From the Laboratorium für Molekulare Biologie, Genzentrum der Universität München, Feodor-Lynen-Strasse 25, 81377 München, Germany
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ABSTRACT |
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Signal transduction through
phosphoinositide 3-OH kinase (PI 3-kinase) has been implicated in the
regulation of lymphocyte adhesion mediated by integrin receptors.
Cellular phosphorylation products of PI 3-kinases interact with a
subset of pleckstrin homology (PH) domains, a module that has been
shown to recruit proteins to cellular membranes. We have recently
identified cytohesin-1, a cytoplasmic regulator of
2 integrin adhesion to intercellular adhesion
molecule 1. We describe here that expression of a constitutively active
PI 3-kinase is sufficient for the activation of Jurkat cell adhesion to
intercellular adhesion molecule 1, and for enhanced membrane
association of cytohesin-1. Up-regulation of cell adhesion by PI
3-kinase and membrane association of endogenous cytohesin-1 is
abrogated by overexpression of the isolated cytohesin-1 PH domain, but
not by a mutant of the PH domain which fails to associate with the
plasma membrane. The PH domain of Bruton's tyrosine kinase (Btk),
although strongly associated with the plasma membrane, had no effect on
either membrane recruitment of cytohesin-1 or on induction of adhesion
by PI 3-kinase. Having delineated the critical steps of the
2 integrin activation pathway by biochemical and
functional analyses, we conclude that PI 3-kinase activates inside-out
signaling of
2 integrins at least partially through cytohesin-1.
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INTRODUCTION |
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Integrins are a diverse family of heterodimeric transmembrane adhesion receptors that are present on most vertebrate cell types. They are known to play important roles either in development, or in somatic functions such as wound healing, and the regulation of complex cell-cell or cell-matrix interactions within the immune system (1-3).
The avidity of integrins for their ligands is dependent on the
activation state of the cell on which they are expressed (4). This type
of regulation of cell adhesion has been termed inside-out signaling,
because intracellular signaling pathways, triggered by,
e.g., protein-tyrosine kinase or G-protein-coupled
receptors, have been shown to contribute to integrin-mediated
adhesiveness (5, 6). The mechanisms by which cytoplasmic signals are transmitted across the plasma membrane through integrin receptors remain unclear, but compelling evidence suggests that the intracellular domains of both (7-11) and
chains participate in this process (12-17).
Previous studies have attempted to elucidate these signaling pathways.
In T lymphocytes, a variety of cell surface receptors have been shown
to regulate PI 3-kinase1
activity by recruiting the p85/110 isoform via SH2-phosphotyrosine interactions, including the T cell antigen receptor, CD2, and CD28
(reviewed in Ref. 18). All of these receptors are capable of inducing
integrin activation, and PI 3-kinase has therefore been implicated in
the up-regulation of cell adhesiveness (19-23). PI 3-kinase has also
been strongly implicated in the activation of the
aIIb3 integrin in platelets and
megakaryocytic cells, respectively (24, 25). However, the precise
nature of the underlying mechanisms remained unknown because, first,
the proximal regulatory elements of integrin affinity modulation were
not characterized, and, second, the cellular mode of action of PI
3-kinase was not well understood.
Recently, candidate cytoplasmic regulatory factors of integrin
activation have been identified, either by biochemical methods or with
the help of the two-hybrid system (26-29). One of them, cytohesin-1,
is a 47-kDa intracellular protein that interacts specifically in
several systems with the cytoplasmic domain of the leukocyte integrin
L
2 (CD11a/18, LFA-1) (29). Cytohesin-1 bears a short amino-terminal domain that may aid in oligomerization, an
extended central homology region that is similar to the yeast Sec7
protein, and a carboxyl-terminal pleckstrin homology (PH) domain.
Overexpression of cytohesin-1 or subdomain constructs in the Jurkat T
cell line was shown to have pronounced in vitro effects on
the binding of
L
2 to its ligand, the
intercellular adhesion molecule 1 (ICAM-1). Whereas the overexpression
of full-length cytohesin-1 resulted in a constitutive adhesion of
L
2, expression of the PH domain construct
specifically inhibited the activation of LFA-1 in a dominant negative
fashion. Since the PH domain was not found to be mediating the
interaction with the integrin cytoplasmic domain, it has been
postulated that its unidentified cellular ligand may be an upstream
component of the inside-out signaling pathway of
L
2. The finding that the overexpressed,
isolated Sec7 domain acted only as a partial agonist pointed in the
same direction (29).
PH domains are structural modules present in more than 100 proteins that play known or postulated roles in signal transduction. It is a commonly found thread that PH domains may aid in membrane recruitment of proteins through their interactions with phosphorylated ligands present at the inner leaflet of cellular membranes (30-32). Although a subgroup of PH domains is capable of interacting with tyrosine-phosphorylated proteins (30), much reminiscent of the SH2 domain function, several isolated PH domains have been shown to bind to phosphoinositides such as phosphatidylinositol (4,5)-bisphosphate in vitro (33-37). Interestingly, certain PH domains show in vitro binding preference to lipid compounds, which are in vivo phosphorylation products of PI 3-kinase. The PH domains of Akt, Btk, and of GRP-1, a close homolog of cytohesin-1, have been demonstrated to belong to the latter group because they bind either phosphatidylinositol (3,4)-bisphosphate (Akt), or phosphatidylinositol (3,4,5)-trisphosphate (PIP3) with high affinity (38-41).
One of the important topics that remain to be addressed is the
characterization of the lymphocyte inside-out signaling pathway proximal to the integrin cytoplasmic domains. In this study, we show
for the first time that a constitutively active version of PI 3-kinase
suffices to activate the L
2 adhesion
pathway in a T cell line. Functional, biochemical, and cell biological
evidence is provided, which suggests that cytohesin-1 is located
downstream of PI 3-kinase and that it is regulated by the recruitment
of its PH domain to the plasma membrane.
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MATERIALS AND METHODS |
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Constructs--
Cytoplasmic immunoglobulin fusion proteins have
been described previously (29). A cDNA fragment for the PH domain
of murine Btk (corresponding to amino acids 8-170) has been subcloned
into pcIgTkg vaccinia expression vector (29). The cDNAs for
Myc-P110* and Myc-P110*(917-950) were subcloned into the pTkg
vaccinia expression vector (70).
Adhesion Assay--
Jurkat E6 cells were infected with
recombinant vaccinia viruses as described (29). 6 h after
infection, cells were labeled with 12 µg/ml bisbenzimide H33342
fluorochrome trihydrochloride (Calbiochem) for 30 min at 37 °C,
collected by centrifugation, resuspended in Hanks' buffered saline
solution (HBSS), and delivered to 96-well plates (Nunc, Maxisorp) at
1.5 × 105/well. Prior to adhesion, plates were coated
with goat anti-human IgG (Fc-specific) antibody at 0.85 µg/well
for 90 min at 25 °C, blocked with 1% (w/v) bovine serum albumin in
PBS, incubated with culture supernatants from COS cells expressing
ICAM-1-Rg fusion protein, and subsequently used in the assay. Where
indicated in the figures, cells were incubated with 100 nM
wortmannin (Sigma) 0.5 h prior to the adhesion assay. Cells were
then allowed to adhere for 1 h at 37 °C, and unbound cells were
carefully washed off with 3 × 300 µl of HBSS. Bound cells were
assayed in 100 µl of 2% (v/v) formaldehyde in PBS using a
fluorescence plate reader (Cytofluor II, PerSeptive). The signal of
1.5 × 105 cells/well at 490 nM
corresponds to 100% adhesion. Each value is the mean of two
determinations carried out in triplicate.
Ligand Displacement Analysis-- Displacement assay for [3H]IP4 binding activity was carried out according to Ref. 71 with modifications; 5 µM of purified, polyhistidine-tagged PH domains derived from expression in Escherichia coli BL21 (29) were rebound to 10 µl of nickel-agarose at 20 nM imidazole in MBSD (50 mM Mops, pH 6.8, 100 mM NaCl, 0.1% Igepal CA-630) in a final volume of 40 µl. After incubation with 10 nM [3H]IP4 for 10 min on ice, increasing amounts of unlabeled inositol phosphate were added for 15 min. Then samples were transferred to ultraspin microfilters (Mr cut-off: 105) and washed with 50 µl of MBSD by centrifugation at 1500 × g. Protein-bound radioactivity was eluted by MBSD, containing 200 mM imidazole. Samples were collected by centrifugation and counted by solid phase scintillation. Values were corrected for nonspecific binding of 10 nM [3H]IP4 to nickel-agarose. Each value represents the mean of two determinations carried out in duplicate.
Gel Filtration Assay-- Purified polyhistidine-tagged full-length cytohesin-1 derived from expression in E. coli BL21 at 10 µM was mixed with 10 nM [3H]IP4 and [3H]D-Ins(1,4,5)P3, respectively, and with 20 µM of the appropriate unlabeled isomer in MBSD. The mixture was applied to an Amersham Pharmacia Biotech Fast Desalting Column HR. The elution position of the protein was monitored at 215 nm with the SMART system (Amersham Pharmacia Biotech). Scintillation counting was used to detect the elution position of the radioactively labeled inositol phosphates. Binding specificity was assessed with a mixture of 10 nM [3H]IP4 and purified GST.
Cellular Fractionation-- Cells which had been infected with recombinant vaccinia viruses or uninfected cells were collected by centrifugation and resuspended on ice in 0.5 ml of ice-cold hypotonic solution (HS: 10 mM Hepes, pH 7.5, 10 mM KCl, 10 mM MgCl2, 0.5 mM dithiothreitol) containing 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Then cells were sheared, the nuclei were removed, and the supernatant cytosol was collected as described (72). The cytosolic fraction was brought to a final concentration of 1% (v/v) Igepal CA-630 and 150 mM NaCl and used directly for immunoprecipitation. The pellet was resuspended, washed with HS, and centrifuged at 15,000 × g for 15 min. The resulting pellet was resuspended in HS containing 1% (v/v) Igepal CA-630 and 150 mM NaCl, centrifuged, and the supernatant representing the particulate fraction was subjected to immunoprecipitation at Protein A-Sepharose employing an antigen-purified antibody directed against cytohesin-1 (29). Immunoprecipitates were analyzed by standard Western blot techniques. To assess that cytoplasmic contents were not trapped in the particulate fraction, lactate dehydrogenase activities were monitored as described (59).
Indirect Immunofluorescence--
Six hours after infection of
Jurkat E6 cells with recombinant vaccinia viruses, cells were placed on
poly-L-lysine-covered microscope slides for 1 h in a
humidified chamber at 37 °C. Non-adherent cells were then washed off
with HBSS, and adherent cells were fixed and immobilized with freshly
prepared 2% (w/v) paraformaldehyde in PBS overnight at 4 °C.
Subsequently, cells were permeabilized for 15 min with 0.2% (v/v)
Triton X-100 in PBS, blocked with 2% (w/v) glycine in PBS, and
incubated with a fluorescein isothiocyanate-labeled goat anti-human IgG
(Fc-specific) antibody (Dianova) in PBS for 2 h at room
temperature. In double labeling experiments, tetramethylrhodamine B
isothiocyanate-coupled phalloidin (Sigma) at 1 µg/ml was included. After the final wash with PBS, slides were mounted on a 9:1 mixture of
glycerol and 100 mM Tris/HCl, pH 9.0, containing
n-propyl-gallate at 20 mg/ml as antifading reagent. Then
samples were either examined on a Zeiss Axiophot microscope using a
Zeiss Neofluar 40 × 1.3 oil immersion objective, or on a confocal
laser scanning apparatus (Leica TCS-NT system, Leica) attached to
a Leica DM IRB inverted microscope with a PLAPO 63 × 1.32 oil
immersion objective. Conventional immunofluorescence images were
recorded on Kodak T-MAX 400ASA film. Confocal images were
collected as 512 × 512 pixel files and processed with the help of
the Photoshop program (Adobe).
p110* Expression and Activity-- Six hours after infection of Jurkat E6 cells with recombinant Myc-P110* and Ig control, cells were lysed in 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 10 mM KCl, 1 mM MgCl2, 0.5 mM dithiothreitol, 0.1 mM sodium orthovanadate, containing 1% (v/v) Igepal CA-630, protease inhibitors leupeptin and aprotinin at 10 µg/ml, and 1 mM phenylmethylsulfonyl fluoride. After collection of the supernatant by centrifugation at 15,000 × g, the samples were subjected to immunoprecipitation with mouse anti-c-Myc antibody (monoclonal antibody 9E10) for 12 h at 4 °C, followed by Protein A-Sepharose 6 MB (Amersham Pharmacia Biotech) for 1 h at room temperature. For detection of p110* -expression, precipitates were washed three times with lysis buffer and subjected to immunoblot analysis, using monoclonal antibody 9E10 as first antibody.
The assay for immunoprecipitated PI 3-kinase activity was essentially carried out according to Ref. 73 with modifications. Following washing of the precipitates with lysis buffer, subsequent washes were performed with 10 mM Tris/HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA three times and with kinase buffer containing 50 mM Hepes, pH 7.6, 5 mM MgCl2, 1 mM EDTA twice. Finally, the beads were resuspended in 100 µl of kinase buffer containing 20 µg of sonicated phosphatidylinositol (PI). The reaction was started by adding 1 µCi of [Phospholipid Binding to GST-Cytohesin-1 Measured by IAsys
Biosensor Technology--
Large unilamellar liposomes were generated
by dialysis according to (74). Liposomes contained 60% (w/w)
-palmitoyl-
-oleoyl-L-
-phosphatidylcholine, 30%
(w/w) dioleoyl-L-
-phosphatidyl-DL-glycerol,
obtained from Sigma and 10% (w/w) PI(3,4,5)P3 (Mantreya,
Inc.). Lipids were dried and resuspended in 10 mM Hepes, pH
7.4, 80 mM KCl, 15 mM NaCl, 0.7 mM
NaH2PO4, 1 mM EGTA, containing 50 mM n-octyl-
-D-glucopyranoside. After removal of detergent by dialysis, liposomes were deposited onto
an IAsys surface (FCS-0301, not derivatized) at 0.1 mg of lipid/ml
of PBS. Subsequently, remaining unspecific binding sites were blocked
by GST and binding of either GST-cytohesin-1 or its mutant
GST-cytohesin-1 (R281C) was analyzed at 2.5 µg/ml. Cuvette was
regenerated by subsequent washing with 1 N NaOH/1% (w/v)
SDS, water, 1 N HCl, water, and methanol.
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RESULTS |
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Expression of a Constitutively Active PI 3-Kinase in Jurkat Cell
Induces Binding of the L
2 Integrin to
ICAM-1--
Previous reports implicated a role of PI 3-kinase in the
regulation of integrin-mediated adhesiveness (see Introduction and references therein). We therefore investigated whether a constitutively active PI 3-kinase can up-regulate lymphocyte adhesion directly. Overexpression of the catalytic subunit of mammalian PI 3-kinase is
usually not sufficient for a constitutively active phenotype, but three
groups have recently generated chimeric, mutant, or membrane-associated
versions of PI 3-kinase, which were shown to result in an activation of
cellular signaling pathways of fibroblast-like cells (42-45). A
chimera (p110*) that comprises the catalytic subunit (p110) of murine
PI 3-kinase, fused at the amino terminus to a regulatory domain derived
from the p85 subunit (42), was used in this study (Fig.
1A). A kinase deficient
variant of p110*, bearing a short in-frame deletion (33 amino acids;
917-950) within the kinase domain, served as control (Fig.
1A).
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The PH Domain of Cytohesin-1 Associates with Cellular Membranes-- How is PI 3-kinase coupled to the regulation of integrin activity? We postulated that the PH domain of cytohesin-1 may provide a link between the activation of cell adhesion mediated by PI 3-kinase and the recruitment of cytohesin-1 to the plasma membrane. Therefore, membrane recruitment of cytohesin-1 in the Jurkat cell line was assessed biochemically by the separation of crude cell extracts. Localization of either overexpressed cytohesin-1 or the isolated PH domain in the cytosolic or the particulate fractions was detected by Western blot analysis. We found that a substantial fraction of both full-length cytohesin-1 and the isolated PH domain was associated with membranes (Fig. 2, A and B).
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Membrane Association of the Cytohesin-1 PH Domain Is Required for
Its Role in the Regulation of Cell Adhesion--
Is the membrane
recruitment mediated by the cytohesin-1 PH domain associated with its
cellular function? To answer this question, we generated a point mutant
(R281C) of the PH domain, which corresponds to a residue in the PH
domain of Btk that had previously been shown to important for
PIP3 binding and cellular function (39, 47). Accordingly,
we found that introduction of the R281C mutation abolished the membrane
association of the cytohesin-1 PH domain (Fig. 2B). We then
investigated the effect of this mutation on the ability of the
cytohesin-1 PH domain to inhibit 2 integrin-mediated adhesion of Jurkat cells. Fig. 2C shows that the R281C
mutant had lost the dominant inhibitory potential, which corresponds directly to its inability to associate with membranes.
Cytohesin-1 Localizes to the Plasma Membrane--
To which
cellular membranes does cytohesin-1 bind? We have shown that
cytohesin-1 can be co-precipitated with the
L
2 integrin from Jurkat cells (23) and
therefore postulated that the PH domain of cytohesin-1 predominantly
associates with the plasma membrane. Immunofluorescence studies were
performed to test this hypothesis. To this end, a full-length
cytohesin-1 fusion protein or the respective wild type or mutant PH
domain constructs were expressed in the Jurkat line. The cells were
subsequently immobilized on poly-L-lysine coated slides,
fixed, permeabilized, and treated with an fluorescein-isothiocyanate
conjugated antibody directed against the cIg portion. In addition to
conventional immunofluorescence microscopy (Fig.
3, panels D, G,
F, and M) subcellular distributions of the
various cytohesin-1 fusion proteins were examined using the confocal
laser scanning method. Measurement of the pixel intensity along a
transect through a cell which was double stained for the respective Ig
fusion protein and actin revealed colocalization of the full-length
cytohesin-1 (panels D-F) or the PH-domain fusion construct
(panels J-L) with the plasma membrane. By contrast, the
cytosolic Ig control protein (panels G-I) or the PH (R281C) mutant showed diffuse cytoplasmic expression. The observed plasma membrane association of cytohesin-1 through the PH domain is consistent with its role in the regulation of
2 integrin
activity.
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The R281C Mutation of the Cytohesin-1 PH Domain Abrogates Binding
Either to Inositol (1,3,4,5)-Tetrakisphosphate or
PIP3--
Is the plasma membrane association of
cytohesin-1 through its PH domain maintained by a ligand that may be
generated by PI 3-kinase? One of the products of PI 3-kinase,
phosphatidylinositol (3,4)-bisphosphate, has been shown to bind to the
PH domain of the proto-oncogene Akt-1 and to regulate its kinase
activity (38, 48, 49). Cellular effector functions of Akt-1 include
cell survival (50). Recently, a murine member of the cytohesin family, GRP-1, was identified by its ability to bind a different product of PI
3-kinase, PIP3, when a cDNA expression library was
screened with inositol phospholipid ligands (40). In the same study, it
was shown that the PH domains of GRP-1 and cytohesin-1 also have strong
binding preference to the soluble compound inositol (1,3,4,5)-tetrakisphosphate (IP4). IP4 bears
the same headgroup as PIP3 and has been postulated to play
a role in detachment of the PH domain of cytohesin-1 and GRP-1 from the
plasma membrane (40). We therefore investigated whether the R281C
mutation of the cytohesin-1 PH domain interfered with its specific
binding to inositol phosphate ligands. Using a gel filtration assay
(51), we confirmed that IP4 co-migrated with purified,
E. coli-derived full-length cytohesin-1 or with the isolated
PH domain, whereas a control compound, inositol (1,4,5)-trisphosphate,
did not co-elute with the purified proteins (Fig.
4A). By contrast,
IP4 did not co-migrate with a control protein, GST (Fig.
4A). More quantitative analyses, employing a ligand
displacement assay revealed that the PH domain of cytohesin-1 binds to
IP4, which is half-maximally displaced at a concentration
of 2.5 µM (Fig. 4B). As expected, the R281C
mutant of the cytohesin-1 PH domain does not bind to IP4
in vitro (Fig. 4B). No binding to
IP4 was observed for the PH domain of the -adrenergic
receptor kinase (
ark, Fig. 4B), which corresponds to its
inability to block integrin adhesion in T cells (29).
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Expression of p110* in Jurkat Cells Induces Membrane Association of
Endogenous Cytohesin-1, but Overexpression of the PH Domain Abrogates
Membrane Localization of Cytohesin-1--
All data presented above
suggested that PI 3-kinase regulates the activity of
L
2 indirectly through membrane
recruitment of cytohesin-1. We therefore overexpressed p110* in the
Jurkat line, fractionated the cells, and assessed the subcellular
distribution of endogenous cytohesin-1. We found that membrane
association of cytohesin-1 was ~2.5-fold enhanced in the presence of
P110*, as compared with cells in which the control constructs had been expressed (Fig. 5). Semiquantitative
analysis of 12 independent experiments confirmed these results; the
mean specific induction of cytohesin-1 in the membrane fraction
following p110* expression was found to be 2-3-fold (Fig. 5 and data
not shown). Moreover, overexpression of the PH domain construct
interfered with membrane association of endogenous cytohesin-1 (Figs. 5
and 6E). This result is consistent with the observed
dominant negative inhibition of Jurkat cell adhesion to ICAM-1 by
the PH domain fusion protein.
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A PH Domain Construct of Cytohesin-1 Blocks Up-regulation of
L
2 Integrin Adhesion Mediated by p110*,
whereas the R281C Mutant or the PH Domain of Bruton's Tyrosine Kinase
Have No Effect--
Adhesion assays as well as biochemical analyses
were then performed to study the functional relationship of PI 3-kinase
and cytohesin-1 in Jurkat cells directly. We had previously shown that
dominant negative inhibition of LFA-1 adhesion to ICAM-1 by the
cytohesin-1 PH domain was relatively specific (29), but it was not
shown whether the PH domain controls used in this were capable of
entering the same compartment. In order to examine this in greater
detail, the PH domain of Btk was included in our present investigation.
Therefore, a cytoplasmic immunoglobulin expression construct of the PH
domain of Btk was made. The PH domain of Btk had previously been shown
to bind PIP3 in vitro, and to be associated with
membranes in vivo (39, 52). Strong membrane association of
the PH domain of Btk was confirmed in our system (Fig.
6, A and B).
However, the Btk-PH domain had no effect on LFA-1 dependent
adhesion to ICAM-1 (Fig. 6C), suggesting that the PH domains
of cytohesin-1 and Btk bind distinct ligands in Jurkat cells.
Biochemical analyses confirmed this interpretation. The Btk-PH
domain did not compete with membrane association of endogenous
cytohesin-1, which on the other hand was almost completely dislodged
from the particular fraction when the PH domain of cytohesin-1 was
overexpressed (Fig. 6E).
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DISCUSSION |
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In this study, we show by both functional and biochemical analyses
that PI 3-kinase is a candidate upstream regulator of cytohesin-1, an
intracellular mediator of integrin activation, which has previously been shown to interact directly with the cytoplasmic domain of the
2 chain. Our experiments indicate that cytohesin-1 is
recruited to the plasma membrane through its carboxyl-terminal PH
domain following PI 3-kinase activation and that membrane association of cytohesin-1 through its PH domain appears to be a functional prerequisite for
2 integrin activation.
PH domains have been found in many signaling molecules. In an attempt
to explain the finding that certain PH domains bind PIP2
in vitro, it was postulated that they may serve as
membrane recruitment modules (30, 31). Broad evidence exists supporting this hypothesis (37, 53-58). However, some PH domains may have other
functions, because in the case of the pleckstrin protein it was
described that membrane localization is provided by the amino-terminal
but not by the carboxyl-terminal PH domain (59). Membrane recruitment
of proteins mediated by PH domains is not necessarily constitutive, and
it appears unlikely that PIP2, although it may be the
physiological ligand for the PLC- PH domain (32), is a
physiologically relevant interaction structure for most PH domains.
Evidence points to the possibility that membrane association of PH
domains is regulated by signal transduction events (37, 49, 60).
Since some PH domains show a binding preference for PIP3 or
phosphatidylinositol (3,4)-bisphosphate in vitro, PI
3-kinase has been implicated in the regulation of membrane recruitment of PH domains. At least three types of PI 3-kinase isoform have been
found in mammalian cells to date (61). One of them, PI 3-kinase , is
predominantly coupled to tyrosine kinase-activated pathways, whereas a
different enzyme, PI 3-kinase
, is induced by heterotrimeric
G-proteins. Binding specificity is conferred by the regulatory
subunits, p85 and p101, respectively. Receptors that are involved in
lymphocyte activation, such as the T cell receptor or CD28, couple to
signal transduction through tyrosine kinase-activated pathways, and
these are also known to induce PI 3-kinase function (46). Our findings
presented here are consistent with the view that PI 3-kinase
is
involved in the up-regulation of cell adhesion by hematopoietic
receptors and we provide evidence for a mechanism. First, a
constitutively active PI 3-kinase induces membrane association of
cytohesin-1 as well as up-regulation of
2 integrin
adhesion to ICAM-1; and second, overexpression of a PH domain construct
of cytohesin-1 or treatment of the cells with PI 3-kinase inhibitors
block cell adhesion stimulated by T cell receptor activation.
Co-expression studies in Jurkat cells finally showed that expression of
a cytohesin-1 PH domain construct but not the introduction of a mutant
PH domain, incapable of membrane association, blocked p110* induced
adhesion. Induction of cell adhesion to ICAM-1 was neither blocked by
expression of the PH domain of Bruton's tyrosine kinase, which was
shown previously to bind PIP3 in vitro and to
mediate membrane association in vivo. Therefore, cytohesin-1
appears to be coupling PI 3-kinase to the activation of cell adhesion
by
2 integrin receptors. On the basis of our data,
however, we still cannot fully exclude that other proteins, which are
regulated by PI 3-kinase, may also play important roles in the
regulation of
2 integrin-mediated cell adhesion. In fact
we found that adhesion mediated by overexpressed cytohesin-1 was
partially blocked by PI 3-kinase inhibitors, suggesting that PI
3-kinase may also contribute to a fully adhesive phenotype by other
cellular functions. Incubating Jurkat cells with PI 3-kinase inhibitors
for extended periods of time completely inhibited their ability to
spread on ICAM-1-coated surfaces (data not shown), and this inability
interfered with tight adhesion, irrespective of which stimulus was
used.
Since integrin-mediated adhesiveness can also be triggered by chemokine receptors that signal through G-proteins (62, 63), an intriguing thought is that other members of the PI 3-kinase family may also be involved in the regulation of cell adhesion. This has in fact been suggested by a recent study (23).
PI 3-kinase appears to activate membrane recruitment of other signaling
molecules, such as the Akt proto-oncogene, through their PH domains.
How is signaling specificity regulated in vivo? One possible
answer is that ligand specificity is not exclusively conferred by PI
3-kinase at the level of phosphoinositide phosphorylation. Akt, for
example, preferentially binds to phosphatidylinositol (3,4)-bisphosphate. It has therefore been postulated that PI 3-kinase as well as a phosphatidylinositol (5)-phosphatase are required to
generate the Akt ligand in vivo (38). However, the PH
domains of cytohesin-1, GRP-1, Bruton's tyrosine kinase, and maybe
more, all bind PIP3 with high affinity. A second layer of
specificity may therefore be provided by additional PH domain ligands.
The PH domain of Bruton's tyrosine kinase can apparently bind protein kinase C (64), and a carboxyl-terminal portion of the PH domain of the
ark is required for the interaction of
ark with
-
subunits of heterotrimeric G-proteins (35). In our study, the PH domain of
Bruton's tyrosine kinase did not interfere with Jurkat cell adhesion
nor did it compete with membrane association of endogenous cytohesin-1,
although it was strongly associated with the plasma membrane. This
points indeed to a highly selective ligand usage of these two PH
domains in vivo. Finally, many PH domains are expressed in
proteins that do also contain other interaction modules such as SH2 or
SH3 domains, so that signal specificity may be achieved by
combinatorial means. We have shown previously for cytohesin-1 that the
Sec7 homology region interacts with the cytoplasmic domain of the
2 integrin (29). Fine specificity of recruitment may
therefore be conferred by at least two elements, the PH- and the Sec7
domain.
Recent studies have shown that cytohesin-1 (65) as well as the very closely related proteins cytohesin-2/ARNO and GRP-1 (66-68) exert guanine nucleotide exchange factor activity for small G-proteins of the Arf family in vitro. The catalytic activity is included in the Sec7 domain but was shown to be regulated by the PH domain. PI 3-kinase has been implicated in the regulation of Arf proteins (69), and this is consistent with a recent finding, namely that Arf exchange activity of GRP-1 is regulated by PIP3 (67). Whether Arf activity correlates with the regulation of integrin-mediated cell adhesion is subject to further analysis, but since Arf-6, cytohesin-2/ARNO, and cytohesin-1 appear to localize to an at least similar compartment, this is an intriguing thought.
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ACKNOWLEDGEMENTS |
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We thank Anke Klippel, Lewis Williams, and Julian Downward for the kind donation of PI 3-kinase cDNAs; Pascalis Sideras and Michael Reth for the Btk cDNA; and Ernst-Ludwig Winnacker for continuous support.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft and by the Bundesministerium für Forschung, Bildung, und Technologie.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.
To whom correspondence should be addressed. Tel.:
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1
The abbreviations used are: PI
3-kinase, phosphoinositide 3-OH kinase; SH, Src homology; PH,
pleckstrin homology; ark,
-adrenergic receptor kinase; GST,
glutathione S-transferase; PBS, phosphate-buffered saline;
HBSS, Hanks' buffered salt solution; ICAM-1, intercellular adhesion
molecule 1; HS, hypotonic solution; MBSD, Mops/NaCl/Igepal CA-630;
IP4, inositol (1,3,4,5)-tetrakisphosphate;
PIP3, phosphatidylinositol (3,4,5)-trisphosphate; Mops,
4-morpholinepropanesulfonic acid; PMA, phorbol 12-myristate
13-acetate.
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