged PI3K activation in p65PI3K-expressing cells. We also considered that 3-poly-PtdIns binds to the C-SH2 domain of p85 with greater affinity than analogues of Tyr-phosphorylated residues (30). It is therefore possible that once PI3K is activated, the 3-poly-PtdIns products bind to the C-SH2 domain of p85, displacing it from the associated Tyr-phosphorylated residues (9). As p65PI3K lacks the C-SH2 domain, this mutant could remain associated to Tyr-phosphorylated GFR for prolonged periods. To test this hypothesis, we analyzed the kinetics of p65PI3K and p85 dissociation from the activated PDGFR. While the amount of p85-associated PDGFR returned to basal levels at 3 h post-stimulation, a modest amount of p65PI3K remained PDGFR-associated. This small difference may contribute to prolong PI3K/PKB activation but seems insufficient to account for the significantly more stable activation of PI3K/PKB in cells expressing p65PI3K. In the case of IL-2R stimulation, it is also not obvious how the lack of an SH2 domain would affect PI3K activation kinetics, as p85 binding to the activating Src kinases has not been shown to involve the p85 SH2 domains. p85 activation by Src kinases nonetheless promotes formation of an intramolecular complex of the C- and the N-SH2 domains, required for PI3K activation (27). It is thus possible that 3-poly-PtdIns binding to the p85C-SH2 domain contributes to reducing the intramolecular association of the N- and C-SH2 domains, thereby down-regulating PI3K activation. Despite these open questions, the distinct PI3K/PKB down-regulation kinetics of p85- and p65PI3K-expressing cells demonstrates that the p85 C-terminal region contributes to control PI3K signal termination.

The other difference between p85- and p65PI3K-expressing cells is the increased activation intensity of PI3K/PKB following receptor stimulation in the latter cells. To identify the mechanism underlying this enhanced p110 activation, we considered the following two possible routes that normally induce class IA PI3K activation: stimulation of Tyr kinases and activation of Ras (1-4, 9, 29). Two groups of Tyr kinases activate PI3K, the receptors with intrinsic Tyr kinase activity such as PDGFR (9), and cytosolic Tyr kinases, such as Src family kinase members (27, 41), which are activated in response to cytokine receptor stimulation (42, 43). In the case of PDGFR, PI3K activation requires that the Tyr-phosphorylated receptor bind to the p85 SH2 domains (9). In the case of IL-2R, however, PI3K is constitutively bound (24, 25), and the Src kinases associate PI3K through a different mechanism involving its SH3 domain and the p85 Pro-rich regions (41). This interaction between Src kinases and p85 could trigger PI3K activation directly. It has nonetheless also been shown that phosphorylation of p85 Tyr-688 by Abl or Src kinases activates PI3K (27).

By using a constitutive active form of the Src family kinase Lck, we show that this pathway induces PI3K activation in p65PI3K- and p85-expressing cells, despite the lack of Tyr-688 in p65PI3K. This suggests that in addition to Tyr-688 phosphorylation, Src kinases use alternative mechanisms to mediate PI3K activation. Addition of a Tyr-phosphorylated peptide representing the activated PDGFR enhanced both p65PI3K-p110 and p85-p110 activities, although p85-p110 was activated more efficiently. Because neither in vitro phosphopeptide addition nor in vivo P505-Lck expression yielded significantly more active p65PI3K-p110 than p85-p110, we concluded that Tyr kinase activation is not responsible for enhanced p65PI3K-p110 activation in response to GFR stimulation. In contrast, the greater V12-Ras-induced PI3K activation in p65PI3K-expressing cells, as well as the reduced V12-Ras-induced p110 activation in p85-expressing cells (compared with control cells), suggests that p85 interferes with V12-Ras-induced p110 activation. We also examined V12-Ras action on purified in vitro translated p65PI3K-p110 and p85-p110 complexes. V12-Ras addition induced significantly greater activation in p65PI3K-p110 than in p85-p110 complexes. As class IA PI3K activation is selectively induced by receptors that mediate Tyr kinase activation (1-4), we proposed that Tyr kinase stimulation may be a prerequisite for p85-p110 response to active Ras forms. To test this hypothesis and mimic Tyr kinase pathway activation, we used a peptide representing the region of PDGFR associating with p85, and we show that incubation of p85-p110 with the PDGFR phosphopeptide led to partial p110 stimulation and allowed further p110 activation by V12-Ras. In conclusion, p85 protects p110 from active Ras-induced activation. This protection is affected via the C-terminal domain residues 572-724, which are absent in p65PI3K. Tyr kinase stimulation nonetheless releases p110 from p85 inhibition, yielding p85-p110 susceptible to Ras action.

p110 activation following binding of Tyr-phosphorylated peptides to p85 (or by Tyr phosphorylation of p85) may depend on a conformational change in p85, which could then be transmitted to p110, as elegantly discussed by Dhand et al. (15). Such changes have been reported in p85 in response to phosphopeptide binding (19-21). In addition, Layton et al. (44) show that these peptides induce oligomerization, a mechanism that may also be involved in enzyme activation. The structures of the isolated SH2, SH3, and BH domains have been resolved by crystallography (reviewed in Ref. 45), although only a sequence-based structural prediction has been reported for the inter-SH2 region (15). According to this prediction, the inter-SH2 region folds as an anti-parallel coiled-coil of two alpha -helices, helix 1 and helix 2 (15). Helix 1 (residues 478-492 of p85alpha ) would mediate primarily the interaction with p110, and helix 2 would stabilize helix 1 structure or orientation. Amino acid sequence comparison showed that the inter-SH2 region has a short basic motif similar to the gelsolin PI(4,5)P2-binding domain (46), which may be part of the lipid-binding pocket of the heterodimeric p85-p110 complex. The conformational change in p85 could unmask the PI(4,5)P2 binding pocket of the PI3K heterodimer. The p65PI3K deletion mutant includes helix 1 and 2 but lacks the terminal inter-SH2 region as well as the remaining p85 C terminus, including the C-SH2 domain (32). This difference is likely to affect the structure and behavior of the inter-SH2 region. According to our observations, Ras action necessarily affects p110 substrate recognition and/or phosphate transfer activity via structural changes in p85. It is therefore possible that p65PI3K-p110 has a structure permissive to Ras action, which is only acquired by p85-p110 complexes after Tyr kinase activation.

The results presented here show how the p65PI3K mutant affects p110 activation, a subject of interest considering that a number of human tumors were recently shown to express p65PI3K-like mutations (33-35). p65PI3K affects p110 activation in several ways. First, p65PI3K yields higher basal PI3K activity. The consequences for cell transformation of increased basal PI3K activity have been demonstrated by examining the phenotype of Pten+/- mice. Pten is a phosphatase that dephosphorylates protein substrates as well as 3-poly-PtdIns. A number of observations nonetheless suggest that the increase in 3-poly-PtdIns in cells with heterozygous Pten loss induces transformation (reviewed in Ref. 47). In support of this view, transgenic mice expressing p65PI3K in T cells show a lymphoproliferative disorder similar to that developed by Pten+/- mice (48, 49). p65PI3K also enhances receptor-induced PI3K activation, facilitating Ras action on p110. Ras activation in p65PI3K-expressing cells would thus yield PI3K enzyme-active. p65PI3K also prolongs PI3K activation kinetics. As enhanced PI3K activation facilitates cell cycle entry (50), this action probably contributes to the growth advantage of tumor cells expressing this mutation. However, because p65PI3K action on p110 still requires GFR-derived signals, it does not result in high constitutive PI3K/PKB activation. This property of p65PI3K also confers a cell division advantage, as constitutively high activation of PI3K/PKB interferes with activation of Forkhead transcription factors, required for mitosis progression (50).

In conclusion, we find that the C-terminal region of p85alpha (residues 572-724) regulates the sequential activation of PI3K by Tyr kinases and by Ras and controls PI3K signal termination. In addition, we present novel mechanisms by which p65PI3K enhances p110 activity, contributing to induction of cell transformation.

    ACKNOWLEDGEMENTS

We thank Drs. S. Levers and B. Vanhaesebroek for peptide column preparation protocols, Dr. J. Downward for the PDGFR phosphotyrosine peptide, and C. Mark for editorial assistance. The Department of Immunology and Oncology was founded and is supported by the Spanish Council for Scientific Research (CSIC) and by the Pharmacia Corporation.

    FOOTNOTES

* This work was supported by grants from the European Union, the Community of Madrid, and the Spanish Dirección General de Ciencia y Desarrollo Tecnológico.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Carretera de Colmenar Km 15, Cantoblanco, Madrid E-28049, Spain. Tel.: 34-91-585-4849; Fax: 34-91-372-0493; E-mail: acarrera@cnb.uam.es.

Published, JBC Papers in Press, August 23, 2002, DOI 10.1074/jbc.M205893200

    ABBREVIATIONS

The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; 3-poly-PtdIns, 3-poly-phosphoinositide; GFR, growth factor receptor; PKB, protein kinase B; BH, BcR homology domain; SH, Src homology; PDGFR, platelet-derived growth factor receptor; IL-2R, interleukin-2 receptor; HA, hemagglutinin; Ab, antibody; PDGF, platelet-derived growth factor; IL, interleukin; IL-2R, interleukin-2 receptor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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