Article |
Address correspondence to Staffan Strömblad, Karolinska Institutet, Department of Microbiology, Pathology, and Immunology, Huddinge University Hospital F46, SE-141 86 Huddinge, Sweden. Tel.: 46-8-585-81032. Fax: 46-8-585-81020. E-mail: staffan.stromblad{at}impi.ki.se
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Abstract |
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Key Words: cell motility; cell signaling; integrin; lamellipodia; p21-activated kinase
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Introduction |
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PAK4 is the first identified member of the group II PAKs, and is implicated in cytoskeletal reorganization and filopodia formation (Abo et al., 1998). Importantly, PAK4 was recently found to be overexpressed in 78% of a variety of human cancer cell lines, an overexpression that might be mediated by gene amplification and may play a role in Ras-mediated transformation (Callow et al., 2002). In addition, overexpression of a hyperactive PAK4 mutant can protect cells from apoptosis induced by TNF (Gnesutta et al., 2001). The capability of PAK4 to promote cell survival is shared with PAK1, but the anti-apoptotic properties of PAK1 and PAK4 may be mediated by distinct mechanisms (Schurmann et al., 2000; Gnesutta et al., 2001). In addition, hyperactive PAK4 is able to transform fibroblasts to grow in soft agar in an anchorage-independent manner (Qu et al., 2001), perhaps in part due to it's ability to promote cell survival.
Integrins are heterodimeric transmembrane receptors and the major group of receptors for ECM proteins. Integrins are essential during development, in tissue homeostasis, and in the progression of various diseases (Hynes, 1992; Giancotti and Ruoslahti, 1999). By mediating cellular attachment to ECM, integrins are also a central part of the cellular motility machinery, where they are regulated by intracellular signaling molecules, which influence integrin localization, clustering, and binding to the ECM. In addition, integrin engagement to the ECM initiates various signaling events, e.g., activation of the ERK1/2 pathway, which is also important for the regulation of cell motility (Giancotti and Ruoslahti, 1999).
Previous studies have shown that v integrins are up-regulated or activated in migratory and invasive mechanisms in vivo, including wound healing, angiogenesis, and metastasis (Felding-Habermann and Cheresh, 1993; Brooks et al., 1994; Friedlander et al., 1995; Strömblad et al., 1996; Brooks et al., 1997). Integrin
vß5 is the predominant vitronectin (VN) receptor for carcinoma cells in vivo, because most carcinoma specimens from patients express
vß5 but not
vß3 (Lehmann et al., 1994; Jones et al., 1997). Importantly, integrin
vß5 has been found to be functionally involved in growth factorinduced carcinoma cell migration in vitro and metastasis in vivo (Klemke et al., 1994; Yebra et al., 1996; Brooks et al., 1997). Furthermore, activation of integrin
vß5 is implicated in VEGF-induced angiogenesis (Friedlander et al., 1995). In this report, we present a novel role for PAK in cell motility. We found that PAK4 directly interacts with the integrin ß5 subunit and specifically regulates
vß5-mediated cell migration.
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Results |
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Relocalization of PAK4 to lamellipodia does not require its kinase activity, integrin interaction, or Cdc42/Rac binding
Given that PAK4 undergoes membrane relocalization upon attachment onto VN, it was of interest to elucidate whether the relocalization of PAK4 is dependent on its integrin or Cdc42/Rac interaction and/or its kinase activity. Therefore, we constructed Flag-tagged PAK4 mutants that lack the binding capacity for Cdc42/Rac (PAK4-L19, 22), the IBD (PAK4-IBD), or PAK4 kinase activity (PAK4-M350) as illustrated in Fig. 5 A. Human M21 melanoma cells were transfected with these PAK4 mutants and compared with cells transfected with wild-type (wt) FlagPAK4 and a vector containing a nonrelated Flag-tagged BAP protein. Under normal culture conditions, wt PAK4 mainly localized in the cytosol (Fig. 5 B). However, upon cell replating onto VN, the majority of cells transfected with PAK4 displayed a relocalization to lamellipodia (Fig. 5 C). A similar relocalization to lamellipodia upon replating was also observed for the kinase-dead and
IBD PAK4 mutants, both of them lacking kinase activity (unpublished data) and PAK4-
IBD also lacking integrin-binding capacity (Fig. 3 B). However, the PAK4-L19, 22 that lacks GTPase-binding capacity was found in lamellipodia in almost half of the cells in regular culture and was then redistributed to the membrane in the remaining cells upon replating onto VN. A quantification of the PAK4 relocalization by counting the number of cells with membrane-localized PAK4 is displayed in Fig. 5 D. Taken together, these results suggest that PAK4 relocalization to lamellipodia does not require its kinase activity or integrin or Cdc42/Rac binding. However, the Cdc42/Rac binding capacity of PAK4 might be inhibitory for PAK4 localization in lamellipodia.
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In a haptotactic cell migration assay, we found that transient expression of EGFPPAK4 in MCF-7 cells specifically induced MCF-7 cell migration on VN, but not integrin ß1mediated cell migration on collagen type I (Fig. 7 A). Furthermore, EGFPPAK4-induced cell migration was blocked by a functional blocking anti-vß5 mAb, but not by an anti-
vß3 mAb (Fig. 7 A, left). Taken together, this demonstrates that PAK4 specifically induces integrin
vß5mediated cell motility. Moreover, stable expression of EGFPPAK4 in MCF-7 cells yielded numerically almost identical results on induction of
vß5-mediated cell migration as transient PAK4 expression, but did not influence cell motility on collagen (Fig. 7 B). Similarly, stable overexpression of a Flag-tagged constitutively active PAK4 mutant (S474E; Callow et al., 2002) induced MCF-7 cell migration to VN to the same degree as EGFPPAK4 (unpublished data). This indicates that overexpression of EGFPPAK4 may saturate PAK4-inducible motility in this cell type, which might be explained by the observation that a large GST fusion partner at the NH2 terminus of PAK1 causes constitutive PAK1 activation, suggesting that the EGFP fusion to PAK4 may cause PAK4 activation.
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Discussion |
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Intriguingly, interactions between PAK family members and integrins may be conserved, because the IBD within PAK family members is highly conserved (Fig. 3 C) and because we found that in addition to PAK4, both hPAK1 and the Drosophila PAK4 homologue MBT are able to interact with various integrin ß subunits (unpublished data). The wide interaction spectra between integrins and PAK family members of man and Drosophila suggest that the capacity for these interactions might be highly conserved during evolution and may thus fulfill vital functions in various species.
Relocalization of PAK4 to lamellipodia
PAK4 has been found to be localized in the cytosol and in the Golgi apparatus (Abo et al., 1998; Callow et al., 2002). In the current study, we found that PAK4 relocalized to motile structures in the cell membrane upon replating onto VN, including relocalization of PAK4 to lamellipodia and ruffles. In comparison, PAK1 can localize to focal adhesions upon replating (unpublished data; Manser et al., 1997). The NH2-terminal regulatory domain of PAK1:1329, including its Cdc42/Rac-binding motif, is sufficient to localize PAK1 to focal adhesions, and PAK1 has been indicated to be recruited to focal adhesions dependent on its binding to Cdc42/Rac (Manser et al., 1997; Brown et al., 2002). However, in a substantial part of the cells (>40%), a PAK4 mutant deficient in binding to Cdc42/Rac localized to the membrane before cell replating onto VN, whereas wt PAK4 only localized to the membrane in a few cells before replating. This suggests that binding to Cdc42/Rac might negatively regulate PAK4 membrane relocalization, which is consistent with the finding by Abo et al. (1998) that overexpression of activated Cdc42 results in localization of PAK4 in the Golgi apparatus. In addition, binding to Nck has been shown to mediate PAK1 membrane localization (Lu et al., 1997), whereas Pix, but not Nck, binding is required for PAK1:1329 localization to focal adhesions (Brown et al., 2002). However, the Nck- and Pix-binding regions of PAK1 are not conserved in PAK4, which might explain why PAK1 can be readily detected in focal adhesions (unpublished data; Manser et al., 1997), whereas PAK4 instead mainly relocalizes to lamellipodia upon replating and is rarely found in focal adhesions (unpublished data). It is unclear how PAK4 relocalization is mediated, but it appears to be independent of its catalytic activity, Cdc42/Rac binding, and integrin binding capacity, because PAK4 mutants deficient in these capacities all still relocalized to lamellipodia upon replating. However, it will be highly interesting to elucidate how relocalization of PAK4 is regulated, because the PAK4 localization at motile cellular structures may be important for its function in motility.
Role of PAK4 in the regulation of cell motility
Cell migration is important in many physiological and pathological processes and the regulation of cell motility is complicated, including both extracellular and intracellular events. Among these, PAK1 has been found to regulate cell motility in mouse fibroblasts (Sells et al., 1999), endothelial cells (Kiosses et al., 1999; Master et al., 2001), and tracheal smooth muscle cells (Dechert et al., 2001). Previous studies have suggested that the regulation of phosphorylation of myosin light chain kinase and LIM kinase (LIMK) by PAK1 and PAK2 might account for the role of these PAK family members in regulation of cell motility on various ECM substrates, but without displaying any apparent integrin specificity (Edwards et al., 1999; Sanders et al., 1999; Goeckeler et al., 2000). However, in this report, we demonstrate that PAK4 specifically induces vß5-mediated cell migration on VN, whereas ß1 integrinmediated cell migration on collagen type I is not influenced.
A recent study indicated that unlike PAK1, PAK4 is unable to phosphorylate myosin light chain kinase (Qu et al., 2001), which may rule out one possible route by which PAK4 could stimulate cell migration. Therefore, the mechanisms for induction of cell motility by PAK1 and PAK4 may be distinct, which is also supported by their distinct localization in cell adhesive structures of focal adhesions and lamellipodia, respectively. PAK4 was recently found to interact with LIMK1, to phosphorylate LIMK1 and stimulate the ability of LIMK1 to phosphorylate cofilin (Dan et al., 2001a). As a consequence, PAK4 and LIMK1 may cooperatively regulate cytoskeletal changes that impact cell motility. However, the PAK4 interaction with integrin ß5 cytoplasmic domain may also directly modulate the extracellular motility machinery, including cell adhesion to ECM for which PAK4 has been indicated to play a functional role (Qu et al., 2001). PAK4 binding to integrin might directly effect the integrin function, and thereby cell motility, and/or localize PAK4 effects to integrin-proximal sites of migratory regulation. For example, we found that PAK4 can phosphorylate the integrin ß5 cytoplasmic domain and this way might affect the integrin vß5 extracellular binding capacity (unpublished data).
Taken together, our study suggests a model where PAK4 binds to integrin ß5 cytoplasmic domain in motile cellular structures and modulates integrin vß5mediated cell migration. This may be brought about by PAK4 regulation of cytoskeletal components and/or by directly influencing integrin
vß5 function, thereby facilitating cell migration.
Possible role of PAK4 in tumor progression and metastasis
Intriguingly, PAK4 was recently found to be overexpressed in 78% of an array of human cancer cell lines where its function may be to promote cell transformation (Callow et al., 2002). In addition to this potential function, our study indicates a role for overexpressed PAK4 in breast carcinoma cell migration, suggesting a potential role also in metastasis. The predominant VN receptor in human carcinomas in vivo is integrin vß5 (Lehmann et al., 1994; Jones et al., 1997), an integrin that can be activated by growth factors for cell migration (Klemke et al., 1994; Yebra et al., 1996). In fact, growth factor stimulation of breast and pancreatic carcinoma cells has been shown to cause tumor dissemination and metastasis in vivo, which was functionally linked to activation of integrin
vß5mediated cell migration (Brooks et al., 1997). Given that PAK4 stimulated
vß5-mediated cell migration in breast carcinoma cells, elucidation of its potential role in growth factor signaling pathways governing integrin
vß5 activation will be very interesting; for example if PAK4 might effect src and/or FAK pathways recently implicated in integrin
vß5 activation (Eliceiri et al., 2002). In addition, stimulation of angiogenesis by VEGF or TGF-
depends on integrin
vß5 activation (Friedlander et al., 1995). Therefore, it will also be interesting to assess the potential role of PAK4 in in vivo progression of carcinoma metastasis as well as angiogenesis.
In conclusion, we report a novel cell motility pathway mediated by the serine/threonine kinase PAK4 that directly interacts with integrin vß5 and selectively induces
vß5-mediated cell motility, a mechanism previously demonstrated to mediate carcinoma dissemination.
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Materials and methods |
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Yeast two-hybrid screening and yeast mating tests
Integrin ß5 cytoplasmic domain (aa 753799; Ramaswamy and Hemler, 1990) was fused to Lex A-DBD in the pEG202 vector (OriGene). The insert sequence and reading frame were confirmed by sequencing. Approximately 2 x 107 yeast transformants of a 19-d mouse whole embryo expression library (OriGene) were pooled and screened by the activation of leucine and ß-galactosidase marker genes. 98 clones were His+ Leu+ Trp+ Ura+ ß-gal+ and 25 of them specifically interacted with ß5 cytoplasmic domain in yeast mating tests. Sequence analysis and bioinformatics studies indicated that 6 out of 25 ß5 cytoplasmic domaininteracting sequences represented mPAK4. For yeast mating tests, different regions of the integrin ß5 cytoplasmic domain were cloned into the pEG202 bait vector using yeast strain RFY206 as host, and different regions of the KD of hPAK4 were cloned into pJG4-5 prey vector using yeast strain EGY48 as host.
Proteinprotein interaction assays
COS-7 cells were transfected with 4 µg expression vector HAPAK4-SR3 by LipofectAmine plus (Life Technologies). Cells were harvested 48 h after transfection and lysed in RIPA buffer (1x PBS, pH 7.4, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1% SDS) with protease inhibitors (0.5 µg/ml leupeptin, 1 mM EDTA, 1 µg/ml pepstatin A, 0.2 mM PMSF). Approximately 500 µg of precleared lysates were immunoprecipitated by 4 µg rabbit anti-HA pAb Y11 (Santa Cruz Biotechnology, Inc.), anti-
vß5 mAb P1F6 (Life Technologies), or anti-ß3 mAb AP3 (GTI) and were separated and probed with rabbit antihuman integrin ß5 cytoplasmic pAb, antiintegrin
v pAb (Chemicon), or antiHA tag mAb F-7 (Santa Cruz Biotechnology, Inc.) and then with an HRP-conjugated secondary pAb (Jackson ImmunoResearch Laboratories) and visualized by ECL technique as previously described (Bao et al., 2002). Alternatively, IP of endogenous PAK4 was performed using 2 µg anti-PAK4 pAb. For testing the in vivo association of PAK4-
IBD mutant with integrin
vß5, COS-7 cells were transfected with FlagPAK4 and FlagPAK4-
IBD. Precleared lysates (100 µg) were immunoprecipitated by 6 µl ascites fluid of antiintegrin
vß5 mAb P1F6 (Life Technology) or rabbit IgG or
-Rab pAb (C-19; Santa Cruz Biotechnology, Inc.) followed by Western blot analysis using anti-Flag tag mAb M2 (Sigma). As positive controls for the Western blot analyses, 1015 µg lysates were applied without IP. To make GST fusion proteins, PAK4 KD (aa 324591) and ß5 (aa 753799) cytoplasmic domains were cloned separately into the GST fusion protein expression vector pGEM-1
T (Amersham Biosciences). GST fusion proteins were purified using glutathione-Sepharose beads (Amersham Biosciences) according to the manufacturer's protocol. To pull down hPAK4, 2 µg of GST ß5 tail fusion protein was mixed with 500 µg cell lysate containing HAhPAK4 in RIPA buffer. Reciprocally, to pull down the endogenous integrin ß5 subunit, 200 µg COS-7 cell lysate was mixed with 5 µg purified GSThPAK4 KD fusion protein in RIPA buffer and incubated overnight at 4°C. Glutathione-Sepharose beads (Amersham Biosciences) were used to capture the GST fusion proteins and the interacting proteins. The bound proteins were visualized by Western blotting with mAb F-7 or rabbit antihuman integrin ß5 cytoplasmic domain pAb (Chemicon), respectively.
Fluorescent microscopy and time-lapse video microscopy
MCF-7 cells stably expressing EGFPPAK4 or EGFP were established under the selection of G418 (0.5 mg/ml). Cells were fixed by 4% paraformaldehyde after attachment on VN. For staining of endogenous PAK4 in MCF-7 cells, rabbit anti-PAK4 pAb was used. For integrin vß5 staining, cells were replated in the absence of FCS and Mn2+ (RPMI 1640, 2 mM CaCl2, 1 mM MgCl2, and 0.5% BSA), and antiintegrin
vß5 mAb clone 15F11 (Chemicon) was used for staining. Anti-Flag mAb M2 (Sigma-Aldrich) was used for Flag tag staining. For quantification of cells with lamellipodial PAK4 localization, six microscopic fields were chosen randomly and counted directly through 20x objective. Statistical analysis was performed using Origin version 6.0 (Microcal Software, Inc.). Stained cells were photographed by fluorescent microscopy using a digital camera. Time-lapse studies were performed
30 min after plating EGFPPAK4 stably transfected MCF-7 cells onto VN-coated chamber slides in the absence of FCS using a fluorescent microscope (Leica). Pictures were captured every minute using Slidebook software version 2.06 (Intelligent Imaging Innovations, Inc.). The acquired pictures were further processed and assembled using Adobe Photoshop® 5.0 and Adobe Illustrator® 8.0.
Cell migration and cell adhesion assays
Haptotactic cell migration assays were performed using Transwell chambers (Costar Inc.) with 8.0 µm pore size. The Transwell membranes were coated with VN (10 µg/ml), collagen type I (10 µg/ml), or 1% BSA at the bottom surfaces for 2 h at 37°C. MCF-7 cells were transfected with EGFP or EGFPPAK4 for 48 h, and then cells were trypsinized, washed, and counted in the presence of soybean trypsin inhibitor (0.25 mg/ml). Cells (1 x 105) were then added on the top of Transwell membranes and allowed to migrate toward VN or collagen type I in the presence or absence of antiintegrin vß3 mAb LM609 (Chemicon) or anti-
vß5 mAb P1F6 (25 µg/ml) for 6 h at 37°C in migration buffer (RPMI 1640, 2 mM CaCl2, 1 mM MgCl2, 0.2 mM MnCl2, and 0.5% BSA). After thoroughly cleaning the upper chambers of the Transwells, the migrated cells expressing EGFP or EGFPPAK4 were counted using a fluorescent microscope; typically 12 microscopic fields were randomly chosen and counted. For comparison, the number of migrating cells was calibrated to the transfection efficiency within the cell population as determined by flow cytometry. Quantification of stably transfected cells was performed by staining using crystal violet followed by counting of random microscopy fields. For the cell adhesion assay, nontreated 48-well plates (Corning Costar Corp.) were used. Wells were coated with 0.510 µg/ml VN overnight at 4°C. 1% heat-denatured BSA was applied to block nonspecific adhesion. MCF-7 cells stably transfected with EGFPPAK4 or EGFP control were plated into the wells in triplicate at 5 x104 cells/well in cell adhesion buffer (RPMI 1640, 2 mM CaCl2, 1 mM MgCl2, 0.2 mM MnCl2, and 0.5% BSA) and allowed to attach for 60 min. After careful washing of nonbound cells using adhesion buffer, MTT was used to quantify the number of stably transfected cells attached.
Flow cytometry analyses
The efficiency for cell transfections and the cell surface expression levels of integrins were analyzed by measurement of EGFP content and phycoerythrin staining intensity, respectively, by FACScan® flow cytometer using CellQuest software (Becton Dickinson) after staining with antiintegrin vß5 mAb P1F6 and a phycoerythrin-conjugated secondary goat antimouse pAb (Jackson ImmunoResearch Laboratories).
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Footnotes |
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Acknowledgments |
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This study was supported by grants to S. Strömblad from the Swedish Cancer Society, the Swedish Science Research Council, the Swedish Strategic Research Foundation, and the Magnus Bergvall Foundation, and to H. Zhang from the Swedish Society of Medicine. H. Zhang was also supported by the Wenner-Gren Foundation.
Submitted: 1 July 2002
Revised: 19 August 2002
Accepted: 20 August 2002
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References |
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