Document Detail

Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells.
Jump to Full Text
MedLine Citation:
PMID:  24597785     Owner:  NLM     Status:  Publisher    
Oncogenic mutations in PIK3CA lead to an increase in instrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3Kα to its plasma membrane localised substrate is also required for increased levels of downstream PIP3/Akt signalling. We have studied the subcellular localisation of wild type and the two most common oncogenic mutants of PI3Kα in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3Kα is pre-formed as a 1:1 p110α:p85α complex in vitro then introduced into live cells by microinjection. Oncogenic E545K and H1047R mutants did not constitutively interact with membrane lipid in vitro or in cells maintained in 10% FCS. Following stimulation of receptor tyrosine kinases, microinjected PI3Kα was recruited to the plasma membrane, but oncogenic forms of PI3Kα were not recruited to the plasma membrane to a greater extent and did not reside at the plasma membrane longer than wild type PI3Kα. Instead, the E545K mutant specifically bound activated Cdc42 in vitro and microinjection of E545K was associated with the formation of cellular protrusions, providing some preliminary evidence that changes in protein-protein interactions may play a role in the oncogenicity of the E545K mutant in addition to the well-known changes in lipid kinase activity.
Meredith J Layton; Natalie Rynkiewicz; Ivan Ivetac; Kristy A Horan; Christina A Mitchell; Wayne A Phillips
Related Documents :
24703895 - Involvement of na/k-atpase in hydrogen peroxide-induced activation of src/erk pathway i...
17088245 - G protein-coupled receptor (gpcr) kinase 2 regulates agonist-independent gq/11 signalin...
19473245 - Sensing the environment: response of candida albicans to the x factor.
17229475 - Map kinase pathways: the first twenty years.
18930955 - Dissociation of tau toxicity and phosphorylation: role of gsk-3beta, mark and cdk5 in a...
19001025 - Novel cardiac apoptotic pathway: the dephosphorylation of apoptosis repressor with casp...
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2014-3-5
Journal Detail:
Title:  Bioscience reports     Volume:  -     ISSN:  1573-4935     ISO Abbreviation:  Biosci. Rep.     Publication Date:  2014 Mar 
Date Detail:
Created Date:  2014-3-6     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8102797     Medline TA:  Biosci Rep     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-ta): Biosci Rep
Journal ID (iso-abbrev): Biosci. Rep
Journal ID (pmc): bsr
Journal ID (publisher-id): BSR
ISSN: 0144-8463
ISSN: 1573-4935
Publisher: Portland Press Ltd.
Article Information
Download PDF
© 2014 The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Licence (CC-BY)( which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.
Received Day: 10 Month: 12 Year: 2013
Revision Received Day: 18 Month: 2 Year: 2014
Accepted Day: 4 Month: 3 Year: 2014
epreprint publication date: Day: 5 Month: 3 Year: 2014
Electronic publication date: Day: 14 Month: 4 Year: 2014
collection publication date: Year: 2014
Volume: 34 Issue: 2
E-location ID: e00104
PubMed Id: 24597785
ID: 3985441
Publisher Id: e00104
DOI: 10.1042/BSR20130133

Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells Title for RRH:Microinjection of mutant PI3K into live cells Alternate Title:M. J. Layton and others
Meredith J. Layton*
Natalie K. Rynkiewicz*
Ivan Ivetac
Kristy A. Horan*
Christina A. Mitchell*
Wayne A. Phillips*§1
*Department of Biochemistry and Molecular Biology, Monash University, VIC 3800, Australia
†Surgical Oncology Research Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC 3002, Australia
‡Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC 3010, Australia
§Department of Surgery (St. Vincent's Hospital), University of Melbourne, Parkville, VIC 3010, Australia
Correspondence: 1To whom correspondence should be addressed (email


The PIK3CA gene encodes the p110α subunit of the Class 1A PI3K (phosphoinositide 3-kinase). The prototypic Class 1A PI3K exists as a heterodimer of a catalytic p110α subunit and a regulatory p85α subunit (p110α/p85α or PI3Kα) [1,2] and phosphorylates the phosphoinositide lipid, PIP2 (phosphoinositide-4,5-disphosphate), at the 3′ position of the inositide ring to form PIP3 (phosphoinositide-3,4,5-trisphosphate) [3]. Somatic, mono-allelic, single base mutations in PIK3CA that result in single amino acid substitutions are found frequently in breast and colon cancers [47] and have been shown to be oncogenic [811].

The p110 and p85 subunits of PI3Kα contain several functional domains. p110α contains a p85α-binding domain, a Ras-binding domain, a C2 domain, a helical domain and a kinase domain. The p85α subunit contains an SH3 (Src homology 3) domain, a GAP (GTPase-activating protein)-like domain, an nSH2 (N-terminal SH2) domain, an iSH2 (inter-SH2) domain that binds p110α and a cSH2 (C-terminal SH2) domain. The most common oncogenic PIK3CA mutations are E545K in the p110α helical domain and H1047R in the p110α kinase domain [8,12]. These mutated forms of PI3Kα (p110αE545K/p85α and p110αH1047R/p85α) are associated with increased PIP3 levels [9,10,13,14] and up-regulation of Akt [also called PKB (protein kinase B)] signalling [9,15]. PI3K/PIP3 signalling regulates a wide range of fundamental cellular processes including cell proliferation, survival, glucose metabolism and cell migration [13].

PI3Kα is not an integral membrane protein and so must be recruited to the PM (plasma membrane) to gain access to its PM-localized substrate, PIP2. Binding to a number of PM-associated proteins, such as activated RTKs (receptor tyrosine kinases), activated Ras, SH3 domain-containing proteins and small GTPases, has been reported to activate PI3Kα [1618]. However, the extent to which these interactions activate the intrinsic lipid kinase activity or activate PI3Kα by translocating it to the PM is not clear [19,20]. Some oncogenic mutations are thought to primarily up-regulate enzymatic activity. For example, p110α is both inhibited and structurally stabilized by tight binding to the p85α subunit [21] and it has been proposed that the intrinsic kinase activity of PI3Kα can be activated by disruption of an inhibitory contact between the p85α nSH2 domain and the p110α catalytic domain, which can occur due to the binding of the nSH2 and cSH2 domains to specific pY (phosphotyrosine)-containing motifs (pYXXM) present in RTKs [2224] or due to the E545K mutation [18,25]. Other oncogenic mutations are proposed to primarily mediate an interaction with the PM [25,26]. For example, from the X-ray crystal structure of p110αH1047R in complex with the iSH2 and nSH2 domains of p85α, it has been proposed that the p110α C2 domain, along with a region of the iSH2 domain, forms a positively charged contact surface for negatively charged membrane lipids [25,26] and that the H1047R mutation alters the conformation of 13 residues near the C-terminus of p110α to form a loop that cooperates with the C2 and iSH2 domains to mediate a constitutive interaction with the PM and thus increases lipid kinase activity by allowing easier access to PIP2 [25].

Although p110αE545K/p85α and p110αH1047R/p85α have been reported to bind lipids better than p110αwt/p85α in vitro [27], the subcellular localization of the wild-type and mutant PI3Kα, and their distribution between the cytosol and PM, has not been studied. Here, we have used a novel approach of microinjection of fluorescently labelled, highly purified, recombinant p110α/p85α complexes to quantify the degree of PM localization of wild-type and oncogenic mutant PI3Kα in cells maintained in growth media, and starved or stimulated cells. We found no difference in the interaction of the wild-type versus mutant PI3Kα with PM lipids in vitro or in its subcellular distribution in intact cells. Instead, we observed increased numbers of cell protrusions in cells microinjected with p110αE545K/p85α and a higher affinity binding of p110αE545K/p85α to activated Cdc42, providing some preliminary evidence that point mutations in PIK3CA may affect protein–protein interactions in addition to its enzymatic activity.


MDCK3 (Madin–Darby canine kidney 3) epithelial cells [28], a gift from Professor Anne Ridley, King's College, London, U.K., were grown in DMEM (Dulbecco's modified Eagle's medium) containing 10% (v/v) FBS. Caco2(C2BBe1) colon epithelial cells were obtained from A.T.C.C. (#CRL-2102) and were grown in RPMI supplemented with 10% FBS. iMMEC (immortalized mouse mammary epithelial cells) were prepared as previously described [29] and grown in DMEM (Dulbecco's modified Eagle's medium)/F-12 (Invitrogen) supplemented with 10% FBS, 1 μg/ml hydrocortisone (Sigma), 5 μg/ml insulin (Sigma) and 5 ng/ml human EGF (epidermal growth factor; BD Transduction Labs). Antibodies used were GST (glutathione transferase; #71–7500, Invitrogen), p85α (#610045; BD Transduction Labs) and E cadherin (#610181; BD Transduction Labs). HRP (horseradish peroxidase)-conjugated secondary antibodies used for immunoblotting were from Chemicon. DAPI (4′,6-diamidino-2-phenylindole) and CellLight™ PM-GFP (green fluorescent protein) BacMam 2.0 used for immunofluorescence were from Molecular Probes. Recombinant human EGF (BD Transduction Laboratories) and insulin (Sigma) used for stimulation were reconstituted as a 10 × stock in FBS (500 ng/ml and 50 μg/ml, respectively). Lipids were purchased from either Sigma [PS (phosphatidylserine) #P5660, PI (phosphatidylinositol) #P2517, PI-(4,5)-P2 #P9763, Folch Fraction IV #B1502 and mixed PIs #P6023] or Echelon Biosciences [PI-3-P #P-3016, PI-(3,4)-P2 #P-3416 and PI-(3,4,5)-P3 #P-3916].

Generation of recombinant proteins

Wild-type or mutant p110αEE/p85α complexes were expressed in insect (Sf9) cells and purified as previously described [30]. Plasmids encoding GST fusions of the PH (pleckstrin homology) domain of Akt1 and Grp1 or a GFP fusion of the PH domain from Btk (courtesy of Professor Tamas Balla, National Institute of Child Health and Human Development, Bethesda, MD, U.S.A.), constitutively active (V12) forms of H-Ras, Rac1 and Cdc42 (courtesy of Professor Anne Ridley) or control GST alone (pGEX-4T1, GE Healthcare) were transformed into the BL21 (DE3) Escherichia coli strain and protein expression was induced with 0.5 mM IPTG (isopropyl β-D-thiogalactopyranoside) for 4 h at 37°C. GST fusion proteins were purified from clarified bacterial cell lysates using glutathione agarose affinity chromatography. The concentrations of purified, recombinant proteins were quantified by UV spectroscopy using a molar extinction coefficient calculated from their amino acid composition (ProtParam).

Fluorescent labelling of purified recombinant proteins

Purified, recombinant wild-type or mutant p110αEE/p85α complexes were buffer exchanged into 20 mM Bicine pH 8.2, 150 mM NaCl containing 10 mM 2-ME (2-mercaptoethanol) using Sephadex G-25 (GE Life Sciences), then incubated with a 100-fold molar excess of the maleimide mono-reactive form of the fluorophore Cy3 (GE Healthcare #PA23031) for 60 min at room temperature (22°C). Unreacted dye was inactivated by the addition of Tris pH 8.0 to a final concentration of 100 mM. GST fusion proteins were buffer exchanged into PBS and labelled with Alexa488 (Molecular Probes #A20000) as per the manufacturer's instructions. Cy3-labelled p110αEE/p85α complexes and Alexa488-labelled GST-fusion proteins were separated from free fluorophore and buffer exchanged into microinjection buffer [20 mM Tris (pH 7.5), 100 mM KCl, 50 mM NaCl, 0.1% (v/v) 2-ME in 40% (v/v) glycerol] using Sephadex G-25 and concentrated using centrifugal filters (Amicon Ultra15 10000 NMWL, Millipore). The number of coupled fluorophore molecules per protein molecule was determined by measuring the ratio of the absorbances at 280 and 550 nm for Cy3 or 495 nm for Alexa488 using a scanning UV spectrophotometer and molar extinction coefficients calculated from the amino acid composition or specified by the manufacturer (150 000 M−1cm−1 for Cy3 and 73 000 M−1cm−1 for Alexa488).

Gel electrophoresis and immunoblotting

Purified recombinant proteins were separated by SDS–PAGE using 10% Tris–glycine gels. Gels of fluorophore-labelled proteins were fixed in 20% (v/v) methanol, 7% (v/v) acetic acid then scanned using a Typhoon scanner (GE Life Sciences) using a 532 nm laser and emission filters for Cy3 and Alexa488. For immunoblots, SDS–PAGE separated proteins were transferred onto a PVDF membrane, blocked and probed with antibodies that recognize either GST or p85α [1/1000 in 20 mM Tris pH7.5, 150 mM NaCl [TBS (Tris-buffered saline)] containing 0.01% (v/v) Tween 20] followed by HRP-labelled secondary antibodies (1/20 000) and detection using chemiluminescence (Pierce). Scanned immunoblots were quantified using ImageQuant (GE Life Sciences) software.

PI3K assays

PI3K assays were carried out as previously described [3133] in TBS containing 5 mM 2-ME. Assays contained 2 mM MgCl2, 2 mM MnCl2, 0.2 mM ATP, 5–10 μCi [32P]γATP, 500 μg/ml of PI and 250 μg/ml of PS. Extracted phospholipids were separated by TLC in 65% (v/v) 1-propanol, 0.7 M acetic acid, 50 mM phosphoric acid, exposed to a phosphor screen (Molecular Dynamics) and analysed using ImageQuant software.


Fluorescently labelled, purified recombinant proteins in microinjection buffer were microinjected into adherent cells with Femtotips II microcapilliaries using a Femtojet microinjector coupled to an Injectman micromanipulator (Eppendorf) essentially as described [34] and according to the manufacturer's instructions. Cells to be microinjected were grown in 6 mm diameter × 1 mm deep wells made using a CultureWell Gasket (#CW-8R-1.0, Grace Biolabs) on a 24×60 mm Nr 1.5 glass coverslip (Menzel) or 35 mm μ-dishes with low walls and an imprinted 500 μm grid (#80156, Ibidi). Cells were microinjected in a media containing 10% FBS with an injection time of 1 s and a pressure of between 100 and 250 hPa depending on cell type, then left to recover for 2 h at 37°C in the same media before fixing or starving and stimulating. Alternately, to measure the effect of stimulation, microinjected cells were starved for 4 h in media containing no FBS at 37°C then stimulated for 10 min with 10% FBS, 50 ng/ml EGF and 5 μg/ml insulin to activate RTKs. For measurement of PM localization, cells (≤10% confluent) were infected with the PM marker CellLight™ PM-GFP according to the manufacturer's instructions 3 days prior to microinjection.


Microinjected cells were fixed with 4% (v/v) formaldehyde in PBS for 10 min. Nuclei were visualized by staining with DAPI (50 μg/ml, 5 min). For images of fixed cells, cells on glass coverslips were mounted using DPX (Sigma) and confocal images were collected using a Leica SP5 confocal microscope equipped with a ×100 [NA (numerical aperture) 1.45] oil immersion lens using standard filter sets and laser lines for Alexa488, Cy3 and DAPI.

For 3D image analysis of fixed cells, unmounted cells in 35 mm μ-dishes were washed and stored in PBS then imaged using a Nikon C1 motorized confocal microscope equipped with a ×60 (NA 1.35) water immersion lens. Cells were microinjected with different fluorescently labelled, purified recombinant proteins at specific grid locations. Immunofluorescence derived from Cy3 and GFP was measured at pre-defined grid locations in 80 successive focal planes between the top and the bottom surface of the cell using standard filter sets and laser lines.

For 3D image analysis of live cells, microinjected cells that had been allowed to recover post-microinjection for 2 h in DME containing 10% FBS in 35 mm μ-dishes were washed and starved in phenol red-free DME with no FBS for 4 h then imaged using a Nikon C1 motorized confocal microscope as above except that 25 successive focal planes of cells microinjected with different proteins at pre-defined grid locations were imaged prior to stimulation and then at 5, 15, 25, 35, 45 and 65 min post-stimulation with 10% FBS, 50 ng/ml EGF and 5 μg/ml insulin. Confocal images were processed and displayed using ImageJ (v 1.43) Software. Fluorescence intensity along a line was calculated and graphed using Plot Profile. Numbers of protrusions were counted manually.

Quantification of PM localization

Co-incidence of Cy3-PI3Kα and GFP–PM marker fluorescence was quantified in reconstructed 3D images of individual cells using Imaris v7.4 software (Bitplane Scientific Software) by measuring how much red immunofluorescence (Cy3-PI3Kα) overlaps with green (GFP–PM marker). 3D image files were cropped to a single cell, then surfaces were created for the red and green channels using settings of Smooth=0.5 μm and Background subtraction=2.5 μm. The green surface was masked and sum of the voxel intensities of the red immunofluorescence inside the mask (i.e. that overlaps with green surface) was calculated as a percentage of the sum of the voxel intensities of all red immunofluorescence to give the percentage of Cy3-PI3Kα that is co-localized with the PM marker. For individual cells in time course experiments, the ratio of the percentage of Cy3-PI3Kα that is co-localized with the PM marker at each time point compared with the percentage of Cy3-PI3Kα that is co-localized with the PM marker at time zero was calculated.

Lipid overlay assay

Lipid overlay assays were performed according to the method of Dowler [35]. Lipids were reconstituted and diluted in methanol:chloroform:water (2:1:0.8) and 1 μl aliquots were spotted onto Hybond C Extra nitrocellulose membrane (GE Healthcare) at concentrations of between 4 and 500 pmol/μl. Purified recombinant proteins were diluted in PBS before spotting onto Hybond C. Dried membranes were blocked in blocking buffer [50 mM Tris (pH 7.5) containing 150 mM NaCl, 0.1% (v/v) Tween 20 and 2 mg/ml BSA] for 1 h, then incubated with purified recombinant wild-type or mutant PI3Kα or GST-fusion proteins (1 nM in blocking buffer). Membranes were washed in TBS containing 0.1% (v/v) Tween 20, then bound proteins were detected using p85α or GST antibodies, HRP-conjugated secondary antibodies and chemiluminescence as described above.

Liposome co-sedimentation assays

Liposome sedimentation assays were carried out essentially as described [36]. Lipid mixtures were dried under nitrogen then resuspended in liposome buffer (25 mM HEPES pH 7.5, 100 mM NaCl, 0.5 mM EDTA) by sonication for 10 min to form liposomes. Purified recombinant wild-type or mutant PI3Kα or GST-fusion proteins were diluted in liposome buffer containing 2 mg/ml BSA and pre-centrifuged at 300000 g for 20 min at 4°C to remove protein aggregates. Clarified proteins (0.5 μg) and sonicated liposomes (50 μg) were mixed and incubated in liposome buffer containing 1 mg/ml casein, to reduce non-specific binding, for 30 min at room temperature with gentle agitation. Liposome bound proteins were separated from unbound by centrifugation at 17000 g for 30 min at 4°C then the liposome pellet was washed once with 0.5 ml liposome buffer and recentrifuged at 17000 g for 30 min at 4°C. 10% of the supernatant and 10% of the pellet were separated by SDS–PAGE and the presence of PI3Kα or GST-Grp1-PH in the supernatant or pellet fractions was detected by immunoblotting using p85α and GST antibodies.

Affinity precipitations

Purified, recombinant wild-type or mutant p110αEE/p85α complexes (100–3200 ng) and GST fusion proteins (1 μg) were incubated in TBS containing 10% (v/v) FBS to reduce non-specific binding and 25 μl of a 50% slurry of glutathione agarose for 1 h at 4°C with gentle agitation. Beads and bound proteins were pelleted by low-speed centrifugation for 10 s and the supernatant removed. Pelleted beads were washed three times with 1 ml ice-cold TBS containing 0.1% Tween 20 and bound proteins were eluted using SDS–PAGE sample buffer. Bound PI3Kα was detected by immunoblotting using p85α antibodies.

Fluorescently labelled wild-type and mutant PI3Kα are active in vitro and in vivo

Complexes of full-length recombinant human p110α and p85α, the prototypic form of Class 1A PI3K, were expressed and purified essentially as described previously for bovine PI3Kα [32] using the strategy of selecting for the p110α/p85α complex by placing a 6 amino acid epitope tag, known as the glu- or EE-tag [37], at the C-terminus of the p110α, the subunit with limiting expression levels [30], which allowed the purification of an enzyme complex with a 1:1 ratio of catalytic:regulatory subunits. The addition of the C-terminal EE tag to p110α did not affect PI3K, as recombinant p110αEE/p85α had high stability and the expected activity and substrate specificity [30].

High purity wild-type or mutant p110αEE/p85α complexes (p110αEEWT/p85α, p110αEEE545K/p85α and p110αEEH1047R/p85α) were covalently labelled in vitro with the fluorophore Cy3 (Figure 1A) as previously described [32]. Between 2 and 4 Cy3 molecules were incorporated per PI3Kα heterodimer. To ensure that covalent labelling of PI3Kα did not disrupt its structure, wild-type and mutant Cy3-PI3Kα complexes were assayed for their phosphoinositide kinase activity in vitro, which were found to be not significantly different to that for unlabelled PI3Kα (Figure 1B). The in vitro phosphoinositide kinase activities of the oncogenic mutants, p110αEEE545K/p85α and p110αEEH1047R/p85α, were higher than that for p110αEEWT/p85α (Figure 1B), as previously reported [30,38,39].

Fluorescently labelled wild-type and mutant Cy3-PI3Kα (Cy3-p110αEEWT/p85α, Cy3-p110αEEE545K/p85α and Cy3-p110αEEH1047R/p85α) were introduced into live cells by microinjection [34]. 0.5–2 μM Cy3-PI3Kα was microinjected into each cell, which equates to 3000–100000 molecules of Cy3-PI3Kα per cell assuming that 1–10% of the cell volume (1–2 pl for MDCK cells, [40]) was injected. To ensure that microinjected, fluorescently labelled wild-type and mutant PI3Kα retained enzymatic activity, production of PIP3 at the PM was measured using the PH domain from Grp1, a PIP3 sensor [41,42]. A purified recombinant fusion protein of GST and the PH domain from Grp1 (GST–Grp1PH) was covalently labelled in vitro with Alexa488 (Figure 1A). Equal amounts of Alexa488–GST–Grp1PH and Cy3-PI3Kα (or Cy3-ovalbumin as a control) were mixed (Figure 1A) and co-microinjected into MDCK cells in media containing 10% FBS (Figure 1C). Cells were incubated in the same media for 2 h post-microinjection then fixed and co-stained with DAPI to visualize the cell nuclei. Co-microinjection of Alexa488–GST–Grp1PH with a Cy3-labelled control, Cy3-ovalbumin (Figure 1C), did not lead to detectable recruitment of Alexa488–GST–Grp1PH to the PM. Co-microinjection of Alexa488–GST–Grp1PH with Cy3-p110αEEWT/p85α, Cy3-p110αEEE545K/p85α and Cy3-p110αEEH1047R/p85α resulted in a small but detectable accumulation of Alexa488–GST–Grp1PH at the PM, which was detected as increased Alexa488 fluorescence at the edge of a cross sectional profile of the cell (Figure 1C). These data demonstrate that microinjection of fluorescently labelled forms of PI3Kα leads to the production of low levels of PIP3 at the PM of cells maintained in the growth medium, which indicates that Cy3-labelled recombinant PI3Kα remains active when microinjected into adherent cells.

Although the in vitro phosphoinositide kinase activities of p110αEEE545K/p85α and p110αEEH1047R/p85α were higher than that for p110αEEWT/p85α (Figure 1B), the amount of Alexa488–GST–Grp1PH, and therefore PIP3, at the PM was not measurably greater in p110αEEE545K/p85α and p110αEEH1047R/p85α-injected cells compared with p110αEEWT/p85α-injected cells. Overexpression of mutant PI3Kα has been shown to result in increased levels of pAkt [9,10,14] downstream of PIP3; however, lower levels of mutant p110α expression are correlated with lower pAkt [43]. The level of Alexa488–GST–Grp1PH, and therefore PIP3, at the PM was very low in PI3Kα-microinjected cells, suggesting that the amount of PI3Kα injected was also very low. An advantage of microinjection is that the amount of protein injected per cell is consistent between cells and between experiments and can be kept low relative to overexpression by transfection of cDNAs, which may better reflect the reported low levels of endogenous p110α and p85α [44]. Low levels of micro-injected PI3Kα may therefore result very small differences in the low levels of PM-associated Alexa488–GST–Grp1PH between cells injected with wild-type and mutant PI3Kα.

Oncogenic mutant PI3Kα does not associate with PM lipids

Because the lipid substrate for PI3Kα, PIP2, is located on the inner leaflet of the PM, recruitment of cytoplasmic PI3Kα to the PM through its binding to PM localized proteins, such as activated RTKs or activated Ras, is an important step in the activation of its lipid kinase activity [1,45]. It has been proposed that the increased activity of the oncogenic H1047R mutant is at least partly due to increased direct binding to PM lipids [2527,46] and therefore greater access to its substrate. To further test this idea, we measured the association of p110αEEWT/p85α, p110αEEE545K/p85α and p110αEEH1047R/p85α with membrane lipids in vitro and in live cells.

The interaction of purified, recombinant wild-type and mutant PI3Kα with liposomes composed of cellular lipids was measured using a liposome co-sedimentation assay (Figure 2A) under conditions in which PI3Kα was capable of phosphorylating liposomes as shown in Figure 1(B). The specificity of the assay was confirmed using GST–Grp1PH and GST alone as a control. GST did not interact with liposomes composed of either brain-derived cellular lipids (Folch fraction IV) or brain-derived cellular lipids supplemented with extra mixed phosphoinositides (mixed PIs+Folch fraction IV). GST–Grp1PH co–sedimented with both types of liposomes but did not aggregate or sediment on its own as it was not found in the pellet fraction in the absence of liposomes. In comparison, PI3Kα did not co-sediment with either brain-derived lipid liposomes or liposomes containing phosphoinositides to a significant extent. There was no evidence for increased association of liposomes composed of cellular lipids with p110αEEE545K/p85α or p110αEEH1047R/p85α compared with p110αEEWT/p85α.

Because it has been proposed that H1047R interacts with membrane lipids via its C2 domain [47] and C2 domains from a number of proteins have been shown to specifically bind phosphoinositides [4850], the interaction of purified, recombinant wild-type and mutant PI3Kα with phosphoinositides was measured using a Protein Lipid Overlay assay [35] (Figure 2B) with detection of phosphoinositide-bound proteins by p85α or GST antibodies, both of which could detect 1ng of PI3Kα or GST fusion protein that had been directly spotted on to the membrane (Figure 2B, lower panel). Purified GST fusion proteins of the PH domains from Grp1 and Akt1 (GST–Grp1PH and GST–AktPH) specifically bound to PIP3 and PIP3 plus PI-(3,4)-P2 respectively as expected [51]. However, there was little detectable interaction of either wild-type or oncogenic mutant PI3Kα with any of the purified phosphorylated phosphoinositides or with PS. A low-level signal for PI3Kα binding to PI-(3,4)-P2 was detectable at longer exposures (Supplementary Figure S1 available at, but even after a 30 min exposure, there was no evidence for increased binding of p110αEEE545K/p85α or p110αEEH1047R/p85α to phosphoinositides compared with p110αEEWT/p85α.

Next, the amount of microinjected Cy3-PI3Kα located at the PM was measured in cells maintained in growth medium (Figure 2C). Confluent MDCK cells, that had been infected with a construct that leads to expression of PM-targeted GFP, were microinjected with Cy3-labelled PI3Kα (or Cy3-ovalbumin as a control), incubated in media containing 10% FBS for 2–4 h then fixed. Unmounted cells were imaged using confocal microscopy (Supplementary Figure S2 available at, and 80 Z sections were collected. The amount of Cy3-derived fluorescence that was co-incident with GFP-derived fluorescence was quantified in 3D using Imaris software. The Cy3-labelled control protein, Cy3-ovalbumin, would not be expected to specifically localize to the PM, thus the percentage of Cy3-ovalbumin that is coincident with PM-localized GFP (PM-GFP) provides a measure of the background co-incident fluorescence. The percentage of Cy3-p110αEEWT/p85α, Cy3-p110αEEE545K/p85α and Cy3-p110αEEH1047R/p85α that was coincident with PM-GFP was not significantly greater than that for Cy3-ovalbumin, indicating that the majority of PI3Kα is cytoplasmic in cells cultured continuously in media containing 10% FBS. Importantly, the proportion of p110αEEE545K/p85α or p110αEEH1047R/p85α localized at the PM was not significantly higher than that of p110αEEWT/p85α, indicating that oncogenic mutant PI3Kα is not constitutively localized at the PM in MDCK cells, and therefore suggesting that increased phosphorylation of PIP2 and Akt in cells expressing oncogenic mutant PI3Kα is not directly attributable to increased substrate access due to membrane lipid binding.

It has been reported that oncogenic mutant PI3K must be activated by pY binding to the nSH2 domain of p85α before increased binding of mutant PI3Kα to PM lipids in vitro is apparent [27]. To test whether greater amounts of mutant Cy3-PI3Kα are recruited to the PM or whether mutant Cy3-PI3Kα is recruited to the PM for a longer period of time upon RTK activation in stimulated cells, the percentage of Cy3-p110αEEWT/p85α, Cy3-p110αEEE545K/p85α or Cy3-p110αEEH1047R/p85α that was co-incident with PM-GFP was measured in the live cells that had been starved for 4 h and at a number of time points post-stimulation (Figure 3). Stimulation of MDCK cells resulted in production of PIP3 at the PM, as evidenced by the recruitment of a GFP-PH domain reporter to the PM (Supplementary Figure S3 available at Cy3-ovalbumin was not recruited to the PM to a significant extent over a 65 min time-course post-stimulation, confirming that the microinjected Cy3-ovalbumin control remains cytoplasmic as expected. The background level of the coincident Cy3-ovalbumin and PM-GFP fluorescence was the same as in fixed cells (Figure 2C). The level of coincident Cy3 and PM-GFP fluorescence in Cy3-PI3Kα-microinjected cells in starved, unstimulated cells was also the same as for the Cy3-ovalbumin control, indicating that Cy3-PI3Kα is predominantly cytoplasmic both in cells maintained in the growth medium containing 10% FBS (Figure 2C) and in serum-starved cells (Figure 3). Membrane recruitment of microinjected Cy3-PI3Kα was clearly detected using this assay. Upon stimulation, the level of co-incident Cy3-p110αEEWT/p85α and PM-GFP increased, peaking at a between 1.5- and 2-fold increase in the percentage of Cy3-PI3Kα that is co-localized with the PM marker at approximately 5 min post-stimulation and declining to near baseline levels after approximately 40 min (Figure 3), which correlates well with the known timecourse for appearance of pAkt post-stimulation [52]. Similarly, the levels of Cy3-p110αEEE545K/p85α and Cy3-p110αEEH1047R/p85α fluorescence co-incident with PM-GFP fluorescence increased upon stimulation. Although only a very small proportion of total cytoplasmic Cy3-p110αEEWT/p85α was recruited to the PM at 5 min post-stimulation (Supplementary Figure S4 available at, the maximum levels of Cy3-p110αEEE545K/p85α and Cy3-p110αEEH1047R/p85α recruited to the PM were not significantly greater than that of Cy3-p110αEEWT/p85α. In addition, a time course showed that recruitment of p110αEEE545K/p85α or Cy3-p110αEEH1047R/p85α to the PM was not significantly extended temporally compared with that of Cy3-p110αEEWT/p85α. This suggests that increased PI3Kα activity in cells expressing oncogenic mutant PI3Kα is not a direct result of increased substrate access associated with increased membrane lipid binding and concomitant increased localization at the PM in either unstimulated cells or upon RTK activation.

Microinjection of PI3Kα containing the E545K oncogenic mutation leads to increased numbers of cell protrusions

During microinjection experiments to assess PM recruitment of wild-type and mutant PI3Kα, it was observed that cells microinjected with p110αEEE545K/p85α exhibited increased numbers of cell protrusions compared with cells injected with either the control Cy3-ovalbumin, p110αEEWT/p85α or p110αEEH1047R/p85α. This effect was greater in stimulated cells compared with either cells maintained in 10% FBS or starved cells. In confluent MDCK epithelial cells, which form a typical epithelial sheet with a cobblestone morphology [28], microinjection of Cy3-ovalbumin did not lead to an observable change in cell shape (Figure 4A). In addition, Cy3-ovalbumin was not observed to be recruited to the PM in stimulated MDCK cells, which supports the 3D quantitative data (Figure 3). For microinjected Cy3-p110αEEWT/p85α, Cy3-p110αEEE545K/p85α and Cy3-p110αEEH1047R/p85α, stimulation resulted in a small proportion of cytoplasmic Cy3-labelled protein accumulating at the margins of the cell, which is indicative of recruitment to the PM (Figure 4A). Microinjection of Cy3-p110αEEWT/p85α did not result in significant morphology changes to either starved or stimulated MDCK cells. However, microinjection of Cy3-p110αEEH1047R/p85α, resulted in a proportion of cells that were more angular than the typical MDCK morphology, and stimulation led to an increased proportion of these angular and asymmetrical cells. Microinjection of Cy3-p110αEEE545K/p85α resulted in an increased number of cells with distinct cell protrusions, and stimulation of these cells both increased the proportion of cells with protrusions and the length of the protrusions. In some stimulated cells, Cy3-p110αEEE545K/p85α was observed to accumulate at the tips of the cell protrusions.

To simplify quantification of cell morphology, cells were scored for the absence, presence of 1 protrusion or greater than 1 protrusion per microinjected cell (Figure 4B). The number of protrusions varied between cell lines due to their different morphology. MDCK epithelial cells and Caco2 colon epithelial cells form a polarized epithelial monolayer in cell culture, while iMMEC did not form tight cell–cell adhesions and grew as single cells. In addition, cell stimulation increased the proportion of cells with protrusions in all cell types and the morphology of microinjected cells varied depending on whether they were in the centre or at the edge of a cell sheet, thus between 10 and 100 cells were scored for each cell type. Non-microinjected or Cy3-ovalbumin microinjected MDCK and Caco2 cells formed no or few protrusions (Figure 4B), while a number of iMMEC had discernible protrusions associated with their more spiky, single-cell morphology. Microinjection of Cy3-p110αEEE545K/p85α resulted in an increased proportion of cells with observable cell protrusions in all three epithelial cell types, and this was exacerbated in stimulated cells. Microinjection of Cy3-p110αEEWT/p85α or Cy3-p110αEEH1047R/p85α resulted in a small increase in the proportion of cells with protrusions, which was also further increased in stimulated cells; however, the difference between p110αEEWT/p85α and Cy3-p110αEEH1047R/p85α was marginal and probably not significant. Introduction of p110αEEE545K/p85α into live cells is therefore associated with a change in cell morphology and increased numbers of cells with membrane protrusions.

The E545K oncogenic mutation leads to increased PI3Kα binding to Rho-subfamily small GTPases

A number of lines of evidence suggest that at least part of the action of PI3Kα on the actin cytoskeleton that leads to cell membrane protrusions such as lamellopodia and filopodia, cell polarization and ultimately cell migration is mediated through the Ras- and Rho-subfamily GTPases [16,21,5356]. In addition, the p110α subunit of PI3Kα binds to Ras through the Ras-binding domain [57], and the p85 subunit has been reported to bind the Rho-subfamily GTPases, Rac and Cdc42, through its N-terminal GAP-like or BH (BCR-homology) domain [18,58]. More recently, Rac1 has also been shown to bind to the Ras-binding domain of p110β [59]. To test whether the E545K oncogenic mutation alters interactions with GTPases, which may be implicated in the observed change in morphology of cells microinjected with p110αEEE545K/p85α, we measured the interaction of wild-type and mutant purified, recombinant PI3Kα with GST fusion proteins of the constitutively active (V12) forms of human H-Ras, Rac1 and Cdc42Hs. Pull-down assays were carried out in the presence of high protein concentrations (~6–8 mg/ml FBS) to minimize non-specific binding, and under these conditions, the amount of recombinant PI3Kα that bound to GST alone was negligible (Figure 5A). p110αEEWT/p85α and Cy3-p110αEEH1047R/p85α did not bind to GST-V12 Cdc42, GST-V12 Rac1 or GST-V12 Ras to a greater extent than to GST, while more p110αEEE545K/p85α bound to GST-V12 Cdc42, GST–V12 Rac1 and GST-V12 Ras compared with GST (Figure 5A).

To estimate the relative affinities of GST-V12 Cdc42, GST-V12 Rac1 and GST-V12 Ras for p110αEEE545K/p85α, the amount of each GST fusion protein bound to increasing concentrations of PI3Kα was measured and the binding isotherms were plotted (Figure 5B). The amounts of GST, GST-V12 Cdc42, GST-V12 Rac1 and GST-V12 Ras used in each assay were similar, as shown. The level of non-specific binding of wild-type and oncogenic PI3Kα to the GST control was similar. Binding of p110αEEE545K/p85α to GST-Ras and GST-Rac1 was not saturated at 3200 ng (~250 nM) PI3Kα and therefore these are likely to be relatively low-affinity interactions. In contrast, the binding of p110αEEE545K/p85α to GST-Cdc42 was saturable and of a higher affinity compared with that of GST-V12 Rac1 and GST-V12 Ras. The E545K oncogenic mutant therefore specifically binds to constitutively active Cdc42 with appreciable affinity compared with wild-type PI3Kα or the H1047R oncogenic mutant.


Although the subcellular localization and dynamics of PIP3 synthesis have been investigated using PIP3 sensors that are recruited to the PM when PIP3 is present, the subcellular localization of PI3Kα, which produces PIP3, has not been well characterized. Currently available antibodies that recognize p110α or p85α do not appear to be of sufficient quality to reliably detect the subcellular location of these proteins by immunofluorescence [23,60]. Expression of PI3Kα by transfection of cDNAs encoding p110α and p85α leads to unequal expression levels of the regulatory and catalytic subunits. The p85α regulatory subunit is generally expressed at high levels relative to p110α and excess free p85α has been shown to act as a dominant negative that inhibits PI3Kα signalling [61,62], whereas the p110α subunit has been shown to be thermodynamically unstable in the absence of p85 [21]. In addition, altering levels of one PI3K subunit alters the relative expression of other subunits [63]. Endogenous PI3K is reported to always be a 1:1 heterodimer of p110 and p85 subunits [64] but the inability to co-express equal levels of p110 and p85 by transient or stable overexpression suggests that ectopically expressed PI3K may not behave like endogenous PI3K.

To test whether increased PM localization is a factor in the higher activity of oncogenic mutant PI3Kα, we chose to take advantage of our ability to express and purify the recombinant p110α/p85α heterodimer in such a way that the ratio of subunits is 1:1 [30]. Highly purified, recombinant p110αEE/p85α was fluorescently labelled in vitro with a low molecular mass (MW=767 Da) CyDye that did not interfere with the enzymatic activity of recombinant PI3Kα (Figure 1B), then introduced into cells by microinjection. This novel technique of pre-forming a protein complex in vitro prior to introducing it into cells bypasses the limitations of traditional transfection and overexpression strategies. In addition, microinjection of labelled protein allows imaging of fluorescent proteins within minutes of their introduction into cells, thus minimizing secondary effects due to changes in expression levels of other proteins [34]. The amount of the different PI3Kα complexes and the control protein microinjected was controlled by injecting the same concentrations of recombinant protein under the same conditions, and the amount of protein injected per cell was kept low relative to overexpression by transfection of cDNAs. It was estimated that between 3000 and 50000 molecules of PI3Kα were injected per cell, which is within the range of the reported concentrations of other signalling proteins [40]. Microinjected, fluorescently labelled PI3Kα retained enzymatic activity, as a PIP3 sensor was recruited to the PM, albeit at low levels, when PI3Kα, but not a control protein, was microinjected (Figure 1C).

It has been proposed that oncogenic mutants of PI3Kα interact directly with the PM lipid bilayer. The p110 subunit of PI3K contains a C2 domain which, in a number of other proteins, has been shown to bind phosphoinositides [65,66]. However, it is not clear, from the X-ray crystal structure of p110αH1047R in complex with the nSH2 and iSH2 domains of p85α (p85αnSH2−iSH2) [25], whether the H1047R mutation alters the accessibility of the putative lipid-binding surface of the C2 domain. Instead, two loops in the kinase domain of p110αH1047R change conformation relative to p110αWT and form a flat, negatively charged surface that was proposed to contact the cell membrane [25]. In addition, the ratio of p110αH1047R/p85αnSH2−iSH2 to p110αWT/p85αnSH2−iSH2 PIP2 kinase activity was different when PIP2-containing liposomes were formed from membrane lipids from different sources, which was interpreted as reflecting the different lipid compositions of the vesicles, suggesting that the H1047R mutation alters the interaction of the kinase domain with the cell membrane.

Even though PI3Kα activity has been shown to be higher in tumour cells containing mutant compared with wild-type PI3Kα [9,10,14], oncogenic mutant forms of PI3Kα were not detected at the PM in cells maintained in the growth medium (Figure 2C). This suggests either that oncogenic forms of PI3Kα do not constitutively interact with the PM or that membrane-associated PI3Kα is below the threshold level of detection in this system. PIP3 and pAkt levels in resting or starved tumour cells with oncogenic PI3Kα have been shown to be similar to those in cells with wild-type PI3Kα that have been stimulated with growth factors that activate RTKs [9,10]. PM recruitment of wild-type and mutant PI3Kα was clearly detected in stimulated MDCK cells (Figure 3), suggesting that if the mutant PI3Kα does constitutively bind to PM lipids but cannot be detected in this system, the steady-state levels are much lower than those when wild-type PI3Kα is recruited to the PM post-stimulation. It is not clear whether such a low level of constitutive PM localization could directly result in the same functional outcome, in terms of PIP3 and Akt phosphorylation, as does the levels of PM localized PI3Kα detected in stimulated cells (Figure 3).

Interactions of p110α/p85αnSH2−iSH2−cSH2 with PIP2-containing lipid vesicles have been detected in vitro by DXMS (deuterium exchange mass spectrometry) and protein lipid FRET (fluorescence resonance energy transfer) [46]. The interaction of regions of the kinase domain of p110α surrounding the catalytic cleft with membrane vesicles required engagement of the p85α SH2 domains with pY-containing peptides [27,46]. In our system, binding to phosphoinositides or to lipid vesicles was not carried out in the presence of pY-containing peptides in order to test whether there was a significant constitutive interaction between oncogenic mutant PI3Kα and membrane lipids, but no interaction was detected between the wild-type or mutant PI3Kα and membrane lipids in vitro and no significant level of PM-localized wild-type or mutant PI3Kα was detected in cells maintained in the growth medium (Figure 2). In growth factor-stimulated cells in which RTKs are activated, PI3Kα was presumably transiently recruited to the PM via interactions with PM-localized pY-containing RTKs or other protein–protein interactions. Only 5–10% of the total PI3Kα was coincident with the PM marker at the peak at 5 min post-stimulation (Supplementary Figure S4), which is similar to the proportion that was shown to co-fractionate with the transmembrane PDGF (platelet-derived growth factor) receptor in PDGF-stimulated cells [67]. However, despite the pY-containing peptide-bound H1047R and E545K oncogenic mutants being shown to bind to lipid vesicles better in vitro by DXMS and protein lipid FRET [46], oncogenic mutants of PI3Kα did not persist longer at the PM than wild-type PI3Kα (Figure 3), suggesting that any PI3Kα–lipid interactions that may occur do not outweigh p85αSH2-pY/RTK interactions in determining the amount of time that oncogenic or wild-type PI3Kα is resident at the PM. It is possible that the interaction between the p110α kinase domain and PIP2-containing lipid vesicles observed previously [27,46] represents transient enzyme–substrate interactions that would not be sufficiently long-lived to be observed as a change in subcellular localization. However, if this was the case, it would be expected that increased enzyme–substrate affinity leading to increased lipid kinase activity would be reflected in an increased Michaelis constant (Km) for oncogenic mutant PI3Kα compared with wild-type. The Km for PIP2 has not been measured directly; however, oncogenic mutants have not been reported to have a higher Km for ATP compared with wild-type PI3Kα but instead display a higher maximum reaction rate (Vmax) [30,38,39], suggesting that oncogenic mutant forms of PI3Kα phosphorylate and turn over their lipid substrate more rapidly.

The functional significance of PI3Kα–lipid interactions remains to be determined. While oncogenic mutations increase the intrinsic phosphoinositide kinase activity of PI3Kα by 1.5- to 4-fold [30,38,39], they do not appear to increase its access to its PM-localized substrate (Figure 3). PI3Kα signalling is also tightly regulated by a number of PIP3 phosphatases including PTEN (phosphatase and tensin homologue deleted on chromosome 10) and 5-phosphatases [68,69], which are thought to be responsible for the short-lived nature of the PI3Kα product, PIP3. Although some lipid phosphatases, notably PTEN, are known as tumour suppressors [69], it is not yet clear whether the magnitude of increase in the intrinsic lipid kinase activity of PI3Kα can override phosphatase regulation to produce the observed increase in Akt signalling without prolonged substrate access.

This raises the possibility that other mechanisms may contribute to the oncogenicity of PI3Kα mutations. p110α and p85α both contain a number of protein–protein interaction domains that can contribute to PI3Kα signalling. In support of this idea, differences between ‘knockout’ and ‘kinase-dead’ mouse models suggest that PI3K may have functions that are independent of its kinase activity [22]. Mutation-specific protein–protein interactions could propagate signalling directly or could conceivably function to localize PI3Kα and PIP3 to regions of the cell where PIP3 phosphatases are less active. Our observation that microinjection of p110αEEE545K/p85α resulted in a morphological change in which an increased proportion of cells had observable protrusions (Figure 4) led us to hypothesize that Rho-subfamily GTPases, such as Rac and Cdc42 [55], could be involved in mediating the effects of the E545K mutation in specific regions of the cell. p110αEEE545K/p85α specifically bound to activated Cdc42 (Figure 5), providing some preliminary evidence that this protein–protein interaction may contribute to the oncogenicity of the E545K mutation. Cdc42 is thought to bind to PI3Kα through the p85α GAP-like or BH domain [18,58]. This domain is not present in any of the X-ray crystal structures [18,25,47] or lipid-binding studies [27,46] of PI3Kα but our observation that the E545K mutation is associated with Cdc42 binding suggests that the E545K mutation alters the conformation of PI3Kα to expose the Cdc42-interaction surface on the p85α BH domain. Recently, E545K-mutant PI3Kα was shown to gain the ability to associate with IRS1 [70] but the interaction was between p110α and non-phosphorylated IRS1 rather than the previously reported mechanism of interaction of these proteins, which involved the p85α–SH2 domains and tyrosine phosphorylated IRS1 [71]. It is therefore also possible that Cdc42 interacts with p110αE545K, possibly via the Ras-binding domain.

During the initiation of directed cell migration, protrusions form at the leading edge of the cell. Extension of both lamellipodial and filopodial protrusions requires actin polymerization beneath the PM [55], which in the case of filopodia is co-ordinated by Cdc42 [21]. PIP3 is found at the leading edge of migrating cells, and PI3K is required for cell polarization [72] and the early steps in EGF- or PDGF-induced cell migration [16]. p110α and Ras have both been shown to be required for EGF-stimulated protrusion formation [16]. Interestingly, Cdc42-regulated filopodia formation has been reported to be dependent on p85α but independent of PI3Kα lipid kinase activity [73]. p110α has also been shown to regulate invadopodia formation [74] and PIK3CA mutations are more common in high- versus low-grade colorectal and lung tumours [75] and are associated with increased invasiveness [14]. The finding that p110αEEE545K/p85α interacts specifically with constitutively active Cdc42 leads us to speculate that during the initiation of migration or invasion, the activation of both PI3Kα and Cdc42 and their subsequent interaction could be required to propagate a signal that contributes to protrusion formation at the leading edge, and that the E545K mutation may pre-activate PI3Kα, so it is ready to switch on the pathway when Cdc42 becomes activated. Interestingly, PIK3CA mutations are associated with K-Ras mutations in advanced tumours [76], which may result in constitutively active Cdc42 in these cells. The p110αEEE545K/p85α–Cdc42-binding interface may represent a therapeutic target that could inhibit PI3Kα E545K signalling without affecting wild-type PI3Kα signalling in non-tumour cells.

Online data Supplementary data


We thank Mike Adams from Bitplane for assistance with the Imaris software.


Meredith Layton designed, conducted and analysed the experiments and wrote the paper with contributions from the other authors. Natalie Rynkiewicz optimized the method for the lipid overlay assay. Kristy Horan conducted the initial experimental optimization of the microinjection procedure. Ivan Ivetac provided the iMMEC cells. Christina Mitchell and Wayne Phillips supervised the work and provided critical input into the paper.


This research was supported by a Grant-in-Aid from the Cancer Council Victoria.

1. Vanhaesebroeck B.,Stephens L.,Hawkins P.. PI3K signalling: the path to discovery and understandingNat. Rev. Mol. Cell. Biol.Year: 20121319520310.1038/nrm329022358332
2. Cantley L. C.. The phosphoinositide 3-kinase pathwayScienceYear: 20022961655165710.1126/science.296.5573.165512040186
3. Hawkins P. T.,Anderson K. E.,Davidson K.,Stephens L. R.. Signalling through Class I PI3Ks in mammalian cellsBiochem. Soc. Trans.Year: 20063464766217052169
4. Bachman K. E.,Argani P.,Samuels Y.,Silliman N.,Ptak J.,Szabo S.,Konishi H.,Karakas B.,Blair B. G.,Lin C.,et al. The PIK3CA gene is mutated with high frequency in human breast cancersCancer Biol. Ther.Year: 2004377277515254419
5. Campbell I. G.,Russell S. E.,Choong D. Y.,Montgomery K. G.,Ciavarella M. L.,Hooi C. S.,Cristiano B. E.,Pearson R. B.,Phillips W. A.. Mutation of the PIK3CA gene in ovarian and breast cancerCancer Res.Year: 2004647678768110.1158/0008-5472.CAN-04-293315520168
6. Velho S.,Oliveira C.,Ferreira A.,Ferreira A. C.,Suriano G.,Schwartz S. Jr.,Duval A.,Carneiro F.,Machado J. C.,Hamelin R.,Seruca R.. The prevalence of PIK3CA mutations in gastric and colon cancerEur. J. CancerYear: 2005411649165410.1016/j.ejca.2005.04.02215994075
7. Miyaki M.,Iijima T.,Yamaguchi T.,Takahashi K.,Matsumoto H.,Yasutome M.,Funata N.,Mori T.. Mutations of the PIK3CA gene in hereditary colorectal cancersInt. J. CancerYear: 20071211627163010.1002/ijc.2282917546593
8. Samuels Y.,Wang Z.,Bardelli A.,Silliman N.,Ptak J.,Szabo S.,Yan H.,Gazdar A.,Powell S. M.,Riggins G. J.,et al. High frequency of mutations of the PIK3CA gene in human cancersScienceYear: 200430455410.1126/science.109650215016963
9. Kang S.,Bader A. G.,Vogt P. K.. Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenicProc. Natl. Acad. Sci. U.S.A.Year: 200510280280710.1073/pnas.040886410215647370
10. Isakoff S. J.,Engelman J. A.,Irie H. Y.,Luo J.,Brachmann S. M.,Pearline R. V.,Cantley L. C.,Brugge J. S.. Breast cancer-associated PIK3CA mutations are oncogenic in mammary epithelial cellsCancer Res.Year: 200565109921100010.1158/0008-5472.CAN-05-261216322248
11. Bader A. G.,Kang S.,Vogt P. K.. Cancer-specific mutations in PIK3CA are oncogenic in vivoProc. Natl. Acad. Sci. U.S.A.Year: 20061031475147910.1073/pnas.051085710316432179
12. Liu P.,Cheng H.,Roberts T. M.,Zhao J. J.. Targeting the phosphoinositide 3-kinase pathway in cancerNat. Rev. Drug Discov.Year: 2009862764410.1038/nrd292619644473
13. Ikenoue T.,Kanai F.,Hikiba Y.,Obata T.,Tanaka Y.,Imamura J.,Ohta M.,Jazag A.,Guleng B.,Tateishi K.,et al. Functional analysis of PIK3CA gene mutations in human colorectal cancerCancer Res.Year: 2005654562456710.1158/0008-5472.CAN-04-411415930273
14. Samuels Y.,Diaz L. A. Jr.,Schmidt-Kittler O.,Cummins J. M.,Delong L.,Cheong I.,Rago C.,Huso D. L.,Lengauer C.,Kinzler K. W.,et al. Mutant PIK3CA promotes cell growth and invasion of human cancer cellsCancer CellYear: 2005756157310.1016/j.ccr.2005.05.01415950905
15. Zhao J. J.,Liu Z.,Wang L.,Shin E.,Loda M. F.,Roberts T. M.. The oncogenic properties of mutant p110α and p110β phosphatidylinositol 3-kinases in human mammary epithelial cellsProc. Natl. Acad. Sci. U.S.A.Year: 2005102184431844810.1073/pnas.050898810216339315
16. Rodriguez-Viciana P.,Warne P. H.,Dhand R.,Vanhaesebroeck B.,Gout I.,Fry M. J.,Waterfield M. D.,Downward J.. Phosphatidylinositol-3-OH kinase as a direct target of RasNatureYear: 199437052753210.1038/370527a08052307
17. Pleiman C. M.,Hertz W. M.,Cambier J. C.. Activation of phosphatidylinositol-3′ kinase by Src-family kinase SH3 binding to the p85 subunitScienceYear: 19942631609161210.1126/science.81282488128248
18. Tolias K. F.,Cantley L. C.,Carpenter C. L.. Rho family GTPases bind to phosphoinositide kinasesJ. Biol. Chem.Year: 1995270176561765910.1074/jbc.270.30.176567629060
19. Klippel A.,Reinhard C.,Kavanaugh W. M.,Apell G.,Escobedo M. A.,Williams L. T.. Membrane localization of phosphatidylinositol 3-kinase is sufficient to activate multiple signal-transducing kinase pathwaysMol. Cell. Biol.Year: 199616411741278754810
20. Rodriguez-Viciana P.,Warne P. H.,Vanhaesebroeck B.,Waterfield M. D.,Downward J.. Activation of phosphoinositide 3-kinase by interaction with Ras and by point mutationEMBO J.Year: 199615244224518665852
21. Cain R. J.,Ridley A. J.. Phosphoinositide 3-kinases in cell migrationBiol. CellYear: 2009101132910.1042/BC2008007919055486
22. Costa C.,Hirsch E.. More than just kinases: the scaffolding function of PI3KCurr. Top. Microbiol. Immunol.Year: 201034617118120563705
23. Kapeller R.,Chakrabarti R.,Cantley L.,Fay F.,Corvera S.. Internalization of activated platelet-derived growth factor receptor-phosphatidylinositol-3′ kinase complexes: potential interactions with the microtubule cytoskeletonMol. Cell. Biol.Year: 199313605260638413207
24. Carpenter C. L.,Auger K. R.,Chanudhuri M.,Yoakim M.,Schaffhausen B.,Shoelson S.,Cantley L. C.. Phosphoinositide 3-kinase is activated by phosphopeptides that bind to the SH2 domains of the 85-kDa subunitJ. Biol. Chem.Year: 1993268947894837683653
25. Mandelker D.,Gabelli S. B.,Schmidt-Kittler O.,Zhu J.,Cheong I.,Huang C. H.,Kinzler K. W.,Vogelstein B.,Amzel L. M.. A frequent kinase domain mutation that changes the interaction between PI3Kα and the membraneProc. Natl. Acad. Sci. U.S.A.Year: 2009106169961700110.1073/pnas.090844410619805105
26. Gabelli S. B.,Huang C. H.,Mandelker D.,Schmidt-Kittler O.,Vogelstein B.,Amzel L. M.. Structural effects of oncogenic PI3Kα mutationsCurr. Top. Microbiol. Immunol.Year: 2010347435320593314
27. Hon W. C.,Berndt A.,Williams R. L.. Regulation of lipid binding underlies the activation mechanism of class IA PI3-kinasesOncogeneYear: 2012313655366610.1038/onc.2011.53222120714
28. Ridley A. J.,Comoglio P. M.,Hall A.. Regulation of scatter factor/hepatocyte growth factor responses by Ras, Rac, and Rho in MDCK cellsMol. Cell. Biol.Year: 199515111011227823927
29. Tikoo A.,Roh V.,Montgomery K. G.,Ivetac I.,Waring P.,Pelzer R.,Hare L.,Shackleton M.,Humbert P.,Phillips W. A.. Physiological levels of PIK3CA (H1047R) mutation in the mouse mammary gland results in ductal hyperplasia and formation of ERα-positive tumorsPLoS ONEYear: 20127e3692410.1371/journal.pone.003692422666336
30. Layton M. J.,Saad M.,Church N. L.,Pearson R. B.,Mitchell C. A.,Phillips W. A.. Autophosphorylation of serine 608 in the p85 regulatory subunit of wild type or cancer-associated mutants of phosphoinositide 3-kinase does not affect its lipid kinase activityBMC Biochem.Year: 2012133010.1186/1471-2091-13-3023270540
31. Whitman M.,Kaplan D. R.,Schaffhausen B.,Cantley L.,Roberts T. M.. Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformationNatureYear: 198531523924210.1038/315239a02987699
32. Layton M. J.,Harpur A. G.,Panayotou G.,Bastiaens P. I.,Waterfield M. D.. Binding of a diphosphotyrosine-containing peptide that mimics activated platelet-derived growth factor receptor β induces oligomerization of phosphatidylinositol 3-kinaseJ. Biol. Chem.Year: 1998273333793338510.1074/jbc.273.50.333799837914
33. Arcaro A.,Volinia S.,Zvelebil M. J.,Stein R.,Watton S. J.,Layton M. J.,Gout I.,Ahmadi K.,Downward J.,Waterfield M. D.. Human phosphoinositide 3-kinase C2β, the role of calcium and the C2 domain in enzyme activityJ. Biol. Chem.Year: 1998273330823309010.1074/jbc.273.49.330829830063
34. Garg R.,Ridley A. J.. Mammalian cell microinjection assay to study the function of Rho family guanosine triphosphatasesMethods Mol. Biol.Year: 200633225726716878698
35. Dowler S.,Kular G.,Alessi D. R.. Protein lipid overlay assaySci. STKEYear: 20022002pI6
36. Hasegawa J.,Tokuda E.,Tenno T.,Tsujita K.,Sawai H.,Hiroaki H.,Takenawa T.,Itoh T.. SH3YL1 regulates dorsal ruffle formation by a novel phosphoinositide-binding domainJ. Cell. Biol.Year: 201119390191610.1083/jcb.20101216121624956
37. Porfiri E.,Evans T.,Chardin P.,Hancock J. F.. Prenylation of Ras proteins is required for efficient hSOS1-promoted guanine nucleotide exchangeJ. Biol. Chem.Year: 199426922672226778077219
38. Chaussade C.,Cho K.,Mawson C.,Rewcastle G. W.,Shepherd P. R.. Functional differences between two classes of oncogenic mutation in the PIK3CA geneBiochem. Biophys. Res. Commun.Year: 200938157758110.1016/j.bbrc.2009.02.08119233141
39. Carson J. D.,Van Aller G.,Lehr R.,Sinnamon R. H.,Kirkpatrick R. B.,Auger K. R.,Dhanak D.,Copeland R. A.,Gontarek R. R.,Tummino P. J.,Luo L.. Effects of oncogenic p110α subunit mutations on the lipid kinase activity of phosphoinositide 3-kinaseBiochem. J.Year: 200840951952410.1042/BJ2007068117877460
40. Tan C. W.,Gardiner B. S.,Hirokawa Y.,Layton M. J.,Smith D. W.,Burgess A. W.. Wnt signalling pathway parameters for mammalian cellsPLoS ONEYear: 20127e3188210.1371/journal.pone.003188222363759
41. Balla T.,Varnai P.. Visualization of cellular phosphoinositide pools with GFP-fused protein-domainsCurr. Protoc. Cell Biol.Year: 2009Chapter 24Unit 2424
42. Varnai P.,Bondeva T.,Tamas P.,Toth B.,Buday L.,Hunyady L.,Balla T.. Selective cellular effects of overexpressed pleckstrin-homology domains that recognize PtdIns(3,4,5)P3 suggest their interaction with protein binding partnersJ. Cell. Sci.Year: 20051184879488810.1242/jcs.0260616219693
43. Yuan T. L.,Wulf G.,Burga L.,Cantley L. C.. Cell-to-cell variability in PI3K protein level regulates PI3K–AKT pathway activity in cell populationsCurr. Biol.Year: 20112117318310.1016/j.cub.2010.12.04721256021
44. Schwanhausser B.,Busse D.,Li N.,Dittmar G.,Schuchhardt J.,Wolf J.,Chen W.,Selbach M.. Global quantification of mammalian gene expression controlNatureYear: 201147333734210.1038/nature1009821593866
45. Huang C. H.,Mandelker D.,Gabelli S. B.,Amzel L. M.. Insights into the oncogenic effects of PIK3CA mutations from the structure of p110α/p85αCell CycleYear: 200871151115610.4161/cc.7.9.581718418043
46. Burke J. E.,Perisic O.,Masson G. R.,Vadas O.,Williams R. L.. Oncogenic mutations mimic and enhance dynamic events in the natural activation of phosphoinositide 3-kinase p110α (PIK3CA)Proc. Natl. Acad. Sci. U.S.A.Year: 2012109152591526410.1073/pnas.120550810922949682
47. Huang C. H.,Mandelker D.,Schmidt-Kittler O.,Samuels Y.,Velculescu V. E.,Kinzler K. W.,Vogelstein B.,Gabelli S. B.,Amzel L. M.. The structure of a human p110α/p85α complex elucidates the effects of oncogenic PI3Kα mutationsScienceYear: 20073181744174810.1126/science.115079918079394
48. Corbalan-Garcia S.,Garcia-Garcia J.,Rodriguez-Alfaro J. A.,Gomez-Fernandez J. C.. A new phosphatidylinositol 4,5-bisphosphate-binding site located in the C2 domain of protein kinase CαJ. Biol. Chem.Year: 20032784972498010.1074/jbc.M20938520012426311
49. Coudevylle N.,Montaville P.,Leonov A.,Zweckstetter M.,Becker S.. Structural determinants for Ca2+ and phosphatidylinositol 4,5-bisphosphate binding by the C2A domain of rabphilin-3AJ. Biol. Chem.Year: 2008283359183592810.1074/jbc.M80409420018945677
50. Dunn R.,Klos D. A.,Adler A. S.,Hicke L.. The C2 domain of the Rsp5 ubiquitin ligase binds membrane phosphoinositides and directs ubiquitination of endosomal cargoJ. Cell. Biol.Year: 200416513514410.1083/jcb.20030902615078904
51. Lemmon M. A.. Membrane recognition by phospholipid-binding domainsNat. Rev. Mol. Cell. Biol.Year: 200899911110.1038/nrm232818216767
52. Williams M. R.,Arthur J. S.,Balendran A.,van der Kaay J.,Poli V.,Cohen P.,Alessi D. R.. The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cellsCurr. Biol.Year: 20001043944810.1016/S0960-9822(00)00441-310801415
53. Hill K. M.,Huang Y.,Yip S. C.,Yu J.,Segall J. E.,Backer J. M.. N-terminal domains of the class IA phosphoinositide 3-kinase regulatory subunit play a role in cytoskeletal but not mitogenic signalingJ. Biol. Chem.Year: 2001276163741637810.1074/jbc.M00698520011278326
54. Vanhaesebroeck B.,Jones G. E.,Allen W. E.,Zicha D.,Hooshmand-Rad R.,Sawyer C.,Wells C.,Waterfield M. D.,Ridley A. J.. Distinct PI(3)Ks mediate mitogenic signalling and cell migration in macrophagesNat. Cell Biol.Year: 19991697110.1038/904510559867
55. Ridley A. J.. Life at the leading edgeCellYear: 20111451012102210.1016/j.cell.2011.06.01021703446
56. Sahai E.,Marshall C. J.. RHO-GTPases and cancerNat. Rev. CancerYear: 2002213314210.1038/nrc72512635176
57. Rodriguez-Viciana P.,Warne P. H.,Khwaja A.,Marte B. M.,Pappin D.,Das P.,Waterfield M. D.,Ridley A.,Downward J.. Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by RasCellYear: 19978945746710.1016/S0092-8674(00)80226-39150145
58. Zheng Y.,Bagrodia S.,Cerione R. A.. Activation of phosphoinositide 3-kinase activity by Cdc42Hs binding to p85J. Biol. Chem.Year: 199426918727187308034624
59. Fritsch R.,de Krijger I.,Fritsch K.,George R.,Reason B.,Kumar M. S.,Diefenbacher M.,Stamp G.,Downward J.. RAS and RHO families of GTPases directly regulate distinct phosphoinositide 3-kinase isoformsCellYear: 20131531050106310.1016/j.cell.2013.04.03123706742
60. Johanson S. O.,Naccache P. A.,Crouch M. F.. A p85 subunit-independent p110α PI 3-kinase colocalizes with p70 S6 kinase on actin stress fibers and regulates thrombin-stimulated stress fiber formation in Swiss 3T3 cellsExp. Cell Res.Year: 199924822323310.1006/excr.1999.440510094829
61. Engelman J. A.,Luo J.,Cantley L. C.. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolismNat. Rev. Genet.Year: 2006760661910.1038/nrg187916847462
62. Luo J.,Field S. J.,Lee J. Y.,Engelman J. A.,Cantley L. C.. The p85 regulatory subunit of phosphoinositide 3-kinase down-regulates IRS-1 signaling via the formation of a sequestration complexJ. Cell. Biol.Year: 200517045546410.1083/jcb.20050308816043515
63. Vanhaesebroeck B.,Ali K.,Bilancio A.,Geering B.,Foukas L. C.. Signalling by PI3K isoforms: insights from gene-targeted miceTrends Biochem. Sci.Year: 20053019420410.1016/j.tibs.2005.02.00815817396
64. Geering B.,Cutillas P. R.,Nock G.,Gharbi S. I.,Vanhaesebroeck B.. Class IA phosphoinositide 3-kinases are obligate p85–p110 heterodimersProc. Natl. Acad. Sci. U.S.A.Year: 20071047809781410.1073/pnas.070037310417470792
65. Corbalan-Garcia S.,Gomez-Fernandez J. C.. The C2 domains of classical and novel PKCs as versatile decoders of membrane signalsBiofactorsYear: 2010361720049899
66. Farah C. A.,Sossin W. S.. The role of C2 domains in PKC signalingAdv. Exp. Med. Biol.Year: 201274066368310.1007/978-94-007-2888-222453964
67. Domin J.,Dhand R.,Waterfield M. D.. Binding to the platelet-derived growth factor receptor transiently activates the p85α–p110α phosphoinositide 3-kinase complex in vivoJ. Biol. Chem.Year: 1996271216142162110.1074/jbc.271.35.216148702949
68. Ooms L. M.,Horan K. A.,Rahman P.,Seaton G.,Gurung R.,Kethesparan D. S.,Mitchell C. A.. The role of the inositol polyphosphate 5-phosphatases in cellular function and human diseaseBiochem. J.Year: 2009419294910.1042/BJ2008167319272022
69. Song M. S.,Salmena L.,Pandolfi P. P.. The functions and regulation of the PTEN tumour suppressorNat. Rev. Mol. Cell. Biol.Year: 20121328329622473468
70. Hao Y.,Wang C.,Cao B.,Hirsch B. M.,Song J.,Markowitz S. D.,Ewing R. M.,Sedwick D.,Liu L.,Zheng W.,Wang Z.. Gain of interaction with IRS1 by p110α-helical domain mutants is crucial for their oncogenic functionsCancer CellYear: 20132358359310.1016/j.ccr.2013.03.02123643389
71. Backer J. M.,Myers M. G. Jr.,Shoelson S. E.,Chin D. J.,Sun X. J.,Miralpeix M.,Hu P.,Margolis B.,Skolnik E. Y.,Schlessinger J.,et al. Phosphatidylinositol 3′-kinase is activated by association with IRS-1 during insulin stimulationEMBO J.Year: 199211346934791380456
72. Janetopoulos C.,Devreotes P.. Phosphoinositide signaling plays a key role in cytokinesisJ. Cell. Biol.Year: 200617448549010.1083/jcb.20060315616908667
73. Jimenez C.,Portela R. A.,Mellado M.,Rodriguez-Frade J. M.,Collard J.,Serrano A.,Martinez A. C.,Avila J.,Carrera A. C.. Role of the PI3K regulatory subunit in the control of actin organization and cell migrationJ. Cell. Biol.Year: 200015124926210.1083/jcb.151.2.24911038173
74. Yamaguchi H.,Yoshida S.,Muroi E.,Yoshida N.,Kawamura M.,Kouchi Z.,Nakamura Y.,Sakai R.,Fukami K.. Phosphoinositide 3-kinase signaling pathway mediated by p110α regulates invadopodia formationJ. Cell. Biol.Year: 20111931275128810.1083/jcb.20100912621708979
75. Stephens L.,Williams R.,Hawkins P.. Phosphoinositide 3-kinases as drug targets in cancerCurr. Opin. Pharmacol.Year: 2005535736510.1016/j.coph.2005.03.00215963759
76. Janku F.,Lee J. J.,Tsimberidou A. M.,Hong D. S.,Naing A.,Falchook G. S.,Fu S.,Luthra R.,Garrido-Laguna I.,Kurzrock R.. PIK3CA mutations frequently coexist with RAS and BRAF mutations in patients with advanced cancersPLoS ONEYear: 20116e2276910.1371/journal.pone.002276921829508
77. Varnai P.,Rother K. I.,Balla T.. Phosphatidylinositol 3-kinase-dependent membrane association of the Bruton's tyrosine kinase pleckstrin homology domain visualized in single living cellsJ. Biol. Chem.Year: 1999274109831098910.1074/jbc.274.16.1098310196179


[Figure ID: F1]
Figure 1  Fluorescently labelled PI3Kα retains lipid kinase activity in vitro and in vivo

(A) 2 μg each Cy3-labelled ovalbumin (Cy3-ovalbumin) and Cy3-labelled PI3Kα (Cy3-p110αEEWT/p85α, Cy3-p110αEEE545K/p85α and Cy3-p110αEEH1047R/p85α) were mixed with 2 μg Alexa488-labelled GST-Grp1PH (Alexa488-GST-Grp1PH) then separated by SDS–PAGE. The fixed, unstained gel was visualized using a fluorescence gel scanner with a 532 nm laser and filters for Alexa488 (green) and Cy3 (red). (B) 200 ng purified, recombinant Cy3-labelled or unlabelled p110αEEWT/p85α, p110αEEE545K/p85α or p110αEEH1047R/p85α were assayed for phosphoinositide kinase activity. Reactions were stopped after 40 min using 1 M HCl. The difference in phosphoinositide kinase activity between each unlabelled and Cy-labelled PI3Kα was not significant (p>0.1). (C) 0.5 μM Cy3-ovalbumin, Cy3-p110αEEWT/p85α, Cy3-p110αEEE545K/p85α or Cy3-p110αEEH1047R/p85α were mixed with 0.5 μM Alexa488-Grp1PH and co-microinjected into adherent MDCK cells in DMEM containing 10% FBS. Cells were allowed to recover for 2 h post-microinjection, then fixed and stained with the nuclear stain, DAPI, and fluorescence associated with Cy3 (middle panel) or Alexa488 (lower panel) was visualized using confocal microscopy. Merged images of Z series projections of Cy3 (red), Alexa488 (green) and DAPI (blue) are shown (top panel). Graphs of Alexa488 fluorescence intensity along the yellow line (lower panel) demonstrate accumulation of Alexa488-GST-Grp1PH at the boundaries of the cell (green arrows). Scale bar=20 μm.

[Figure ID: F2]
Figure 2  Mutant or wild-type PI3Kα does not bind PM lipids in vitro or in vivo

(A) Liposomes derived from 25 μg/ml brain-derived cellular lipids (Folch fraction IV) or 12.5 μg/ml brain-derived cellular lipids plus 12.5 μg/ml mixed PIs were incubated with 5 μg/ml wild-type or mutant (E545K or H1047R) PI3Kα (upper panel) or 5 μg/ml GST-Grp1PH or GST (lower panel). Liposomes were pelleted by centrifugation and liposome-bound protein in 10% of the pellet fraction (P) and free/unbound protein in 10% of the supernatant fraction (S) was detected by immunoblotting using antibodies that recognize p85α or GST. (B) 4–500 pmol purified PS or a phosphoinositide was spotted onto a nitrocellulose membrane. Membranes were blocked then incubated with 10 nM wild-type or mutant (E545K or H1047R) PI3Kα (upper panel) or 10 nM GST-Grp1PH or GST-Akt PH (middle panel). Bound PI3Kα, GST-Grp1PH or GST-AktPH was detected by immunoblotting using antibodies that recognize p85α or GST. 0.2–5 ng PI3Kα or GST fusion proteins spotted directly onto nitrocellulose and detected using p85α or GST antibodies (lower panel) were used as positive controls for immunoblotting. (C) 0.5–2 μM Cy3-ovalbumin, Cy3-p110αEEWT/p85α, Cy3-p110αEEE545K/p85α or Cy3-p110αEEH1047R/p85α was microinjected into adherent MDCK cells expressing PM-localized GFP in DMEM containing 10% FBS. Cells were allowed to recover for 2 h post-microinjection, then fixed and washed in PBS. Fluorescence associated with Cy3 (red) or GFP (green) was visualized using confocal microscopy (upper panel) (scale bar=10 μm). The percentage of Cy3-PI3Kα fluorescence that was co-incident with GFP-PM marker fluorescence was quantified in 80 successive focal planes in individual cells using Imaris v7.4 software. The median, 25th and 75th percentiles of the percentage of Cy3-PI3Kα co-incident with the PM were calculated from measurements of a minimum of 25 individual cells from three independent experiments and plotted as a box-and-whiskers plot.

[Figure ID: F3]
Figure 3  Mutant PI3Kα is not localized to the PM to a greater extent or for an extended time upon cell stimulation compared with wild-type PI3Kα

0.5–2 μM Cy3-ovalbumin (○), Cy3-p110αEEWT/p85α (●), Cy3-p110αEEE545K/p85α (▲) or Cy3-p110αEEH1047R/p85α (■) was microinjected into adherent MDCK cells expressing membrane-localized GFP in DMEM containing 10% FBS. Cells were allowed to recover for 2 h post-microinjection, then starved for 4 h and imaged prior to stimulation and at 5, 15, 25, 35, 45 and 65 min post-stimulation with 10% FBS, 50 ng/ml EGF and 5 μg/ml insulin. The percentage of Cy3-PI3Kα fluorescence that was coincident with GFP-PM marker fluorescence was quantified in 25 successive focal planes in individual cells at each time point using Imaris v7.4 software and expressed as the ratio of the percentage of Cy3-PI3Kα that is co-localized with the PM marker at each time point compared with the percentage of Cy3-PI3Kα that is co-localized with the PM marker at time zero (T/T0). The means±S.E.M. of the T/T0 ratio was calculated from measurements of a minimum of five individual cells from three independent experiments at each time point. **=P<0.05 *=P<0.1 compared with time 0.

[Figure ID: F4]
Figure 4  The E545K oncogenic mutation is associated with increased number of cells with cell membrane protrusions

(A) 0.5–2 μM Cy3-ovalbumin, Cy3-p110αEEWT/p85α, Cy3-p110αEEE545K/p85α or Cy3-p110αEEH1047R/p85α were microinjected into adherent MDCK cells in DMEM containing 10% FBS. Cells were allowed to recover for 2 h post-microinjection, then starved for 4 h before stimulation with 10% FBS, 50 ng/ml EGF and 5 μg/ml insulin for 10 min. Unstimulated and stimulated cells were fixed, and the fluorescence associated with Cy3 was visualized using confocal microscopy. Merged images of Z series projections are shown. Arrows indicate protrusions. Scale bar=10 μm (B) 0.5–2 μM Cy3-ovalbumin, Cy3-p110αEEWT/p85α, Cy3-p110αEEE545K/p85α or Cy3-p110αEEH1047R/p85α were microinjected into adherent MDCK, Caco2 or iMMEC epithelial cells in media containing 10% FBS. Cells were treated as described in Figure 4(A) and the number of cells with either 0, 1 or greater than one protrusion was counted. Between 10 and 100 individual cells from at least two independent experiments were scored for each condition.

[Figure ID: F5]
Figure 5  The E545K oncogenic mutated form of PI3Kα specifically binds to constitutively active Cdc42

(A) The amount of purified, recombinant wild-type (wt) or oncogenic mutant (E545K or H1047R) PI3Kα bound to 1 μg GST, GST-V12 Cdc42, GST-V12 Rac1 or GST-V12 Ras in the presence of 10% FBS was measured by immunoblotting using an antibody that recognizes p85α. (B) 1 μg GST, GST-V12 Cdc42, GST-V12 Rac1 or GST-V12 Ras was incubated with increasing amounts of p110αEEWT/p85α (●), p110αEEE545K/p85α (▲) or p110αEEH1047R/p85α (■). The amount of bound PI3Kα was measured by immunoblotting using an antibody that recognizes p85α and the density of each band was quantified using ImageQuant software. Duplicate points were plotted as means±S.E.M. 1% of the amount of each PI3Kα and GST-fusion protein used in the binding assays (Inputs) was measured by immunoblotting using antibodies that recognize p85α or GST or ensure that equivalent levels of protein were used in each assay.

Article Categories:
  • Original Paper
Article Categories:
  • S2

Keywords: Cdc42, microinjection, mutation, oncogene, PI3K, PIK3CA.
Keywords: 2-ME, 2-mercaptoethanol, Akt, also called PKB (protein kinase B), BH, BCR-homology, cSH2, C-terminal SH2, DAPI, 4′,6-diamidino-2-phenylindole, DXMS, deuterium exchange mass spectrometry, EGF, epidermal growth factor, GAP, GTPase-activating protein, GFP, green fluorescent protein, GST, glutathione transferase, HRP, horseradish peroxidase, iMMEC, immortalized mouse mammary epithelial cells, iSH2, inter-SH2, MDCK, Madin–Darby canine kidney, NA, numerical aperture, nSH2, N-terminal SH2, PDGF, platelet-derived growth factor, PH, pleckstrin homology, PI, phosphatidylinositol, PI3K, phosphoinositide 3-kinase, PIP2, phosphoinositide-4,5-disphosphate, PIP3, phosphoinositide-3,4,5-trisphosphate, PM, plasma membrane, PS, phosphatidylserine, PTEN, phosphatase and tensin homologue deleted on chromosome 10, pY, phosphotyrosine, RTK, receptor tyrosine kinase, SH, Src homology.

Previous Document:  Total Synthesis of the Akuammiline Alkaloid Picrinine.
Next Document:  Effect of Ceramic Thickness and Shade on Mechanical Properties of a Resin Luting Agent.