From the 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
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ABSTRACT |
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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 (LL5 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 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 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 PKB 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 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
Cloning of Human LL5 Cell Culture--
PAE cells expressing wild-type or mutant
(Y740F/Y751F) PDGF- 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-LL5 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- 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 LL5 Phosphoinositide Binding Specificities of LL5 Purification of LL5
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).
Cloning, Tissue Distribution, and Lipid Binding Properties of
LL5
Expression plasmids encoding NH2-terminal Myc- and
GFP-tagged LL5
We analyzed the lipid binding specificity of LL5 Distribution of LL5
Interestingly, with PAE cells transiently expressing GFP-LL5
We examined the distribution of GFP-LL5
We examined the distribution of GFP-LL5
We also tested whether Myc-LL5
Together these results suggest that overexpressed LL5
We consider the best working explanation for these data is that even at
relatively low cellular levels of PtdIns(3,4,5)P3 LL5 The Nature of the Vesicle Compartment That Can be Decorated by
LL5
We used latrunculin B to disassemble actin fibers in PAE cells. It had
no effect on the distribution of GFP-LL5 LL5
We tested whether These results imply that LL5 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 LL5 LL5). The protein bound PtdIns(3,4,5)P3
selectively in vitro. Strikingly, a substantial proportion
of LL5
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 LL5
to
this vesicle population. Importantly, pleckstrin homology domain
mutants of LL5
that could not bind PtdIns(3,4,5)P3 were
constitutively localized to this vesicle population. At increased
PtdIns(3,4,5)P3 levels, LL5
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 LL5
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, LL5
bound to the cytoskeletal adaptor,
-filamin, tightly and in a PI3K-independent fashion, both in vitro
and in vivo. This interaction could co-localize
heterologously expressed
-filamin with GFP-LL5
in the
unidentified vesicles.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Phe mutants. A
number of receptor tyrosine kinases (relevant here, the PDGF
-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-
receptors or (Y740F/Y751F) PDGF-
receptors (6) that have allowed the impact of selectivity blocking type
IA PI3K activation to be assessed in vivo (7).
-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).
) as a result of PtdIns(3,4,5)P3
binding (21). For DAPP-1, which has been claimed to bind PLC-
(11), it is presumably the relocation of the PLC-
, to the cell surface and
the location of its phospholipid substrate that could be relevant.
-,
-, and
-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.
-filamin and undergoes a novel redistribution in response to
reductions in PtdIns(3,4,5)P3 levels.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and Relevant Constructs--
The
cDNA encoding the full-length open reading frame of LL5
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 LL5
(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),
LL5
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
-filamin cDNA was a kind gift from Dominic Chung (University of Washington).
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.
, 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.
-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).
was used as a probe for
Northern blot analysis. The probe was labeled with
[
-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.
--
1 × 107 COS-7 cells were transfected by electroporation with 20 µg of the DNA construct encoding Myc-tagged LL5
. 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
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)).
Interacting Partners--
Recombinant
proteins, GST-LL5
and GST-LL5
PH, were expressed in bacteria
and purified on GS-Sepharose beads. The glutathione-Sepharose beads
bound to GST LL5
and GST-LL5
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 LL5
and GST-LL5
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
-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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
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 LL5
and a closely related molecule LL5
, 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 LL5
. LL5
and LL5
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 LL5
, which would not recognize LL5
, 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 LL5 . A, amino acid sequence of
human LL5
. 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 LL5
by Northern blot
analysis. A multiple tissue Northern blot
(Clontech) containing polyadenylated RNA from the
indicated human tissues was probed for LL5
expression using a
32P-labeled, unique, NH2-terminal fragment of
LL5
(745 bp). The LL5
probe was observed to hybridize to a
transcript of the predicted size (6 kb).
(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
LL5 . A, immunoblot of Myc-tagged
full-length LL5
. Myc-LL5
was transiently transfected into PAE
cells. Cell lysates were immunoblotted with anti-Myc antibodies.
B, in vitro lipid binding specificity of
LL5
. Recombinant, Myc-tagged LL5
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-LL5
equivalent to 1% of that included in the binding assays. Myc-tagged
LL5
was detected by immunoblotting with anti-Myc antibodies.
C, binding of Myc-LL5
PH and
Myc-LL5
-K1162A/R1163A expressed in COS-7 cell lysates to
PtdIns(3,4,5)P3 beads. Proteins were detected by
immunoblotting with anti-Myc antibodies.
in
vitro. Cytosolic fractions were prepared from COS-7 cells
transfected with Myc-LL5
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 LL5
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 LL5
from the beads (see Fig 2B);
indicating that LL5
can bind PtdIns(3,4,5)P3 selectively
under these assay conditions. Mutations in the PH domain predicted to
disrupt lipid binding (LL5
PH and LL5
-K1162A/R1163A) abolished
LL5
binding to the PtdIns(3,4,5)P3 beads (Fig.
2C).
in Cells--
We transiently expressed Myc-
or GFP-LL5
in PAE cells stably overexpressing the PDGF
receptor.
In the presence of serum or after only short periods of serum
starvation (up to 6 h), the LL5
constructs appeared
predominantly cytosolic in fixed cells. After prolonged serum
starvation (8 h or more) a significant proportion of Myc- or GFP-LL5
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-LL5
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 LL5
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 LL5
.
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-LL5
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-LL5
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-LL5
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-LL5 in PAE
cells. PAE cells were transiently transfected with GFP-LL5
.
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-LL5 .
A, PAE cells were transiently transfected with
GFP-LL5
. 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-LL5
-K1162A/R1163A. C, PAE cells were transiently
transfected with GFP-LL5
. 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-LL5
localization in the presence of serum
(2 h + 2 h detached). The panel on the right
shows GFP-LL5
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-LL5
. The conditions and time of detachment are
indicated. The values shown are mean ± S.D. from three different
experiments.
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-LL5
from the vesicular pool into
the cytoplasmic fraction without a clearly detectable accumulation near
the plasma membrane. Transient expression of GFP-LL5
in a PAE cell
line expressing (Y740F/Y751F)-PDGF-
-receptors (unable to bind and activate type I PI3Ks) revealed that GFP-LL5
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 LL5
constructs into a particulate
compartment, we serum-starved and/or detached PAE cells transiently
expressing GFP-LL5
and held them in suspension. Both treatments
significantly increased the proportion of GFP-LL5
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).
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).
PH and
GFP-LL5
-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).
-K1162A/R1163A (detected with a
RITC-labeled 2° antibody) co-localized with GFP-LL5
in
wortmannin-treated PAE cells that express the above constructs. The two
constructs co-localized (not shown).
can
translocate to the plasma membrane in response to PI3K activation but
that under conditions of low PtdIns(3,4,5)P3, LL5
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 LL5
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
PH
and LL5
-K1162A/R1163A constructs suggest that it is likely that this
process represents the association of LL5
with a pre-existing
organelle (unless they are formed specifically in the presence of these
constructs) and that the key event is that LL5
"perceives" that
cellular PtdIns(3,4,5)P3 is low, rather than the levels of
PtdIns(3,4,5)P3 are actually low.
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
LL5
(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 LL5
can
undergo a net translocation to the plasma membrane in response to
receptor activation of type I PI3Ks.
--
The work we described above suggests that the
"vesicular" LL5
is unlikely to be a aggregated/denatured
protein. This view is also supported by the dynamic, jittering
movements of GFP-LL5
-associated structures in the presence of
wortmannin or of GFP-LL5
-K1162A/R1162A decorated structures. We
attempted to co-localize GFP-LL5
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-LL5
did not co-localize
with any of these markers (see Supplementary Material).
-decorated vesicles (see
Supplementary Material). We co-transfected cells with Arf-6 and
GFP-LL5
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-LL5
-K1162A/R1163A, stimulated with PDGF and detected the LL5
construct via an anti-Myc antibody and a RITC secondary antibody. The
internalized GFP-DAPP-1 did not co-localize with the vesicular LL5
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-LL5
-decorated structures in the presence of
wortmannin (data not shown). We have not yet positively identified the
vesicle compartment that is labeled by LL5
constructs although the
number markers we have failed to co-localize suggest that it is a
tightly defined subpopulation.
-binding Proteins--
In the context of our hypothesis that
the PI3K-dependent redistribution of LL5
to a vesicular
compartment might be dependent/blocked by an LL5
-associated protein
we attempted to isolate potential binding partners. We prepared
GST-LL5
and GST-LL5
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-LL5
and GST-
PH-LL5
(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
-filamin. This
assignment was confirmed by a
-filamin-specific monoclonal antibody
and a pan-filamin antibody preparation in immunoblots of the 260-kDa
protein eluted from the GST-LL5
affinity supports (Fig.
5C).
View larger version (42K):
[in a new window]
Fig. 5.
Identification of a LL5
interacting protein. A, isolation of an
LL5
-binding protein from 35S-GST, GST-SHIP-1,
GST-LL5
PH, and GST-LL5
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-LL5
PH and GST-LL5
but not GST nor GST-SHIP-1.
B, Coomassie detection of the LL5
interacting
protein affinity purified from COS-7 cell lysate. C,
immunodetection of LL5
interacting protein with independent
anti-filamin antibodies. LL5
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
-filamin (right
panel).
-filamin could interact with LL5
in
vivo. (Glu-Glu)-tagged LL5
and
-filamin were transfected
individually and together into COS-7 cells (note, transfection with
-filamin did not substantially increase the amount of total
-filamin in the cells). Lysates were prepared and immunoprecipitated
with anti-(Glu-Glu) monoclonal antibody covalently attached to protein G-Sepharose.
-Filamin was only immunoprecipitated in the presence of
(Glu-Glu)-LL5
(Fig. 6A).
About 5% of the total
-filamin was recovered in the washed
(Glu-Glu)-LL5
immunoprecipitates. This indicates that
-filamin
and LL5
can interact in vivo. We examined the
distribution of
-filamin and its relationship to LL5
in COS-7
(Fig. 6, B and C) and PAE cells (data not shown).
We transiently transfected cells with GFP-LL5
and/or
-filamin
(detected by
-filamin-specific antibody and a RITC-labeled secondary
antibody). In cells co-transfected with GFP-LL5
and
-filamin in
the presence of wortmannin, it was clear that 30-40% of the
-filamin-positive structures were also positive for the GFP-LL5
vesicular compartment (Fig. 6B). In cells transfected with
-filamin alone, or co-transfected with GFP-LL5
and
-filamin
but not treated with wortmannin, the
-filamin adapted a punctate
distribution that was insensitive to wortmannin and were clearly
smaller than those that contained both GFP-LL5
and
-filamin (Fig.
6C). In cells co-transfected with
-filamin and GFP-LL5
and treated with wortmannin, the structures that were only positive for
-filamin were the same size as the
-filamin-positive structures
in cells transfected with
-filamin alone. These results suggest that
LL5
and
-filamin can co-localize in both COS-7 and PAE cells in
the presence of wortmannin and that
-filamin localization is
dictated by LL5
and wortmannin. This indicates that the interaction
between LL5
and
-filamin (in the presence of wortmannin) leads to
the targetting of
-filamin into the vesicular compartment by LL5
and that
-filamin is not responsible for directing or blocking the
movement of LL5
into a vesicular compartment.
View larger version (37K):
[in a new window]
Fig. 6.
Interaction of
-filamin and LL5
in
vivo assessed by immunoprecipitation and
immunocytochemistry. A, interaction of
-filamin with
(Glu-Glu)-tagged LL5
in vivo. (Glu-Glu)-LL5
and
-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)-LL5
with an anti-(Glu-Glu) antibody and
the lower panel shows detection of
-filamin with a
-filamin-specific antibody. B, colocalization of
GFP-LL5
and
-filamin in wortmannin-treated COS-7 cells.
GFP-LL5
and
-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-LL5
following wortmannin treatment. The panel in red shows
-filamin distribution in the same cells using a
-filamin-specific
antibody. The panel on the right is a merged
image and shows an enlarged area with colocalization of GFP-LL5
and
-filamin in the vesicular structures. C,
localization of GFP-LL5
and
-filamin in COS-7 cells. GFP-LL5
and
-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-LL5
. The panel in red shows distribution
of
-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
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
LL5
is unable to bind PtdIns(3,4,5)P3, LL5
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 LL5
blocks targeting of LL5
to a vesicular compartment. This signal could be a modification to LL5
(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 LL5
shows a dramatic change in distribution as the cellular levels of
PtdIns(3,4,5)P3 alter in the low basal range.
redistributes relatively (cf. events like apoptosis) rapidly under these conditions; however, it is completely unclear whether the changes in LL5
distribution have a cause/effect relationship with these survival pathways.
can bind
-filamin. Our results indicate that this is not a
PI3K-regulated interaction and that it appears to serve to redistribute
-filamin, although we have not yet established whether LL5
can
recruit
-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
-filamin serves as a stabilizer and
organizer of the actin cytoskeleton, this interaction may be important
for the role of
-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 LL5
on
-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
-filamin cDNA, Louis M. Kunkel (Howard Hughes Medical Institute)
for the
-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.
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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REFERENCES |
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---|
1. | Vanhaesebroeck, B., Leevers, S. J., Ahmadi, K., Timms, J., Katso, R., Driscoll, P. C., Woscholski, R., Parker, P. J., and Waterfield, M. D. (2001) Annu. Rev. Biochem. 70, 535-602[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Yano, H.,
Nakanishi, S.,
Kimura, K.,
Hanai, N.,
Saitoh, Y.,
Fukui, Y.,
Nonomura, Y.,
and Matsuda, Y.
(1993)
J. Biol. Chem.
268,
25846-25856 |
3. |
Vlahos, C. J.,
Matter, W. F.,
Hui, K. Y.,
and Brown, R. F.
(1994)
J. Biol. Chem.
269,
5241-5248 |
4. | Wennstrom, S., Hawkins, P., Cooke, F., Hara, K., Yonezawa, K., Kasuga, M., Jackson, T., Claesson-Welsh, L., and Stephens, L. (1994) Curr. Biol. 4, 385-393[Medline] [Order article via Infotrieve] |
5. | Kazlauskas, A., and Cooper, J. A. (1990) EMBO J. 9, 3279-3286[Abstract] |
6. | Wennstrom, S., Siegbahn, A., Yokote, K., Arvidsson, A. K., Heldin, C. H., Mori, S., and Claesson-Welsh, L. (1994) Oncogene 9, 651-660[Medline] [Order article via Infotrieve] |
7. | Hawkins, P. T., Eguinoa, A., Qiu, R. G., Stokoe, D., Cooke, F. T., Walters, R., Wennström, S., Claesson-Welsh, L., Evans, T., Symons, M., and Stephens, L. (1995) Curr. Biol. 5, 393-403[Medline] [Order article via Infotrieve] |
8. | Burgering, B. M., and Coffer, P. J. (1995) Nature 376, 599-602[CrossRef][Medline] [Order article via Infotrieve] |
9. | Franke, T. F., Yang, S. I., Chan, T. O, Datta, K., Kazlauskas, A., Morrison, D. K., Kaplan, D. R., and Tsichlis, P. N. (1995) Cell 81, 727-736[Medline] [Order article via Infotrieve] |
10. | Anderson, K. E., Lipp, P., Bootman, M., Ridley, S. H., Coadwell, J., Ronnstrand, L., Lennartsson, J., Holmes, A. B., Painter, G. F., Thuring, J., Lim, Z., Erdjument-Bromage, H., Grewal, A., Tempst, P., Stephens, L. R., and Hawkins, P. T. (2000) Curr. Biol. 10, 1403-1412[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Marshall, A. J.,
Niiro, H.,
Lerner, C. G.,
Yun, T. J.,
Thomas, S.,
Disteche, C. M.,
and Clark, E. A.
(2000)
J. Exp. Med.
191,
1319-1332 |
12. | Dowler, S., Montalvo, L., Cantrell, D., Morrice, N., and Alessi, D. R. (2000) Biochem. J. 349, 605-610[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Rao, V. R.,
Corradetti, M. N.,
Chen, J.,
Peng, J.,
Yuan, J.,
Prestwich, G. D.,
and Brugge, J. S.
(1999)
J. Biol. Chem.
274,
37893-37900 |
14. | Anderson, K. E., Coadwell, J., Stephens, L. R., and Hawkins, P. T. (1998) Curr. Biol. 8, 684-691[Medline] [Order article via Infotrieve] |
15. | Venkateswarlu, K., Oatey, P. B., Tavare, J. M., and Cullen, P. J. (1998) Curr. Biol. 8, 463-466[Medline] [Order article via Infotrieve] |
16. | Krugmann, S., Anderson, K. E., Ridley, S. H., Risso, N., McGregor, A., Coadwell, J., Davidson, K., Eguinoa, A., Ellson, C. D., Lipp, P., Manifava, M., Ktistakis, N., Painter, G., Thuring, J. W., Cooper, M. A., Lim, Z. Y., Holmes, A. B., Dove, S. K., Michell, R. H., Grewal, A., Nazarian, A., Erdjument-Bromage, H., Tempst, P., Stephens, L. R., and Hawkins, P. T. (2002) Mol. Cell. 9, 95-108[Medline] [Order article via Infotrieve] |
17. | Venkateswarlu, K., Gunn-Moore, F., Oatey, P. B., Tavare, J. M., and Cullen, P. J. (1998) Biochem. J. 335, 139-146[Medline] [Order article via Infotrieve] |
18. | Haslam, R. J., Koide, H. B., and Hemmings, B. A. (1993) Nature 363, 309-310[Medline] [Order article via Infotrieve] |
19. | Lemmon, M. A., and Ferguson, K. M. (2000) Biochem. J. 350, 1-18[CrossRef][Medline] [Order article via Infotrieve] |
20. | Ferguson, K. M., Kavran, J. M., Sankaran, V. G., Fournier, E., Isakoff, S. J., Skolnik, E. Y., and Lemmon, M. A. (2000) Mol. Cell 6, 373-384[Medline] [Order article via Infotrieve] |
21. | Vanhaesebroeck, B., and Alessi, D. R. (2000) Biochem. J. 15, 561-576 |
22. | Marte, B. M., and Downward, J. (1997) Trends Biochem. Sci. 22, 355-358[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Khwaja, A.,
Rodriguez-Viciana, P.,
Wennstrom, S.,
Warne, P. H.,
and Downward, J.
(1997)
EMBO J.
16,
2783-2793 |
24. | Stossel, T. P., Condeelis, J., Cooley, L., Hartwig, J. H., Noegel, A., Schleicher, M., and Shapiro, S. S. (2001) Nat. Rev. Mol. Cell. Biol. 2, 138-145[CrossRef][Medline] [Order article via Infotrieve] |
25. | Van der Flier, A., and Sonnenberg, A. (2001) Biochim. Biophys. Acta. 1538, 99-117[Medline] [Order article via Infotrieve] |
26. |
Isakoff, S. J.,
Cardozo, T.,
Andreev, J., Li, Z.,
Ferguson, K. M.,
Abagyan, R.,
Lemmon, M. A.,
Aronheim, A.,
and Skolnik, E. Y.
(1998)
EMBO J.
17,
5374-5387 |
27. | Levi, L., Hanukoglu, I., Raikhinstein, M., Kohen, F., and Koch, Y. (1993) Biochim. Biophys. Acta 1216, 342-344[Medline] [Order article via Infotrieve] |
28. | Dowler, S., Currie, R. A., Campbell, D. G., Deak, M., Kular, G., Downes, C. P., and Alessi, D. R. (2000) Biochem. J. 351, 19-31[CrossRef][Medline] [Order article via Infotrieve] |
29. | Welch, H. C., Coadwell, W. J., Ellson, C. D., Ferguson, G. J., Andrews, S. R., Erdjument-Brromage, H., Tempst, P., Hawkins, P. T., and Stephens, L. R. (2002) Cell. 108, 809-821[Medline] [Order article via Infotrieve] |
30. |
Dyson, J. M.,
O'Malley, C. J.,
Becanovic, J.,
Munday, A. D.,
Berndt, M. C,
Coghill, I. D.,
Nandurkar, H. H.,
Ooms, L. M.,
and Mitchell, C. A.
(2001)
J. Cell Biol.
155,
1065-1079 |
31. | Nagano, T., Yoneda, T., Hatanaka, Y., Kubpta, C., Murakami, F., and Sato, M. (2002) Nat. Cell Biol. 4, 495-501[CrossRef][Medline] [Order article via Infotrieve] |