Class IA PI3Ks have many roles in health and disease. The rules that govern intersubunit and receptor associations, however, remain unclear. We engineered mouse lines in which individual endogenous class IA PI3K subunits were C-terminally tagged with 17aa that could be biotinylated in vivo. Using these tools we quantified PI3K subunits in streptavidin or PDGFR pull-downs and cell lysates. This revealed that p85α and β bound equivalently to p110α or p110β but p85α bound preferentially to p110δ. p85s were found in molar-excess over p110s in a number of contexts including MEFs (p85β, 20%) and liver (p85α, 30%). In serum-starved MEFs, p110-free-p85s were preferentially, compared with heterodimeric p85s, bound to PDGFRs, consistent with in vitro assays that demonstrated they bound PDGFR-based tyrosine-phosphorylated peptides with higher affinity and co-operativity; suggesting they may act to tune a PI3K activation threshold. p110α-heterodimers were recruited 5-6× more efficiently than p110β-heterodimers to activated PDGFRs in MEFs or to PDGFR-based tyrosine-phosphorylated peptides in MEF-lysates. This suggests that PI3Kα has a higher affinity for relevant tyrosine-phosphorylated motifs than PI3Kβ. Nevertheless, PI3Kβ contributes substantially to acute PDGF-stimulation of PIP
The equine conceptus is surrounded by a fibrous capsule that persists until about day 20 of pregnancy, whereupon the capsule is lost, the conceptus attaches to the endometrium and placentation proceeds. Before attachment, the endometrium secretes in abundance a protein of the lipocalin family, uterocalin. The cessation of secretion coincides with the end of the period during which the conceptus is enclosed in its capsule, suggesting that uterocalin is essential for the support of the embryo before direct contact between maternal and foetal tissues is established. Using recombinant protein and fluorescence-based assays, we show that equine uterocalin binds the fluorescent fatty acids 11-(dansylamino)undecanoic acid, dansyl-d,l-α-amino-octanoic acid and cis-parinaric acid, and, by competition, oleic, palmitic, arachidonic, docosahexaenoic, γ-linolenic, cis-eicosapentaenoic and linoleic acids. Uterocalin also binds all-trans-retinol, the binding site for which is coincident or interactive with that for fatty acids. Molecular modelling and intrinsic fluorescence analysis of the wild-type protein and a Trp → Glu mutant protein indicated that uterocalin has an unusually solvent-exposed Trp side chain projecting from its large helix directly into solvent. This feature is unusual among lipocalins and might relate to binding to, and uptake by, the trophoblast. Uterocalin therefore has the localization and binding activities for the provisioning of the equine conceptus with lipids including those essential for morphogenesis and pattern formation. The possession of a fibrous capsule surrounding the conceptus might be an ancestral condition in mammals; homologues of uterocalin might be essential for early development in marsupials and in eutherians in which there is a prolonged preimplantation period.
We describe a novel approach to the relative quantification of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] and its application to measure, in neutrophils, the activation of phosphoinositide 3-kinase (PI3K). This protein-lipid overlay-based assay allowed us to confirm and extend the observations, first, that N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation of primed human neutrophils leads to a transient and biphasic increase in PtdIns(3,4,5)P3 levels and, second, that the ability of fMLP to stimulate PtdIns(3,4,5)P3 accumulation in neutrophils isolated from mice carrying a Ras-insensitive ('DASAA') knock-in of PI3Kγ (p110γDASAA/DASAA) is substantially dependent on the Ras binding domain of PI3Kγ. We describe a novel approach to the relative quantification of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] and its application to measure, in neutrophils, the activation of phosphoinositide 3-kinase (PI3K). This protein-lipid overlay-based assay allowed us to confirm and extend the observations, first, that N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation of primed human neutrophils leads to a transient and biphasic increase in PtdIns(3,4,5)P3 levels and, second, that the ability of fMLP to stimulate PtdIns(3,4,5)P3 accumulation in neutrophils isolated from mice carrying a Ras-insensitive ('DASAA') knock-in of PI3Kγ (p110γDASAA/DASAA) is substantially dependent on the Ras binding domain of PI3Kγ. The phosphoinositide 3-kinases (PI3Ks) are a family of ubiquitous multi-domain signaling proteins that phosphorylate the 3-hydroxyl of phosphoinositides. Mammalian PI3Ks are divided into three classes based on their structure and substrate specificity. Class I PI3Ks are acutely activated by a variety of cell surface receptors, and are responsible for synthesis of intracellular phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] (1Vanhaesebroeck B. Leevers S.J. Ahmadi K. Timms J. Katso R. Driscoll P.C. Woscholski R. Parker P.J. Waterfield M.D. Synthesis and function of 3-phosphorylated inositol lipids. Annu. Rev. Biochem. 2001; 70: 535-602Crossref PubMed Scopus (1376) Google Scholar). Thus, stimulation of many cells results in a PI3K-dependent accumulation of PtdIns(3,4,5)P3 in the inner leaflet of the plasma membrane. This usually transient production of PtdIns(3,4,5)P3 initiates membrane recruitment of protein effectors bearing a subset of domains, most commonly pleckstrin homology (PH) domains, that specifically bind PtdIns(3,4,5)P3. Typically, this results in a colocalization of enzymes and substrates that stimulates further downstream signaling cascades. PI3Ks are critical enzymes in numerous signaling pathways involved in the control of cell proliferation, trafficking, metabolism, cell motility, and immune cell responses. There are four class I PI3Ks in mammalian cells. Class I PI3Ks can be subdivided further into class Ia and class Ib, based on their structure and regulation. The single class Ib isoform, PI3Kγ, is highly expressed in neutrophils and comprises a regulatory subunit, either p101 (2Stephens L.R. Eguinoa A Erdjument-Bromage H. Lui M. Cooke F. Coadwell J. Smrcka A.S. Thelen M. Cadwallader K. Tempst P. The Gβγ sensitivity of a PI3K is dependent upon a tightly associated adaptor, p101. Cell. 1997; 89 (et al.): 105-114Google Scholar) or p84 (3Suire S. Coadwell J. Ferguson G.J. Davidson K. Hawkins P. Stephens L. p84, a new Gβγ-activated regulatory subunit of the type IB phosphoinositide 3-kinase p110γ. Curr. Biol. 2005; 15: 566-570Google Scholar), and the p110γ catalytic subunit. p110γ is activated through dual regulation by both Ras (4Pacold M.E. Suire S. Perisic O. Lara-Gonzalez S. Davis C.T. Walker E.H. Hawkins P.T. Stephens L. Eccleston J.F. Williams R.L. Crystal structure and functional analysis of Ras binding to its effector phosphoinositide 3-kinase gamma. Cell. 2000; 103: 931-943Google Scholar, 5Suire S. Condliffe A.M. Ferguson G.J. Ellson C.D. Guillou H. Davidson K. Welch H. Turner M. Chilvers E.R. Hawkins P.T. Gβγs and the Ras binding domain of p110γ are both important regulators of PI3Kγ signalling in neutrophils. Nat. Cell Biol. 2006; 8 (et al.): 1303-1309Google Scholar) and Gβγ. Gβγ-mediated stimulation of p110γ is dependent on the p101 subunit, whereas GTP-Ras activates p110γ directly through its interaction with the p110γ Ras binding domain (RBD) (5Suire S. Condliffe A.M. Ferguson G.J. Ellson C.D. Guillou H. Davidson K. Welch H. Turner M. Chilvers E.R. Hawkins P.T. Gβγs and the Ras binding domain of p110γ are both important regulators of PI3Kγ signalling in neutrophils. Nat. Cell Biol. 2006; 8 (et al.): 1303-1309Google Scholar). Structural analysis has shown that the introduction of five point mutations ('DASAA') into the RBD of p110γ renders it unable to bind to and be activated by GTP-Ras (4Pacold M.E. Suire S. Perisic O. Lara-Gonzalez S. Davis C.T. Walker E.H. Hawkins P.T. Stephens L. Eccleston J.F. Williams R.L. Crystal structure and functional analysis of Ras binding to its effector phosphoinositide 3-kinase gamma. Cell. 2000; 103: 931-943Google Scholar, 5Suire S. Condliffe A.M. Ferguson G.J. Ellson C.D. Guillou H. Davidson K. Welch H. Turner M. Chilvers E.R. Hawkins P.T. Gβγs and the Ras binding domain of p110γ are both important regulators of PI3Kγ signalling in neutrophils. Nat. Cell Biol. 2006; 8 (et al.): 1303-1309Google Scholar). In neutrophils, the activity of p110γ contributes to the control of many cellular responses, such as adhesion, chemotaxis, phagocytosis, and superoxide production (6Cantley L.C. The phosphoinositide 3-kinase pathway. Science. 2002; 296: 1655-1657Google Scholar). Given their established role in various pathologies (7Wymann M.P. Marone R. Phosphoinositide 3-kinase in disease: timing, location, and scaffolding. Curr. Opin. Cell Biol. 2005; 17: 141-149Crossref PubMed Scopus (179) Google Scholar), PI3Ks represent an attractive target for drug discovery. However, measuring PtdIns(3,4,5)P3 represents an important methodological challenge for progress in this field. Although PI3K signaling pathways have been extensively studied in cultured cells, the number of assays available for quantification of PtdIns(3,4,5)P3 in primary cells is limited (8Rusten T.E. Stenmark H. Analyzing phosphoinositides and their interacting proteins. Nat. Methods. 2006; 3: 251-258Google Scholar). Most of the existing methods are time-consuming and expensive and require specific equipment and expertise. Here we have investigated whether a specific phosphoinositide (PI) binding domain could enable the development of a novel assay to quantitate PtdIns(3,4,5)P3 in cell extracts as an alternative to the use of radioactive tracers or microscopy-based assays. Divecha and colleagues (9Divecha N. Roefs M. Los A. Halstead J. Bannister A. D'Santos C. Type I PIPkinases interact with and are regulated by the retinoblastoma susceptibility gene product-pRB. Curr. Biol. 2002; 12: 582-587Google Scholar) provided evidence that lipid overlay assays using the PH domain of phospholipase Cδ1 could be used to quantify PtdIns(4,5)P2 purified from cell extracts using neomycin beads. The PH domain of the general receptor for phosphoinositides-1 (GRP1) specifically binds PtdIns(3,4,5)P3 (10Venkateswarlu K. Gunn-Moore F. Oatey P.B. Tavaré J.M. Cullen P.J. Nerve growth factor- and epidermal growth factor-stimulated translocation of the ADP-ribosylation factor-exchange factor GRP1 to the plasma membrane of PC12 cells requires activation of phosphatidylinositol 3-kinase and the GRP1 pleckstrin homology domain. Biochem. J. 1998; 335: 139-146Google Scholar) and has been used as a probe to quantify PtdIns(3,4,5)P3, mainly using microscopy-based assays (11Gray A. Van der Kaay J. Downes C.P. The pleckstrin homology domains of protein kinase B and GRP1 (general receptor for phosphoinositides-1) are sensitive and selective probes for the cellular detection of phosphatidylinositol 3,4-bisphosphate and/or phosphatidylinositol 3,4,5-trisphosphate in vivo. Biochem. J. 1999; 344: 929-936Google Scholar, 12Gray A. Olsson H. Batty I.H. Priganica L. Downes C.P. Nonradioactive methods for the assay of phosphoinositide 3-kinases and phosphoinositide phosphatases and selective detection of signaling lipids in cell and tissue extracts. Anal. Biochem. 2003; 313: 234-245Google Scholar, 13Furutani M. Itoh T. Ijuin T. Tsujita K. Takenawa T. Thin layer chromatography-blotting, a novel method for the detection of phosphoinositides. J. Biochem. (Tokyo). 2006; 139: 163-170Google Scholar). Here we have developed a novel assay for PtdIns(3,4,5)P3 and have used it to measure transient changes in PtdIns(3,4,5)P3 abundance in human neutrophils and the impact on PtdIns(3,4,5)P3 accumulation in neutrophils from mice expressing the DASAA-mutated PI3Kγ. This novel assay is rapid and sensitive and could be used to design high-throughput assays for PtdIns(3,4,5)P3 and possibly other low-abundance inositol lipids using alternative PI-specific domains. Tumor necrosis factor α (TNFα) was from R & D; N-formyl-methionyl-leucyl-phenylalanine (fMLP) and HBSS were from Sigma. Neomycin beads were a generous gift of Dr. Robin Irvine (Cambridge University). The GFP-GRP1 PH domain fusion, initially cloned into EGFP C1 vector (11Gray A. Van der Kaay J. Downes C.P. The pleckstrin homology domains of protein kinase B and GRP1 (general receptor for phosphoinositides-1) are sensitive and selective probes for the cellular detection of phosphatidylinositol 3,4-bisphosphate and/or phosphatidylinositol 3,4,5-trisphosphate in vivo. Biochem. J. 1999; 344: 929-936Google Scholar), was subcloned into pFbac A. This construct allows expression of the GFP-GRP1 PH domain in the context of an N-terminal 6 × histidine tag. A mutant version of the GRP1 PH domain unable to interact with phosphoinositides was obtained by site-directed mutagenesis, changing K273 to A. The sequences of the wild-type and mutant constructs were verified by sequencing. Baculovirus DNA was obtained by transfecting DH10 BAC cells with the above constructs according to the Invitrogen protocols. The PH domain of dual adaptor for phosphotyrosine and 3-phosphoinositide 1 (DAPP1) fused to GFP (14Anderson K.E. Lipp P. Bootman M. Ridley S.H. Coadwell J. Ronnstrand L. Lennartsson J. Holmes A.B. Painter G.F. Thuring J. DAPP1 undergoes a PI 3-kinase-dependent cycle of plasma-membrane recruitment and endocytosis upon cell stimulation. Curr. Biol. 2000; 10 (et al.): 1403-1412Google Scholar) was cloned into a modified version of pQE30 and sequenced. The new construct allowed expression of a 6 × histidine-tagged version of the GFP-DAPP1 PH domain in BL-21DE3PlysS cells. Sf9 cells were transfected using Insectin (Invitrogen) liposomes with linearized baculo gold DNA (BD Biosciences) and the relevant baculovirus transfer vectors. The recombinant baculoviruses were plaque purified and amplified. After infection, cells were harvested into ice-cold 0.41% KCl, 2.66% sucrose, 20 mM MgCl2, and 8 mM NaH2PO4 (pH 6.2, 25°C) containing 1 mM di-isopropylfluorophosphate. The cells were then washed and frozen in liquid N2 and stored at −80°C. The GFP-fused PH domains were purified using a metal-ion chelation column (Talon, Clontech). Cell pellets were thawed and sonicated into 0.1 M NaCl and 50 mM sodium phosphate (pH 8.0, 4°C), 10 mM Tris-HCl (pH 8.0, 4°C), 1 mM MgCl2, and antiproteases (10 μg/ml each of pepstatin A, aprotinin, leupeptin, antipain, and bestatin and 0.1 mM PMSF). A 120,000 g cytosolic fraction was supplemented with Tween 20 and betaine (0.05%, w/v, and 1%, respectively) and loaded onto Talon resin, which was subsequently washed sequentially with 20 column volumes each of buffers A, B, C, and D. Buffer A contained 50 mM sodium phosphate (pH 8.0, 4°C), 10 mM Tris-HCl (pH 8.0, 4°C), 0.15 M NaCl, 1% betaine, and 0.5% Tween 20 (w/v). Buffer B contained buffer A plus Triton X-100 (1%, w/v). Buffer C contained buffer A but was at pH 7.1 and 4°C. Buffer D contained buffer A but was at pH 7.5 and contained 0.02% Tween 20 (w/v), ethylene glycol (0.05%, v/v), and 1 mM MgCl2. The Talon resin was then washed with 8 column volumes of buffer E, which contained buffer D supplemented with 10 mM imidazole (pH 7.5) and buffer F, which contained buffer D supplemented with 70 mM imidazole (pH 7.5, final concentration). Typically, 1 ml fractions were immediately collected and supplemented with 1 mM DTT and 1 mM EGTA (final concentrations). This yielded 3 mg of recombinant protein per liter of Sf9 culture. The DAPP1 PH domain fused to a 6 × histidine and GFP was expressed in BL-21DE3PlysS after induction with IPTG (0.1 mM) for 16 h at 25°C and purified with Talon resin as described above. Radiolabeled PtdIns(3,4,5)P3 was prepared as described previously (15Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R. Reese C.B. McCormick F. Tempst P. Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B. Science. 1998; 279 (et al.): 710-714Google Scholar). Briefly, phosphatidylserine (PtdS, 250 μM) and PtdIns(4,5)P2 (50 μM) were dried and resuspended by sonication in a buffer containing 0.1 M NaCl, 20 mM HEPES, and 1 mM EGTA. Conversion of PtdIns(4,5)P2 to radiolabeled PtdIns(3,4,5)P3 was achieved by mixing the PtdS-PtdIns(4,5)P2 liposomes with [γ32P]ATP and recombinant p110γ (5Suire S. Condliffe A.M. Ferguson G.J. Ellson C.D. Guillou H. Davidson K. Welch H. Turner M. Chilvers E.R. Hawkins P.T. Gβγs and the Ras binding domain of p110γ are both important regulators of PI3Kγ signalling in neutrophils. Nat. Cell Biol. 2006; 8 (et al.): 1303-1309Google Scholar) in a buffer containing 0.1 M NaCl, 1 mM EGTA, 1 mM DTT, 2 mM MgCl2, and 20 mM HEPES for 20 min at 30°C. The reaction was terminated by addition of chloroform-methanol (2:1), and the lipid fraction containing the radiolabeled PtdIns(3,4,5)P3 was subsequently extracted. Human neutrophils (purity ≥95% by cytospin) were isolated from the peripheral blood of healthy volunteers by centrifugation over plasma/Percoll gradients (16Haslett C. Guthrie L.A. Kopaniak M.M. Johnston Jr., R.B. Henson P.M. Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide. Am. J. Pathol. 1985; 119: 101-110Google Scholar). The blood was centrifuged at 316 g for 20 min to pellet erythrocytes and leukocytes. The supernatant was collected and centrifuged at 1,912 g for 30 min, with the resultant supernatant yielding platelet-poor plasma (PPP). Dextran was added to the pelleted erythrocytes and leukocytes [2.5 ml of 6% dextran (Amersham Biosciences)], and saline (Baxters Healthcare) was added so that the volume was equal to the original volume of blood. The erythrocytes were left to sediment at room temperature until a clean interface between the erythrocytes and the leukocyte-rich plasma could be seen. The leukocyte-rich plasma was centrifuged at 316 g for 6 min, and the pelleted leukocytes were resuspended in PPP and loaded onto a discontinuous 42%/51% percoll gradient (Amersham Biosciences) (percoll diluted 9:1 with saline to give a 90% solution, and further dilutions made in PPP), which was centrifuged at 262 g for 10 min. Neutrophils were harvested from the 42%/51% interface, diluted into the remaining PPP, and centrifuged at 316 g for 6 min. The cells were then centrifuged and washed twice in HBSS. Murine bone marrow-derived neutrophils were prepared essentially as described previously (17Condliffe A.M. Davidson K. Anderson K.E. Ellson C.D. Crabbe T. Okkenhaug K. Vanhaesebroeck B. Turner M. Webb L. Wymann M.P. Sequential activation of class IB and class IA PI3K is important for the primed respiratory burst of human but not murine neutrophils. Blood. 2005; 106 (et al.): 1432-1440Google Scholar). Briefly, murine bone marrow was dispersed in HBSS (without Ca2+ and Mg2+) with 0.25% fatty acid-free BSA (HBSS/BSA) and centrifuged (1,256 g, 30 min, room temperature) over discontinuous gradients comprised of 81, 62, and 55% Percoll in HBSS. Mature neutrophils were obtained from the 55%/62% interface (purity 75–85% by cytospin), and contaminating red blood cells were removed by ammonium chloride lysis; cells were washed twice in HBSS/BSA. Neutrophils (1.106 for human, 2.106 for mouse) were washed twice in HBSS and incubated with TNFα (200 U/ml−1) for 30 min at 37°C. Aliquots (200 μl) were warmed to 37°C in the presence or absence of 100 nM wortmannin (⩽0.01% final concentration of DMSO) for 5 min and then challenged with 10 μl fMLP (100 nM and 10 μM final concentrations for human and mouse cells, respectively) for indicated times. Incubations were terminated by addition of solvents. The lipid extraction from neutrophils was performed as initially described in (18Shacht J. Purification of polyphosphoinositides by chromatography on immobilized neomycin. J. Lipid Res. 1978; 19: 1063-1067Google Scholar). After stimulation, 750 μl chloroform-methanol-HCl (1 mM; 484:242:23.55) was added to 170 μl of neutrophil sample, followed by vigorous vortexing. The single phase was split by the addition of chloroform (725 μl) followed by 170 μl of HCl (2 mM containing 1 mM Tetrabutylammonium sulphate). After mixing, the samples were centrifuged (10,000 g, 5 min), and the lower organic phase was transferred to a new tube and dried under vacuum. Preliminary experiments indicated that PtdIns(3,4,5)P3 needed to be enriched from a total cellular lipid extract for efficient detection by subsequent protein-lipid overlay. Neomycin beads previously had been used to purify PIs from a complex lipid mixture (18Shacht J. Purification of polyphosphoinositides by chromatography on immobilized neomycin. J. Lipid Res. 1978; 19: 1063-1067Google Scholar). We used them to purify PIs from human and mice neutrophil lipid extracts. Dried lipid extracts corresponding to 1.106 cells for human neutrophils and 2.106 cells for mouse neutrophils were dissolved in 500 μl of a mixture containing 100 ml chloroform, 200 ml methanol, 3.2 ml ammonium formate (2 mM), and 16.8 ml H2O. Packed neomycin beads (10 μl) were then added, and samples were mixed end-over-end on a rotating wheel for 20 min. After the beads were washed three times with the above mixture, PIs were eluted by the addition of 950 μl of methanol-chloroform-HCl (2.4 mM; 500:250:200) and transferred to a new tube. Chloroform (750 μl) and water (125 μl) were added, and samples were centrifuged (10,000 g, 5 min) after vigorous vortexing. The lower phase containing purified PIs was collected and dried under vacuum. The purified PIs were dissolved in 3 μl of chloroform-methanol-HCl (12 mM; 200:100:1) prior to spotting onto nitrocellulose membranes (19Dowler S. Kular G. Alessi D.R. Protein lipid overlay assay. Sci. STKE. 2002; 129: PL6Google Scholar). After blocking the nonspecific binding sites, the membranes were incubated overnight at 4°C with a GFP-PH domain (from GRP1; 0.5 μg/ml−1 in TBS containing 2% BSA and 0.05% Tween 20). The blots were washed in the above buffer without BSA and incubated with a rabbit anti-GFP polyclonal antibody (a gift from Dr. L. Roderick, The Babraham Institute). After washing, the blots were incubated with anti-rabbit antibody conjugated to HRP, and visualization was carried out using ECL (Amersham). Quantification was achieved by direct visualization of the chemiluminescence using a charge-coupled device (CCD) camera (Image Reader LAS-1000; Fujifilm). Densitometry was performed with AIDA software with local background subtraction. We tested the ability of neomycin beads to reproducibly purify PtdIns(3,4,5)P3 from a complex lipid extract prepared from human neutrophils. In vitro radiolabeled PtdIns(3,4,5)P3 was added to a total lipid extract from 1.106 human neutrophils. [32P]PtdIns(3,4,5)P3 was reproducibly recovered to 70% of the total input (Fig. 1 ), indicating that these beads can be used to enrich PtdIns(3,4,5)P3 from a cellular extract. We chose to use GRP1-PH domain as a probe to detect PtdIns(3,4,5)P3 on blots because it had been reported to be highly specific for PtdIns(3,4,5)P3 (10Venkateswarlu K. Gunn-Moore F. Oatey P.B. Tavaré J.M. Cullen P.J. Nerve growth factor- and epidermal growth factor-stimulated translocation of the ADP-ribosylation factor-exchange factor GRP1 to the plasma membrane of PC12 cells requires activation of phosphatidylinositol 3-kinase and the GRP1 pleckstrin homology domain. Biochem. J. 1998; 335: 139-146Google Scholar). The recombinant protein was assembled in the context of both a 6 × histidine tag and a GFP tag. The 6 × histidine tag allowed purification of the recombinant protein after expression in Sf9 cells. The purified protein was subsequently used in a protein-lipid overlay-based procedure (20Dowler S. Currie R.A. Campbell D.G. Deak M. Kular G. Downes C.P. Alessi D.R. Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities. Biochem. J. 2000; 351: 19-31Google Scholar) to detect PtdIns(3,4,5)P3 immobilized on a nitrocellulose filter. This probe was, in turn, decorated with anti-GFP antibody followed by an anti-IgG coupled to HRP. The relative amount of PtdIns(3,4,5)P3 was then estimated by direct visualization of the light emitted in a chemiluminescence assay using a CCD camera. Consistent with data published by others (20Dowler S. Currie R.A. Campbell D.G. Deak M. Kular G. Downes C.P. Alessi D.R. Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities. Biochem. J. 2000; 351: 19-31Google Scholar), the GRP1 reporter showed high specificity for PtdIns(3,4,5)P3 over PtdIns(3,4)P2 and PtdIns(4,5)P2 when used in our protein-lipid overlay assay compared with a GFP-DAPP1 PH domain construct used under the same conditions (Fig. 2 ). As expected, mutation of lysine K273 in the GRP1 PH domain, previously shown to be critical for PtdIns(3,4,5)P3 binding (21Ferguson K.M. Kavran J.M. Sankaran V.G. Fournier E. Isakoff S.J. Skolnik E.Y. Lemmon M.A. Structural basis for discrimination of 3-phosphoinositides by pleckstrin homology domains. Mol. Cell. 2000; 6: 373-384Google Scholar), attenuated the interaction of the probe with PtdIns(3,4,5)P3 in a protein-lipid overlay assay (Fig. 3A ), thus providing further evidence for the specificity of the GRP1 probe.Fig. 3.Specificity and sensitivity of the GRP1 PH domain probe and its use for detection of PtdIns(3,4,5)P3 in a PtdIns(3,4,5)P3/PtdIns(4,5)P2 mixture. A: 0 to 2 pmol of PtdIns(3,4,5)P3 were mixed with a constant amount of PtdIns(4,5)P2 (100 pmol), dried under vacuum, resuspended in 3 μl of chloroform-methanol-HCl (12 mM; 200:100:1), spotted onto nitrocellulose membranes, and analyzed by a protein-lipid overlay procedure with GFP-(K273A)-GRP1(mutant) or GFP-GRP1 (WT) PH domain as the primary probe (both probes used at the same concentration of 0.5 μg/ml−1 in TBS containing 2% BSA and 0.05% Tween 20). B: 0, 1, 5, 10, and 20 pmol of PtdIns(3,4,5)P3 were mixed with 100 pmol of PtdIns(4,5)P2, dried under vacuum, resuspended in 3 μl of chloroform-methanol-HCl (12 mM; 200:100:1), spotted onto nitrocellulose membranes, analyzed by a protein-lipid overlay procedure using the GFP-GRP1 PH domain as the primary probe, and subjected to analysis by densitometry. Data are means ± SD (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Use of neomycin beads, as described by Shacht (18Shacht J. Purification of polyphosphoinositides by chromatography on immobilized neomycin. J. Lipid Res. 1978; 19: 1063-1067Google Scholar), allows purification of all polyphosphoinositides from a complex lipid mixture. Because PIs purified using neomycin are likely to be composed mainly of PtdIns(4,5)P2, we tested the ability of our assay to quantitate PtdIns(3,4,5)P3 in the presence of an excess of PtdIns(4,5)P2. In human neutrophils, we have previously shown that the level of PtdIns(4,5)P2 is 10 (stimulated cells) to 100 (unstimulated cells) times higher than PtdIns(3,4,5)P3 (22Stephens L.R. Hughes K.T. Irvine R.F. Pathway of phosphatidylinositol(3,4,5)-trisphosphate synthesis in activated neutrophils. Nature. 1991; 351: 33-39Google Scholar). We show that our assay is linear, with quantities of PtdIns(3,4,5)P3 up to 20 pmol when mixed with 100 pmol of PtdIns(4,5)P2 (Fig. 3A). Furthermore, we were able to detect as little as 0.2 pmol of PtdIns(3,4,5)P3 mixed in 100 pmol of PtdIns(4,5)P2 (Fig. 3B). PI3Ks contribute to the control of numerous neutrophil functions. Using 32P-PI radiolabeling of neutrophils, we had shown previously that human neutrophils transiently synthesize PtdIns(3,4,5)P3 when challenged with agonists such as bacterially derived fMLP (17Condliffe A.M. Davidson K. Anderson K.E. Ellson C.D. Crabbe T. Okkenhaug K. Vanhaesebroeck B. Turner M. Webb L. Wymann M.P. Sequential activation of class IB and class IA PI3K is important for the primed respiratory burst of human but not murine neutrophils. Blood. 2005; 106 (et al.): 1432-1440Google Scholar). Consistent with this previous report, we showed that the GRP1 probe allowed detection of PtdIns(3,4,5)P3 accumulation in human neutrophils stimulated with fMLP (Fig. 4 ). Increased PtdIns(3,4,5)P3 synthesis was detected using the wild-type probe but not the point-mutated probe (Fig. 4). Furthermore, the increase in PtdIns(3,4,5)P3 detected by the GRP 1 probe was abolished when cells were preincubated with wortmannin, a potent inhibitor of PI3K, further supporting the specificity of our PtdIns(3,4,5)P3 assay. We also measured the increase in PtdIns(3,4,5)P3 in fMLP-stimulated TNFα-primed human neutrophils over time. We observed a transient biphasic increase in PtdIns(3,4,5)P3 level peaking at 10 s and 60 s after addition of fMLP (Fig. 5 ). These results were consistent with those obtained previously using metabolic labeling of cells (17Condliffe A.M. Davidson K. Anderson K.E. Ellson C.D. Crabbe T. Okkenhaug K. Vanhaesebroeck B. Turner M. Webb L. Wymann M.P. Sequential activation of class IB and class IA PI3K is important for the primed respiratory burst of human but not murine neutrophils. Blood. 2005; 106 (et al.): 1432-1440Google Scholar), presented as a dotted line for reference in Fig. 5. As shown in Fig. 2, the GRP1 PH domain binds to PtdIns(3,4,5)P3 and, to a much lesser extent, to PtdIns(3,4)P2. The similarity between the kinetics of PtdIns(3,4,5)P3 accumulation measured by two different techniques (Fig. 5) further suggested that the protein-lipid overlay assay faithfully reported PtdIns(3,4,5)P3 levels from the complex PI mixture purified from the cells with neomycin beads because of their clear difference compared with the slower monophasic accumulation of PtdIns(3,4)P2 reported in the radiolabeling studies (17Condliffe A.M. Davidson K. Anderson K.E. Ellson C.D. Crabbe T. Okkenhaug K. Vanhaesebroeck B. Turner M. Webb L. Wymann M.P. Sequential activation of class IB and class IA PI3K is important for the primed respiratory burst of human but not murine neutrophils. Blood. 2005; 106 (et al.): 1432-1440Google Scholar). To extend our findings, we used our assay to measure PtdIns(3,4,5)P3 accumulation in bone marrow-derived neutrophils from wild-type mice and mice carrying a homozygous knock-in of PI3Kγ mutated in its Ras binding site ('DASAA') (5Suire S. Condliffe A.M. Ferguson G.J. Ellson C.D. Guillou H. Davidson K. Welch H. Turner M. Chilvers E.R. Hawkins P.T. Gβγs and the Ras binding domain of p110γ are both important regulators of PI3Kγ signalling in neutrophils. Nat. Cell Biol. 2006; 8 (et al.): 1303-1309Google Scholar) after stimulation with fMLP. As expected, fMLP increased PtdIns(3,4,5)P3 levels by over 4-fold in wild-type neutrophils. In neutrophils derived from mutant mice, PtdIns(3,4,5)P3 accumulation was attenuated. Our present study shows that fMLP-stimulated accumulation of the mass of PtdIns(3,4,5)P3 [as opposed to the 32P content described in (5Suire S. Condliffe A.M. Ferguson G.J. Ellson C.D. Guillou H. Davidson K. Welch H. Turner M. Chilvers E.R. Hawkins P.T. Gβγs and the Ras binding domain of p110γ are both important regulators of PI3Kγ signalling in neutrophils. Nat. Cell Biol. 2006; 8 (et al.): 1303-1309Google Scholar)] is markedly reduced in neutrophils prepared from p110γDASAA/DASAA mice as compared with their wild-type controls (Fig. 6 ). Both Gβγ and Ras contribute to activation of PI3Kγ and PtdIns(3,4,5)P3 accumulation in mouse neutrophils (5Suire S. Condliffe A.M. Ferguson G.J. Ellson C.D. Guillou H. Davidson K. Welch H. Turner M. Chilvers E.R. Hawkins P.T. Gβγs and the Ras binding domain of p110γ are both important regulators of PI3Kγ signalling in neutrophils. Nat. Cell Biol. 2006; 8 (et al.): 1303-1309Google Scholar). Consistent with the ability of Gβγ to activate PI3Kγ independently of Ras, we observed that in neutrophils prepared from p110γDASAA/DASAA mice, the level of PtdIns(3,4,5)P3 accumulation was reduced but not completely abolished (Fig. 6). The data presented in this report validate a novel assay for PtdIns(3,4,5)P3 in cell extracts. The assay is simple, rapid, highly sensitive, and specific for PtdIns(3,4,5)P3, and could possibly be developed as a higher-throughput assay if, for instance, the neomycin-based purification step could be adapted for use on immobilized surfaces.
Abstract Circulating neutrophils are, by necessity, quiescent and relatively unresponsive to acute stimuli. In regions of inflammation, mediators can prime neutrophils to react to acute stimuli with stronger proinflammatory, pathogen-killing responses. In neutrophils G protein-coupled receptor (GPCR)-driven proinflammatory responses, such as reactive oxygen species (ROS) formation and accumulation of the key intracellular messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3), are highly dependent on PI3K-γ, a Ras-GTP, and Gβγ coincidence detector. In unprimed cells, the major GPCR-triggered activator of Ras is the Ras guanine nucleotide exchange factor (GEF), Ras guanine nucleotide releasing protein 4 (RasGRP4). Although priming is known to increase GPCR–PIP3 signaling, the mechanisms underlying this augmentation remain unclear. We used genetically modified mice to address the role of the 2 RasGEFs, RasGRP4 and son of sevenless (SOS)1/2, in neutrophil priming. We found that following GM-CSF/TNFα priming, RasGRP4 had only a minor role in the enhanced responses. In contrast, SOS1/2 acquired a substantial role in ROS formation, PIP3 accumulation, and ERK activation in primed cells. These results suggest that SOS1/2 signaling plays a key role in determining the responsiveness of neutrophils in regions of inflammation.
Class I phosphoinositide 3-kinases (PI3Ks) are important regulators of neutrophil migration in response to a range of chemoattractants. Their primary lipid products PtdIns(3,4,5)P3 and PtdIns(3,4)P2 preferentially accumulate near to the leading edge of migrating cells and are thought to act as an important cue organizing molecular and morphological polarization. We have investigated the distribution and accumulation of these lipids independently in mouse neutrophils using eGFP-PH reportersand electron microscopy (EM). We found that authentic mouse neutrophils rapidly polarized their Class I PI3K signalling, as read-out by eGFP-PH reporters, both at the up-gradient leading edge in response to local stimulation with fMLP as well as spontaneously and randomly in response to uniform stimulation. EM studies revealed these events occurred at the plasma membrane, were dominated by accumulation of PtdIns(3,4,5)P3, but not PtdIns(3,4)P2, and were dependent on PI3Kγ and its upstream activation by both Ras and Gβγs.
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