Signaling between focal adhesion kinase and trio.

The Trio guanine nucleotide exchange factor functions in neural development in Caenorhabditis elegans and Drosophila and in the development of neural tissues and skeletal muscle in mouse. The association of Trio with the Lar tyrosine phosphatase led us to study the role of tyrosine phosphorylation in Trio function using focal adhesion kinase (FAK). The Lar-interacting domain of Trio is constitutively tyrosine-phosphorylated when expressed in COS-7 cells and was highly phosphorylated when it was co-transfected with FAK. Co-precipitation studies indicated that Trio binds to the FAK amino-terminal domain and to the FAK kinase domain via its SH3 and kinase domains, respectively. Tyrosine-phosphorylated FAK and Trio were present mainly in the detergent-insoluble fraction of cell lysates, and co-expression of Trio and FAK resulted in increased amounts of Trio present in the detergent-insoluble fraction. Immunofluorescence of cells co-transfected with FAK and Trio revealed significant co-localization of the proteins at the cell periphery, indicating that they form a stable complex in vivo. A FAK phosphorylation site, tyrosine residue 2737, was identified in subdomain I of the Trio kinase domain. Additionally, in vitro phosphorylation assays and in vivo co-expression studies indicated that Trio enhances FAK kinase activity. These results suggest Trio may be involved in the regulation of focal adhesion dynamics in addition to effecting changes in the actin cytoskeleton through the activation of Rho family GTPases.

The Trio guanine nucleotide exchange factor functions in neural development in Caenorhabditis elegans and Drosophila and in the development of neural tissues and skeletal muscle in mouse. The association of Trio with the Lar tyrosine phosphatase led us to study the role of tyrosine phosphorylation in Trio function using focal adhesion kinase (

FAK). The Lar-interacting domain of Trio is constitutively tyrosine-phosphorylated when expressed in COS-7 cells and was highly phosphorylated when it was co-transfected with FAK. Co-precipitation studies indicated that Trio binds to the FAK amino-terminal domain and to the FAK kinase domain via its SH3 and kinase domains, respectively. Tyrosinephosphorylated FAK and Trio were present mainly in the detergent-insoluble fraction of cell lysates, and coexpression of Trio and FAK resulted in increased amounts of Trio present in the detergent-insoluble fraction. Immunofluorescence of cells co-transfected with FAK and Trio revealed significant co-localization of the proteins at the cell periphery, indicating that they form a stable complex in vivo.
A FAK phosphorylation site, tyrosine residue 2737, was identified in subdomain I of the Trio kinase domain. Additionally, in vitro phosphorylation assays and in vivo co-expression studies indicated that Trio enhances FAK kinase activity. These results suggest Trio may be involved in the regulation of focal adhesion dynamics in addition to effecting changes in the actin cytoskeleton through the activation of Rho family GTPases.
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 phos-phatase, and the adaptor proteins Dock and Ena (3)(4)(5)(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)(24)(25)(26), as well as undergoing autophosphorylation (27,28). Autophosphorylated FAK recruits the p60 src (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.

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 (GenBank TM accession number UF091395). Trio.SIK D refers to amino acids 2485-3038 with the a catalytically inactive kinase domain caused by a K2756A mutation, and Trio.K T refers to amino acids 2730 -2994. Cell Transfections-COS-7 cell transient transfections were done by the DEAE-dextran/Me 2 SO method using ϳ2 g of plasmid DNA/2 ϫ 10 5 cells, and the cells were harvested ϳ20 h after transfection. The proteins were metabolically labeled with [ 35 S]methionine and [ 35 S]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 [ 35 S]methionine and [ 35 S]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 MgCl 2 ) containing 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 10 g/ml aprotinin. Insoluble material was pelleted by centrifugation at 16,000 ϫ g, and the supernatant was used for immunoprecipitation of soluble protein. The cell lysates were then precleared once with 25 l of protein A-Sepharose (Amersham Biosciences) for 1-2 h. For immunoprecipitations 1 l of anti-HA mAb 12CA5 ascites fluid and 25 l 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 MgCl 2 . 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 ϫ g centrifugation step were boiled in equal volumes of 1ϫ 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.

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 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 ϫ g, and the supernatants and pellets were boiled in equal volume of 1ϫ 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. 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).
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 tyrosinephosphorylation (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)(36)(37). However, Trio constructs were also phosphorylated by the HA-FAK Y397F 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 tyrosinephosphorylated 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.K D ) 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.K D ) 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.  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.K Y2737F constructs, processed as described above, and subjected to immunoblotting analysis using anti-Tyr(P) and anti-Myc tag antibodies. WB, Western blot.

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.
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-K T ). The GST and GST-K T 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 [␥-32 P]ATP. FAK kinase domain phosphorylation was increased 2.5-fold when assayed in the presence of active Trio kinase domain (GST-Trio.K T ) 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.K T 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.K D ) with Trio.K T re-sulted 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.SIK D 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. 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 tyrosinephosphorylated 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 detergentinsoluble 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 neces-sary. 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 cotransfected 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)(36)(37). The inability of kinase-dead Trio.SIK D to activate FAK autophosphorylation indicates a possible role for Trio kinase activity in the regulation of FAK. Although little The cells were then processed as described under "Experimental Procedures," and Myc-FAK.K and Myc-FAK.K D were immunoprecipitated using anti-Myc antibody and protein A-Sepharose. GST and GST-Trio.K T were precipitated using glutathione-Sepharose and washed extensively, and the GST and the GST-Trio.K T were eluted using 20 mM glutathione in 50 mM Tris pH 7.5 buffer. The Myc-FAK.K and Myc-FAK.K D immunoprecipitates were assayed with the eluted GST and GST-Trio.K T as described under "Experimental Procedures," boiled in 1ϫ 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.SIK D . 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. 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.