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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON
Department of Biochemistry and Molecular Biology and the Miles and Shirley Fiterman Center for Digestive Diseases, Mayo Clinic, Rochester, Minnesota
Submitted 1 May 2008 ; accepted in final form 28 August 2008
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ABSTRACT |
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cell migration; phosphorylation
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A current gap in our understanding of cortactin function is an apparent disconnect between some of the observations made using in vitro assays, such as the negative effects of Src-mediated phosphorylation of cortactin on actin assembly, and those made in living cells. For example, endothelial cells overexpressing cortactin were observed to be exceptionally motile, while migration of cells into a wound was inhibited in cells expressing a cortactin mutant unable to be phosphorylated on key tyrosine residues (11). Similarly, overexpression of cortactin in breast cancer cells resulted in increased bone metastasis when these cells were injected into a nude mouse model, whereas injection of cells expressing a tyrosine phospho-inhibitory cortactin mutant resulted in fewer bone metastases as compared with the control group (14). Thus, the functions of cortactin appear to be regulated in a complex manner through phosphorylation at multiple sites. Indeed, a recent mass spectrometry analysis of cortactin purified from human embryonic kidney HEK-293 cells identified 17 new sites of cortactin phosphorylation for a total of 22 sites (4 tyrosine, 14 serine, and 4 threonine) when combined with the previously identified phosphorylation sites (17). However, the functional implications of this differential phosphorylation on actin dynamics with relation to migration of living cells need to be tested and compared.
Here, using the expression of phospho-mimetic and phospho-inhibitory cortactin tyrosine and serine mutants in cultured tumor cells and fibroblasts, we provide a detailed analysis of the mechanisms by which serine and tyrosine phosphorylation of cortactin may differentially contribute to cell migration. Interestingly, while phosphorylation at either site promotes cell migration, tyrosine phosphorylation appears to increase focal adhesion dynamics and lead to stress fiber disassembly. In contrast, serine phosphorylation of cortactin promotes the assembly of extensive branched actin networks. Thus these findings provide novel insights into how distinct types of phosphorylation may differentially regulate the effects of cortactin on specific processes to mediate cell migration and metastasis.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Constructs, transfection, and other reagents.
Cells were transfected with the indicated constructs using either the Lipofectamine Plus Transfection Reagent (Invitrogen) or Genejammer Transfection Reagent (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The cDNA sequence corresponding to rat cortactin isoform B (GenBank accession no. NM_021868) was used for generating wild-type and phospho-mutant cortactin constructs. Generation of wild-type red fluorescent protein (RFP)-tagged cortactin has been previously described (4). RFP-cortactin tyrosine and serine mutants were generated on the basis of known tyrosine (11) and serine (3, 18) phosphorylation sites using the Stratagene QuikChange Site-Directed Mutagenesis kit (Stratagene). The Myc-tagged type 1 phosphatidylinositol-4-phosphate 5-kinase-
(PIP5K1) construct was a gift from Laura M. Machesky (University of Birmingham, Birmingham, UK), and the green fluorescent protein (GFP)-paxillin construct was a gift from Christopher E. Turner (State University of New York, Syracuse, NY). Monoclonal anti-paxillin antibody was from BD Biosciences (San Jose, CA), monoclonal anti-vinculin antibody was from Sigma (St. Louis, MO), and FITC-conjugated phalloidin and Alexa-conjugated secondary antibodies were from Invitrogen. EGF was from Invitrogen, and nocodazole was from Sigma.
Microscopy. Cells were processed for fluorescence microscopy as previously described (10), except that cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100 for 2 to 5 min, depending on the cell type. ProLong (Invitrogen) antifade reagent was used as mounting medium. Live imaging experiments were performed using previously described conditions (21). Microscopy was performed on a Zeiss Axiovert 200 epifluorescent microscope (Zeiss, Thornwood, NJ). Digital images were acquired and analyzed using a cooled-CCD Orca II camera (Hamamatsu Photonics, Hamamatsu City, Japan) and the software program IPLab (Scanalytics, Fairfax, VA). All images were processed using Adobe Photoshop software (Adobe Systems, Mountain View, CA).
To analyze comets using electron microscopy, cells containing PIP5K1-induced comets positive for RFP-tagged cortactin were first identified using fluorescence microscopy. These cells were then processed for electron microscopy analysis using standard procedures and observed with a JEOL 1200 transmission electron microscope (JEOL USA, Peabody, MA).
Lamellipodial protrusion. Transfected cells were plated on glass coverslips and serum starved (0.2% FBS) for 16 h. Subsequently, cells were either mock stimulated with carrier or stimulated with EGF (30 ng/ml) for 20 min, processed for fluorescence microscopy analysis, and stained with FITC-conjugated phalloidin. The percentage of transfected cells extending a lamellipodium was determined for each condition.
Wound healing. Transfected PANC-1 cells were grown to confluency on gridded glass coverslips, serum starved (0.2% FBS) for 16 h, and then wounded with a pipette tip. After thorough washing, low serum medium was replaced with full serum medium, and the migration of cells into the wound was followed over a 14-h time period. At time 0, multiple images of each slip were taken, focusing on the coverslip grid. At regular time intervals afterward, images were taken in the exact same locations using the grid as a reference to track the migration of cells in the wound. Image integration was performed using IPLab software, and individual fluorescent cells were tracked. All fluorescent cells within a 100-µm distance from the wound front at time 0 were tracked. Approximately 20 cells were analyzed for each condition. The total distance each cell traveled was measured, and since under some conditions the cells traveled in aberrant pathways, the net distance into the wound perpendicular to the wound front was also measured.
Comet formation.
FR cells were plated on glass coverslips and were cotransfected with RFP-cortactin and Myc-PIP5K1 constructs. Twenty-four hours after transfection, cells were processed for fluorescence microscopy analysis, staining with FITC-conjugated phalloidin, or correlative electron microscopy analysis. Additionally, transfected cells were plated in imaging dishes, and comets were imaged in living cells via the RFP-cortactin in their tails.
Focal adhesion dynamics. Focal adhesion size and morphology were assessed in fixed FR cells that had been allowed to spread for 6 h on coverslips coated with 1 µg/ml fibronectin (Calbiochem, San Diego, CA) by staining with a monoclonal anti-paxillin antibody or on the basis of GFP-paxillin signal.
To monitor focal adhesion turnover in living cells, MEFs and PANC-1 cells were cotransfected with GFP-paxillin and RFP-cortactin constructs. Twenty-four hours after transfection, cells were trypsinized and resuspended in serum-containing medium. Subsequently, cells were replated on coverslips coated with 1 µg/ml fibronectin and allowed to spread for 30 min before being imaged using either time-lapse confocal microscopy or total internal reflection fluorescence (TIRF) microscopy.
Focal adhesion reassembly following microtubule-mediated focal adhesion disassembly was assayed in MEFs and PANC-1 cells as follows. First, cells were treated with 10 µg/ml nocodazole for 3 h at 37°C to induce microtubule disassembly. The drug was then washed out, and microtubules were allowed to repolymerize over various time intervals. Subsequently, cells were fixed and processed for immunofluorescence, staining with a monoclonal antibody against vinculin. Cells were imaged using fluorescence microscopy, and the presence or absence of focal adhesions was quantitated.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In addition to generating the RFP-tagged cortactin mutant mimicking Src-mediated phosphorylation (CortYE) indicated above, cortactin phospho-mutants designed to inhibit (Y384/429/445F; CortYF) Src-mediated phosphorylation, or inhibit (S368/381A; CortSA) or mimic (S368/381D; CortSD) ERK-mediated phosphorylation were constructed on the basis of previously identified sites of tyrosine (11) and serine (3, 18) phosphorylation. These mutants, RFP-tagged wild-type cortactin (CortWT) or vector alone, were then expressed in BxPC-3 (Fig. 1, C–G) or HPAF-II cells (supplemental material Fig. S1; the online version of this article contains supplemental data), and the effects on the percentage of cells that formed lamellipodia in the absence and presence of EGF were quantitated. As expected, EGF treatment resulted in an approximately 3.5-fold stimulation of lamellipodial protrusion in vector- and CortWT-expressing cells (Fig. 1H and supplemental Fig. S1F). Interestingly, cells expressing the cortactin phospho-mimetic mutants CortSD and CortYE exhibited an approximately 2.5- to 3-fold increase in the percentage of cells that extended lamellipodia in the absence of EGF, as compared with control and CortWT-expressing cells (Fig. 1H and supplementary Fig. S1F; gray bars). Furthermore, this EGF-dependent extension of lamellipodia was reduced in cells expressing the cortactin phospho-inhibitory mutant CortYF. The baseline lamellipodial formation seen in cells expressing the phospho-inhibitory CortSA mutant may be attributed to the prolonged activation of ERK caused by overexpression of cortactin as demonstrated by Timpson et al. (29).
We next tested whether the increase in lamellipodial protrusion exhibited by the cortactin tyrosine and serine phospho-mimetic mutants (CortSD and CortYE) may translate into increased cell motility. We observed PANC-1 cells to be more motile than HPAF-II or BxPC-3 cells (A. E. Kruchten, E. W. Krueger, and M. A. McNiven, unpublished observations); thus, wound-healing assays were performed using PANC-1 cells. Expression of CortWT enhanced the migratory phenotype of cells (Fig. 2, A and D), in agreement with previous reports that overexpression of cortactin in fibroblasts and endothelial cells enhances cell migration (11, 23). Similarly, cells expressing CortSD or CortYE also exhibited an enhancement in cell migration, although not above that of CortWT-expressing cells (Fig. 2, C and D, and data not shown). In contrast, cells expressing CortSA or CortYF migrated only approximately half as far as cells expressing CortWT or their respective phospho-mimetic mutant (Fig. 2, B and D, and data not shown). Thus, both tyrosine and serine phosphorylation of cortactin appear to be important not only for lamellipodial protrusion, but also for the stimulatory effects of cortactin on cell migration.
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(PIP5K1; 20, 25). These comets are easily visualized and act to push vesicles forward using an actin network also containing cortactin (20). Thus we could readily quantify the number and dimensions of actin comet tails formed behind vesicles in fixed or living cells coexpressing Myc-tagged PIP5K1 and either wild-type or phospho-mutants of RFP-tagged cortactin based on the fluorescent cortactin incorporated into the tail and/or FITC-conjugated phalloidin, used to label actin. Most notably, in comparison to the number of comets formed in cells expressing CortWT (Fig. 3A) or CortSA (data not shown), CortSD-expressing cells (Fig. 3C) showed a substantial increase in comet number (
2-fold; Fig. 3J), suggesting an increase in actin polymerization. In contrast, expression of cortactin tyrosine mutants, either phospho-inhibitory (CortYF; Fig. 3B) or phospho-mimetic (CortYE; data not shown), only slightly increased the number of comets per cell in comparison to CortWT-expressing cells (Fig. 3J).
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To confirm the differences in actin comet tail dimensions observed in cells expressing the different cortactin phospho-mutants at a higher resolution, comets were viewed using electron microscopy (Fig. 3, E–I). In support of the fluorescence imaging, the shorter, thinner comets formed in the CortSA-expressing cells exhibited an actin network that appeared to surround only a small portion of the vesicle ( 30–40%; Fig. 3, F and G), whereas in cells expressing CortSD, a larger portion of the vesicle was surrounded by actin filaments (50–70%; Fig. 3, H and I). In addition, the comet base was wider in cells expressing CortSD, as compared with cells expressing CortSA.
Tyrosine phosphorylation of cortactin alters focal adhesion morphology and dynamics. Observation of actin comet tails indicated that serine phosphorylation of cortactin preferentially promotes actin assembly in cells; however, both tyrosine and serine phospho-mimetic cortactin mutants promote lamellipodial protrusion and cell migration (Figs. 1 and 2 and supplemental Fig. S1). This paradox suggests that Src-mediated tyrosine phosphorylation of cortactin may be contributing to these dynamic processes in a way distinct from actin assembly. Some insights into this possibility come from a recent study showing that knockdown of cortactin protein levels reduces the rate of focal adhesion assembly (1) and our observation that some cells expressing cortactin appear to exhibit prominent focal contact-like structures (Fig. 6). Thus, because Src-mediated phosphorylation is known to play a role in regulating focal adhesion dynamics (8, 34), we tested whether tyrosine phosphorylation of cortactin may contribute to lamellipodial protrusion not by actin assembly at the leading edge, but by regulating focal adhesion turnover.
First, focal adhesion size was analyzed in two different cell types: FR cells, chosen because of the large adhesions made by these cells (Fig. 4A, A'), and PANC-1 cells, which exhibit smaller, more ephemeral adhesions (Fig. 4F,F'). To mark focal adhesions, cells were transfected to express RFP-tagged CortWT or the cortactin phospho-mutants in combination with GFP-tagged paxillin, a widely used marker for monitoring focal adhesion dynamics (32, 34), or alternatively, cells were stained with an anti-paxillin antibody. As shown in Fig. 4, FR cells expressing CortYE exhibited small, sparsely populated focal adhesions (Fig. 4B,B'), compared with cells expressing CortWT (Fig. 4A,A'), whereas CortYF-expressing cells exhibited much larger, more densely packed focal adhesions that appeared to fill the cell border (Fig. 4C,C'). Quantitation of focal adhesion size in FR cells expressing wild-type cortactin or the various cortactin phospho-mutants revealed a slight reduction in size in cells expressing CortYE, CortSA, or CortSD, as compared with CortWT-expressing cells, whereas expression of CortYF resulted in an increase in focal adhesion size (Fig. 4D). Indeed, a 2.5-fold difference in focal adhesion size was determined in cells expressing CortYF versus CortYE. While more difficult to quantitate, similar effects on focal adhesion size were observed in PANC-1 tumor cells expressing CortYE (Fig. 4G,G') and CortYF (Fig. 4H,H') compared with cells expressing CortWT (Fig. 4F,F').
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10–15 min and subsequently almost completely disassembled over the following 5 min (Fig. 4E). Interestingly, CortYE-expressing cells assembled and disassembled their small focal adhesions much faster, as they both appeared and disassembled over an 15-min time period. In strong contrast, the large focal adhesions present in CortYF-expressing cells assembled at a rate similar to that of CortWT-expressing cells, but persisted at 70% of the peak GFP-paxillin fluorescence intensity beyond 30 min (Fig. 4E). PANC-1 cells demonstrated focal adhesion dynamics similar to those of MEFs, with cells expressing CortYF taking 1.5–2 times as long as CortWT-expressing cells to assemble or disassemble focal adhesions (Fig. 4I). Compilation of this focal adhesion assembly-disassembly data from multiple PANC-1 cells into a single representative curve for each condition (CortWT, CortYE or CortYF expression) provides another way of depicting that focal adhesion dynamics are slowed in PANC-1 cells expressing CortYF (Fig. 4J). As a second method to assess a role for cortactin phosphorylation in regulating focal adhesion size and turnover, we used a microtubule-mediated focal adhesion disassembly assay similar to that established by Ezratty et al. (7). Here, MEFs and PANC-1 cells expressing wild-type or phospho-mutant cortactin were treated with nocodazole to disassemble microtubules and induce formation of focal adhesions. Subsequently, following washout of the drug, cells were allowed to recover for 2 h and were then stained for the focal adhesion marker vinculin. During the first 60 min of the recovery period, >90% of the focal adhesions are disassembled, possibly by an endocytic mechanism (7), while focal adhesion reassembly occurs during the second 60 min. It is predicted that ephemeral, dynamic focal adhesions will not form as quickly during the allotted recovery period, while stable adhesions will reassemble more readily. Consistent with this premise, both MEFs and PANC-1 cells expressing CortYE showed a marked decrease in focal adhesion recovery (Fig. 5, A, A' and C,C', respectively, asterisks) compared with the CortWT- or CortYF-expressing cells (Fig. 5, B,B' and D,D', asterisks; and data not shown). In contrast, focal adhesions in CortYF-expressing cells (Fig. 5, B,B' and D,D', asterisks) recovered more readily and were noticeably larger than those in adjacent control cells as well as those in the CortYE-expressing cells. Semiquantitative counting of the number of focal adhesions in these cell populations (Fig. 5E) suggested modest differences in focal adhesion reassembly between cells expressing wild-type cortactin and either of the cortactin tyrosine phospho-mutants, whereas a more noticeable difference existed between cells expressing CortYE versus CortYF.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Serine phosphorylation of cortactin promotes actin assembly in cells.
Serine phosphorylation of cortactin has been reported to stimulate N-WASP-Arp2/3-mediated actin polymerization in vitro, and this stimulatory effect was prevented by subsequent tyrosine phosphorylation (18). More recently, another in vitro study using purified components demonstrated that tyrosine phosphorylation of cortactin does amplify the generation of actin barbed ends; however, this effect requires the adaptor protein Nck1 as an additional component of the actin assembly mixture. In addition, it is further enhanced by including WASP-interacting protein (WIP) (28). These latter components were not included in the earlier study. Our primary goal was to provide a useful extension of these in vitro observations to the context of living cells, something that has been lacking to date. To this end, we analyzed the effects of different cortactin phospho-mutants on the number, length, and girth of PIP5K1-induced actin comet tails as an in vivo indicator of the assembly of actin networks. Indeed, our observations of a marked increase in comet numbers and dimensions in cells expressing CortSD are in strong support of the observations made in vitro. However, an increase in comet length was also observed in cells expressing CortWT and CortYE, indicating a complex interplay of cortactin phosphorylation in the regulation of actin dynamics within cells. Interestingly, unusually wide, bifurcated comets were observed at the light microscopy level in CortSD-expressing cells (Fig. 3D'), and using electron microscopy, a wide base of actin nucleation that appeared to wrap around more than half of the vesicle membrane could be detected at the membrane interface. Such robust actin branching would be particularly useful at the leading edge of a migrating tumor cell to extend a lamellipodium forward.
Cortactin as a key regulator of focal adhesion dynamics? Cortactin has been implicated in the regulation of focal adhesion dynamics in migrating cells (1). Because expression of CortYE did not appear to alter actin assembly appreciably in vivo (Fig. 3) and Src-mediated phosphorylation is known to play a key role in regulating focal adhesion dynamics (8, 34), we tested whether tyrosine phosphorylation of cortactin may preferentially affect focal adhesion size and/or dynamics (Figs. 4 and 5). Here, we observed a marked increase in the size of focal adhesions in cells expressing either CortWT or CortYF, consistent with observations of others that cortactin promotes focal adhesion stability (1). Moreover, the marked increase in the size of focal adhesions in cells expressing CortYF (Fig. 4) suggests a role for cortactin tyrosine phosphorylation in focal adhesion disassembly. Indeed, observation of GFP-paxillin in living cells (Fig. 4) using TIRF microscopy revealed a substantial difference in focal adhesion turnover in cells expressing CortYE (turnover in 5–15 min) versus CortWT (turnover in 15–25 min) or CortYF (stable >25 min). Results from an assay using a distinct approach to measure focal adhesion dynamics— the ability of focal adhesions to reassemble following microtubule-mediated disassembly—provided further support to these findings (Fig. 5).
All of these observations are consistent with an important role for cortactin phosphorylation in regulating focal adhesion dynamics, and in turn, lamellipodial protrusion. Furthermore, our findings are consistent with the premise that active Src (favoring tyrosine-phosphorylated cortactin and corresponding to CortYE) leads to smaller focal adhesions (8). Conversely, cells lacking Src family kinases or expressing a kinase-dead Src (corresponding to CortYF) have enlarged peripheral focal adhesions with impaired disassembly (8, 13, 33). Thus, Src-mediated phosphorylation of cortactin may be important in regulating focal adhesion size and dynamics, and thereby modulate cell migration.
As illustrated in Fig. 6C, we propose that differential cortactin phosphorylation plays an important role in the regulation of two distinct processes, actin and focal adhesion dynamics, both of which can contribute to cell migration. How these processes are affected by differences in the phosphorylation status of cortactin is unclear, although alterations in protein-protein interactions are likely to be involved. For example, serine phosphorylation of cortactin by ERK appears to allow for an increased association with N-WASP and facilitate branched actin assembly (18), whereas tyrosine phosphorylation of cortactin increases its association not only with Nck1 (28), but also with dynamin (37), an interaction which might contribute to focal adhesion disassembly (7). Based on the findings described here, in combination with those from other groups (1, 18, 28), we predict that phospho-regulation of cortactin contributes to cell motility in two major ways. First, serine phosphorylation by ERK promotes the formation of branched actin networks, which supports lamellipodial formation and extension. Second, tyrosine phosphorylation by Src may also promote some actin assembly in vivo, although most conspicuously triggers turnover of focal adhesions and a reduction in the capping or anchoring of actin stress fibers at these structures.
In the future it will be important to determine how the specific phospho-forms of cortactin are regulated by different cellular stimulations, the interplay among the various types of cortactin phosphorylation, and the distinct effects of the various cortactin phospho-forms on the variety of cellular processes involving cortactin.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* A. E. Kruchten and E. W. Krueger contributed equally to this work. 
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REFERENCES |
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