Assembly of the p85α homodimer 1 Assembly and Molecular Architecture of the Phosphoinositide 3-Kinase p85α homodimer

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein coupled receptors, and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85α, a modular protein consisting of 5 domains, binds and inhibits the enzymatic activity of Class IA PI3Ks. Here, we describe the structural states of the p85α dimer, based on data from in vivo and in vitro solution characterization. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85α that is functionally equivalent to native p85α. Analytical ultracentrifugation (AUC) studies showed that p85α undergoes rapidly reversible monomer-dimer assembly that is highly exothermic in nature. In addition to the documented SH3-PR1 dimerization interaction, we identified a second intermolecular interaction mediated by cSH2 domains at the Cterminal end of the polypeptide. We have http://www.jbc.org/cgi/doi/10.1074/jbc.M115.689604 The latest version is at JBC Papers in Press. Published on October 16, 2015 as Manuscript M115.689604 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Assembly of the p85α homodimer 2 demonstrated in vivo concentration-dependent dimerization of p85α using fluorescence fluctuation spectroscopy (FFS). Finally, we have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle X-ray scattering (SAXS) and chemical cross-linking structural analysis of the discrete dimer. These experimental data have been used for the integrative structure determination of the p85α dimer. Our study provides new insight into the structure and assembly of the p85α homodimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to transiently associate with multiple binding proteins. p85α (PIK3R1) is one of 5 regulatory subunits that inhibit and stabilize the p110 catalytic subunits of Class IA PI3 kinases (PI3K)(1). The p85α protein consists of 5 domains (Fig. 1): an Nterminal Src-homology 3 (SH3) domain, a BCR homology Rac/Cdc42-binding domain flanked by two proline-rich motifs (PR1 and PR2), and two Src-homology 2 domains (nSH2 and cSH2) that flank the coiled-coil inter-SH2 domain (iSH2). The iSH2 domain binds to the N-terminal adaptorbinding domain (ABD) of p110α, p110β, and p110δ, yielding a stable low-activity heterodimer. Activation of p85/p110 heterodimers by receptor tyrosine kinases (RTKs) requires binding of the nSH2 and cSH2 domains to phosphorylated YXXM motifs in the RTKs themselves, or in adaptors like IRS-1, which relieves inhibitory contacts between the SH2 domains and p110 (2). In cells, activated p85/p110 heterodimers phosphorylate PI(4,5)P2 to generate PI(3,4,5)P3. The lipid phosphatase PTEN antagonizes PI3kinase signaling by dephosphorylating PI(3,4,5)P3 (3). Given their modular structure, it is not surprising that p85 subunits participate in intraand intermolecular interactions in addition to binding p110. p85α/p110 dimers are activated by binding of the p85α BCR domain to GTP-bound Rac (4) and Cdc42 (5) as well as the binding of proline-rich domains to SH3 domains from Srcfamily kinases (6). The binding of the influenza protein NS1 similarly activates p85β/p110 dimers (7). The iSH2 domains of both p85α and p85β bind the tumor suppressor BRD7, resulting in nuclear translocation of p85α and its sequestration from cytosolic p110 (8). Independently of p110, the N-terminus of p85α binds PTEN, protecting the latter from degradation and negatively regulating PIP3 production in cells (9-11). The proline-rich motifs that flank the BCR domain both contain consensus SH3-binding sequences (12). Dimerization of p85α as well as the SH3-PR1-BCR fragment of p85α (residues 1333; Fig. 1B) has been reported, and peptides derived from the PR1 motif disrupt p85α dimerization (13). These results show that intermolecular SH3-PR1 interactions in the native protein are involved in p85α dimerization. In vivo dimerization has been demonstrated by reciprocal immunoprecipitation of differentially epitopetagged p85α in several cell lines (11). A p85α mutation, identified in a human endometrial carcinoma, truncates p85α midway through the BCR domain at residue 160; expression of this mutant activates PI3K signaling by inhibiting homodimerization of endogenous p85α, thereby blocking its stabilization of PTEN. The pathological consequence of this mutation highlights the biological importance of the p85α dimer (11). Because p85α plays a central regulatory role in the PI3K signaling pathway, characterization of its structure and assembly dynamics is crucial to understanding its function in both normal physiology and disease. Our study combines in vivo and in vitro solution characterization of p85α dimerization with integrative multi-state modeling of its global architecture using complementary analytical approaches (14-20). We have characterized the reversible monomer-dimer equilibrium of p85α, showing that dimerization is mediated by multiple domain contacts. Further, we have defined solution conditions under which the protein is monomeric or predominantly dimeric, and used these conditions for small angle X-ray scattering (SAXS) and chemical cross-linking studies that have informed the structural modeling of the p85α dimer. Our study provides new insight into p85α dimerization, suggesting that p85α is a highly dynamic molecule whose conformational flexibility allows it to efficiently exchange among multiple binding partners. Assembly of the p85α homodimer 3 EXPERIMENTAL PROCEDURES Expression and purification of cysteine-free p85α—Wild-type human p85α was cloned into the pGEX-6P-1 bacterial expression vector (GE Healthcare) using the BamHI-EcoRI sites. Its six cysteines were mutated (C146S, C167S, C498S, C656S, C659V, and C670L) to generate cysteinefree p85α. Hereafter, we refer to ‘cysteine-free p85α’ as p85α and the wild type protein as ‘native p85α’. Similarly, truncation mutants will be referred to as p85α with superscripts denoting the residues included in the fragment. The construct coding for p85α was expressed in BL21CodonPlus competent cells (Agilent Technologies), which were induced overnight with 0.4 mM IPTG (isopropyl β-D-1thiogalactopyranoside) at 25 °C. The cells were harvested by centrifugation and the pellets resuspended on ice in lysis buffer (PBS containing 4 mM DTT, 2 mM EDTA, 2 mM PMSF, 2.5 U/ml Pierce universal nuclease for cell lysis (Thermo Scientific) and Roche cOmplete, Mini Protease Inhibitor Tablets (Roche Diagnostics). The cells were lysed by sonication in an ice bath using a Branson Sonicator with a microprobe tip at output level 5 for 30 s followed by 30 s on ice, for 5 cycles. The lysate was brought to 1% Triton-X, incubated for 20 min at 4 °C on a rotating wheel, and centrifuged at 15,000 rpm in a SS-34 rotor for 20 min at 4 °C. The supernatant was incubated with Pierce glutathione agarose (Thermo Scientific) for 2 4 h at 4 °C on a rotating wheel. The resin was washed 3 times by resuspension in 10 column volumes of 50 mM Tris, 150 mM NaCl, pH 8.0. p85α was cleaved from the GST with PreScission protease (GE Healthcare) overnight at 4 °C in cleavage buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM DTT at pH 8.0) on a rotating wheel. The resin was transferred to a chromatography column and supernatant containing the cleaved protein was collected, along with 2 washes of 1 column volume cleavage buffer each. The PreScission cleavage reaction leaves five residues (GPLGS) at the N-terminus preceding the p85α sequence. The resultant p85α was dialyzed overnight into Mono Q buffer (20 mM Tris, 20 mM NaCl, pH 8.0), and loaded onto a Mono Q 10/100 GL anion exchange column (GE Healthcare) and eluted with a 0 – 350 mM NaCl gradient over 40-column volumes. The peak fractions were analyzed by SDS-PAGE, pooled, concentrated and loaded onto a HiLoad 26/60 Superdex 200 prep grade gel filtration column (GE Healthcare) equilibrated in gel filtration buffer (50 mM Tris, 300 mM NaCl, pH 8.0). Fractions were analyzed by SDS-PAGE and those containing >95% pure p85α were pooled and concentrated for use or storage at -80 °C. Stored protein was thawed on ice and centrifuged using a TLA-120.2 rotor (Beckman Coulter) at 80,000 rpm for 15 min at 4 °C prior to use. Protein concentrations were measured using UV absorbance at 280 nm (Nanodrop 2000 UV-Vis Spectrophotometer, Thermo Scientific) and corresponding extinction coefficients calculated from protein sequences using Expasy ProtParam (http://web.expasy.org/protparam/). Protein mass was confirmed via mass spectrometry to be 83,938.6 Da (based on the sequence the calculated mass is 83,951.6 Da). Truncated fragments of p85α (Fig. 1) were expressed from the corresponding cDNAs that were synthesized by PCR and ligated into pGEX6P-1 using the BamHI-EcoRI sites. All constructs were verified by sequencing. The truncated fragments of p85α were expressed and purified using the same protocol described for the fulllength protein. For binding assays, native GSTp85α and GST-p85α were purified as described above except that p85α was not cleaved from the glutathione agarose. SDS-PAGE and Coomassie staining were used to quantitate bead-bound GST fusion proteins. Beads were either stored at 4 °C for up to 1 week or frozen in 50% glycerol at 20°C. In vitro binding assays—HEK293T cells were transfected with wild-type N-myc-p110α using Fugene HD for 48 hr. Cells were lysed on ice in lysis buffer [20 mM Tris HCI, (pH 8.1), 137 mM NaCI, 1 mM MgC12, 1 mM CaC12, 10%o (v/v) glycerol, Nonidet P-40; 150,M vanadate; 1 mM phenylmethylsulfonyl fluoride, Roche tablet, and phosphatase cocktails (Sigma)], followed by incubation on a rotating wheel for 20 min at 4 °C and centrifugation at 13,000 rpm for 10 min. The clarified supernatant was then incubated with rotation for 2 hr at 4 °C with glutathione Sepharose beads or beads complexed with GSTp85α or native GST-p85α. The beads were washed Assembly of the p85α homodimer 4 3 times with PBS containing 1% NP-40, once with PBS, and boiled in 2X Laemmli sample buffer for analysis by SDS-PAGE. Membranes were blotted with an N-myc antibody (Cell Signaling Technologies) and developed with ECL western blotting substrate (Pierce). For Rac binding, GST-Rac was bacterially purified and bound to glutathione Sepharose, followed by loading without or with GTPγS (21) and incubated with rotation for 2 hr at 4 °C with p85α. The glutathione Sepharose beads were washed 3 times with PBS containing 1% NP-40, once with PBS, and boiled in 2X Laemmli sample buffer for analysis by SDS-PAGE. Membranes were blotted with in-house anti-p85α-nSH2 antibody and developed with ECL western blotting substrate (Pierce). Lipid kinase assay—N-myc-p110α was immunopurified from transiently transfected HEK293T cells as above. Protein-G pellets were washed, incubated with bacterially purified GSTp85α or native GST-p85α, and assayed for lipid kinase activity as previously described (22). SH2-phosphopeptide binding—GST-p85αcSH2 and native GST-p85α-cSH2 constructs (residues 617-724) were expressed in BL-21 E. coli, processed as above, and partially purified by elution with 20 mM glutathione from glutathione Sepharose beads. A tyrosine phosphopeptide containing the photo-activatable amino acid Benzoyl Phenylalanine (Bpa) (Gly-Asp-Gly-pTyrBPA-Pro-Met-Ser-Pro-Lys-Ser) was N-terminally labeled with [I]Bolton-Hunter reagent and desalted by chromatography on Sephadex G-10. The labeled peptide (4.7 μM final, 3 x 10 CPM/assay) was incubated with 2 μg of GSTcSH2 domain in the absence or presence of 250 μM unlabeled peptide. The samples were irradiated on ice with a 200 nm UV lamp at a distance of 1 cm for 1 hr, boiled in Laemmli sample buffer and analyzed by SDS-PAGE and autoradiography. Analytical ultracentrifugation (AUC)—AUC studies were conducted with a Beckman Optima XL-I centrifuge using the AN-60Ti rotor and the absorption optics set to 280 nm. Protein samples were run in either the low or high salt SAXS buffers described below at temperatures ranging from 4 to 37 °C. An SH3-binding peptide (RPLPPRPGA) used to inhibit dimerization was synthesized by GenScript and kept at -20 °C as a concentrated stock solution that was thawed and diluted into the buffer appropriate for each experiment. Sednterp version 20120828 Beta (http://sednterp.unh.edu) was used to calculate the partial specific volume of the proteins from their sequence and the density and viscosity of the buffers. The sedimentation parameters were corrected to standard conditions (20,w) using these