LL5beta Is a Phosphatidylinositol (3,4,5)-Trisphosphate Sensor That Can Bind the Cytoskeletal Adaptor, gamma -Filamin*,

Varuni ParanavitaneDagger §, W. John Coadwell, Alicia Eguinoa||, Phillip T. HawkinsDagger **, and Len StephensDagger DaggerDagger

From the Dagger  Inositide Laboratory,  Bioinformatics Group, The Babraham Institute, Cambridge CB2 4AT, United Kingdom and the || Department of Immunology and Oncology, National Centre for Biotechnology, Madrid 28045, Spain

Received for publication, August 15, 2002, and in revised form, October 3, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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We identified a potential phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) binding pleckstrin homology domain in the data bases and have cloned and expressed its full coding sequence (LL5beta ). The protein bound PtdIns(3,4,5)P3 selectively in vitro. Strikingly, a substantial proportion of LL5beta became associated with an unidentified intracellular vesicle population in the context of low PtdIns(3,4,5)P3 levels produced by the addition of wortmannin or LY294002. In addition, expression of platelet-derived growth factor-receptor mutants unable to activate type 1A phosphoinositide 3-kinase (PI3K) or serum starvation in porcine aortic endothelial cells lead to redistribution of LL5beta to this vesicle population. Importantly, pleckstrin homology domain mutants of LL5beta that could not bind PtdIns(3,4,5)P3 were constitutively localized to this vesicle population. At increased PtdIns(3,4,5)P3 levels, LL5beta was redirected to a predominantly cytoplasmic distribution, presumably through a PI3K-dependent block on its targeting to the vesicular compartment. Furthermore, at high, hormone-stimulated PtdIns(3,4,5)P3 levels, it became significantly plasma-membrane localized. The distribution of LL5beta is thus dramatically and uniquely sensitive to low levels of PtdIns(3,4,5)P3 indicating it can act as a sensor of both low and hormone-stimulated levels of PtdIns(3,4,5)P3. In addition, LL5beta bound to the cytoskeletal adaptor, gamma -filamin, tightly and in a PI3K-independent fashion, both in vitro and in vivo. This interaction could co-localize heterologously expressed gamma -filamin with GFP-LL5beta in the unidentified vesicles.

    INTRODUCTION
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Phosphoinositide 3-kinases (PI3Ks)1 3-phosphorylate phosphoinositides. There are three classes of PI3Ks. The type I enzymes seem to act as PtdIns(4,5)P2 3-kinases in vivo; they can be activated by a variety of close-to-receptor transduction events and drive accumulation of PtdIns(3,4,5)P3 in the inner leaflet of the plasma membrane. This PtdIns(3,4,5)P3 serves as signal recruiting proteins from the cytosol that possess modules, typically PH domains, capable of binding its head group (1).

There are a variety of reagents that can be used to inhibit PI3K activity. Most widely used are wortmannin (2) and LY294002 (3); both of which potently inhibit nearly all classes of PI3K and hence cannot generally be used to implicate a particular PI3K in a process. More specific are receptor tyrosine kinase Tyr right-arrow Phe mutants. A number of receptor tyrosine kinases (relevant here, the PDGF beta -receptor) are capable of binding type IA PI3Ks at specific tyrosine residues that become phosphorylated following ligand binding (4). Mutation of these tyrosines to phenylalanine blocks type IA PI3K binding and activation but does not affect association of other effectors (1, 5). Stable, clonal cell lines have been created overexpressing wild-type PDGF-beta receptors or (Y740F/Y751F) PDGF-beta receptors (6) that have allowed the impact of selectivity blocking type IA PI3K activation to be assessed in vivo (7).

There is now a substantial family of PtdIns(3,4,5)P3-binding proteins that have been shown to translocate to the plasma membrane in response to receptor stimulation of type I PI3K activity, including PKB (8, 9), DAPP-1 (10-13), PDK-1 (14), ARNO (15), ARAP-3 (16), and GRP1 (17). All of the above PI3K effectors bind 3-phosphorylated lipids via a PH domain. PH domains are protein modules of ~100 amino acids that bind a variety of ligands ranging from inositol phosphates and phosphoinositides to possibly G-protein beta gamma -subunits (18, 19). Those PH domains that bind phosphoinositides specifically form a subset that can be recognized via a consensus sequence of basic residues implicated in binding. Initially this concept was based solely on a limited number of sequence alignments, however, as more phosphoinositide binding PH domains were characterized the early consensus has been evolved and further validated by work that has described the structure of a number of PH domains, some with phosphoinositide-based ligands bound (20). Many different types of proteins seem to use PH domains as phosphoinositide binding modules including enzymes (e.g. PKB, BTK, Vav, and PDK-1) and adaptor proteins (e.g. DAPP-1).

It is generally thought that phosphoinositide-dependent shifts in signaling proteins from predominantly cytosolic to membrane distributions are, in some way, activating. In the case of PKB this seems to result from co-localization with its upstream regulator PDK-1 combined with increased availability of the site PDK phosphorylates (threonine 308 in PKBalpha ) as a result of PtdIns(3,4,5)P3 binding (21). For DAPP-1, which has been claimed to bind PLC-gamma (11), it is presumably the relocation of the PLC-gamma , to the cell surface and the location of its phospholipid substrate that could be relevant.

Through the application of PI3K inhibitors, some of which have been described above, it has become clear that type I PI3K signaling regulates a huge variety of cellular responses. One of the most widely important of these is cell survival (22). In essence PI3Ks and PKB are thought to supply a signal from some receptors that block cells from undergoing apoptosis (23). These signals operate constitutively in the presence of relatively low levels of survival factors but their inactivation upon factor withdrawal leads rapidly to apoptosis (1).

Filamins are actin-binding proteins that act to stabilize large three-dimensional actin networks, through their ability to dimerize (24, 25). Mammals can make alpha -, beta -, and gamma -filamins that possess different tissue distributions. The filamins also seem to bind to a variety of membrane-associated structural or signaling proteins, typically via a region of the molecule that does not interfere with the actin-binding or dimerization domains and these include, cytoplasmic tails of integrins and receptors. Hence filamins can be seen as structural proteins contributing directly to the mechanical properties of the cytoskeleton but also as points at which a variety of cell-surface signals can converge on the actin cytoskeleton.

In this article we describe the identification, cloning, and expression of a PH domain-containing protein that binds PtdIns(3,4,5)P3 and behaves as a PtdIns(3,4,5)P3-effector but also associates with gamma -filamin and undergoes a novel redistribution in response to reductions in PtdIns(3,4,5)P3 levels.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Cloning of Human LL5beta and Relevant Constructs-- The cDNA encoding the full-length open reading frame of LL5beta was obtained via the I.M.A.G.E. clones 531882 (accession numbers, aa116053), 208876 (accession number, h63748), and 82052 (accession number, t68150), which were all obtained from the I.M.A.G.E Consortium (UK HGMP Resource Centre, Hinxton, United Kingdom). The full-length open reading frame of LL5beta (3762 bp) was ligated in-frame with an amino-terminal Myc or Glu-Glu tag into pCMV3, pEGFP (Clontech), or the pGEX 4T1 (Amersham Biosciences) bacterial expression vectors. Point mutants at the PtdIns(3,4,5)P3 binding motif (K1162A and R1163A), LL5beta Delta PH (residues 1-1138), and the isolated PH domain (residues 1138-1253) were generated by a PCR-based mutagenic strategy and ligated in-frame with an amino-terminal Myc or Glu-Glu (Glu-Glu) tag into pCMV3, pEGFP, and the pGEX4T1 expression vectors. All inserts were verified by sequence analysis (Babraham Technix). Full-length gamma -filamin cDNA was a kind gift from Dominic Chung (University of Washington).

Cell Culture-- PAE cells expressing wild-type or mutant (Y740F/Y751F) PDGF-beta receptor were maintained in F-12 nutrient mixture (Ham's F-12, Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS, Invitrogen). COS-7 cells were maintained in DMEM (Invitrogen) supplemented with 10% HI-FBS (Invitrogen). All cells were maintained at 37 °C in a 5% CO2 humidified atmosphere and were not allowed to reach confluence.

Transient Transfection-- PAE and COS-7 cells were transfected by electroporation with 20 µg of total plasmid DNA as described previously (10). Transfected PAE cells were plated onto glass coverslips (~8 × 104 cells per coverslip) in Ham's F-12 media containing 10% HI-FBS for 12 h, then serum starved for 6-8 h in F-12 media supplemented with 0.5% fatty acid-free BSA, in the presence of 1 units/ml penicillin and 0.1 mg/ml streptomycin (Invitrogen). Transfected COS-7 cells were plated onto glass coverslips (~8 × 104 cells per coverslip) in DMEM containing 10% FBS for 12-16 h, then serum starved for 12 h in DMEM supplemented with 0.5% fatty acid-free BSA, in the presence of 1 unit/ml penicillin and 0.1 mg/ml streptomycin.

Cell Detachment-- Twelve hours following transfection of PAE cells with GFP-LL5beta , cells were washed with F-12 media either containing 10% FBS or 0.1% fatty acid-free BSA for 2 h. Cells were trypsinized, treated with trypsin inhibitor (Sigma), and left in suspension (end-on end rotation) for either 30 min or 2 h at 37 °C in Hepes-buffered F-12 media (pH 7.2). Cells were then washed, resuspended in PBS, and cytospun (300 rpm for 3 min) onto coverslips, fixed in paraformaldehyde, and examined under fluorescence microscopy.

Immunofluorescence-- Transfected cells were grown on coverslips and serum-starved as described above. Cells were then washed and incubated at 37 °C for 30 min in F-12 media containing 0.5% fatty acid-free BSA, 30 mM Hepes (pH 7.4), and 1 unit/ml penicillin and 0.1 mg/ml streptomycin prior to stimulation for the indicated times. Cells were treated with human PDGF (B-B10 ng/ml) (Autogen Bioclear) for 5 min, latrunculin B (10 µg/ml) (Sigma) for 10 min, or wortmannin (100 nM, Sigma) for 15 min. Following treatment, cells were promptly fixed by incubating with buffer containing 4% paraformaldehyde for 15 min at room temperature followed by 3 washes with 150 mM Tris (pH 7.4). Depending on the requirement of the primary antibody, in some cases, cells were fixed in ice-cold 100% methanol for 5 min and rinsed in dH2O. For cells transfected with GFP constructs, coverslips were then rinsed in dH2O and mounted on slides using Aqua Polymount (Polysciences Inc.). A series of dyes for detection of mitochondria (mitotracker red, 100 nM, 15 min; Molecular Probes), lysosomes (lysotracker red, 50 nM, 30 min; Molecular Probes), and the transferrin receptor conjugated to Texas Red dye (marker for endosomes, 3 min for early endosomes and 10 min for late endosomes) were added to live cells expressing GFP contructs prior to fixation. For all other immunofluorescence studies, cells were fixed and permeabilized in PBS, 0.1% Triton X-100 for 10 min, washed three times in PBS, then incubated with PBS + 1% BSA (w/v) for 30 min at room temperature before being incubated with anti-Myc monoclonal antibody anti-clathrin polyclonal antibody (Santa Cruz Biotechnology), anti-EEA1 monoclonal antibody (Transduction Laboratories), anti-caveolin-1 antibody (Santa Cruz Biotechnology), TRITC-phalloidin (Sigma), anti-alpha -tubulin (Sigma), anti-vinculin (Sigma), or anti-PMP70 antibody (Sigma), as indicated for 1 h at room temperature. Coverslips were washed three times for 5 min in PBS + 0.5% BSA and then incubated with the appropriate secondary fluorescein isothiocyanate/RITC-conjugated antibody (1 h at room temperature). Coverslips were then washed four times in PBS + 0.5% BSA (5 min), PBS (5 min), then rinsed in dH2O before being mounted onto slides, allowed to dry, and viewed under a Zeiss Axiophot fluorescence microscope. Images were captured using a SPOT digital camera (Diagnostic Instruments).

Confocal Image Analysis of Live Cells-- Cells were transfected, cultured on sterile glass coverslips, and treated as described above. For imaging, coverslips were mounted on the stage of an Olympus 1 × 70 microscope interfaced with an UltraView confocal system. The cells were imaged at 37 °C using a thermostatically controlled cell chamber and incubated in PAE salt solution (25 mM Hepes, pH 7.4, 1.8 mM CaCl2, 5.37 mM KCl, 0.81 mM MgSO4, 112.5 mM NaCl, 25 mM D-glucose, 1 mM NaHCO3, and 0.1% (w/v) fatty acid-free BSA).

Time-lapse images of GFP-transfected cells were obtained using an UltraView confocal microscope (PerkinElmer Life Sciences). GFP fluorescence was excited at 488 nm and the emission was collected at wavelengths >505 nm using a long pass filter. Typically, 12 bit ~600 × 400 pixel images were captured every 2-3 s.

Northern Blot Analysis-- A 745-bp fragment encoding the unique NH2 terminus of LL5beta was used as a probe for Northern blot analysis. The probe was labeled with [gamma -32P]dCTP (Amersham Biosciences) and the Prime-a-Gene labeling system (Promega). The radioactive probe was applied to multiple tissue Northern blots containing RNA from various human tissues obtained from Clontech and carried out according to the recommended protocol.

Phosphoinositide Binding Specificities of LL5beta -- 1 × 107 COS-7 cells were transfected by electroporation with 20 µg of the DNA construct encoding Myc-tagged LL5beta . Cells were allowed to recover in DMEM containing 10% HI-FBS in two 15-cm diameter dishes for 48 h and were washed and lysed with 5 ml/dish of lysis buffer (1% Nonidet P-40, 20 mM Hepes (pH 7.5), 0.12 M NaCl, 5 mM EDTA, 5 mM EGTA, 5 mM beta  glycerophosphate, 1 mM orthovanadate, 10 mM NaF). Lysates were centrifuged (190,000 × gav for 30 min). Samples of the supernatants (0.5 ml) were mixed with 50 µM free dipalmitoyl forms of competing phosphoinositides (16) on ice for 10 min. They were transferred to 5-µl packed PtdIns(3,4,5)P3 beads (16) in lysis buffer and mixed gently for 1 h. Sedimented beads were washed (4 times, <15 min) in modified lysis buffer (0.1% Nonidet P-40 rather than 1% Nonidet P-40). Proteins were eluted with SDS sample buffer, separated by SDS-PAGE, transferred onto polyvinylidene difluoride membrane, and detected by immunoblotting (anti-Myc (monoclonal)).

Purification of LL5beta Interacting Partners-- Recombinant proteins, GST-LL5beta and GST-LL5beta Delta PH, were expressed in bacteria and purified on GS-Sepharose beads. The glutathione-Sepharose beads bound to GST LL5beta and GST-LL5beta Delta PH were used to "pull down" interacting proteins from COS-7 cell lysates. Previously seeded COS-7 cells were washed twice in PBS and left in Met- and Cys-free DMEM for 35 min. Then, 0.2 mCi of [35S]methionine and [35S]cysteine (Amersham Pharmacia Biotech) was added 16 h prior to lysis. Lysates were centrifuged for 10 min at 4 °C at 13,000 × gav and supernatants were transferred to 2 µg of GST LL5beta and GST-LL5beta Delta PH purified on 30 µl of glutathione-Sepharose beads, and allowed to mix for 2 h at 4 °C. Four washes were then carried out in lysis buffer (30 mM Hepes (pH 7.4), 10 mM NaF, 5 mM beta -glycerophosphate, 1 mM MgCl2, 1 mM EGTA, 1% Nonidet P-40, 110 mM NaCl, 1 mM dithiothreitol) followed by a final wash in modified lysis buffer containing 0.25% Nonidet P-40 instead of 1% Nonidet P-40. Proteins were eluted in SDS sample buffer, separated by SDS-PAGE, the gel dried down onto Whatman paper, and exposed to x-ray film for 2 days at -70 °C.

In preparation for the trypsin digestion of the interacting protein, the above pull-down method was carried out on a larger scale without 35S labeling; the SDS-PAGE gel was stained with Coomassie Blue and the relevant band cut out and further cut into gel slices of ~1 mm3. This was washed in 3× 50% acetonitrile, 25 mM NH4 bicarbonate (pH 8), then soaked in 100% acetonitrile for 5 min, and dried in a 5200 centrifugal concentrator for 20 min. 10 µg of excision grade trypsin (Sigma) in 25 mM NH4 bicarbonate (pH 8) was added to the dried gel slices and digested at 37 °C for 16 h. Extraction of peptides was carried out by soaking the gel slice in 50% acetonitrile, 5% trifluoroacetic acid for 30 min with gentle agitation. A second extraction was carried out as above and the combined extracts were dried as before for 1 h. The dried sample was then reconstituted by adding 4 µl of 50% acetonitrile, 0.1% trifluoroacetic acid. The generated peptides were then analyzed at Applied Biosystems by a mass spectrometer (Qstar Pulsar I).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Cloning, Tissue Distribution, and Lipid Binding Properties of LL5beta -- Using the consensus Lys-Xaa-Gly/Ser-Xaa(6-11)-Arg/Lys-Xaa-Arg-Phe/Leu in 1996 (26) we identified a partial human protein sequence in the NCB1/EMBL protein expressed sequence tag data base encoding the COOH-terminal domain of a protein, previously called LL5 (rat) (27), that we predicted would bind PtdIns(3,4,5)P3. Sequencing the relevant Image clone (82052) revealed upstream overlaps with further expressed sequence tags and iteration identified a potential upstream start codon with an in-frame stop immediately upstream. Although we began searching for the human orthologue of rat LL5, we ended up with the open reading frame of a paralogue of the human orthologue of rat LL5. The predicted open reading frame was for a 160-kDa protein (see Fig. 1A) and a full-length clone (accession number AJ496194) was created from Image clones 531883, 208876, and 82052. The protein contains a single spectrin repeat and a COOH-terminal PH domain. The same PH domain was also identified as a potential PtdIns(3,4,5)P3-binding protein in a screen by Isakoff et al. (26) and subsequently by Dowler et al. (28) (who called the host protein LL5beta and a closely related molecule LL5alpha , which is the human orthologue of the rat LL5). We will retain the nomenclature Dowler et al. (28) applied to the PH domain of this protein and hence will term it LL5beta . LL5alpha and LL5beta are different proteins that occur at different locations and have less than 70% identity at the protein level. A 745-bp probe from the NH2-terminal region of LL5beta , which would not recognize LL5alpha , was used to analyze a Northern blot prepared from human tissues. A 6-kb band was detected in a number of tissues with the highest levels found in heart, kidney, and placenta (see Fig. 1B).


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Fig. 1.   Amino acid sequence and tissue distribution of LL5beta . A, amino acid sequence of human LL5beta . The sequence is shown in single-letter code and residue numbers are indicated. The single spectrin repeat (residues 671-778) is in red and the PH domain (residues 1143-1246) is highlighted in gray with the key conserved residues in the PtdIns(3,4,5)P3 binding motif in pink. B, tissue distribution of LL5beta by Northern blot analysis. A multiple tissue Northern blot (Clontech) containing polyadenylated RNA from the indicated human tissues was probed for LL5beta expression using a 32P-labeled, unique, NH2-terminal fragment of LL5beta (745 bp). The LL5beta probe was observed to hybridize to a transcript of the predicted size (6 kb).

Expression plasmids encoding NH2-terminal Myc- and GFP-tagged LL5beta (and various constructs, see below) were prepared and transiently transfected into COS-7 and PAE cells. Anti-Myc immunoblots of appropriately transfected PAE or COS-7 cells revealed a 160-kDa protein (see Fig. 2A).


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Fig. 2.   Immunoblot of Myc-tagged full-length and in vitro lipid binding specificity of LL5beta . A, immunoblot of Myc-tagged full-length LL5beta . Myc-LL5beta was transiently transfected into PAE cells. Cell lysates were immunoblotted with anti-Myc antibodies. B, in vitro lipid binding specificity of LL5beta . Recombinant, Myc-tagged LL5beta prepared in COS-7 cells were mixed with 50 µM dipalmitoyl forms of free, competing phosphoinositides, PtdIns(3,4,5)P3, PtdIns(3,4)P2, PtdIns(4,5)P2, PtdIns(3,5)P2, PtdIns(3)P, PtdIns(4)P, and PtdIns(5)P for 10 min and then transferred to PtdIns(3,4,5)P3 beads, allowed to mix for 1 h, washed, and proteins were eluted with SDS sample buffer. The first lane was loaded with a sample of Myc-LL5beta equivalent to 1% of that included in the binding assays. Myc-tagged LL5beta was detected by immunoblotting with anti-Myc antibodies. C, binding of Myc-LL5beta Delta PH and Myc-LL5beta -K1162A/R1163A expressed in COS-7 cell lysates to PtdIns(3,4,5)P3 beads. Proteins were detected by immunoblotting with anti-Myc antibodies.

We analyzed the lipid binding specificity of LL5beta in vitro. Cytosolic fractions were prepared from COS-7 cells transfected with Myc-LL5beta and mixed with Affi-Gel beads covalently attached to PtdIns(3,4,5)P3 (16). The beads were washed, and bound proteins were eluted and immunoblotted with anti-Myc antibody. This revealed that ~1-2% of input LL5beta was recovered on the beads. Various competing, free phosphoinositides were added to the binding reactions and this revealed that PtdIns(3,4,5)P3 most effectively displaced LL5beta from the beads (see Fig 2B); indicating that LL5beta can bind PtdIns(3,4,5)P3 selectively under these assay conditions. Mutations in the PH domain predicted to disrupt lipid binding (LL5beta Delta PH and LL5beta -K1162A/R1163A) abolished LL5beta binding to the PtdIns(3,4,5)P3 beads (Fig. 2C).

Distribution of LL5beta in Cells-- We transiently expressed Myc- or GFP-LL5beta in PAE cells stably overexpressing the PDGFbeta receptor. In the presence of serum or after only short periods of serum starvation (up to 6 h), the LL5beta constructs appeared predominantly cytosolic in fixed cells. After prolonged serum starvation (8 h or more) a significant proportion of Myc- or GFP-LL5beta become particulate apparently at the expense of the cytosolic pool in both living or fixed cells. After approximately 6 h of serum starvation, when the protein was predominantly cytosolic, stimulation with PDGF resulted in a partial translocation of both Myc- and GFP-LL5beta to the edge of the cell (Fig. 3). This event was observed in both living and fixed cells using confocal and standard epifluorescence microscopes. The translocation was apparently more prolonged than that displayed by proteins such as DAPP-1 or PKB studied under similar conditions. Furthermore, it appeared that the peripheral accumulation of LL5beta constructs correlated with reductions in both its cytosolic and particulate pools (also see below). PAE cells were similarly transfected with a GFP-tagged form of the isolated PH domain of LL5beta . It did not translocate to the edge of the cell in response to PI3K stimulation. However, we note that although full-length GAP1m translocates to the cell membrane in a PI3K-dependent manner, the isolated PH domain does not translocate when expressed as an independent module.2 In an attempt to assess the PI3K dependence of this response we preincubated PAE cells transiently expressing Myc- or GFP-LL5beta with LY294002 or wortmannin, however, there was a profound redistribution of both constructs in response to the PI3K inhibitors alone that was observed in both living (GFP) or fixed cells (both tags). The cytosolic levels of GFP-LL5beta were very substantially reduced and an intracellular vesicular compartment, apparently identical to that seen in cells after prolonged serum starvation, became decorated (Fig. 4A, see also Supplementary Material for a video showing the effects of wortmannin on the distribution of GFP-LL5beta in living PAE cells). We have not seen this phenomenon before in the context of similar experiments with PAE cells studying proteins such as PDK-1, PKB, ARAP-3, PRex-1, and DAPP-1 (10, 14, 16, 29).


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Fig. 3.   The effects of PDGF stimulation on the subcellular localization of GFP-LL5beta in PAE cells. PAE cells were transiently transfected with GFP-LL5beta . After recovery and serum starvation, the cells were stimulated with PDGF (10 ng/ml). A, live cells were viewed with a confocal microscope. Images were captured at the indicated times after PDGF stimulation commenced. B, cells were fixed at the indicated times after PDGF stimulation commenced and viewed under a confocal microscope.


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Fig. 4.   Effects of inhibition of PI3K activity and serum starvation on the localization of GFP-LL5beta . A, PAE cells were transiently transfected with GFP-LL5beta . After recovery and serum starvation, the cells were either untreated, or treated with wortmannin (100 mM for 15 min), then fixed in paraformaldehyde and viewed with a fluorescence microscope. B, PAE cell transiently transfected with GFP-LL5beta -K1162A/R1163A. C, PAE cells were transiently transfected with GFP-LL5beta . Following 12 h recovery, cells were washed with media containing either 10% serum or 0.1% fatty acid-free BSA. After 2 h, cells were detached by trypsinization and left in suspension in media either containing 10% serum or 0.1% fatty acid-free BSA for a further 30 min or 2 h. Cells were then washed and cytospun onto coverslips, fixed in paraformaldehyde, and examined with a fluorescence microscope. The panel on the left shows GFP-LL5beta localization in the presence of serum (2 h + 2 h detached). The panel on the right shows GFP-LL5beta localization to vesicles in the absence of serum (0.1% fatty acid free-BSA for 2 h + 2 h detached). D, a summary of the data from the experiment described in B. 300 cells were counted and scored for the presence of vesicular GFP-LL5beta . The conditions and time of detachment are indicated. The values shown are mean ± S.D. from three different experiments.

Interestingly, with PAE cells transiently expressing GFP-LL5beta in the presence of 30 µM LY294002 (where the inhibition of PI3Ks was substantial but not complete), PDGF stimulation caused a significant redistribution of GFP-LL5beta from the vesicular pool into the cytoplasmic fraction without a clearly detectable accumulation near the plasma membrane. Transient expression of GFP-LL5beta in a PAE cell line expressing (Y740F/Y751F)-PDGF-beta -receptors (unable to bind and activate type I PI3Ks) revealed that GFP-LL5beta was constitutively associated with intracellular vesicles in the absence of wortmannin or LY294002. In an attempt to assess whether other procedures, potentially capable of reducing cellular PtdIns(3,4,5)P3 levels, could cause this shift of LL5beta constructs into a particulate compartment, we serum-starved and/or detached PAE cells transiently expressing GFP-LL5beta and held them in suspension. Both treatments significantly increased the proportion of GFP-LL5beta in the vesicular compartment, this was seen most clearly in cells that were detached and in the absence of serum (Fig. 4, C and D).

We examined the distribution of GFP-LL5beta in transiently transfected COS-7 cells to check whether this phenomenon is cell-type specific. We found that the construct was very largely cytosolic in both living and fixed cells and treatment with PI3K inhibitors wortmannin or LY294002 lead to a reduction in cytosolic staining and the decoration of an intracellular vesicular compartment (data not shown).

We examined the distribution of GFP-LL5beta Delta PH and GFP-LL5beta -K1162A/R1163A (double point mutation in the PH domain predicted to abolish PtdIns(3,4,5)P3 binding) in PAE (Fig. 4B) and COS-7 cells. Both constructs adopted a constitutive vesicular distribution in both PAE and COS-7 cells. These distributions in PAE cells were unaffected by PDGF (not shown).

We also tested whether Myc-LL5beta -K1162A/R1163A (detected with a RITC-labeled 2° antibody) co-localized with GFP-LL5beta in wortmannin-treated PAE cells that express the above constructs. The two constructs co-localized (not shown).

Together these results suggest that overexpressed LL5beta can translocate to the plasma membrane in response to PI3K activation but that under conditions of low PtdIns(3,4,5)P3, LL5beta becomes associated with a vesicular compartment. This does not appear to be an artifact of inhibition of PI3K activity as a number of inhibitory strategies are effective nor is the vesicular compartment made up of aggregated protein because LL5beta enters the compartment rapidly (15 min), can apparently move back into the cytosolic compartment under certain conditions, and the decorated vesicles move actively around the cell in a manner akin to vesicles like endosomes (see Supplementary Materials). Furthermore, our results with the Delta PH and LL5beta -K1162A/R1163A constructs suggest that it is likely that this process represents the association of LL5beta with a pre-existing organelle (unless they are formed specifically in the presence of these constructs) and that the key event is that LL5beta "perceives" that cellular PtdIns(3,4,5)P3 is low, rather than the levels of PtdIns(3,4,5)P3 are actually low.

We consider the best working explanation for these data is that even at relatively low cellular levels of PtdIns(3,4,5)P3 LL5beta can cycle on and off the plasma membrane from the cytosol in a PH domain-dependent manner (without substantial accumulation at the plasma membrane) and that this process leads to modification of LL5beta (e.g. phosphorylation or association of a protein) that prevents it becoming localized into the vesicular compartment and has a lifetime of roughly 15 min. The cytosolic pool of LL5beta can undergo a net translocation to the plasma membrane in response to receptor activation of type I PI3Ks.

The Nature of the Vesicle Compartment That Can be Decorated by LL5beta -- The work we described above suggests that the "vesicular" LL5beta is unlikely to be a aggregated/denatured protein. This view is also supported by the dynamic, jittering movements of GFP-LL5beta -associated structures in the presence of wortmannin or of GFP-LL5beta -K1162A/R1162A decorated structures. We attempted to co-localize GFP-LL5beta with a variety of markers in wortmannin-treated PAE cells. We used Texas Red-conjugated transferrin to label early (3 min incubation with cells) and late (10 min incubation with cells) endosomes, an antibody against early endosomal autoantigen 1 as an alternative marker for early endosomes, lysotracker as a marker for lysosomes, anti-caveolin antibodies to identify caveolae, anti-clathrin antibodies to decorate clatherin-coated pits, anti-PMP70 to label peroxysomes, mitotracker to label mitochondria, anti-vinculin antibodies to identify focal adhesions, and phalloidin to identify filamentous actin fibers. The GFP-LL5beta did not co-localize with any of these markers (see Supplementary Material).

We used latrunculin B to disassemble actin fibers in PAE cells. It had no effect on the distribution of GFP-LL5beta -decorated vesicles (see Supplementary Material). We co-transfected cells with Arf-6 and GFP-LL5beta and localized the Arf-6 with anti-Arf-6 antibodies and RITC secondary antibodies; the constructs did not co-localize (see Supplementary Material). We have previously observed that DAPP-1 becomes localized to an endosomal compartment in the presence of PDGF. We co-transfected PAE cells with GFP-DAPP-1 and Myc-LL5beta -K1162A/R1163A, stimulated with PDGF and detected the LL5beta construct via an anti-Myc antibody and a RITC secondary antibody. The internalized GFP-DAPP-1 did not co-localize with the vesicular LL5beta construct (data not shown). Finally we used a dominant-negative dynamin construct that we have previously used to establish that DAPP-1 is internalized in a dynamin-sensitive fashion and is an effective inhibitor of dynamin-mediated membrane internalization (dynamin and dynamin mutant was a kind gift of H. McMahon). In an experiment where the dynamin point mutant blocked internalization of co-transfected GFP-DAPP-1 in response to PDGF it had no effect on the formation or distribution of GFP-LL5beta -decorated structures in the presence of wortmannin (data not shown). We have not yet positively identified the vesicle compartment that is labeled by LL5beta constructs although the number markers we have failed to co-localize suggest that it is a tightly defined subpopulation.

LL5beta -binding Proteins-- In the context of our hypothesis that the PI3K-dependent redistribution of LL5beta to a vesicular compartment might be dependent/blocked by an LL5beta -associated protein we attempted to isolate potential binding partners. We prepared GST-LL5beta and GST-LL5beta Delta PH in bacteria and derived glutathionine-Sepharose beads loaded with them, GST alone, or GST-SHIP-1 as a control. These protein-loaded beads were mixed with aliquots of lysates made from [35S]methionine-labeled COS-7 cells, washed, the bound protein was resolved by SDS-PAGE, and the gel was dried and autoradiographed. A 260-kDa protein was recovered specifically with GST-LL5beta and GST-Delta PH-LL5beta (Fig. 5A). Two-dimensional isoelectric focusing and SDS-PAGE could not further resolve the 260-kDa protein band (not shown). The preparation was scaled up without [35S]methionine and the final one-dimensional SDS-PAGE gel was stained with Coomassie Brilliant Blue (Fig. 5B). The 260-kDa protein band was excised, in-gel digested with trypsin, and the resulting peptides were ultimately analyzed by electrospray mass spectrometry (Q star Pulsar i). Four peptides were selected for fragmentation and the patterns of the m/z ratios were used to reconstruct their sequences. Those sequences were used to interrogate the NRDB (nonredundant data base maintained by the European Bioinformatics Institute) and they identified gamma -filamin. This assignment was confirmed by a gamma -filamin-specific monoclonal antibody and a pan-filamin antibody preparation in immunoblots of the 260-kDa protein eluted from the GST-LL5beta affinity supports (Fig. 5C).


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Fig. 5.   Identification of a LL5beta interacting protein. A, isolation of an LL5beta -binding protein from 35S-GST, GST-SHIP-1, GST-LL5beta Delta PH, and GST-LL5beta were expressed and purified from bacteria on glutathione-Sepharose beads and used to affinity purify any interacting proteins from lysates of 35S-labeled COS-7 cells. Pull-downs were washed, eluted with SDS sample buffer, resolved by SDS-PAGE, and autoradiographed. The autoradiogram shows the interacting protein, above the 220-kDa marker pulled down by both GST-LL5beta Delta PH and GST-LL5beta but not GST nor GST-SHIP-1. B, Coomassie detection of the LL5beta interacting protein affinity purified from COS-7 cell lysate. C, immunodetection of LL5beta interacting protein with independent anti-filamin antibodies. LL5beta interacting protein purified from COS-7 cell lysate was immunoblotted with RR90, a filamin antibody that recognizes all three filamin isoforms (left panel) and antibody that recognizes specifically gamma -filamin (right panel).

We tested whether gamma -filamin could interact with LL5beta in vivo. (Glu-Glu)-tagged LL5beta and gamma -filamin were transfected individually and together into COS-7 cells (note, transfection with gamma -filamin did not substantially increase the amount of total gamma -filamin in the cells). Lysates were prepared and immunoprecipitated with anti-(Glu-Glu) monoclonal antibody covalently attached to protein G-Sepharose. gamma -Filamin was only immunoprecipitated in the presence of (Glu-Glu)-LL5beta (Fig. 6A). About 5% of the total gamma -filamin was recovered in the washed (Glu-Glu)-LL5beta immunoprecipitates. This indicates that gamma -filamin and LL5beta can interact in vivo. We examined the distribution of gamma -filamin and its relationship to LL5beta in COS-7 (Fig. 6, B and C) and PAE cells (data not shown). We transiently transfected cells with GFP-LL5beta and/or gamma -filamin (detected by gamma -filamin-specific antibody and a RITC-labeled secondary antibody). In cells co-transfected with GFP-LL5beta and gamma -filamin in the presence of wortmannin, it was clear that 30-40% of the gamma -filamin-positive structures were also positive for the GFP-LL5beta vesicular compartment (Fig. 6B). In cells transfected with gamma -filamin alone, or co-transfected with GFP-LL5beta and gamma -filamin but not treated with wortmannin, the gamma -filamin adapted a punctate distribution that was insensitive to wortmannin and were clearly smaller than those that contained both GFP-LL5beta and gamma -filamin (Fig. 6C). In cells co-transfected with gamma -filamin and GFP-LL5beta and treated with wortmannin, the structures that were only positive for gamma -filamin were the same size as the gamma -filamin-positive structures in cells transfected with gamma -filamin alone. These results suggest that LL5beta and gamma -filamin can co-localize in both COS-7 and PAE cells in the presence of wortmannin and that gamma -filamin localization is dictated by LL5beta and wortmannin. This indicates that the interaction between LL5beta and gamma -filamin (in the presence of wortmannin) leads to the targetting of gamma -filamin into the vesicular compartment by LL5beta and that gamma -filamin is not responsible for directing or blocking the movement of LL5beta into a vesicular compartment.


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Fig. 6.   Interaction of gamma -filamin and LL5beta in vivo assessed by immunoprecipitation and immunocytochemistry. A, interaction of gamma -filamin with (Glu-Glu)-tagged LL5beta in vivo. (Glu-Glu)-LL5beta and gamma -filamin were transfected individually and together into COS-7 cells. The cell lysates were then immunoprecipitated with anti-(Glu-Glu) beads. The samples of the supernatant and pellet resulting from the immunoprecipitation and 1% of the cell lysate included in each assay were immunoblotted. The upper panel shows detection of (Glu-Glu)-LL5beta with an anti-(Glu-Glu) antibody and the lower panel shows detection of gamma -filamin with a gamma -filamin-specific antibody. B, colocalization of GFP-LL5beta and gamma -filamin in wortmannin-treated COS-7 cells. GFP-LL5beta and gamma -filamin were transiently transfected into COS-7 cells and plated onto coverslips. 12 h later, cells were treated with wortmannin (100 mM, 15 min). The panel in green shows vesicular structures decorated by GFP-LL5beta following wortmannin treatment. The panel in red shows gamma -filamin distribution in the same cells using a gamma -filamin-specific antibody. The panel on the right is a merged image and shows an enlarged area with colocalization of GFP-LL5beta and gamma -filamin in the vesicular structures. C, localization of GFP-LL5beta and gamma -filamin in COS-7 cells. GFP-LL5beta and gamma -filamin were transiently transfected into COS-7 cells and plated on coverslips, left in 10% serum, and fixed 12 h later. The panel in green shows distribution of GFP-LL5beta . The panel in red shows distribution of gamma -filamin (in absence of wortmannin) and the merged image on the right shows the distinct localizations of the two proteins.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

These results imply that LL5beta has the potential to act as a PH domain-containing PI3K effector that can translocate to the plasma membrane in response to receptor activation of type I PI3Ks. However, at low levels of PtdIns(3,4,5)P3 or when the PH domain of LL5beta is unable to bind PtdIns(3,4,5)P3, LL5beta is directed to a vesicular compartment. We consider the simplest explanation for these events, bearing in mind that unstimulated cells contain low levels of PtdIns(3,4,5)P3 and that PH domain/PtdIns(3,4,5)P3-mediated membrane recruitment is probably a dynamic process with turnover times of the order of a maximum of 1-105, that PH domain/PtdIns(3,4,5)P3-mediated signaling through LL5beta blocks targeting of LL5beta to a vesicular compartment. This signal could be a modification to LL5beta (e.g. phosphorylation, dephosphorylation, or association of a protein) that is reversed in the absence of reinforcing signals in a time scale of 10-30 min. The outcome is that LL5beta shows a dramatic change in distribution as the cellular levels of PtdIns(3,4,5)P3 alter in the low basal range.

Much literature has shown how many cells require PI3K signals to survive in several different contexts. Notable among these are serum or growth factor starvation and detachment from a substrate. We have noted that LL5beta redistributes relatively (cf. events like apoptosis) rapidly under these conditions; however, it is completely unclear whether the changes in LL5beta distribution have a cause/effect relationship with these survival pathways.

LL5beta can bind gamma -filamin. Our results indicate that this is not a PI3K-regulated interaction and that it appears to serve to redistribute gamma -filamin, although we have not yet established whether LL5beta can recruit gamma -filamin to the plasma membrane in a PI3K-dependent manner. This contrasts with a number of examples of proteins that bind filamins and as a result are targetted to the actin-containing cytoskeleton e.g. SHIP-1 (30). In the light of the fact that gamma -filamin serves as a stabilizer and organizer of the actin cytoskeleton, this interaction may be important for the role of gamma -filamin. This view is strengthened by the result of a recent study that has suggested that filamin A is effectively down-regulated and as a result cell migration is reduced by interaction with L-FILIP (31). Interestingly, L-FILIP targets filamin A into an undefined punctate intracellular organelle, where the filamin A is degraded. It will be important to investigate the effects of LL5beta on gamma -filamin degradation and whether FILIP family proteins target filamins into a related intracellular compartment.

    ACKNOWLEDGEMENTS

We thank Philip Jackson of Applied Biosystems for identification of the interacting protein peptides by electrospray-sequencing technology, Peter Lipp for use of confocal microscopes, and Nick Ktistakis for discussions and useful reagents. We also thank Dominic Chung (University of Washington) for the gift of gamma -filamin cDNA, Louis M. Kunkel (Howard Hughes Medical Institute) for the gamma -filamin-specific antibody, Dieter O. Fürst (University of Potsdam) for filamin antibody, and Harvey McMahon (LMB, Cambridge) for the dynamin constructs.

    FOOTNOTES

* This work was supported in part by the Biotechnology and Biological Sciences Research Council SAIN initiative.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a movie (file A) and an additional figure.

§ Supported by a Biotechnology and Biological Sciences Research Council studentship.

** Advanced Biotechnology and Biological Sciences Research Council fellow.

Dagger Dagger To whom correspondence should be addressed. Tel.: 44-0-1223-496615; Fax: 44-0-1223-496043; E-mail: len.stephens@bbsrc.ac.uk.

Published, JBC Papers in Press, October 9, 2002, DOI 10.1074/jbc.M208352200

2 P. J. Cullen, personal communication.

    ABBREVIATIONS

The abbreviations used are: PI3K, phosphoinositide 3-kinase; PH, pleckstrin homology; GFP, green fluorescent protein; PAE, porcine aortic endothelial; PDGF, platelet-derived growth factor; TRITC, tetramethylrhodamine isothiocyanate; HI-FBS, heat-inactivated fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; PBS, phosphate-buffered saline; RITC, rhodamine isothiocyanate; GST, glutathione S-transferase; PtdIns(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3, 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate.

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