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Department of Cell Biology,1 Division of Hematology and Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294-00052
Received 13 September 2004/ Returned for modification 26 November 2004/ Accepted 11 April 2005
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ABSTRACT |
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INTRODUCTION |
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TRIP6 (thyroid receptor-interacting protein 6), also known as ZRP-1 (zyxin-related protein 1), is a member of the zyxin family that has been implicated in the regulation of actin dynamics and signal transduction involved in cell adhesion and migration (22, 40, 42). Originally discovered as an interacting protein of the nuclear thyroid hormone receptor in a yeast two-hybrid system (18), TRIP6 was later identified as a focal adhesion molecule with the capability to shuttle between cell surface and nucleus (36). The zyxin family members, including zyxin, TRIP6, LPP (lipoma preferred partner), and Ajuba, possess a proline-rich region and nuclear export signals at their N terminus and three LIM domains at their carboxyl terminus (3, 14, 22, 26). The LIM domains (named by the initials of three homeodomain proteins, Lin-11, Isl-1, and Mec-3) contain two cysteine-rich zinc finger motifs, which are critical for protein-protein interaction (2). Several LIM domain-containing proteins, such as the zyxin family members, paxillin, and other related proteins, have been shown to localize at focal adhesion plaques and associate with actin cytoskeleton (9, 19, 28, 40). They serve as scaffold or adaptor proteins for the assembly of multiple protein complexes involved in actin rearrangement, cell adhesion, and motility.
Previously it has been shown that zyxin and TRIP6 associate with the Cas family members, including p130cas and CasL/HEF1, and may cooperate in cell motility (42). Very recently, we have demonstrated that LPA stimulation promotes the recruitment of TRIP6 to the activated LPA2 receptor and induces the association of TRIP6 with the components of focal complexes, including paxillin, p130cas, FAK, and c-Src (40). Overexpression of TRIP6 enhances LPA-induced cell migration; in contrast, suppression of the endogenous TRIP6 expression by its specific small interfering RNA (siRNA) inhibits it in SKOV-3 ovarian cancer cells (40). Similarly, another zyxin family member, LPP, has been reported to promote epidermal growth factor-induced cell migration in vascular smooth muscle cells (11). In contrast to zyxin knockout mice, which do not exhibit significant phenotypes (12), the Ajuba-null cells show a reduction of tyrosine phosphorylation of p130cas, Crk, and Dock180 at nascent focal complexes, which leads to the inhibition of Rac activation and impaired cell migration (27). All of these results suggest that these zyxin family members are involved in cell motility. However, the detailed mechanism(s) by which these zyxin family members regulate cell migration is not yet clear.
Despite all of the data suggesting a role for TRIP6 in LPA-induced cell migration, very little is known about how TRIP6 function is regulated in this dynamic process. In the present study, we demonstrate that c-Src mediates LPA-induced TRIP6 phosphorylation at the Tyr-55 residue. This phosphorylation does not regulate its focal adhesion targeting or association with actin cytoskeleton. However, it is critical for the recruitment of Crk and p130cas and for c-Src-mediated ERK activation. Ultimately, these signaling events regulate the process of LPA-induced morphological changes and cell migration.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Phosphorylation of TRIP6 by c-Src in vitro and in cells. pGEX-6P-3-TRIP6 was transformed into E. coli BL21(DE3)(LysS). GST-TRIP6 was purified and further digested with PreScission protease (Amersham Biosciences) to cleave GST. Purified TRIP6 was phosphorylated by recombinant p60c-Src (Upstate Biotechnology Inc.) at 30°C for 30 min and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gel slices containing phosphorylated TRIP6 were cut out and digested in situ with trypsin. After purification, the phosphorylation sites of TRIP6 were identified by mass spectrometry analysis (Protein Chemistry Core Facility, Baylor College of Medicine). Similarly, 1 µg of TRIP6 or TRIP6-Y55F was purified and incubated with recombinant p60c-Src. The phosphoproteins were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and detected with a horseradish peroxidase (HRP)-conjugated antiphosphotyrosine antibody (PY20H; Santa Cruz Biotechnology).
To detect tyrosine phosphorylation of TRIP6 in cells, NIH 3T3 cells, Src-null SYF cells, or c-Src-reconstituted SYF+c-Src cells (American Type Culture Collection) were transiently transfected with an empty vector or the expression vector of TRIP6 or TRIP6-Y55F. Cells were starved in 0.1% fatty acid-free bovine serum albumin (BSA)-containing Dulbecco's modified Eagle's medium (DMEM) for 8 h or overnight. Cells were incubated without or with 1 or 2 µM LPA for various times and harvested in lysis buffer (1% Triton X, 10% glycerol, 150 mM NaCl, 10 mM HEPES, 1 mM EDTA, 1 mM EGTA) supplemented with a mixture of protease inhibitors and phosphatase inhibitors. The endogenous or epitope-tagged TRIP6 was immunoprecipitated with a TRIP6-specific monoclonal antibody (Transduction Laboratories) or epitope-specific antibodies and resolved by SDS-PAGE. The tyrosine-phosphorylated TRIP6 was detected by immunoblotting using an HRP-conjugated antiphosphotyrosine antibody (RC20H [Transduction Laboratories] or PY20H [Santa Cruz Biotechnology]).
In vitro binding of Crk I with c-Src-phosphorylated TRIP6. To examine the effect of c-Src-mediated phosphorylation on the binding of TRIP6 with Crk I in vitro, GST, GST-Crk I, GST-TRIP6, GST-TRIP6-Y55F, and GST-TRIP6-P58A were expressed in E. coli and purified by immobilizing the proteins on glutathione-Sepharose 4B beads (Amersham Biosciences). Crk I was further purified by cleaving GST with PreScission protease (Amersham Biosciences). GST and GST fusion proteins of TRIP6, TRIP6-Y55F, and TRIP6-P58A were phosphorylated by c-Src in vitro. Following reaction, GST fusion protein-immobilized glutathione beads were washed four times to remove c-Src. These beads were then incubated with purified Crk I for 3 h at 4°C. Crk I pulled down by GST fusion proteins was resolved by SDS-PAGE, and the immunoblot was probed with a Crk-specific antibody (Transduction Laboratories).
Cellular coimmunoprecipitation and LPA-induced ERK activation. Cellular coimmunoprecipitation was performed as described previously (40). To determine LPA-induced ERK activation, transfected SYF+c-Src cells were starved overnight and then stimulated with 2 µM LPA for 5 or 10 min. The whole-cell lysates were subjected to SDS-PAGE, and the immunoblot was probed with an anti-phospho-ERK polyclonal antibody (Promega). The blot was then stripped and reprobed with an anti-ERK2 monoclonal antibody (Transduction Laboratories) to ensure equal expression of total ERKs. The MEK1-K97A expression vector (30) was cotransfected or not with pEGFP or pEGFP-TRIP6 into SKOV-3 cells. The expression of MEK1 and MEK1-K97A was detected by probing the immunoblot with a MEK1-specific antibody (Transduction Laboratories). The activation of ERKs was quantitated using NIH Image J software.
Immunocytochemistry and time-lapse imaging of live cells. LPA-promoted colocalization of TRIP6 or TRIP6-Y55F with MYC-FAK, vinculin, paxillin, or actin was performed in SYF+c-Src cells or NIH 3T3 cells as described before (40). For live cell imaging, SYF+c-Src cells or SYF cells expressing different GFP constructs as indicated were plated on glass-bottomed 35-mm tissue culture dishes (MatTek Corp.). After starvation for 2 h in DMEM containing 0.1% fatty acid-free BSA and 10 mM HEPES, pH 7.4, GFP-positive cells were visualized with an Olympus IX70 inverted fluorescence microscope using a 100x objective. Ten micromolar LPA was then added at the center of the plates, and the images of GFP-positive cells were acquired every 20 or 30 s for 30 to 40 min with a Photometrics 1400 charge-coupled device camera under the control of IPLab software (Scanalytics, Inc.).
Transwell cell migration assays. SKOV-3 cells expressing GFP, GFP-TRIP6, or GFP-TRIP6-Y55F in the absence or presence of MEK1-K97A were subjected to a transwell cell migration assay as described previously (40). The bottom of the transwell membrane was coated with 10 µg/ml fibronectin overnight before the experiment. Cells at a density of 150,000/well were placed in the upper chamber, 2 µM LPA was added or not to the lower chamber, and the cells were allowed to migrate for 6 h. After removing the nonmigrated cells from the top surface, the migrated GFP-positive cells on the whole filter were counted by fluorescence microscopy. Meanwhile, an aliquot of cells was placed on the coverslips, and GFP-positive cells in each field were counted to estimate transfection efficiency. The relative migration rate was defined as the increase of migrated cells compared to migrated GFP control cells in the absence of LPA treatment and has been adjusted by transfection efficiency.
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RESULTS |
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We then set out to map the relevant sites of TRIP6 phosphorylation. The recombinant TRIP6 purified from E. coli was subjected to phosphorylation by the active c-Src kinase in vitro, digested with trypsin, and sequenced. The result revealed that TRIP6 is predominantly phosphorylated by c-Src at Tyr-55 (Fig. 2A). Another minor phosphorylation site of TRIP6 is Tyr-123. However, mutation of Tyr-123 to Phe did not alter c-Src-mediated TRIP6 phosphorylation in vitro or in cells (data not shown).
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The capability of TRIP6 to target to focal adhesions and colocalize with actin is not affected by Tyr-55 mutation. To investigate how Tyr-55 phosphorylation regulates the function of TRIP6 in LPA signaling, we first examined LPA-dependent recruitment of TRIP6 and the TRIP6-Y55F mutant to focal adhesions and colocalization with actin cytoskeleton. Previously we have shown that TRIP6 is predominantly cytosolic or forms small clusters at sites of adhesions in SKOV-3 cells after serum starvation overnight (40). Similarly, we have found that by serum starvation overnight, both TRIP6 and TRIP6-Y55F were diffusely distributed in the cytoplasm in SYF+c-Src cells or NIH 3T3 cells. Only a small amount of TRIP6 or TRIP6-Y55F colocalized with vinculin, paxillin, and transfected MYC-FAK in SYF+c-Src cells (see Fig. S1A, C, and E in the supplemental material), or with actin in NIH 3T3 cells (see Fig. S1G in the supplemented material). With LPA stimulation for 15 min, substantial amounts of TRIP6 and TRIP6-Y55F have been found to associate with endogenous vinculin, paxillin, and transfected MYC-FAK in SYF+c-Src cells (see Fig. S1B, D, and F in the supplemental material), and colocalize with actin in NIH 3T3 cells (see Fig. S1H in the supplemental material). Thus, the function of TRIP6 in LPA-induced focal adhesion targeting and actin colocalization is not changed by this point mutation. The localization of vinculin, paxillin, transfected MYC-FAK, and actin in GFP control cells treated with LPA or not is shown in Fig. S2 in the supplemental material.
Tyr-55 phosphorylation of TRIP6 regulates its binding to Crk and p130cas. The majority of Src substrates identified so far contain SH2 or SH3 binding domains, or both, which are important for protein-protein interaction during the organization of signaling pathways. When phosphorylated, the phosphotyrosine motifs bind with a high degree of specificity to SH2 domains or phosphotyrosine binding domains contained in different proteins (25). Several such molecules, including p130cas, paxillin, c-Cbl, and Gab1, utilize pYXXP motifs to bind the Crk SH2 domain (10). Here, phosphotyrosine and proline residues are critical for this interaction. Analogously, the Y55-Q-A-P motif of human TRIP6 (Fig. 2A) or Y55-Q-P-P motif of mouse TRIP6 may conform to a binding site for the Crk SH2 domain once Tyr-55 is phosphorylated by c-Src. To test this hypothesis, we first examined the importance of the pY55-Q-A-P58 motif of TRIP6 in Crk I binding in vitro. Thus, GST fusion proteins of wild-type TRIP6, TRIP6-Y55F mutant, and TRIP6-P58A mutant were subjected to in vitro phosphorylation by a recombinant c-Src kinase. Following the reaction, c-Src was removed by extensive washing, and the GST fusion proteins, phosphorylated or not, were used to pull down recombinant Crk I, which contains one SH2 domain and one SH3 domain. As shown in Fig. 3A, c-Src phosphorylated TRIP6 and the TRIP6-P58A mutant equally well, which was greatly eliminated by Y55F mutation. Strikingly, only the c-Src-phosphorylated TRIP6 was able to pull down Crk I, but not nonphosphorylated TRIP6 or the TRIP6-Y55F mutant (Fig. 3B). Although the TRIP6-P58A mutant was phosphorylated by c-Src, it was unable to bind Crk I (Fig. 3B). We further examined the association of TRIP6 with Crk I in HEK 293T cells. In all of the following experiments using GFP constructs to perform coimmunoprecipitation, GFP did not nonspecifically bind to the antibody-conjugated agarose beads (data not shown). As shown in Fig. 4A, LPA promoted the interaction of TRIP6 with Crk I in HEK 293T cells only when c-Src was coexpressed. In contrast to wild-type TRIP6, the TRIP6-Y55F mutant was unable to associate with Crk I in HEK 293T cells even if c-Src was overexpressed (Fig. 4B). Similarly, mutation of Pro-58 to Ala disrupted the interaction of TRIP6 with Crk I in c-Src-expressing HEK 293T cells (see Fig. S3 in the supplemental material). These results indicate that c-Src-mediated phosphorylation of TRIP6 at Tyr-55 renders TRIP6 capable of binding to Crk in vitro and in cells.
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LPA-stimulated ERK activation is regulated by tyrosine phosphorylation of TRIP6. Many G protein-coupled receptors, including the LPA receptors, mediate Ras-dependent activation of ERK cascades. Substantial evidence has shown that recruitment and activation of the Src family kinases regulate these signaling pathways (20). In addition to mediating mitogenic signaling, activated ERKs have been shown to regulate adhesion turnover and cell migration (34, 37). Phosphorylation of MLCK by the activated ERK can modulate actomyosin contractility (15) and promote adhesion disassembly (37). Moreover, the activated Gi/o-ERK pathway has been shown to contribute to LPA-induced pancreatic tumor cell migration (33). To examine if TRIP6 is involved in these signaling pathways, we knocked down the expression of endogenous TRIP6 in SYF and SYF+c-Src fibroblasts by a mouse TRIP6-specific siRNA and assessed its effect on LPA-induced ERK activation. As shown in Fig. 5A, the Src-deficient SYF cells exhibited a lower level of LPA-activated ERKs compared to SYF+c-Src cells (top panel, compare lanes 2 and 6). The TRIP6-specific siRNA significantly knocked down the expression of endogenous TRIP6 (third panel) and reduced LPA-induced ERK activation more dramatically in SYF+c-Src cells (top panel, compare lanes 6 and 7 with lanes 9 and 10) than in SYF cells (top panel, compare lanes 2 and 4), suggesting that TRIP6 is involved in c-Src-mediated ERK activation. This effect was apparently regulated by Tyr-55 phosphorylation, since LPA-induced ERK activation was enhanced in SYF+c-Src cells overexpressing TRIP6 but was reduced in that overexpressing TRIP6-Y55F mutant (Fig. 5B).
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5 to 10 min, GFP-expressing cells started to retract their cell bodies and completely rounded up after LPA treatment for 22.8 ± 1.0 min (n = 6). Subsequently, they displayed cell surface blebbing and pseudopodium formation. Strikingly, cells expressing GFP-TRIP6 exhibited rapid morphological changes and became rounded soon after LPA stimulation for 8.6 ± 1.1 min (n = 9 in three independent experiments; P < 0.01 versus GFP control; Student's t test). In contrast, cells expressing GFP-TRIP6-Y55F required a much longer time to round up, which could be from 16 to 35 min (29.8 ± 2.8 min; n = 5 in three independent experiments; P < 0.01 versus GFP-TRIP6-expressing cells). Moreover, with the concomitant changes of cell morphology, wild-type TRIP6 at sites of adhesions disappeared much faster than TRIP6-Y55F, as shown in videos S1 and S2 in the supplemental material.
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If c-Src-mediated phosphorylation of TRIP6 is important for its function in LPA-induced morphological changes, expression of wild-type TRIP6 in Src-null SYF cells would not promote this process. To test this hypothesis, SYF cells expressing GFP or GFP-TRIP6 were stimulated with LPA, and time-lapse imaging was performed. As shown in Fig. 6C, the GFP control SYF cells rounded up soon after LPA stimulation. Compared to the elongated shapes of SYF+c-Src fibroblasts, the morphology of SYF cells was more square-like. Thus, we did not observe a dramatic retraction of cell bodies in these cells. Interestingly, overexpression of TRIP6 in SYF cells did not promote LPA-induced morphological changes as it did in SYF+c-Src cells, and it even inhibited LPA-induced cell rounding. The turnover of TRIP6 at the sites of adhesions seemed much slower in SYF cells than in SYF+c-Src cells. All of these results suggest that c-Src-mediated phosphorylation of TRIP6 regulates LPA-induced morphological changes and possibly affects the turnover of TRIP6 at sites of adhesions as well.
Previously it was shown that coupling of tyrosine-phosphorylated p130cas and the Crk SH2 domain is critical for cell motility (7, 16). Since Tyr-55 mutation disrupts the coupling of TRIP6 to Crk and p130cas and reduces LPA-induced ERK activation, it might affect LPA-induced cell migration. To understand how Tyr-55 mutation affects TRIP6 function in LPA-induced cell migration, GFP, GFP-TRIP6, or the GFP-TRIP6-Y55F mutant was transiently expressed in SKOV-3 cells, and LPA-induced cell migration was assessed. As shown in Fig. 7, in the absence of ligand stimulation, the relative cell migration rate was slightly higher in cells overexpressing GFP-TRIP6 and the GFP-TRIP6-Y55F mutant than the GFP control cells. After LPA stimulation, cell migration was highly increased by overexpression of TRIP6. However, the ability of TRIP6 to promote LPA-induced cell migration was significantly reduced by Tyr-55 mutation. Similar results were observed in NIH 3T3 cells and SYF+c-Src cells (data not shown). This finding suggests that phosphorylation of TRIP6 at Tyr-55 promotes LPA-induced cell migration. However, the function of TRIP6 in this event is also regulated by another mechanism(s), since TRIP6-Y55F does not act as a dominant-negative mutant.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The zyxin family members share a high degree of sequence homology in the carboxyl-terminal LIM domains; however, the N-terminal proline-rich sequences are much less homologous (22). The Tyr-55 residue is only present in TRIP6 and not other zyxin family members. Thus, they must be regulated either by phosphorylation at other sites or by totally different mechanisms. Whether c-Src-mediated phosphorylation is only specific to TRIP6 but not other zyxin family members remains to be determined. However, our results do reveal some analogies between TRIP6 and another LIM domain-containing focal adhesion molecule, paxillin. First, tyrosine phosphorylation of paxillin is also induced by LPA stimulation (31). Second, this phosphorylation is not required for their localization to focal adhesions; instead, it mediates the recruitment of Crk and promotes their disassembly at the sites of adhesions (35, 37). Consequently, it regulates their functions in cell motility. However, there are also some distinctions between these two molecules. It has been reported that FAK, in association with c-Src, phosphorylates paxillin at Tyr-31 and Tyr-118 (4, 29). Although LPA induces the association of TRIP6 with FAK (40), overexpression of FAK barely increases tyrosine phosphorylation of TRIP6 (data not shown). Instead, TRIP6 is predominantly phosphorylated at Tyr-55 by c-Src (Fig. 2). Previously it was shown that paxillin associates with the protein tyrosine phosphatase PTP-PEST (32). This binding may be necessary for the targeting of PTP-PEST to focal adhesions to facilitate dephosphorylation of the main PTP-PEST substrate, p130cas (1, 8, 32). However, paxillin does not seem to be a direct substrate for PTP-PEST (1). Although it has been shown that the third LIM domain and carboxyl-terminal region of TRIP6 bind to the second PDZ domain of human PTP-1E and its mouse homologue, PTP-BL, the functional significance for this interaction is not known (22). It is also not clear whether TRIP6 is a substrate for PTP-1E or PTP-BL in cells.
In this report, we demonstrate that overexpression of TRIP6, but not the Y55F mutant of TRIP6, promotes LPA-induced morphological changes in SYF+c-Src cells (Fig. 6A). In contrast, overexpression of TRIP6 in SYF cells markedly retards the whole process (Fig. 6C). These results suggest that the effect of TRIP6 in LPA-induced morphological changes is dependent on c-Src-mediated phosphorylation. Since the non-tyrosine-phosphorylated TRIP6 is able to target to focal adhesions and colocalize with actin in SYF cells, it is possible that it forms stable complexes with other signaling molecules, which results in more mature adhesions, thereby perturbing adhesion disassembly. Thus, the balance between the expression of phosphorylated and dephosphorylated TRIP6 would be important for normal control of its function in adhesion turnover and cell motility.
The aberrant regulation of Src family kinases has been shown to associate with the initiation and progression of various cancers. For example, in late-stage ovarian cancers in which the levels of LPA are elevated (41), the Src family kinases are also overexpressed and activated (39). Whether LPA-induced tyrosine phosphorylation of TRIP6 is aberrantly regulated by the activated Src family kinases during ovarian cancer progression and whether it contributes to ovarian tumor invasion and metastasis remain to be investigated in the future.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health grant CA100848 (to F.-T.L).
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
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