William and Karen Davidson Laboratory of Brain Tumor Biology, Hermelin Brain Tumor Center, Department of Neurosurgery, Henry Ford Hospital, 2799 West Grand Blvd, Detroit, MI 48202, USA
* Author for correspondence (e-mail: oliver{at}bogler.net)
Accepted 31 March 2003
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Summary |
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Key words: Glioma, Astrocytes, SETA/CIN85/Ruk, AIP1, Focal adhesion kinase, Cytoskeleton, Electrical cell substrate impedance sensor
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Introduction |
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Recently SETA/CIN85/Ruk has been implicated in participating in the
internalization of activated receptor tyrosine kinases together with Cbl
proteins. Ligand activation of EGFR leads to the binding and phosphorylation
of c-Cbl or Cbl-b proteins via their modified SH2 phosphotyrosine binding
domains, and the recruitment of SETA/CIN85 by binding of its SH3 domains to
the C-terminus of the Cbls (Take et al.,
2000; Soubeyran et al.,
2002
; Szymkiewicz et al.,
2002
). SETA/CIN85 is monoubiquitinated at lysines in its
C-terminus, while the EGFR is polyubiquitinated by the Cbls marking it for
proteasomal degradation (Soubeyran et al.,
2002
; Haglund et al.,
2002
; Szymkiewicz et al.,
2002
). In addition, endophilins are recruited to the complex, by
virtue of a constitutive interaction with SETA/CIN85 mediated by the
endophilin SH3 domain and the SETA/CIN85 proline rich C-terminus
(Soubeyran et al., 2002
;
Szymkiewicz et al., 2002
). It
has been demonstrated that the internalization and ubiquitination of the EGFR
can be mechanistically separated, and the interaction of the SETA/CIN85-Cbl
complex with the receptor may be primarily involved in internalization into
clathrin coated vesicles, while the ubiquitination state may regulate
subsequent sorting into recycling or degradation pathways
(Soubeyran et al., 2002
;
Szymkiewicz et al., 2002
).
While the implication of SETA/CIN85/Ruk in receptor internalization is an
important advance in our understanding of the function of this protein, it is
unlikely to reflect all aspects of it. In addition to the demonstration that
SETA/Ruk can inhibit PI3K activity (Gout
et al., 2000), there is a growing list of interactions with other
signaling molecules including regulators of the cytoskeleton and modulators of
apoptosis including Crk-I, Crk-II, p130(Cas), Grb2, Sos1 and AIP1
(Chen et al., 2000
;
Borinstein et al., 2000
;
Watanabe et al., 2000
). In
addition, localization of SETA in the cell may provide important clues to its
function. We had previously observed that SETA staining overlapped with actin
staining in astrocytes (Chen et al.,
2000
), which prompted further investigation of the association
between SETA and cytoskeletal proteins, focal adhesion kinases and whether it
could modulate cellular adhesion characteristics. In addition we had shown
that AIP1, a binding partner of SETA, also had a cell-staining pattern with
cytoskeletal appearance, and so investigated its association with cytoskeletal
proteins at the molecular level. Here we show by immunocytochemistry and
confocal microscopy that SETA staining overlaps with actin and ß-tubulin
staining in astrocytes, but that there is no direct molecular interaction
between these proteins. Similarly, intense SETA staining at focal adhesions
but the presence of relatively small amounts of SETA in FAK or PYK-2
immunoprecipitates suggests that indirect binding may the most prevalent mode
of interaction between these proteins. In contrast the SETA interacting
protein AIP1 can be found complexed to cytoskeletal proteins and is robustly
present in focal adhesion kinase immunoprecipitates, and so may be directing
SETA to them. In agreement with this is the observation that SETA increases
the amount of AIP1 in these complexes. Furthermore, we show that SETA promotes
adhesion and AIP1 or c-Cbl antagonize adhesion as measured at steady state by
an electrical cell-substrate impedance sensor [ECIS
(Mitra et al., 1991
)],
suggesting that these proteins have an impact on cell behavior. Interestingly,
the presence of AIP1 proteins in PYK-2 complexes correlates with a reduction
in cell adhesion in ECIS experiments, suggesting a direct link between the
interaction between these proteins and cell adhesion. Lastly, we show that
AIP1 can reduce the level of endogenous focal adhesion kinase phosphorylation,
providing a possible mechanism of its ability to regulate these processes.
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Materials and Methods |
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Antibodies used for immunohistochemistry were phalloidin-FITC and
anti-ß-tubulin (Sigma, St Louis, MO). For western blots and
immunoprecipitations we used the following antibodies: monoclonal mouse
anti-HA (F-7) and polyclonal goat anti-actin (I-19) antibodies from Santa Cruz
Biotechnology (Santa Cruz, CA); monoclonal anti-FLAG (M2), anti-GFAP (G-A-5),
anti-MAP1 (HM-1), anti--tubulin (B-5-1-2), anti-ß-tubulin
(2-28-33), anti-
-tubulin (GTU-88), anti-phosphotyrosine tubulin
(TUB-1A2), as well as polyclonal rabbit anti-actin, anti-MAPs and anti-PKC
zeta antibodies from Sigma. Monoclonal mouse anti-phosphotyrosine antibody
(4G10) from Upstate Biotechnology as well as monoclonal anti-AIP1 (49),
anti-FAK (77) and anti-PYK2 (11) antibodies from BD Biosciences, San Jose, CA.
Polyclonal anti-SETA antibodies were as described
(Chen et al., 2000
).
Cell lines and cell transfection
Primary rat cortical astrocytes were isolated as described
(McCarthy and De Vellis, 1980;
Chen et al., 2000
) and were
cultured in DMEM supplemented with antibiotics and 10% fetal calf serum
(DMEMFCS). HEK293 embryonic kidney cells were cultured under standard
conditions in DMEM-FCS. Cells were transfected with plasmids by a modified
calcium-phosphate procedure. The day before transfection either two or five
million cells were plated in 10 cm tissue culture dishes to allow them to
remain non-confluent or become confluent, respectively, two days after
transfection.
Immunoprecipitation and in vitro confrontation
Cells were washed twice with ice-cold PBS and were lysed on ice for 30
minutes in a modified RIPA buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 1%
IGEPAL CA-630 (Sigma), 0.5% deoxycholate, 0.1% SDS, 5 mM EDTA, 1 mM EGTA, 1 mM
DTT, 4 mM sodium azide, 1 mM PMSF, 5 mM benzamidine], as well as a protease
inhibitor cocktail (2 µg/ml aprotinin and leupeptin, 10 µg/ml E-64 and
trypsin inhibitor, 1 µg/ml pepstatin A) and a phosphatase inhibitor
cocktail (2 mM sodium vanadate and sodium fluoride, 5 mM sodium molybdate and
15 mM p-nitrophenylphosphate). Following lysis, the cell suspension was
sheared ten times through an 18G1g needle, ten times through an IM1 needle,
and incubated on ice for another 30 minutes. The cell solution was then
cleared by centrifugation at 20,000 g at 4°C. The
supernatant was used for immunoprecipitation studies. Appropriate
concentrations of primary antibody were added and the solution was rotated at
4°C for at least an hour. Antibody-protein complexes were precipitated
with 50 µl Protein A-agarose solution (Roche) by rotation at 4°C
overnight. The agarose beads were collected by centrifugation at 12,000
g for five minutes at 4°C, and were washed seven times
with precipitation buffer on ice. Finally, the sediment was boiled for five
minutes at 95°C in 2x NuPAGE® LDS Sample Buffer
(Invitrogen) containing 20% ß-mercaptoethanol and transferred to ice
immediately. The solution was cleared of agarose by centrifugation and stored
at -80°C until further analysis by protein electrophoresis. Microtubule
spin-down assays were performed using the Microtubule Associated Protein
Spin-Down Assay Kit BK029 (Cytoskeleton Inc., Denver, CO) according to the
manufacturers instructions. In vitro confrontation experiments were carried
out as previously described (Chen et al.,
2000; Borinstein et al.,
2000
).
Western blotting
Protein samples were analyzed by SDS-PAGE with NuPAGE 4-12% or 10% Bis-Tris
gels (1 mm) according to the manufacturer's guidelines. This gel system alters
the relative mobility of proteins when compared with conventional Laemmli PAGE
gels, and so extra care was taken to confirm the identity of all proteins
identified here, by comparing non-transfected controls and using more than one
antibody when possible. Proteins were blotted to PVDF and were incubated for 1
hour in blocking buffer (5% BSA and 1% Tween-20 in TBS) and overnight with
appropriate dilutions of primary antibody in blocking buffer. Membranes were
washed and incubated for 1 hour with alkaline-phosphatase conjugated secondary
antibody solution in blocking buffer (Sigma; dilutions: anti-mouse antibody
1:3000, anti-rabbit antibody 1:5000, and anti-goat antibody 1:15,000). After
additional washing steps, antibody complexes were visualized on film by
Immun-Star AP substrate (Bio-Rad).
Immunocytochemistry
Primary cortical rat astrocytes were grown on glass coverslips, washed in
phosphate buffered saline (PBS; pH 7.4) and fixed in 4% w/v paraformaldehyde
in PBS for 10 minutes, before being rinsed in Hanks Balanced Salt Solution
(HBSS; buffered with 0.04 M HEPES pH 7.4, 5% v/v calf serum) and incubated in
primary antibodies in HBSS supplemented with 5% v/v goat serum for one hour in
a humidified chamber. After several rinses in HBSS cells were incubated in
species and isotype specific secondary fluorescently labeled antibodies
(Southern Biotechnology, Birmingham, AL) or phalloidin-FITC for another hour.
After the final washing steps, cells were mounted in glycerol supplemented
with 2.5% w/v 1,4-diazobicyclo-2.2.2 octane (Sigma) and viewed on a Bio-Rad
MRC1024 confocal microscope.
ECIS cell attachment assay
The electrical cell-substrate impedance sensor system (ECIS, Applied
Biophysics, Troy, NY) (Mitra et al.,
1991; Tiruppathi et al.,
1992
; Lo et al.,
1995
) was applied to analyze the ability of SETA, AIP1 and c-Cbl
to alter the steady-state cell adhesive properties of HEK293 cells.
Corresponding plasmids were transiently transfected 48 hours prior to each
experiment. Cells were harvested after 2 days and plated at 200,000 cells per
well in ECIS chambers with 400 µl DMEM-FCS to obtain a confluent layer of
cells covering each electrode. Cell attachment behavior was monitored for up
to 10 hours, with resistance values being collected every few seconds by the
ECIS device. Complete single-layer electrode coverage was confirmed
microscopically prior to data analysis. To analyze ECIS results, resistance
values were adjusted by first subtracting the value of each individual
electrode before addition of the cells, when only medium was present to
compensate for minor differences in electrode manufacture. Values were then
normalized by subtraction of the resistance value of lacZ-transfected
control cells at each time-point. At each hour the ten readings leading up to
that hour were averaged and their standard deviation calculated. Finally, the
change relative to the zero time point was calculated for each data series
independently, and graphed as change in resistance over time.
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Results |
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AIP1 but not SETA interacts directly with cytoskeletal proteins
To investigate the interaction between SETA and cytoskeleton proteins,
immunoprecipitation experiments were performed in HEK293 cells, which are more
efficiently transfected than primary astrocytes. Cells were transfected with
full-length SETA and immunoprecipitates made with antibodies against various
cytoskeletal components were analyzed by anti-SETA western blot
(Fig. 2). This analysis failed
to show evidence for a direct interaction between SETA and actin, any of the
tubulins, microtubule associated proteins (MAPs) or GFAP
(Fig. 2, lanes 1-8). In
addition no interaction was demonstrable with the signal transduction
associated molecule protein kinase C (PKC;
Fig. 2, lane 9). SETA could be
recovered by immunoprecipitating the EGFR, as has been shown previously
(Soubeyran et al., 2002).
Because immunoprecipitation experiments of cytoskeletal components may favor
recovery of monomers, a microtubule associated protein spin-down assay was
also performed. Microtubules were polymerized in vitro
(Hyman et al., 1991
) and
confronted with bacterial SETA protein or cell extract that had been
pre-cleared of microtubules but contained SETA, and the synthesized
microtubules were subsequently pelleted by centrifugation. Anti-SETA western
blots performed on the pellets failed to provide evidence for a direct
interaction between SETA and microtubules (data not shown), suggesting that
the co-localization observed in astrocytes occurs indirectly.
|
The SETA binding partner, apoptosis linked gene-2 interacting protein 1
[AIP1 (Chen et al., 2000)], was
present in immunoprecipitates made with antibodies against various
cytoskeletal components, as well as EGFR. Strong AIP1 signals were obtained in
association with actin,
-tubulin, tyrosine phosphorylated tubulin and
with the MAPs (Fig. 2, lanes
1,2,5,6,7), while lower but detectable signals were obtained with
ß-tubulin and GFAP immunoprecipitates
(Fig. 2, lanes 3,8), and a weak
signal was obtained with
-tubulin
(Fig. 2, lane 3). No AIP1
protein was recovered in immunoprecipitates made with PKC antibody,
demonstrating the specificity of the other interactions
(Fig. 2, lane 9). The ability
of AIP1 to bind cytoskeletal proteins allows that it mediates their
interactions with SETA.
SETA and AIP1 interact with focal adhesion kinases
The intense SETA immunoreactivity observed at focal adhesions
(Fig. 1A-F) suggested that SETA
may interact with proteins associated with these specialized structures.
Therefore we next immunoprecipitated the endogenous focal adhesion kinases FAK
and PYK-2 from SETA-transfected HEK293 cells
(Fig. 3). These experiments
revealed that there was a weak interaction between SETA and FAK
(Fig. 3A) and PYK-2
(Fig. 3B). Cell density did not
affect the strength of interaction (shown for PYK-2 in
Fig. 3B) suggesting that SETA
is a weak interacting partner of the focal adhesion kinases under different
physiological conditions. The SETA protein associated with focal adhesion
kinases was detected as a band with an apparent molecular weight of about 160
kDa in these experiments, while other SETA bands, including one at 85 kDa,
that were observed in the lysates were not represented in the
immunoprecipitates (Fig. 3).
The 160 kDa band appeared only when gels are run under weak denaturing
conditions, and likely represents a dimer of SETA mediated by the coiled-coil
domain (Borinstein et al.,
2000; Watanabe et al.,
2000
). To test this directly, we introduced either SETA full
length or SETA
cc, which lacks the C-terminal coiled-coil, into HEK293
cells, and performed western blots under strong or weak denaturing conditions
(Fig. 3C). The 160 kDa SETA
band was only found under weak denaturing conditions when the SETA protein
encoded the coiled-coil motif (Fig.
3C, lane 3).
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To determine whether a stronger interaction could again be observed between
focal adhesion kinases and AIP1, we transfected AIP1 into HEK293 cells and
immunoprecipitated endogeneous FAK and PYK-2 proteins
(Fig. 4). High levels of AIP-1
were present in FAK (Fig. 4A,
lane 1) and PYK-2 (Fig. 4Bb,
lanes 1,2) immunoprecipitates. Interestingly, the amount of AIP1 recovered
from precipitates of the focal adhesion kinases could be somewhat enhanced by
increasing the cell density (shown for PYK-2 in
Fig. 4B, lanes 1,2), while AIP1
expression levels were similar (Fig.
4B, lanes 5,6). In contrast, another binding partner of SETA,
c-Cbl (Borinstein et al., 2000;
Take et al., 2000
;
Soubeyran et al., 2002
), was
not found in FAK immunoprecipitates (Fig.
4A, lane 3), suggesting that not all SETA binding partners are
present in this complex. However, c-Cbl was part of a complex with PYK-2,
indicating an interesting difference between these two focal adhesion kinases.
However, it was only present at a low intensity
(Fig. 4B, lanes 3,4) as
compared to AIP1. Lastly, another protein that is associated with AIP1,
apoptosis linked gene 2 (ALG-2) was examined in this context, and found to be
recoverable in a PYK-2 immunoprecipitate
(Fig. 4C).
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The interaction between focal adhesion kinases and actin is calcium
dependent, and can be strengthened by high levels of intracellular calcium,
which promote PYK-2 activity, autophosphorylation and complex formation
(Lev et al., 1995). To
determine whether this would also mediate an increase in AIP1 association, the
concentration of calcium was increased in cell lysates prior to
immunoprecipitation with anti-PYK-2 antibodies
(Fig. 5). In the presence of 10
mM CaCl2 a noticeably higher level of AIP1 and actin was recovered
from PYK-2 immunoprecipitates (Fig.
5, lane 3).
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SETA promotes the interaction between AIP1 and PYK-2
If SETA, AIP1 and the focal adhesion kinases participate in a multi-protein
complex, it is possible that altering the level of one component may affect
the interaction of the others. To test this hypothesis we first attempted to
create an AIP1 mutant that was incapable of binding SETA. Two deletion
constructs of the AIP1's proline rich C-terminus, where the sequences thought
to interact with SETA's SH3 domains are located, were made
(Fig. 6A): one lacking the
C-terminal half (nonsense codon at position 784; AIP1-784Stop) and the other
lacking the N-terminal half (in-frame deletion from residues 717 to 784
inclusive; AIP1-717-784). In vitro binding assays between bacterial
GST-SETA and in vitro transcribed and translated AIP1 showed that binding of
AIP1-784Stop to SETA was indistinguishable from wild type AIP1
(Chen et al., 2000
), while
AIP1-
717-784 bound at low levels, and did not discriminate between
different SETA SH3 domains (Fig.
6B). This suggests that the N-terminal half of the proline rich
C-terminus of AIP1 contains the SETA binding site, and that this deletion
mutant can be used to probe the role of the SETAAIP1 interaction.
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The effect of SETA on the interaction between AIP1 or AIP1 mutants and FAK or PYK-2 was then tested (Fig. 7). The transfection of SETA did not alter the amount of AIP1 detected in FAK immunoprecipitates, but did strengthen the interaction between AIP1 and PYK-2 (Fig. 7, lanes 1,2). The lower amount of SETA in the lysates of the AIP1-SETA cotransfection (Fig. 7, lane 2) as compared to the other SETA-containing co-transfections (Fig. 7, lanes 4,6,8) was a consistent observation, which we are currently investigating. It is possible that this is the reason we did not observe an effect of SETA on AIP1 in the FAK immunoprecipitations. However, this lower level did not affect the amount of SETA in the PYK-2 complexes, made from the same lysates, nor from SETA having an affect on the amount of AIP1 associated with PYK-2 (Fig. 7, lanes 1,2), implying that the amount of SETA was not necessarily limiting.
|
Interestingly, no interaction with either focal adhesion kinase was
observed for a mutant, AIP1-Y319F, which has an alteration in the single
consensus tyrosine kinase phosphorylation site, shown to be phosphorylated by
src in the Xenopus homolog of AIP1, Xp95
(Che et al., 1999). Reduced
interaction with focal adhesion kinases was also observed for the other AIP1
mutants, AIP1-
717-784 and AIP1-784Stop. Interestingly transfection of
SETA was able to increase the amount of these two AIP1 mutants in both FAK and
PYK-2 immunoprecipitates (Fig.
7, lanes 5-8). In both sets of immunoprecipitations SETA had a
more pronounced effect on the amount of AIP1-784Stop than AIP1-
717-784
recovered (Fig. 7, lanes 6,8),
in line with their relative abilities to interact with SETA. Neither wild-type
AIP1 nor the deletion mutants increased the amount of SETA in focal adhesion
kinase complexes.
SETA and AIP1 modulate cell adhesion via the attenuation of PYK-2
activity
To determine whether the association between SETA and elements of the
cytoskeleton and focal adhesions had any impact on cell behavior we measured
steady-state cell adhesion using an electrical cell-substrate impendence
sensor [ECIS (Mitra et al.,
1991; Tiruppathi et al.,
1992
; Lo et al.,
1995
)]. ECIS allows the measurement of cell-substrate adhesion
both during the cell attachment phase and after cells have become attached and
are in a steady state condition, rather than relying on the transient
attachment of cells that have been trypsinized, are spherical and do not have
well-formed focal adhesions. HEK293 cells were transfected with expression
plasmids encoding SETA, AIP1, AIP1 mutants or the SETA binding partner c-Cbl,
and analyzed by ECIS under confluent conditions over a period of nine hours.
Changes in resistance after subtraction of the measurements obtained from
lacZ-transfected control cells, and after setting the zero time point
to a zero value were calculated over time
(Fig. 8). Transfection of full
length SETA resulted in an increase in resistance, after an initial three hour
period as cells settled on the electrode, and a reaching of a steady state of
close to 1500 ohm over control (Fig.
8A), indicating a pro-adhesive effect of SETA. In contrast,
transfection of c-Cbl or AIP1 had the opposite effect and diminished
resistance and cell adhesion. Again changes were manifested after an initial
period of settlement, and resulted in these cells reaching a steady state
close to -1500 ohm relative to the lacZ-transfected control
(Fig. 8A). This suggests that
AIP1 and c-Cbl have anti-adhesive effects. To compare the impact of AIP1
mutants to AIP1, AIP1-
717-784, AIP1-784Stop and AIP1Y319F were also
analyzed. AIP1Y319F had no prominent effect on adhesion, and a slight increase
over time was observed (Fig.
8B). Similarly, AIP1-
717-784 showed essentially no
difference when compared to control cells
(Fig. 8B). In contrast,
AIP1-784Stop mediated a similar decrease in adhesion to wild-type AIP1. These
data correlated very well with what was observed in
Fig. 7 in terms of AIP1 protein
in PYK-2 immunoprecipitates: a reduction in adhesion was only observed in the
case of AIP1 and AIP1-784Stop, which were present in PYK-2 complexes, but not
in the case of AIP1Y319F or AIP1-
717-784 which were not recruited to
this kinase. These immunoprecipitation studies had also demonstrated the
ability of SETA to recruit AIP1-
717-784 but not AIP1Y319F proteins to
focal adhesion kinase complexes, and so SETA's ability to modulate cell
adhesion decreases by AIP1 proteins was tested by ECIS. This analysis further
supported the correspondence between a reduction of cell adhesion and the
presence of AIP1 protein in PYK-2 complexes. In the case of co-transfection of
SETA and AIP1Y319F, which did not result in the recruitment of the AIP1 mutant
to PYK-2 (Fig. 7, lanes 2,4) an
increase in adhesion similar to that observed when SETA was transfected alone
was observed (Fig. 8A,B),
suggesting that SETA's effect was the dominant phenomenon. Interestingly, when
SETA was co-transfected with AIP1-
717-784 a reduction in cell adhesion
relative to control was observed (Fig.
8B), paralleling the ability of SETA to increase the amount of
AIP1-
717-784 in PYK-2 complexes
(Fig. 7, lanes 5,6).
Co-transfection of SETA and AIP1 or AIP1-784Stop did not further decrease
adhesion, suggesting that it did not promote an effect beyond that already
induced by these AIP1 proteins alone.
|
Finally, to determine whether the presence of AIP1 proteins in focal
adhesion kinases had any consequences on the biochemical nature of these
proteins, cells were transfected with AIP1 proteins, and the endogenous PYK-2
or FAK protein was immunoprecipitated and analyzed for phosphotyrosine levels
(Fig. 9). This study
demonstrated that co-transfection of AIP1 resulted in a significant
attenuation of both PYK-2 and FAK tyrosine phosphorylation levels, suggesting
that this protein may regulate the activity of these protein kinases.
AIP1-784Stop also reduced PYK-2 phosphotyrosine levels, but to a smaller
extent, and appeared to have little if any effect on FAK phosphorylation
levels. This mutant is also present in PYK-2 immunoprecipitates
(Fig. 7) and capable of
reducing adhesion in HEK293 cells (Fig.
8). As expected from the cell adhesion and the PYK-2
immunoprecipitation data, neither AIP1Y319F nor AIP1-717-784 affected
focal adhesion kinase phosphorylation levels
(Fig. 9). This experiment
suggests that negative modulation of PYK-2 phosphorylation levels is a
consequence of AIP1 protein binding and a possible mediator of AIP1 action on
cell adhesion.
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Discussion |
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The localization of SETA to focal adhesions prompted us to further explore
interactions with the focal adhesion kinases FAK
(Cary and Guan, 1999;
Parsons et al., 2000
) and
PYK-2/Cakß/RAFTK/CADTK/FAK2 (Lev et
al., 1995
). Immunoprecipitation studies of endogenous focal
adhesion kinases revealed the presence of small amounts of a higher molecular
weight isoform of transfected SETA, which most likely represented a dimer
(Fig. 3). While it has
previously been demonstrated that the coiled-coil domain of SETA can mediate
multimerization (Borinstein et al.,
2000
; Watanabe et al.,
2000
; Verdier et al.,
2002
) this is the first instance to our knowledge, of a
SETA-binding partner showing a preference for SETA dimers.
Although the association between SETA and FAK or PYK-2 could occur directly
via SETA's SH3 domains and the proline-rich regions of both kinases
(Lev et al., 1995;
Cary and Guan, 1999
), the
amount of SETA observed in FAK or PYK-2 immunoprecipitates was relatively low
and contrasted with the robust signal obtained by cell staining. This suggests
that SETA may also be interacting with focal adhesions via other proteins, and
prompted an investigation of AIP1 and c-Cbl
(Fig. 4). It has previously
been shown that c-Cbl associates with PYK-2 via src
(Sanjay et al., 2001
), and
localizes to actin lamellae in fibroblasts
(Scaife and Langdon, 2000
).
Immunoprecipitation studies showed a stronger association between AIP1 and FAK
or PYK-2 than was found for SETA, mirroring the results obtained for
cytoskeletal proteins, and again allowing for a role for AIP1 in mediating
SETA localization. As reported by others
(Sanjay et al., 2001
) c-Cbl
was associated with PYK-2, however it bound more weakly than AIP1, and did not
associate with FAK. This suggests the interaction between SETA and the focal
adhesion kinases does not resemble SETA's recruitment to the activated EGFR
complex, which occurs via Cbl proteins
(Soubeyran et al., 2002
), but
is likely to involve other molecules including AIP1.
The strong presence of AIP1 in focal adhesion kinase complexes prompted an
investigation of its binding partner ALG-2
(Vito et al., 1999), which was
found to be associated with PYK-2 as well. Interestingly, ALG-2 binding to
AIP1 is Ca2+ regulated (Vito et
al., 1999
) as is PYK-2 activation
(Lev et al., 1995
). ALG-2
shares some sequence homology with the calpain protease family, which
disassembles focal adhesions by cleaving the activated FAK complex, and
reduces cell adhesion (Frame et al.,
2002
), allowing for the possibility that it has a regulatory role,
and that association with AIP1, and perhaps indirectly SETA, modulates its
activity. Furthermore, analysis of the calcium-dependency of the AIP1-PYK-2
interaction (Fig. 5) showed
that a large increase in calcium concentration resulted in an enhanced
attraction of AIP1 and actin to PYK-2. This may reflect the ability of
increased intracellular Ca2+ to stimulate the formation of focal
adhesions in intact cells (Lev et al.,
1995
; Blaukat et al.,
1999
).
To test the role of the interaction between SETA and AIP1 in the presence
of AIP1 in focal adhesion kinase complexes we created mutants in AIP1. All
AIP1 mutants were restricted in their capacity to bind FAK or PYK-2. The
AIP1Y319F mutation, which alters the single consensus tyrosine phosphorylation
site, eliminated interaction between AIP1 and PYK-2 completely, allowing for a
critical role for tyrosine kinase signaling in the formation of this complex.
It has been established that the interaction between activated PYK-2 and src
results in the src-mediated phosphorylation of other associated proteins
(Blaukat et al., 1999;
Parsons et al., 2000
),
allowing for the possibility that the stability of the AIP1-PYK-2 interaction
is dependent on the presence of src and its ability to phosphorylate AIP1 at
Y319F.
Analysis of AIP1 mutants also showed that deletion of the SETA binding site
in AIP1-717-784 strongly diminished the amount of AIP1 in focal
adhesion kinase immunoprecipitations, making it undetectable in the case of
PYK-2. Furthermore, elimination of the other half of the proline rich
C-terminus in AIP1-784Stop also significantly diminished binding to FAK or
PYK-2. Taken together this suggests that the interaction between AIP1 and
focal adhesion kinases is not SETA dependent. However, SETA was capable of
increasing the amount of AIP1 associated with PYK-2 and partially restoring
the interaction of the C-terminal deletion mutants of AIP1 with FAK or PYK-2.
This included AIP1-
717-784, which lacks the SETA binding site,
suggesting that it may be mediated indirectly, and possibly represents the
stabilization of the focal adhesion protein complex via other associated
proteins. It is worth noting in this context that, in addition to the
interactions discussed above, there are other binding partners of the focal
adhesion kinases that are also known to interact with SETA. These include the
adaptor protein Grb2 (Lev et al.,
1995
; Cary et al.,
1998
; Cary and Guan,
1999
; Borinstein et al.,
2000
) and the p85 regulatory subunit of PI3K
(Cary and Guan, 1999
;
Gout et al., 2000
;
Parsons et al., 2000
). In
addition, receptor tyrosine kinases, including the EGFR may play a role, as
they interact with the very N-terminus of FAK and PYK-2
(Parsons et al., 2000
) and are
internalized by a complex that contains SETA/CIN85
(Soubeyran et al., 2002
).
To examine the impact of the molecular events described above on cells we
performed ECIS cell adhesion assays with HEK293 cells transfected with AIP1,
AIP1 mutants, SETA or c-Cbl. First, we compared the impact of AIP1, SETA and
c-Cbl, and found that all three molecules had profound effects on adhesion,
with SETA mediating increased cell adhesion, while AIP1 and c-Cbl caused a
similar decrease (Fig. 8A).
Analysis of AIP1 mutants, and the modulation of their impact by SETA, provided
further evidence that the presence of AIP1 in PYK-2 complexes is an important
negative modulator of adhesion. For example, AIP1Y319F, which was not found in
focal adhesion kinase complexes, did not reduce cell adhesion, and was
incapable of antagonizing SETA's stimulation of cell adhesion in
co-transfection experiments. In contrast, AIP1 or AIP1-784Stop, which bound
PYK-2 in the presence or absence of SETA, reduced adhesion regardless of
whether SETA was co-transfected. Interestingly, AIP1-717-784, which
alone was not detectable in PYK-2 immunoprecipitates did not modulate cell
adhesion. However, when SETA was co-transfected, and increased the amount of
AIP1-
717-784 in the complex (Fig.
7), this resulted in a reduction in adhesion as measured by ECIS
(Fig. 8B). These data
demonstrate a direct connection between the composition of the PYK-2 complex
and cell behavior, and suggest that the presence of AIP1 has a negative impact
on cell adhesion.
The molecular basis for AIP1's negative impact on cell adhesion is not yet
fully understood but has been reported in a different assay for the human
homologue Hp95 (Wu et al.,
2002). Our experiments show that AIP1 is capable of reducing the
level of tyrosine phosphorylation of both FAK and PYK-2
(Fig. 9). Focal adhesion kinase
phosphorylation on tyrosine has been linked to increased cell adhesion in
another cellular context (McDonald et al.,
2000
). Similarly, it has been shown that c-Cbl negatively
regulates cell adhesion in a complex with src and PYK-2, and does so by
binding the auto-phosphorylation site of c-src, which would otherwise be
activated by integrin signaling via focal adhesion kinases
(Blaukat et al., 1999
;
Sanjay et al., 2001
). This
illustrates a possible mechanism for c-Cbl action in our experiments, and
raises the possibility that AIP1 could reduce focal adhesion kinase
phosphorylation on tyrosine by interacting with src. AIP1 is likely to be a
src kinase substrate by analogy to its Xenopus homologue Xp95
(Che et al., 1999
). Indeed, the
apparent requirement for a tyrosine phosphorylation site (Y319) in AIP1 for
interaction with focal adhesion kinases suggests that AIP1 may be an adaptor
protein between src and focal adhesion kinases. An alternative mechanism of
action may relate to AIP1's potential of recruiting of ALG-2, a relative of
calpain proteases that are known to negatively affect focal adhesions
(Frame et al., 2002
).
Similarly SETA's mechanism of action on cell adhesion is not yet clear,
although it appears to work in part independently to promote adhesion when no
AIP1 is present. When AIP1 proteins are present, this effect is masked by its
promotion of AIP1 protein presence in PYK-2 immunoprecipitates. The molecular
structure of SETA, as well as the wide range of other interactions described
for it, allow that SETA may have additional functions in integrating focal
adhesions with other signal transduction pathways
(Chen et al., 2000
;
Borinstein et al., 2000
;
Take et al., 2000
;
Watanabe et al., 2000
;
Dikic, 2002
).
In summary, SETA and AIP1 have been identified as proteins that are
associated with diverse cytoskeletal elements, including focal adhesion
kinases and that have the capacity of modulating cell attachment, which in the
case of AIP1 may relate to its modulation of focal adhesion kinase
phosphorylation levels and activity. The SETA relative CD2AP/CMS has been
reported to co-localize with actin in membrane ruffles
(Kirsch et al., 1999),
suggesting that this family of adaptor molecules is involved in mediating
cytoskeletal function in a variety of settings. Because these proteins have
also been implicated in other important cellular functions, including the
regulation of apoptosis [AIP1 (Vito et
al., 1999
; Chen et al.,
2000
)] and the internalization of receptor tyrosine kinases
[SETA/CIN85 (Take et al.,
2000
; Soubeyran et al.,
2002
)], they may represent novel points of integration between
cell shape, adhesion and motility, and cell division and survival.
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References |
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