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INTRODUCTION |
The Trio Dbl homology
(DH)1 guanine nucleotide
exchange factor (GEF) is essential for late embryonic development and
functions in fetal skeletal muscle formation and in the organization of neural tissue (1). Furthermore, the Trio-like
Caenorhabditis elegans unc-73 gene product is
necessary for both cell migration and axon guidance (2), and
Drosophila Trio also regulates axon guidance.
Drosophila Trio has been functionally linked to
Rac, p21-activated kinase, Abl protein-tyrosine kinase, DLAR
transmembrane protein-tyrosine phosphatase, and the adaptor proteins
Dock and Ena (3-6). Trio is a large and complex protein containing two functional DH GEF domains, which activate Rho-type GTPases, a protein
serine/threonine kinase domain, two SH3-like domains, an Ig-like
domain, and multiple spectrin-like repeats (7). Trio and other DH GEF
family members function to activate Rho GTPases such as RhoA, Rac1, and
Cdc42 by promoting the exchange of GDP for GTP. Once activated, Rho
GTPases function in diverse cellular processes including actin
cytoskeleton organization, mitogen-activated protein kinase cascade
signaling, and gene transcription (8). Targets for Rho GTPases include
protein kinases, lipid kinases, and nonenzymatic proteins (9-12). Trio
itself is a RhoA target and binds the prenyl group of RhoA via the
immunoglobulin-like domain (13). This interaction is promoted by Trio
carboxyl-terminal GEF domain activity and leads to changes in cell
morphology and in the subcellular localization of Trio and RhoA.
The regulation of DH GEFs is incompletely understood, but PH
domain-mediated interactions with phospholipids or proteins, phosphorylation of DH GEF proteins, or interactions of DH GEFs with
other GTPases may be involved (14). The solution structure of the
amino-terminal Trio-GEF/PH region revealed that the GEF and PH domains
interact with one another, and mutational analysis demonstrated that
the PH domain enhances Trio GEF activity for Rac1 in vitro
(15). Trio was isolated through its binding to the intracellular region
of the LAR transmembrane protein-tyrosine phosphatase, and the Trio
segment that binds the LAR PTP-D2 domain was shown to include the
Ig-like and serine/threonine kinase domains (7). The Trio
amino-terminal GEF domain displays Rac1 and RhoG activity (7, 16), and
the carboxyl-terminal GEF domain exhibits RhoA GEF activity in
vitro (7). Trio ectopic expression alters actin cytoskeleton
organization, as well as the distribution of focal contact sites (17,
18).
The FAK (focal adhesion kinase) protein-tyrosine kinase (PTK) is
activated following integrin binding of extracellular matrix and likely
plays a key role in integrin-mediated regulation of cell growth and
survival, as well as in the control of cell spreading and migration.
For instance, FAK-deficient mouse embryonic mesodermal cells (19) and
fibroblasts (20) have reduced cell motility and enhanced focal
adhesions, and expression of a dominant-negative FAK mutant, FRNK,
causes delayed cell spreading and decreases cell motility (20, 21).
Furthermore, ectopic expression of FAK in Chinese hamster ovary cells
enhances cell migration (22). FAK phosphorylates a number of
substrates, including Cas (Crk-associated substrate) family docking proteins and paxillin (23-26),
as well as undergoing autophosphorylation (27, 28). Autophosphorylated FAK recruits the p60src (Src) PTK, the docking proteins Crk and
Nck, phosphatidylinositol 3-kinase (29), as well as Grb2 and Grb7
(reviewed in Ref. 30). Cas and paxillin are involved in coordinating
integrin-mediated regulation of cell proliferation and migration (31,
32). The role of FAK in cell spreading and migration appears to be
mediated at least in part via Cas and paxillin phosphorylation. For
example, increased cell migration associated with ectopic FAK
expression depends on FAK kinase activity and an intact Cas-binding
site in FAK (20).
In this paper, we show that FAK binds Trio and phosphorylates Trio near
the ATP-binding site in its kinase domain and that Trio and FAK
co-localize in vivo at the cell periphery. Finally, Trio
activates FAK autophosphorylation in vitro and in
vivo, indicating that FAK and Trio comprise a novel bi-directional
signaling complex.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructions--
Trio expression constructs were made
using standard techniques and confirmed by restriction mapping and, in
some cases, DNA sequencing. Trio cDNA constructs were cloned into
the pMT.HAtag or pMT.Myctag expression vectors, which encode either a
hemagglutinin (HA) or Myc epitope tag sequence immediately upstream of
the cloning site. The extent of the Trio regions encoded by these
plasmids are schematically shown in Fig. 1. Amino acid numbering of
Trio is according to a full-length Trio of 3,038 amino acids
(GenBankTM accession number UF091395).
Trio.SIKD refers to amino acids 2485-3038 with the a
catalytically inactive kinase domain caused by a K2756A mutation, and
Trio.KT refers to amino acids 2730-2994. The following constructs were used for FAK in the pMT3 vector: FAK.
N (amino acids
384-1052), FAK.K (amino acids 384-706), FAK.KD (amino
acids 384-706, K454R), and FAK.
NK (amino acids 707-1052).
Cell Transfections--
COS-7 cell transient transfections were
done by the DEAE-dextran/Me2SO method using ~2 µg of
plasmid DNA/2 × 105 cells, and the cells were
harvested ~20 h after transfection. The proteins were metabolically
labeled with [35S]methionine and
[35S]cysteine during the final 4 h prior to
harvesting of cells. HeLa cells were transfected by calcium phosphate
precipitation and fixed and stained 3 days after transfection as
described previously (13).
Cell Labeling and Protein Analysis--
The cell proteins were
metabolically labeled with [35S]methionine and
[35S]cysteine essentially as previously described (33).
Following labeling, the cells were washed in PBS, lysed in Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM MgCl2)
containing 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
leupeptin, and 10 µg/ml aprotinin. Insoluble material was pelleted by
centrifugation at 16,000 × g, and the supernatant was
used for immunoprecipitation of soluble protein. The cell lysates were
then precleared once with 25 µl of protein A-Sepharose (Amersham
Biosciences) for 1-2 h. For immunoprecipitations 1 µl of anti-HA mAb
12CA5 ascites fluid and 25 µl of protein A-Sepharose were added per
ml of precleared lysate for 1 h. The precipitates were then washed
with buffer containing 0.1% Nonidet P-40, 150 mM NaCl, 25 mM Tris-HCl, pH 7.5, and 5 mM
MgCl2. The precipitated proteins were analyzed using
SDS-PAGE under reducing conditions followed by autoradiography. The
proteins from cell lysates or co-precipitated proteins from nonlabeled
cells were resolved by SDS-PAGE and then transferred to Immobilon-P
(Millipore, Bedford, MA), and the immunoblots were probed with the
anti-HA mAb HA 11 (BabCO, Richmond, CA) or the anti-GST mAb 112 (ImmunoGen, Cambridge, MA). The immunoblots were developed with rabbit
anti-mouse-horseradish peroxidase-conjugated IgG (Dako, Carpinteria,
CA) and exposed to film using the chemiluminescence reagent, luminol,
essentially as described by the supplier (PerkinElmer Life Sciences).
The immunoblots were quantitated from scanned films using Un-Scan-It gel digitizing software from Silk Scientific, Inc. (Orem, UT). For
experiments in which the detergent-insoluble (P) or detergent-soluble (S) fractions were used, the cells were lysed as above, and the pellet
and supernatant from the 16,000 × g centrifugation
step were boiled in equal volumes of 1× SDS-PAGE sample buffer.
Peptide Mapping--
The tryptic peptide maps were
performed as described previously (34). Briefly, labeled protein was
excised from dried gels, hydrated, washed in MeOH to remove SDS, and
digested 2 days with 0.1 mg/ml trypsin in 100 mM ammonium
bicarbonate. The liberated peptides were dried and separated by
electrophoresis at pH 1.9 in acetic acid/formic acid/water (8:2:90) for
45 min at 800 V and by ascending chromatography in
1-butanol/pyridine/acetic acid/water (37.5:25:7.5:30).
Immunofluorescence--
HeLa cells were rinsed in PBS, fixed in
2% paraformaldehyde/PBS for 15 min, and then permeabilized for 10 min
in 0.1% Triton X-100/PBS containing 2% horse serum. Nonspecific
antibody-binding sites were blocked by a 30-min incubation in blocking
buffer (10% normal goat serum in PBS). To detect HA-Trio and Myc-FAK,
the permeabilized cells were exposed to the anti-HA mAb HA 11 and anti-Myc for 1 h and washed, and then the primary antibodies were detected with a 30-min treatment of Texas Red-linked goat anti-mouse IgG2a (Southern Biotech) and fluorescein isothiocyanate-linked goat
anti-mouse IgG1 (Southern Biotech). Slides were mounted in a polyvinyl
alcohol medium and viewed on a Nikon E800 microscope equipped for epifluorescence.
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RESULTS |
Trio Is Tyrosine-phosphorylated--
Trio was identified by virtue
of its ability to interact with the LAR protein-tyrosine phosphatase
through its carboxyl-terminal Ig-like domain and kinase domain. Because
Trio interacts with LAR and thus could be expected to be a candidate
LAR substrate, we tested whether Trio was tyrosine-phosphorylated.
Immunoblot analysis showed tyrosine phosphorylation of endogenous Trio
precipitated from HeLa cells treated with the tyrosine phosphatase
inhibitor pervanadate, indicating that Trio is a substrate for tyrosine kinases in vivo (data not shown). To map the site(s) of Trio
tyrosine phosphorylation, we initially expressed constructs encoding
various regions of Trio (schematically shown in Fig.
1A): the amino-terminal spectrin repeats (HA-Trio.SP), the two GEF domains (HA-Trio.G1 and
HA-Trio.G2S), the second GEF domain through to carboxyl terminus (HA-Trio.GSIK), and the carboxyl-terminal region of Trio containing the carboxyl-terminal SH3 domain, Ig-like domain, and kinase domain (HA-Trio.SIK). When expressed in COS-7 cells, all of the Trio subregions partitioned to the detergent-soluble fraction
except the HA-Trio.SIK, which partitioned to both the
detergent-soluble (S) and -insoluble fractions (P) (Fig.
1B). Immunoblotting of the detergent-soluble and -insoluble
pellet fractions using an anti-Tyr(P)-specific mAb indicated that
Trio.SIK, the Lar-binding portion of Trio, was the only region of Trio
undergoing significant levels of tyrosine phosphorylation and that
tyrosine-phosphorylated Trio.SIK was almost exclusively localized to
the insoluble fraction (Fig. 1B). Expression of either the
Trio SH3, Ig-like, or kinase domains alone indicated that the kinase
domain was responsible for localization to the Triton-insoluble
cytoskeletal fraction (Fig. 2A
and data not shown).

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Fig. 1.
A, diagram of the Trio constructs used
in this study. The structure of the 3038-amino acid Trio protein is
shown schematically at the top, and below are the
Trio deletion constructs used in this study. The numbers in
parentheses are the Trio residues encoded by the various
constructs. GEF-D, guanine nucleotide exchange factor
domain; SH3-D, Src homology 3 domain. B, Trio is
phosphorylated in vivo. COS-7 cells were transiently
transfected with the following Trio constructs: HA-Trio SP, HA-Trio G1,
HA-Trio.GSIK, HA-Trio.G2S, and HA-Trio.SIK. Two days after
transfection, the cells were lysed in Nonidet P-40 lysis buffer and
centrifuged at 10 000 × g, and the supernatants and
pellets were boiled in equal volume of 1× SDS sample buffer. Equal
aliquots from each fraction were electrophoresed on SDS-polyacrylamide
gels, transferred to Immobilon-P, and subjected to Western blotting
analysis using anti-HA and anti-Tyr(P) antibodies. WB,
Western blot.
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Fig. 2.
Trio is phosphorylated by FAK.
A, COS-7 cells were transiently transfected with the Trio
constructs HA-Trio.SIK, HA-Trio.SI, and HA-Trio.K alone or with either
HA-FAK or HA-FAKY397F. The cells were processed as
described in the legend to Fig. 2 and subjected to immunoblotting
analysis using anti-HA or anti-Tyr(P) antibodies. B, COS-7
cells were transfected with HA-Trio.GSIK and Myc-FAK, and the cells
were processed as described above. C, COS-7 cells were
transiently transfected with the Myc-Trio.K or
Myc-Trio.KY2737F constructs, processed as described above,
and subjected to immunoblotting analysis using anti-Tyr(P) and anti-Myc
tag antibodies. WB, Western blot.
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Phosphorylation of Trio by FAK--
Several properties of the
focal adhesion kinase FAK were consistent with it mediating the Trio
phosphorylation observed in COS-7 cells. Like the Trio constructs, the
majority of the tyrosine-phosphorylated FAK in transiently transfected
COS-7 cells is present in the insoluble fraction (Fig. 2A).
Immunoblot analysis of COS-7 cell lysates also indicated that the FAK
in the insoluble fraction was reactive with antibodies specific for the
FAK Tyr-397 phosphorylation site (data not shown), which is the major
FAK autophosphorylation site (25, 35, 36). The similar localization of
Trio and active autophosphorylated FAK suggested that FAK may
phosphorylate Trio.
To test the phosphorylation of Trio by FAK, the Trio constructs
HA-Trio.SIK, HA-Trio.SI, HA-Trio.K, and HA-Trio.GSIK (Fig. 1A) were all co-expressed with HA-FAK in COS-7 cells (Fig.
2, A and B). The cell extracts were prepared, and
the detergent-soluble and detergent-insoluble fractions were run on
SDS-polyacrylamide gels. Immunoblotting analysis revealed that in the
presence of FAK, all of the Trio constructs present in the insoluble
pellet fraction displayed shifts to lower mobility forms, consistent with increased tyrosine-phosphorylation (Fig. 2, A and
B). Additionally, in the presence of FAK, there was
increased partitioning of the Trio constructs to the
detergent-insoluble fraction of the lysates, especially evident with
the Trio.GSIK (Fig. 2B), suggesting that FAK can regulate
Trio localization. The HA-Trio.GSIK, HA-Trio.SIK, HA-Trio.SI, and
HA-Trio.K constructs in the insoluble fraction of COS-7 lysates were
subsequently found to be tyrosine-phosphorylated, and the constructs
containing the Trio kinase domain were more highly phosphorylated (Fig.
2 and data not shown). There was no observed phosphorylation of Trio
constructs when co-expressed with FAK containing a kinase catalytic
mutation (data not shown). The FAK autophosphorylation site Tyr-397 has
been shown to recruit and bind the Src PTK, forming a complex between
these two active tyrosine kinases (27, 28, 35-37). However, Trio
constructs were also phosphorylated by the HA-FAKY397F
construct in which the Src-binding site is mutated, suggesting that
Trio phosphorylation is mediated by FAK and not by Src that is
associated with FAK (Fig. 2A). Co-transfection of Trio.SIK
and FAK in HeLa cells also demonstrated preferential localization of
tyrosine-phosphorylated Trio.SIK to the insoluble fraction of cell
lysates (data not shown). Taken together, these results indicate that
there are at least two sites of tyrosine phosphorylation in Trio: the
SI region and the kinase domain.
FAK Phosphorylates One Site in the Trio Kinase Domain--
Using
the Trio kinase domain as substrate, we identified by mutational
analysis which amino acid residue was predominantly phosphorylated by
FAK (Fig. 2C and data not shown). Trio constructs in which
tyrosine residues were mutated to phenylalanine were co-transfected
with HA-FAK in COS-7 cells, and the soluble (S) and insoluble (P)
fractions of the lysate were subjected to immunoblotting analysis.
Mutation of Trio Tyr-2737 to phenylalanine (Y2737F) resulted in
elimination of the majority of the tyrosine phosphorylation of the Trio
kinase domain by FAK (Fig. 2C). The identification of one
main phosphorylation site in the kinase domain indicates that the
phosphorylation of Trio by FAK is relatively specific.
Trio Binds FAK--
To determine whether Trio binds FAK, we
performed co-precipitation studies using cell lysates from COS-7 cells
expressing HA-tagged FAK constructs (shown schematically in Fig.
3A) and GST-tagged Trio
constructs. The cells were lysed, the soluble proteins precipitated
with glutathione-Sepharose, and immunoblotting analysis was performed
to identify co-precipitating FAK constructs. Whereas no FAK constructs
shown in Fig. 3A bound to GST expressed alone (data not
shown), the GST-Trio.SIK and GST-Trio.K constructs both bound the
catalytically inactive FAK kinase domain construct (FAK.KD)
and Trio.K also bound active FAK.K kinase domain (Fig. 3B),
indicating that the kinase domains of FAK and Trio interact. The
increased affinity of the FAK kinase mutant (FAK.KD) for
Trio as compared with the active FAK kinase may be further evidence that Trio is a substrate of FAK. GST-Trio.SI bound full-length FAK
efficiently, but the removal of the amino-terminal portion of FAK
(HA-FAK.
N) greatly reduced Trio binding, suggesting that Trio.SI
bound to this region of FAK. Taken together, these results indicate
that Trio binds FAK in two distinct regions: the FAK amino-terminal
portion via the SH3-Ig-like region and the FAK kinase domain via the
kinase domain.

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Fig. 3.
Trio has two distinct FAK-binding sites.
COS-7 cells were transfected with the following HA-tagged FAK
constructs: FAK, FAK. N, FAK.K, FAK.KD, and FAK.
NK (see "Experimental Procedures" for construct
descriptions) and with the GST-tagged Trio constructs Trio.SIK,
Trio.SI, or Trio.K (see legend to Fig. 1 for descriptions) as well as
GST alone. The cells were lysed as described in the Fig. 2 legend, and
the GST-tagged protein was precipitated using glutathione-Sepharose
beads. The beads were washed as described under "Experimental
Procedures" and boiled in 1× SDS sample buffer, and the HA-tagged
FAK construct was detected by immunoblotting analysis using anti-HA
antibody.
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Co-localization of Trio and FAK--
To determine the subcellular
localization of the FAK-Trio complex in cells, HA-Trio.GSIK and Myc-FAK
were expressed either alone or together in HeLa cells and then Trio
(Fig. 4, red) or FAK (Fig. 4,
green) localization was determined by immunofluorescence staining. When expressed alone, both Trio.GSIK and FAK are generally expressed throughout the cell with an increase at the cell edges (Fig.
4, A and B). Co-expressed Trio.GSIK and FAK were
also enriched at the cell periphery where they displayed a high degree
of co-localization (Fig. 4, C and D, see
arrows). In summary, our results indicate that Trio is
phosphorylated by FAK, binds FAK through its SH3/Ig region and kinase
domain, and co-localizes with FAK at the cell periphery.

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Fig. 4.
Transiently expressed Trio and FAK
co-localize in HeLa cells. HeLa cells were transiently transfected
with HA-Trio.GSIK and Myc-FAK, then fixed, and stained with anti-HA and
anti-Myc antibody as described under "Experimental Procedures."
All of the images are of identical magnification.
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Trio Activates FAK Autophosphorylation--
The interaction of the
Trio and FAK kinase domains suggested that, in addition to being a FAK
substrate, Trio may also regulate FAK kinase activity. To test this
hypothesis, we assayed in vitro equal amounts of
immunoprecipitated FAK kinase domain in the presence of either purified
GST or purified GST-tagged Trio kinase domain (GST-KT). The
GST and GST-KT proteins were expressed in COS-7 cells,
precipitated with glutathione-Sepharose, eluted with free glutathione,
and assayed with the FAK kinase domain in the presence of
[
-32P]ATP. FAK kinase domain phosphorylation was
increased 2.5-fold when assayed in the presence of active Trio kinase
domain (GST-Trio.KT) compared with its activity with GST
(Fig. 5A) or with GST-Tara, a
Trio-binding protein (38) (data not shown). Phosphoamino acid analysis
indicated that the majority of the phosphorylation was on tyrosine
(data not shown), suggesting that the Trio kinase domain increases FAK
kinase domain autophosphorylation. Phosphopeptide mapping analysis of
the FAK.K assayed with GST or GST-Trio.KT revealed that the
same phosphopeptides were present (Fig. 5B, labeled
A-F), although the relative amount of the major phosphopeptide A
was increased in the presence of Trio. Based upon its relative position, peptide A is probably the tryptic peptide containing Tyr-397
(28). Kinase assays of kinase-inactive FAK (Myc-FAK.KD) with Trio.KT resulted in limited FAK phosphorylation,
indicating that Trio did not significantly phosphorylate the FAK kinase
domain in vitro (Fig. 5A). We next examined the
in vivo phosphorylation of HA-FAK that was transiently
expressed with the HA-Trio.SIK construct in COS-7 cells (Fig.
5C). Lysates of the cells were prepared, and the levels of
protein expression and tyrosine phosphorylation were determined by
immunoblot analysis. Relative to its expression levels, FAK tyrosine
phosphorylation was 2.5-fold greater in the presence of kinase-active
Trio.SIK as compared with FAK expressed alone, whereas FAK co-expressed
with kinase-inactive Trio.SIKD had phosphotyrosine levels
that were decreased 50%. No tyrosine phosphorylation of FAK was
observed when the FAK kinase catalytic mutant was expressed, suggesting
that the observed tyrosine phosphorylation of FAK was a result of
autophosphorylation. These results suggest that Trio activates FAK
autophosphorylation and may regulate FAK activity in
vivo.

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Fig. 5.
Trio kinase domain activates FAK kinase
autophosphorylation. A, COS-7 cells were transiently
transfected either with Myc-FAK.K, Myc-FAK.KD, GST, or
GST-Trio.KT (corresponding to Trio amino acids 2730-2994).
The cells were then processed as described under "Experimental
Procedures," and Myc-FAK.K and Myc-FAK.KD were
immunoprecipitated using anti-Myc antibody and protein A-Sepharose. GST
and GST-Trio.KT were precipitated using
glutathione-Sepharose and washed extensively, and the GST and the
GST-Trio.KT were eluted using 20 mM glutathione
in 50 mM Tris pH 7.5 buffer. The Myc-FAK.K and
Myc-FAK.KD immunoprecipitates were assayed with the eluted
GST and GST-Trio.KT as described under "Experimental
Procedures," boiled in 1× SDS sample buffer, and electrophoresed.
The proteins were stained by Coomassie Blue, and the labeled proteins
were visualized by autoradiography. B, phosphorylated FAK
kinase domain protein was excised from polyacrylamide gels and digested
with trypsin. The resulting peptides were dried and separated by
electrophoresis at pH 1.9 (arrow E) and ascending
chromatography (arrow C) as described under "Experimental
Procedures." C, COS-7 cells were transiently transfected
with HA-FAK alone or together with either HA-Trio.SIK or
HA-Trio.SIKD. The cells were processed as described under
"Experimental Procedures" and subjected to immunoblotting analysis
using anti-HA and anti-Tyr(P) antibodies. WB, Western
blot.
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DISCUSSION |
We provide evidence that the FAK PTK and the Trio S/TK interact
and possibly regulate one another. FAK and Trio have two binding sites
for each other; both proteins are tyrosine-phosphorylated in the
detergent-insoluble fraction of cell lysates, and both proteins
co-localize at the cell periphery in vivo. FAK
phosphorylates Trio tyrosine residue 2737, which is near the Trio
kinase ATP-binding site, suggesting that FAK-mediated tyrosine of Trio
may serve to regulate Trio kinase activity. Additionally, Trio
increased the FAK PTK activity in vitro and in
vivo, suggesting that bi-directional regulation occurs between
Trio and FAK.
Tyrosine-phosphorylated FAK and Trio are strongly enriched in the
detergent-insoluble fraction, and the insolubility of Trio is increased
when it is co-expressed with FAK. We have mapped two separate sites of
interaction between FAK and Trio on each protein, and it is possible
that Trio becomes preferentially associated with active FAK in
vivo. FAK may therefore regulate the localization of Trio in the
cell and affect its interaction with potential kinase or GEF
substrates. Recent work suggests that Trio regulates neurite outgrowth
in PC12 cells (39), possibly in a pathway triggered by NGF, and Pyk2
and FAK have been shown to be involved with neurite formation in PC12
cells (40). Recently FAK has also been shown to phosphorylate and
activate the guanine exchange factors PDZ-RhoGEF and LARG (41).
The amount of FAK associated with the Triton-insoluble fraction of
cells was previously shown to increase upon integrin cross-linking (42), and the specific activity of FAK in the insoluble fraction was
enhanced compared with the FAK in the soluble fraction (43). Furthermore, endogenous FAK in Schwann cells lysates localized to the
detergent-insoluble fraction, forming a complex with
1-integrin and paxillin (44). Treatment of cells with
latrunculin A, which promotes actin filament disassembly and inhibits
de novo formation of filaments, had no effect on the
association of FAK and Trio with the detergent-insoluble fraction (data
not shown), suggesting either that phosphorylated FAK and Trio are not
associated with the actin cytoskeleton or that they bind to stabilized
actin filaments. Similarly, treatment of Schwann cells with the actin
polymerization inhibitor cytochalasin D did not disassemble the
insoluble complex that bound FAK but rather increased the amount of FAK
in the insoluble fraction (44). Interestingly, death-associated
protein kinase (DAP kinase) and Duet have kinase domains that are
highly homologous to the Trio kinase domain and were also reported to
partition to the detergent-insoluble fraction (45, 46). It is possible that DAP kinase and Duet share a common binding protein or substrate with Trio.
Tyr-2737 of Trio was identified as the main phosphorylation site when
the Trio kinase domain was co-expressed with FAK. This site is in
subdomain I of the Trio kinase domain and near the ATP-binding site.
This phosphorylation site location suggests a possible mechanism for
the regulation of Trio kinase activity by FAK, although experiments to
delineate more precisely any regulation of Trio kinase activity by FAK
are necessary. This phosphorylation site is conserved among many
protein kinases, including those similar to Trio such as Duet (45), Zip
(47), and DAP (48) as well as other kinases such as calmodulin KII,
Polo kinase, and casein kinase I (49). Lar and liprin-
1 were shown
to localize to the distal ends of focal adhesions (33), and FAK-Trio
complexes may also be involved in directing LAR to focal complexes. An
obvious role for Lar would be the dephosphorylation of
tyrosine-phosphorylated proteins at focal adhesions. Indeed, in
vitro assays of homogenized preparations of the
detergent-insoluble fraction of cells containing tyrosine-phosphorylated FAK and Trio indicated that recombinant Lar can
dephosphorylate both proteins (data not shown). These results further
implicate Lar as a potential regulator of focal adhesion turnover or formation.
Our experiments indicate that Trio can regulate FAK activity in
vitro and in vivo. The Trio kinase domain binds
co-transfected FAK kinase domain and is phosphorylated by FAK,
indicating that there is a direct interaction between the kinase
domains of FAK and Trio. Moreover, in in vitro kinase
assays, the FAK kinase domain displayed increased autophosphorylation
activity in the presence of the Trio kinase domain. It is possible that
the binding of Trio induces the FAK kinase domain to adopt a more
active conformation. The co-expression of kinase-active Trio.SIK with
FAK in COS-7 cells also resulted in increased tyrosine phosphorylation
of FAK. A kinase-dead FAK construct was not phosphorylated, suggesting that the increase in FAK phosphorylation was a result of
autophosphorylation. FAK autophosphorylation occurs mainly at Tyr-397,
which recruits Src, leading to further phosphorylation at Tyr-566 and
Tyr-567 of the FAK kinase domain, ultimately increasing FAK activity
(27, 28, 35-37). The inability of kinase-dead Trio.SIKD to
activate FAK autophosphorylation indicates a possible role for Trio
kinase activity in the regulation of FAK. Although little
phosphorylation of the FAK kinase domain by Trio was seen in
vitro, it is possible that Trio phosphorylates FAK in another
region. Previously, the focal adhesion targeting domain of FAK was
shown to be serine-phosphorylated during mitosis, altering its
interaction with Cas (50, 51).
In summary, FAK and Trio bind each other through multiple domains and
co-localize in cells. In addition, Trio activates FAK autophosphorylation and is a substrate for FAK. Overall, the
interaction between FAK and Trio suggests possible new mechanisms by
which focal adhesion dynamics and cell motility are regulated.