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J. Biol. Chem., Vol. 281, Issue 40, 30081-30093, October 6, 2006
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1


2
From the
Department of Veterans Affairs and Department of Biomedical Sciences, Meharry Medical College, Nashville, Tennessee 37208 and the
Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
Received for publication, June 19, 2006 , and in revised form, July 31, 2006.
| ABSTRACT |
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| INTRODUCTION |
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Although ligand binding to CXCR4 triggers various signaling cascades including activation of G proteins, phosphatidylinositol 3-kinase, the Rho-p160 ROCK axis, focal adhesion kinase, and the extracellular signal regulated kinases 1 and 2 (ERK1/2) (10-13), molecular mechanisms underlying CXCR4-mediated chemotaxis are still not fully understood. Increasing lines of evidence support the postulate that CXCR4 internalization, a process that occurs through clathrin-coated pits (14), plays a role of receptor internalization in chemotaxis (15-18). Studies from our group or others demonstrated that inhibition of chemokine receptor internalization resulted in reduction of chemotaxis (15-19), suggesting that membrane trafficking and chemotaxis are somehow connected. This interpretation is also supported by the recent findings that clathrin-dependent endocytosis is a process linking actin dynamics (20), and several actin cytoskeleton-associated proteins, such as cortactin, are involved in this process (21, 22).
Cortactin, an 80- and 85-kDa protein, is a widely expressed actin-binding protein. Cortactin contains 6.5 37-amino acid repeats, of which the 4th is required for filamentous actin binding activity (23). Cortactin also harbors an N-terminal acidic domain, and it binds through a conserved DDW motif, the Arp2/3 complex, and activates it in conjunction with the filamentous actin binding repeat region (23-25). These findings suggest that cortactin plays an important role in the remodeling of the actin cytoskeleton.
The role of cortactin in linking actin cytoskeleton and endocytosis was first recognized in an experiment showing that cortactin directly associates with dynamin, a large GTPase that is involved in the liberation of clathrin-coated pits from the plasma membrane via its Src homolog 3 (SH3) domain and proline-rich domain, respectively (22). Electron microscopy revealed that cortactin associated with endosomes along with the Arp2/3 complex (26). More recently it has been shown that cortactin is distributed over the surface or base of clathrin lattices as well as actin filaments associated with the clathrincoated pits (27). The inhibitory effect of microinjection of anticortactin antibody or transfection of a plasmid encoding the cortactin SH3 domain on endocytosis further supports the role of cortactin in receptor-mediated endocytosis (27). Cortactin, originally identified as a substrate for Src kinase (28), is directly phosphorylated by Src on three tyrosine residues, tyrosine 421, tyrosine 466, and tyrosine 482 (29, 30). However, little is known about the role of each of these phosphorylations in cortactin functions.
In this study we demonstrated that CXCL12 activation of CXCR4 leads to cortactin translocation from endosomes to the cell periphery. Ligand stimulation also induced tyrosine phosphorylation of cortactin in a Src- and dynamin-dependent manner. Overexpression of wild-type cortactin, but not the phosphorylation-deficient cortactin mutant, resulted in enhanced CXCR4 recycling, prolonged ERK1/2 activation, and enhanced CXCR4-mediated chemotaxis. In contrast, RNA interference of cortactin significantly inhibited CXCR4 internalization and reduced CXCR4-mediated ERK1/2 activation and chemotaxis. These data indicate that cortactin plays important roles in CXCR4 signaling and trafficking as well in the receptor-mediated chemotaxis.
| EXPERIMENTAL PROCEDURES |
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Cell Culture and TransfectionHuman embryonic kidney (HEK) 293 cells or HeLa cells were grown in Dulbecco's modified essential medium containing 10% fetal bovine serum, 100 units of penicillin/ml, and 100 µg of streptomycin/ml in 5% CO2, 95% air at 37 °C. Cells were cultured in P-100 dishes or on 22 x 22-mm glass coverslips for transfections and immunofluorescence microscopy, respectively. Transfection was performed with Lipofectamine Plus reagent (Invitrogen). Cells stably expressing CXCR4 or CXCR2 were selected with 560 µg/ml Geneticin and evaluated for receptor expression by radioligand binding assay using 125I-labeled CXCL12 or 125I-labeled CXCL8 (Amersham Biosciences).
Confocal MicroscopyCells grown on coverslips for 1 or 2 days were prepared as described previously (31). HEK293 cells stably expressing Myc-CXCR4 were treated with carrier buffer or CXCL12 for various time intervals and fixed with methanol. Cells were washed with phosphate-buffered saline and incubated with an antibody mixture containing a mouse monoclonal anti-Myc antibody (Santa Cruz Biotechnology) and a rabbit polyclonal cortactin antibody (Santa Cruz Biotechnology) for 30 min. Cells were washed and incubated with a antibody mixture containing a CY3-conjugated anti-mouse antibody (Molecular Probes) and a fluorescein isothiocyanateconjugated anti-rabbit antibody (Molecular Probes) for 30 min. HeLa cells transfected with enhanced green fluorescence protein (EGFP)-Rab5 were treated with CXCL12 for 5 or 30 min before being fixed in methanol. Cells were incubated with a rabbit polyclonal cortactin antibody for 30 min followed by a CY3-conjugated anti-rabbit antibody for 30 min. Confocal microscopy was performed on an LSM-510 laser scanning microscope (Carl Zeiss, Thornwood, NY) with a 63 x 1.3 numerical aperture oil immersion lens using dual excitation (488 nm for fluorescein isothiocyanate (FITC) or EGFP, 568 nm for Cy3) and emission (515-540 nm for FITC or EGFP, 590-610 nm for Cy3) filter sets. All digital images were captured at the same settings to allow direct quantitative comparison of staining patterns. Final images were processed using Adobe Photoshop software. Confocal colocalization between two fluorescent staining was quantified using Optimas 5.2 image analysis software. Briefly, matching green and red images of individual cells were opened sequentially. Fluorescence thresholds were applied to each image to exclude diffuse labeling in the cytoplasm. Punctate sites of positive staining were identified, and each image was converted to binary form. Matching binary images derived from CXCR4, and cortactin images were then subjected to an "and" operation, resulting in the identification of sites containing both CXCR4 and cortactin staining. The following equation was used to determine percentage colocalization per cell: % colocalization = (no. of punctate sites containing both CXCR4 and cortactin) x 100/(no. punctate of sites containing CXCR4). Means ± S.E. were determined and plotted using SigmaPlot software.
Co-immunoprecipitation and Western BlotFor the co-immunoprecipitation of CXCR4 and cortactin, HEK293 cells stably expressing HA-CXCR4 were treated with CXCL12 (10 nM) for various time intervals, washed 3 times with ice-cold phosphate-buffered saline, and lysed in 1 ml of radioimmune precipitation assay buffer containing phosphate-buffered saline (pH 7.0), 0.1% sodium deoxycholate, 0.01% SDS, and 1% Nonidet P-40. The cell debris was removed by centrifugation (15,000 x g, 15 min). The supernatant was precleared by incubation with 40 µl of protein A/G-agarose (Pierce) for 1 h at 4 °C to reduce nonspecific binding. After removing the protein A/G-agarose by centrifugation (15,000 x g, 1 min), the cleared supernatant was collected, and 10 µl of mouse monoclonal anti-HA antibody (Santa Cruz Biotechnology) was added for overnight precipitation at 4 °C. Protein A/G (40 µl) was then added, and incubation was continued at 4 °C for 2 h. The protein A/G-antibody-antigen complex was collected by washing three times with ice-cold immunoprecipitation buffer. The final pellet was resuspended in 40 µl of SDS sample buffer containing 5%
-mercaptoethanol and heated to 50 °C for 10 min. Forty microliters of this preparation was separated by 10% SDS-PAGE, and the proteins on the gel were transferred to nitrocellulose membranes (Bio-Rad). The co-immunoprecipitated cortactin was detected by Western blotting using a rabbit anti-cortactin antibody (Santa Cruz Biotechnology).
Cortactin Phosphorylation AssayHEK293 cells stably expressing HA-CXCR4 were grown in 6-well plates. Cells were treated with CXCL12 for different time intervals before being lysed in radioimmune precipitation assay buffer. Lysates containing equal amounts of protein were subjected to SDS-PAGE. Phosphorylated cortactin was detected by Western blot analysis with phosphospecific cortactin antibodies against tyrosine 421, tyrosine 466, and tyrosine 482 (Santa Cruz Biotechnology).
Mitogen-activated Protein Kinase AssayHEK293 cells stably expressing HA-CXCR4 and transiently transfected with vector or cortactin plasmids were grown in 6-well plates. Agonist-treated cells were lysed by radioimmune precipitation assay buffer. Lysates containing equal amounts of protein were subjected to SDS-PAGE. Phosphorylated ERK1/2 was detected by a phosphospecific ERK1/2 antibody (Santa Cruz Biotechnology).
Densitometry Analysis of Western BlotsThe relative amount of the Western blot bands was measured by densitometry analysis using NIH Image software (rsb.info.nih.gov/nih-image). The relative density of the protein bands was calculated in the area encompassing the immunoreactive protein band and subtracting the background of an adjacent nonreactive area in the same lane of the protein of interest.
Chemotaxis AssayA 96-well chemotaxis chamber (Neuroprobe, Gaithersburg, MD) was used for chemotaxis assays, and the lower compartment of the chamber was loaded with 400-µl aliquots of 1 mg/ml ovalbumin/Dulbecco's modified essential medium (chemotaxis buffer) or CXCL12 diluted in the chemotaxis buffer (0.001-10 nM). Polycarbonate membranes (10-µm pore size) were coated on both sides with 20 µg/µl human collagen type IV, incubated for 2 h at 37 °C, and then stored at 4 °C overnight. To prepare HEK293 cells for chemotaxis assay, cells were removed from the culture dish by trypsinization, washed with Hanks' solution, and incubated in 10% fetal bovine serum/Dulbecco's modified essential medium for 2 h at 37 °C to allow time for restoration of receptors. Cells (5 x 105 in 100 µl) were loaded into the top of each well of a 96-well chemotaxis chamber. The bottom of each well contained 600 µl of prewarmed chemotaxis buffer with different concentrations of ligand. The plate was then incubated for 240 min at 37 °C in a 5% CO2 atmosphere, and cells migrating to the lower chamber were counted after being stained with a Diff-Quik kit. Cell chemotaxis was quantified by counting the number of migrating cells present in 10 microscope fields (20x objective).
Internalization and Recycling AssaysHEK293 cells stably expressing HA-CXCR4 and transiently transfected with vector or cortactin plasmids were grown in 24-well plates that were precoated with 0.1 mg/ml poly-L-lysine (Sigma, Mr 30,000-70,000). For the internalization assay cells were incubated at 4 °C in 0.5 ml of serum-free Dulbecco's modified essential medium containing 125I-labeled CXCL12 (75 nCi/ml) at 4 °C for 1 h with or without excess unlabeled CXCL12 for the determination of nonspecific binding. After removing unbound ligand, the cells were shifted to 37 °C. At each time point, the medium was removed, and the cell surface 125I-labeled CXCL12 was removed by incubating with 1 ml of ice-cold 0.2 M acetic acid and 0.5 M NaCl for 6 min. Internalized CXCL12 (acid-resistant, cell-associated cpm) was determined with a
-counter. For the recycling assay cells were incubated with unlabeled CXCL12 (10 nM) for 1 h at 4°C. After removing unbound ligand, cells were shifted to 37 °C. At each time point, residual bound CXCL12 was removed by acid wash, and surface receptor levels were assessed by binding of 125I-labeled CXCL12.
Fluorescence-activated Cell Sorting AnalysisHEK293 cells stably expressing CXCR4 were transfected with vector (control), cortactin plasmids, control siRNA, or cortactin specific siRNA. Cells were incubated with a monoclonal phycoerythrin-conjugated CXCR4 antibody (BD PharMingen, San Diego, CA) at 4 °C for 60 min. In a parallel experiment, parental HEK293 cells were incubated with the phycoerythrin-conjugated CXCR4 antibody as a control. Cells were washed and fixed in 2% formaldehyde in phosphatebuffered saline and analyzed in a FACScan equipped with CellQuest software (BD Biosciences).
Intracellular Calcium Mobilization AssayHEK293 cells stably expressing CXCR4 were transfected with vector (control), cortactin plasmids, control siRNA, or cortactin specific siRNA. Cells were released by shaking, collected by centrifugation at 300 x g for 5 min, and washed with incubation buffer (Hanks' buffer containing 5 mM HEPES). Cells were resuspended at 2 x 106 cells/ml and incubated with 2.5 µM Fluo-3 (Molecular Probes) for 30 min at 37 °C. After incubation the cells were washed once with the incubation buffer containing 2 mM CaCl2. The cells were finally adjusted to 2 x 106 cells/ml. Cells were stimulated with CXCL12 (10 nM), and intracellular Ca2+ mobilization experiments were performed as described previously (17). The time taken to recover 80% of the mobilized Ca2+ (t0.80) was used as a measure of Ca2+ removal.
| RESULTS |
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The ligand-dependent colocalization of CXCR4 with cortactin suggests that these two proteins may interact with each other. To assess this probable interaction, we treated HEK293 cells stably expressing CXCR4 with CXCL12 (10 nM) for different time intervals, immunoprecipitated CXCR4 from the cell lysate, and assessed the presence of cortactin by Western blot. We observed a basal association of CXCR4 with cortactin before ligand treatment that increased significantly in a time-dependent fashion, peaking at 5 min and lasting for
60 min (Fig. 3). By comparing the density of the immunoblots for cortactin coprecipitated with CXCR4 from 1 ml of cell lysate and the immunoblots of cortactin in 40 µl of cell lysate, we estimated that maximally 10% cortactin proteins were co-immunoprecipitated with CXCR4. Because glutathione S-transferase-CXCR4 did not pull down cortactin in the in vitro binding assay (data not shown), we propose that cortactin may interact with CXCR4 indirectly via association with other proteins in a CXCL12-modulated CXCR4 signaling complex.
Based on the previous findings that CXCR4 signaling involves Src (38), which directly phosphorylates cortactin at tyrosine residues (tyrosine 421, tyrosine 466, and tyrosine 482) (29), we determined whether stimulation of CXCR4 with specific ligand affects cortactin tyrosine phosphorylation. HEK293 cells stably expressing HA-CXCR4 were treated with CXCL12 for different time intervals. Cortactin tyrosine phosphorylation was determined by Western blot analysis using phospho-specific cortactin antibodies against the tyrosine 421, tyrosine 466, and tyrosine 482. As shown in Fig. 4, A and B, stimulation of the CXCR4-expressing cells resulted in cortactin phosphorylation at the tyrosine 421 but not at the tyrosine 466 or tyrosine 482. Overexpression of wild-type c-Src resulted in slight enhancement of cortactin phosphorylation but with no significant difference compared with the control cells (Fig. 4, C and D). However, overexpression of a dominant negative mutant form of c-Src (K295R/Y527F) significantly reduced CXCL12-induced cortactin tyrosine phosphorylation (Fig. 4, C and D). These findings suggest that c-Src is involved in CXCR4-mediated cortactin phosphorylation.
The GTPase dynamin is a cortactin-binding protein that may play a role in recruitment of cortactin to clathrin-coated pits (27). Consequently, we determined whether CXCR4-mediated cortactin tyrosine phosphorylation requires dynamin. As shown in Fig. 4, E and F, overexpression of the wild-type dynamin II did not affect CXCR4-mediated cortactin tyrosine phosphorylation, whereas overexpression of the dominant negative mutant dynamin II (K44A) inhibited tyrosine phosphorylation. These data suggest that the normal function of dynamin is required for CXCR4-mediated cortactin tyrosine phosphorylation.
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Because cortactin overexpression prolonged CXCR4-mediated ERK1/2 activation, we proposed that knockdown of cortactin with RNA interference may exert an opposite effect. To test this hypothesis, HeLa cells transiently transfected with control siRNA or cortactin-specific siRNA were treated with CXCL12 for different time intervals, and phosphorylation of ERK1/2 was assessed. As shown in Fig. 5, C and D, in cells transfected with the cortactin-specific siRNA, ligand stimulation resulted in a modest ERK1/2 phosphorylation. However, cells transfected with the control siRNA exhibited similar time-dependent ERK1/2 activation as the cells transfected with the empty vector (control) as described above. Western blot of cortactin showed that the expression level of cortactin was robustly reduced in cells transfected with the specific siRNA compared with cells transfected with the control siRNA. These data suggest that normal cortactin expression is critical for CXCR4 signaling.
To determine whether other chemokine receptor-mediated ERK1/2 activation is affected by cortactin overexpression or knockdown, we transfected HEK293 cells stably expressing CXCR2, another chemokine receptor, with empty vector (control), cortactin expressing vector, control siRNA, or cortactin specific siRNA. After incubating the cells with or without CXCL8 (10 nM) for 10 min, ERK1/2 phosphorylation was detected by Western blot analysis as described above. As shown in Fig. 5, E and F, CXCL8 treatment induced a marked phosphorylation of ERK1/2 in the control cells and in the cortactin-overexpressing cells but not in the cells with cortactin knockdown. These data suggest that other chemokine receptor-mediated ERK1/2 activation is similarly regulated by cortactin.
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We also determined if CXCR4-mediated early signal transduction is affected by cortactin overexpression or knockdown. HEK293 cells stably expressing CXCR4 were transiently transfected with empty vector (control), cortactin construct, control siRNA, or cortactin specific siRNA, and CXCL12 induced calcium mobilization was assessed as described previously (17). As shown in Fig. 7, in the vector-transfected cells, CXCL12 stimulation induced a remarkable increase of intracellular free Ca2+, suggesting Ca2+ mobilization from intracellular stores. The level of intracellular free Ca2+ nearly returned to the base line within 60 s, with a t0.80 of 52.42 ± 3.28 s. Similar levels of increase in intracellular free Ca2+ were observed in the cells transfected with cortactin construct, control siRNA, and cortactin siRNA, with t0.80 values of 53.15 ± 2.94, 52.48 ± 3.86, and 55.54 ± 5.10 s, respectively. Obviously, no significant difference in ligand-induced Ca2+ mobilization was found among these transfected cells.
Although cortactin has been shown to associate with clathrincoated pits (27), the effect of cortactin on trafficking of any chemokine receptor has not been addressed. Using a radioligand binding assay that allows assessment of cell surface bound versus internalized ligand, we observed that overexpression of cortactin did not affect the maximal CXCR4 internalization (
75%) but significantly increased the rate of the receptor internalization within 10 min (Fig. 8A). Interestingly and in contrast, overexpression of the cortactin-Y421A mutant significantly reduced ligand-induced CXCR4 internalization (Fig. 8A). These data suggest that cortactin is important for ligand-evoked CXCR4 internalization, and Src phosphorylation of cortactin appears to play a role in CXCR4 internalization.
Based on the early/sorting endosomal localization of cortactin (26), we proposed that cortactin may also affect CXCR4 recycling subsequent to internalization. To test this hypothesis, HEK293 cells stably expressing HA-CXCR4 were transiently transfected with empty vector (control) or cortactin-expressing vector. CXCR4 recycling was assessed. As shown in Fig. 8B, after an initial phase of CXCR4 internalization and recovery for 60 min, the reappearance of CXCR4 at the cell surface was observed in both the control and the cortactin-overexpressing cells, presumably due to the recycling of endocytosed receptors. However, more receptors appeared to be re-expressed on the cell surface in the cortactin-overexpressing cells compared with the control cells. These data suggest that cortactin overexpression promotes CXCR4 recycling.
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9.5-fold above basal. Interestingly, compared with the control cells, which exhibited no chemotactic response in response to high concentrations of CXCL12 (10 nM), the cortactin-overexpressing cells still exhibited chemotactic response (3-fold above control) in response to 10 nM CXCL12. These data suggest that overexpression of cortactin can elicit enhanced and prolonged chemotactic response to CXCL12 stimulation. We also determined the effect of overexpression of the cortactin tyrosine phosphorylation-deficient mutant (cortactin-Y421A) on CXCR4-mediated chemotaxis in HEK293 cells stably expressing CXCR4. Overexpression of the cortactin mutant resulted in a reduced chemotactic response compared with that of the control cells, and the chemotactic response was not prolonged as the wild-type cortactin-overexpressing cells. These data suggest that cortactin phosphorylation is important for chemotaxis.
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| DISCUSSION |
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These studies provide the first evidence for the translocation of cortactin from endosomal compartments to cell surface in response to chemokine stimulation. Previous studies have shown that cortactin undergoes membrane translocation in response to other stimuli, such as growth factor signaling, integrin activation, and bacteria entry (32-34). Our data also demonstrate that the translocated cortactin forms a complex with CXCR4. However, our data suggest that cortactin does not bind directly to CXCR4 since we did not obtain evidence for the direct interaction in our in vitro binding (glutathione S-transferase pull down) studies (data not shown). These findings suggest that cortactin associates with and likely modulates a CXCR4-containing complex. There is such a scenario for the somatostatin receptor subtype 2 (SST2) where the cortactin C-terminal SH3 domain binds several intracellular proteins, one of which is cortactin-binding protein 1 (42).
Cortactin translocation to the cell surface is likely required for its Src-dependent tyrosine phosphorylation because Src is a membrane-associated tyrosine kinase and because cortactin phosphorylation occurred within 5 min, which is in the same time frame as the cortactin cell surface translocation. Previous studies have shown that Src directly phosphorylates cortactin in vitro (29) on three tyrosine residues (tyrosine 421, tyrosine 466, and tyrosine 482) located within the proline-rich domain (30). However, CXCR4-mediated cortactin phosphorylation appeared to only occur at the tyrosine 421 residue. The underlying mechanism remains unknown, but the presence of a consensus Src SH2 binding sequence next to tyrosine 421 raises the possibility that this residue may be initially phosphorylated by Src, thereby allowing for the stabilization of Src with cortactin through an interaction between the Src SH2 domain and phosphorylated tyrosine 421. Stabilization of Src at tyrosine 421 may act to further facilitate the phosphorylation of cortactin at tyrosines 466 and 482 (43). Therefore, we cannot exclude the possibility that other tyrosine residues (tyrosine 466 and tyrosine 482) are phosphorylated after the phosphorylation of tyrosine 421 over prolonged CXCL12 treatment. This is highly possible because we observed that once cortactin was phosphorylated by CXCL12 stimulation within 5 min, the phosphorylation lasted over the entire experimental period (60 min), with no sign of decrease. Previous studies have shown that stimulation by the growth factors fibroblast growth factor-1 or epidermal growth factor leads to a rapid induction of cortactin tyrosine phosphorylation (within 5 min) that decreased within 1 h but increased again within 4-7 h and was sustained for 12-24 h (44), presumably due to the dynamic association of cortactin with c-Src (45). In addition to the involvement of Src, dynamin appears to play a role in CXCL12-induced cortactin phosphorylation, since a dominant negative mutant of dynamin (K44A) blocked cortactin phosphorylation. The underlying mechanisms remain unknown. We propose that dynamin may be required for cortactin membrane translocation to interact with c-Src, or it may be critical for the kinase activity of c-Src. It would be worthwhile to investigate if other chemokine receptors also mediate cortactin tyrosine phosphorylation, but it is likely true that not all G protein-coupled receptors mediate cortactin tyrosine phosphorylation, as demonstrated by the previous study that stimulation of
2-adrenergic receptor induced Src activation but not cortactin phosphorylation (46).
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We demonstrated that CXCL12-induced cortactin tyrosine phosphorylation is important for CXCR4-mediated activation of ERK1/2, which is considered to play an important role in cell proliferation and survival. This is based on the observation that overexpression of wild-type cortactin but not the cortactin-Y421A mutant prolonged CXCL12-induced ERK1/2 activation, whereas knockdown of cortactin blocked the receptor-mediated ERK1/2 activation. We showed that the cell surface CXCR4 expression and ligand-induced early signaling such as Ca2+ mobilization were not affected by either cortactin overexpression or cortactin knockdown, suggesting other mechanisms are involved in the effect of cortactin of chemokine receptor-initiated ERK1/2 activation. Recent studies suggest involvement of Src kinase in CXCR4-mediated ERK1/2 activation (39). Because cortactin is a direct substrate of Src kinase, we propose that CXCR4-mediated cortactin tyrosine phosphorylation may link Src with the downstream effector cascades, leading to ERK1/2 activation. In addition, studies on epidermal growth factor receptors have suggested that overexpression of cortactin induced prolonged ERK1/2 activation likely through preventing receptor degradation and promoting receptor recycling, resulting in more receptors on the cell surface to respond to ligand (48). We cannot exclude this possibility in our system since we also observed that overexpression of cortactin promoted CXCR4 recycling. Interestingly, previous studies have demonstrated that cortactin is phosphorylated by ERK1/2 (49), and ERK1/2-mediated cortactin phosphorylation plays an important role in actin polymerization (50). These together with our findings suggest that cortactin and ERK1/2 are mutually regulated, and this mutual regulation plays a critical role in fine tuning actin cytoskeleton.
The evidence for the involvement of cortactin in CXCR4-mediated chemotaxis comes from our observation that overexpression of cortactin robustly enhanced chemotaxis, whereas knockdown of cortactin blocked chemotaxis. This can be explained in different ways. The role of cortactin in chemotaxis may be due to the dual role of cortactin in endocytosis of the receptor, terminating response, and in recycling, resulting in accelerated restoration of receptors to the cell surface to respond to the CXCL12 gradients. Second, cortactin may act as a linker between clathrin-dependent endocytosis and the actin network, thereby regulating CXCR4-mediated chemotaxis. Cortactin is known to be localized in dynamic-actin assembly sites, such as lamellipodia, endosomes, podosomes, and invadopodia (51). Recent studies have shown that cortactin binds to and activate Arp2/3 complex (24), which binds to the side of an actin filament and nucleates daughter filaments (52), to form branched-actin network that produces protructive force necessary for directed cell movement. It is conceivable that overexpression of cortactin may enhance the activation of Arp2/3 proteins and, thus, results in enhanced chemotaxis, whereas cells deficient in cortactin have a selective defect in the persistence of lamellipodial protraction, with impaired cell migration and invasion (41). The precise mechanisms for the involvement of cortactin in CXCR4-mediated chemotaxis remain to be revealed; it should be noted that cortactin tyrosine phosphorylation is important for the receptor-mediated chemotaxis based on the result that overexpression of the cortactin-Y421A reduced chemotaxis significantly. Because cortactin is a direct substrate for Src family kinases, this result strongly supports the previous findings regarding the important role of Src family kinases in CXCR4-mediated chemotaxis (53, 54).
Taken together this study provides new and important evidence that CXCL12 ligand stimulation of CXCR4 induced tyrosine phosphorylation of cortactin, which plays a role in CXCR4 internalization and recycling, CXCR4-mediated ERK1/2 activation, and chemotaxis. Considering the important roles of CXCR4 in the development of immune system and central nervous system and in cancer growth and metastasis, these data may provide significant insight into understanding the mechanisms underlying CXCR4 functions, particularly since cortactin is overexpressed in many cancer types (28, 55, 56) in which CXCR4 is expressed or up-regulated (57, 58).
| FOOTNOTES |
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1 These authors contributed equally to this work. ![]()
2 To whom correspondence should be addressed. Tel.: 615-327-6363; Fax: 615-327-6757; E-mail: gfan{at}mmc.edu.
3 The abbreviations used are: CXCR4, CXC chemokine receptor 4; HEK293 cells, human embryonic kidney 293 cells; ERK1 and -2, extracellular signalregulated kinases 1 and 2; siRNA, short interference RNA; SH3, Src homolog three; EGFP, enhanced green fluorescence protein; HA, hemagglutinin. ![]()
| ACKNOWLEDGMENTS |
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| REFERENCES |
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