Portal de Periódicos da CAPES

Nck Adapters Are Involved in the Formation of Dorsal Ruffles, Cell Migration, and Rho Signaling Downstream of the Platelet-derived Growth Factor β Receptor

2008; Elsevier BV; Volume: 283; Issue: 44 Linguagem: Inglês

10.1074/jbc.m800913200

1083-351X

Aino Ruusala, Tony Pawson, Carl‐Henrik Heldin, Pontus Aspenström,

Wnt/β-catenin signaling in development and cancer

The SH3 and SH2 domain-containing adapter proteins Nck1 and Nck2 are known to function downstream of activated tyrosine kinase receptors, such as the platelet-derived growth factor (PDGF) receptors. The SH2 domain of Nck1 binds to phosphorylated tyrosine residue 751 in PDGFβ receptor and has been suggested to have a role in the PDGF-induced mobilization of the actin filament system. Because Tyr-751 is a site for additional receptor interactors, it has been difficult to discriminate the signaling from Nck from signaling via other molecules. For this reason we have used mouse embryonic fibroblasts derived from mice in which the genes for Nck1 and Nck2 have been inactivated by gene targeting (knock-out (KO) cells). The mutant cells had a reduced ability to form edge ruffles in response to PDGF, and the presence of Nck was obligatory for the formation of dorsal ruffles. In addition, the KO cells had a reduced chemotactic and migratory potential. Importantly, KO cells had reduced cell attachment properties and a reduced ability to form focal adhesions in response to serum stimulation. Moreover, signaling involving the Rho GTPases was defective in KO cells. In summary, our observations suggest that the Nck adapters are needed for signaling to Rho GTPases and actin dynamics downstream of the PDGFβ receptor. The SH3 and SH2 domain-containing adapter proteins Nck1 and Nck2 are known to function downstream of activated tyrosine kinase receptors, such as the platelet-derived growth factor (PDGF) receptors. The SH2 domain of Nck1 binds to phosphorylated tyrosine residue 751 in PDGFβ receptor and has been suggested to have a role in the PDGF-induced mobilization of the actin filament system. Because Tyr-751 is a site for additional receptor interactors, it has been difficult to discriminate the signaling from Nck from signaling via other molecules. For this reason we have used mouse embryonic fibroblasts derived from mice in which the genes for Nck1 and Nck2 have been inactivated by gene targeting (knock-out (KO) cells). The mutant cells had a reduced ability to form edge ruffles in response to PDGF, and the presence of Nck was obligatory for the formation of dorsal ruffles. In addition, the KO cells had a reduced chemotactic and migratory potential. Importantly, KO cells had reduced cell attachment properties and a reduced ability to form focal adhesions in response to serum stimulation. Moreover, signaling involving the Rho GTPases was defective in KO cells. In summary, our observations suggest that the Nck adapters are needed for signaling to Rho GTPases and actin dynamics downstream of the PDGFβ receptor. There is a close correlation between the activities of tyrosine kinase receptors and the dynamic reorganization of the actin filament system (1Rönnstrand L. Heldin C.-H. Int. J. Cancer. 2001; 91: 757-762Crossref PubMed Scopus (130) Google Scholar, 2Tallquist M. Kazlaukas A. Cytokine Growth Factor Rev. 2004; 15: 205-213Crossref PubMed Scopus (327) Google Scholar). Tyrosine kinase receptors, such as the platelet-derived growth factor (PDGF) 2The abbreviations used are: PDGF, platelet-derived growth factor; PDGFRβ, PDGFβ receptor; FBS, fetal bovine serum; GST, glutathione S-transferase; KO, knock out; MEF, mouse embryonic fibroblast, PAE, porcine aortic endothelial; PI3K, phosphoinositide 3-kinase; TRITC, tetramethylrhodamine isothiocyanate; WASP, Wiskott-Aldrich syndrome protein; WT, wild type; siRNA, small interfering RNA; PBS, phosphate-buffered saline. 2The abbreviations used are: PDGF, platelet-derived growth factor; PDGFRβ, PDGFβ receptor; FBS, fetal bovine serum; GST, glutathione S-transferase; KO, knock out; MEF, mouse embryonic fibroblast, PAE, porcine aortic endothelial; PI3K, phosphoinositide 3-kinase; TRITC, tetramethylrhodamine isothiocyanate; WASP, Wiskott-Aldrich syndrome protein; WT, wild type; siRNA, small interfering RNA; PBS, phosphate-buffered saline. β receptor (PDGFRβ), exert their action by ligand-induced receptor dimerization, which induces transphosphorylation of the two receptor molecules. The phosphorylated tyrosine residues in the activated PDGF receptor constitute docking sites for SH2 domain-containing proteins (1Rönnstrand L. Heldin C.-H. Int. J. Cancer. 2001; 91: 757-762Crossref PubMed Scopus (130) Google Scholar, 2Tallquist M. Kazlaukas A. Cytokine Growth Factor Rev. 2004; 15: 205-213Crossref PubMed Scopus (327) Google Scholar). The role of the individual tyrosine residues in PDGF signaling has been extensively studied in cells such as fibroblasts and porcine aortic endothelial (PAE) cells expressing mutant PDGFRβ in which specific tyrosine residues in the cytoplasmic part of the PDGFRβ have been replaced with phenylalanine residues (1Rönnstrand L. Heldin C.-H. Int. J. Cancer. 2001; 91: 757-762Crossref PubMed Scopus (130) Google Scholar, 2Tallquist M. Kazlaukas A. Cytokine Growth Factor Rev. 2004; 15: 205-213Crossref PubMed Scopus (327) Google Scholar). Using this approach, the specific importance of individual tyrosine residues for binding and activation of downstream signaling proteins has been studied. PDGFRβ-mediated mobilization of the actin filament system, seen as the formation of membrane ruffles, has been found to require the activity of the phosphoinositide 3-kinase (PI3K)/Rac signaling pathway (3Wennström S. Siegbahn A. Ykote K. Arvidsson A.K. Heldin C.-H. Mori S. Claesson-Welsh L. Oncogene. 1994; 9: 651-660PubMed Google Scholar, 4Wennström S. Hawkins P. Cooke F. Hara K. Yonezawa K. Kasuga M. Jackson T. Claesson-Welsh L. Stephens L. Curr. Biol. 1994; 4: 385-393Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 5Nobes C.D. Hawkins P. Stephens L. Hall A. J. Cell Sci. 1995; 108: 225-233Crossref PubMed Google Scholar). PDGFRβ with mutated tyrosine residues 740 and 751 (PDGFRβ-Y740F/Y751F) can no longer bind the SH2 domain of the p85 regulatory subunit of PI3K (6Kashishian A. Kazlauskas A. Cooper J.A. EMBO J. 1992; 11: 1373-1382Crossref PubMed Scopus (200) Google Scholar). Moreover, the PDGFRβ-Y740F/Y751F-expressing PAE cells do not form membrane ruffles in response to PDGF-BB (3Wennström S. Siegbahn A. Ykote K. Arvidsson A.K. Heldin C.-H. Mori S. Claesson-Welsh L. Oncogene. 1994; 9: 651-660PubMed Google Scholar, 4Wennström S. Hawkins P. Cooke F. Hara K. Yonezawa K. Kasuga M. Jackson T. Claesson-Welsh L. Stephens L. Curr. Biol. 1994; 4: 385-393Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). Several of the phosphotyrosines in activated PDGFRβ constitute docking sites for multiple substrates, i.e. the tyrosine residue 751 is also the docking site for Nck1, and the phospholipase Cγ- and SHP2-binding site tyrosine 1009 is also a binding site for Nck2 (7Nishimura R. Li W. Kashishian A. Mondino A. Zhou M. Cooper J. Schlessinger J. Mol. Cell. Biol. 1993; 13: 6889-6896Crossref PubMed Scopus (152) Google Scholar, 8Kazlauskas A. Kishishian A. Cooper J.A. Valius M. Mol. Cell. Biol. 1992; 12: 2534-2544Crossref PubMed Google Scholar, 9Kazlauskas A. Feng G.-S. Pawson T. Valius M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6939-6942Crossref PubMed Scopus (188) Google Scholar, 10Rönnstrand L. Mori S. Arvidsson A.K. Eriksson A. Wernstedt C. Hellman U. Claesson-Welsh L. Heldin C.H. EMBO J. 1992; 11: 3911-3919Crossref PubMed Scopus (169) Google Scholar, 11Rönnstrand L. Arvidsson A.K. Kallin A. Rorsman C. Hellman U. Engström U. Wernstedt C. Heldin C.-H. Oncogene. 1999; 18: 3696-3702Crossref PubMed Scopus (62) Google Scholar, 12Chen M. She H. Kim A. Woodley D.T. Li W. Mol. Cell. Biol. 2000; 20: 7867-7880Crossref PubMed Scopus (73) Google Scholar). Hence, studies of cell lines harboring mutant PDGFRβ receptors will not discriminate between the different potential binding partners for the receptor.The Nck family of adapter proteins consists of two gene products, Nck1 and Nck2 (13Lehmann, J. M, Rietmüller G. Johnson J.P. Nucleic Acids Res. 1989; 18: 1048Crossref Scopus (158) Google Scholar, 14Braverman L.E. Quilliam L.A. J. Biol. Chem. 1999; 274: 5542-5549Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Nck was shown to be a substrate for tyrosine kinase receptors and to have tumor promoting activities (15Li W. Hu P. Skolnik E.Y. Ullrich A. Schlessinger J. Mol. Cell. Biol. 1992; 12: 5824-5833Crossref PubMed Scopus (155) Google Scholar, 16Chou M.M. Fajardo J.E. Hanafusa H. Mol. Cell. Biol. 1992; 12: 5834-5842Crossref PubMed Scopus (81) Google Scholar). Nck adaptors have also been implicated in the regulation of the actin filament system (for review, see Refs. 17Li W. Fan J. Woodley D.T. Oncogene. 2001; 20: 6403-6417Crossref PubMed Scopus (130) Google Scholar and 18Buday L. Wunderlich L. Tamas P. Cell. Signal. 2002; 14: 723-731Crossref PubMed Scopus (188) Google Scholar). Many proteins that directly influence the actin polymerization machinery, such as Wiskott-Aldrich syndrome protein (WASP), WASP-interacting protein, and p21-activated kinase (Pak), can directly bind to the SH3 domains of Nck (for review, see Ref. 17Li W. Fan J. Woodley D.T. Oncogene. 2001; 20: 6403-6417Crossref PubMed Scopus (130) Google Scholar). Most studies on the Nck adaptors have so far been focused on Nck1, and it is not known to what extent there exists a functional redundancy between the two paralogues. It is somewhat surprising that Nck1 and Nck2 seem to have separate binding sites on the activated PDGFRβ as the SH2 domains of the two adapters are almost identical. In an effort to study the involvement of the Nck adaptor proteins in PDGF signaling separate from PI3K-mediated signaling, we used mouse embryonic fibroblasts (MEFs) in which the Nck1 and Nck2 genes had been inactivated by gene targeting (KO cells) (19Bladt F. Aippersbach E. Gelkop S. Strasser G.A. Nash P. Tafuri A. Gertler F.B. Pawson T. Mol. Cell. Biol. 2003; 23: 4586-4597Crossref PubMed Scopus (142) Google Scholar).EXPERIMENTAL PROCEDURESAntibodies and Plasmids—Rabbit polyclonal anti-Arp3A antiserum was a generous gift from L. Machesky, Birmingham, UK. Rabbit polyclonal anti-N-WASP antiserum was raised against a peptide, CRFYGPQVNNISHTKEKK, which represents amino acid residues 173–189 of the human N-WASP. In addition, the following antibodies were used: mouse monoclonal anti-Myc (Santa Cruz Biotechnology), mouse monoclonal anti-RhoA (Santa Cruz), anti-phosphotyrosine (PY99, Santa Cruz), mouse monoclonal anti-vinculin and mouse monoclonal anti-α-tubulin (Sigma), mouse monoclonal anti-Rac1 (Upstate Biotechnology, Inc.), mouse monoclonal anti-Nck (BD Transduction Laboratories), rabbit anti-phospho-Akt (serine 473, Cell Signaling Technology), rabbit anti-Akt (Cell Signaling Technology), AlexaFluor 488-conjugated anti-mouse antibodies (Molecular Probes), TRITC-conjugated anti-mouse (Jackson ImmunoResearch Laboratories), and TRITC-conjugated anti-rabbit (DAKO). TRITC-labeled phalloidin, fluorescein isothiocyanate-labeled phalloidin (Sigma), or AlexaFluor 488-conjugated phalloidin (Molecular Probes) were used to visualize filamentous actin. The vectors expressing the constitutively active Rho GTPases have been described before (20Aspenström P. Fransson Å. Saras J. Biochem. J. 2004; 377: 327-337Crossref PubMed Scopus (305) Google Scholar).Cell Cultivation—The establishment of MEFs from Nck1/2-/- mice has been shown in a previous article (19Bladt F. Aippersbach E. Gelkop S. Strasser G.A. Nash P. Tafuri A. Gertler F.B. Pawson T. Mol. Cell. Biol. 2003; 23: 4586-4597Crossref PubMed Scopus (142) Google Scholar). Wild-type, Nck1/2-/-, and Nck1/2-/- cells in which Nck1 had been stably transfected (in this article called WT, KO, and KO/Nck1 rescue cells, respectively) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS. KO/Nck2 rescue cells were created by stably expressing mouse Nck2 in KO MEFs essentially as described before (19Bladt F. Aippersbach E. Gelkop S. Strasser G.A. Nash P. Tafuri A. Gertler F.B. Pawson T. Mol. Cell. Biol. 2003; 23: 4586-4597Crossref PubMed Scopus (142) Google Scholar). Porcine aortic endothelial (PAE) cells stably transfected with the human PDGFβ receptor, harboring point mutations in tyrosine residues 740 or 751, PAE/PDGFRβ-Y740 or PAE/PDGFRβ-Y751 mutant receptor-expressing cells, were cultured in Ham's F-12 medium supplemented with 10% FBS. The cells were cultured at 37 °C in an atmosphere of 5% CO2. Knock-down of Nck expression was triggered by transfection of WT MEFs with double-stranded Nck1- or Nck2-specific small interfering RNA (siRNA) (Sigma) or a control siRNA employing the Silentfect transfection reagent and protocol (Bio-Rad). For immunostaining purposes, cells were seeded on coverslips and transfected by Lipofectamine according to the procedure provided by the manufacturer. For cell staining, the cells on the coverslips were fixed in 2% paraformaldehyde in PBS for 20 min at room temperature. The cells were permeabilized in 0.5% Triton X-100 in PBS for 5 min and thereafter washed again and incubated in the presence of 0.01 m glycine in PBS for 1 h. The slides prepared for visualization of endogenous N-WASP and Arp3A were instead fixed in ice-cold methanol for 10 min and blocked in 0.01 m glycine, PBS for 1 h. Primary and secondary antibodies were diluted in PBS containing 5% FBS. Cells were incubated with primary antibodies followed by secondary antibodies for intervals of 1 h. The coverslips were mounted on object slides in Fluoromount-G mounting medium (Southern Biotechnology Associates). The cells were photographed by a Hamamatsu ORCA CCD digital camera employing the QED Imaging System software using a Zeiss Axioplan2 microscope.For Western blotting analysis, cell lysis was performed essentially as described before (20Aspenström P. Fransson Å. Saras J. Biochem. J. 2004; 377: 327-337Crossref PubMed Scopus (305) Google Scholar). The cell lysates were subjected to SDS-PAGE, and the proteins were then transferred to Hybond-C Extra nitrocellulose filters (GE Healthcare). Western blotting was performed with primary mouse or rabbit antibodies followed by horseradish peroxide-conjugated anti-mouse or anti-rabbit secondary antibodies (GE Healthcare). The Western blots were detected by the Luminol Western blotting substrate (Santa Cruz).Chemotaxis Analysis—A detailed description of the assay has been described before (21Demoulin J.B. Seo J.K. Ekman S. Grapengiesser E. Hellman U. Ronnstrand L. Heldin C.H. Biochem. J. 2003; 376: 505-510Crossref PubMed Scopus (41) Google Scholar). Briefly, chemotaxis was measured using the ChemoTx System 96-well disposable element (Neuro Probe Inc.), 8-μm pore size and 30-μl wells. The wells were coated with 50 μg/ml fibronectin before use. The cells were starved overnight, trypsinized, and washed in the presence of 1% aprotinin. The chemoattractants in starvation medium were added to the lower wells, and the starved cells (50,000 cells per well) were seeded on the upper side of the filter in starvation medium. The chamber was incubated at 37 °C for 4 h, and the cells were fixed to the filter in 96% ethanol, washed in water, and stained with Giemsa solution. The non-migrating cells were removed from the upper site of the filter, and the filter was scanned in a CCD camera (Fuji). Quantifications were performed using Aida Image Analyzer software.Cell Adhesion Assay—The procedure essentially followed the procedure by Wennerberg et al. (22Wennerberg K. Lohikangas L. Gullberg D. Pfaff M. Johansson S. Fässler R. J. Cell Biol. 1996; 132: 227-238Crossref PubMed Scopus (260) Google Scholar). Briefly, wells in a 96-well microtiter plate were coated with human plasma fibronectin (30 μg/ml), 15% FBS or 1% bovine serum albumin (BSA) at 37 °C for 1 h and blocked with 1% BSA at 37 °C for 1 h. Cells in a serum-free medium were seeded and incubated for 1 h at 37 °C. After washing the wells once with PBS, the attached cells were fixed for 10 min in 96% ethanol at room-temperature, stained with crystal violet for 30 min, washed, and lysed in 1% Triton X-100 for 20 min on a shaker. Bound dye was quantified by measuring the absorbance in the enzyme-linked immunosorbent assay reader at 595 nm.Protein Production and Rho Activation Assay—Glutathione S-transferase (GST) fusion proteins were expressed in Escherichia coli and purified on glutathione-Sepharose beads (GE Healthcare). Briefly, the bacteria were lysed in a buffer containing 50 mm Tris-HCl, pH 7.5, 5 mm MgCl2, 50 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 1% aprotinin (Trasylol, Beyer), and 1 mm dithiothreitol. Activity assays for activated, GTP-bound, Cdc42, Rac1, and RhoA were performed essentially as described before (23Edlund S. Landström M. Heldin C.-H. Aspenström P. Mol. Biol. Cell. 2002; 13: 902-914Crossref PubMed Scopus (340) Google Scholar).RESULTSNck Adapters Are Needed for the Formation of Membrane Ruffles—The two members of the Nck family of tyrosine kinase receptor substrates, Nck1 and Nck2, share the same overall primary structure; each of them has three SH3 domains followed by one SH2 domain (Fig. 1A). To study the need for Nck in PDGF-induced cytoskeletal reorganization, we used MEFs derived from mice in which Nck1 and Nck2 were ablated (KO cells) (19Bladt F. Aippersbach E. Gelkop S. Strasser G.A. Nash P. Tafuri A. Gertler F.B. Pawson T. Mol. Cell. Biol. 2003; 23: 4586-4597Crossref PubMed Scopus (142) Google Scholar). We first stimulated MEFs isolated from wild-type mice (WT cells) with PDGF-BB for the time periods depicted in Fig. 1B. Non-stimulated cells appeared spread, with well developed stress fibers. Visualization of the focal adhesions with an antibody specific for the focal adhesion component vinculin detected the presence of arrowhead-shaped focal adhesions in the non-stimulated cells (Fig. 1, B and C). PDGF-BB stimulation resulted in a rapid dissolution of the stress fibers and focal adhesions associated with a relocalization of filamentous actin into edge ruffles and dorsal ruffles (also known as circular ruffles). We found that the WT cells had rather small edge ruffles, and but the dorsal ruffles were prominent. After 10 min of PDGF-BB stimulation, 66% of the cells and after 20 min 80% of the WT cells had developed dorsal ruffles (Fig. 1, B and C). Interestingly, the KO cells behaved in a markedly different manner. The cells were smaller than the wild-type MEFs, and PDGF-BB stimulation did not result in any prominent formation of membrane ruffles, and only 9% of the cells displayed some sort of ruffling activity (Fig. 1, B and C). We also tested KO cells in which the Nck1 or Nck2 genes had been reintroduced (KO/Nck1 and KO/Nck2 rescue cells; supplemental Fig. 1A). These cells were also smaller than the wild-type MEFs, but in contrast to the KO cells, the PDGF-induced ruffling activity in the KO/Nck1 and KO/Nck2 rescue cells was partially recovered. The KO/Nck2 rescue cells displayed an almost normal membrane ruffling activity. However, the ruffles appeared to be present predominantly at the cell edges rather than on the dorsal side of the cells (Fig. 1, B and C). The ruffling activity was more transient than in the WT cells, and there was a clear drop in ruffling activity after 20 min of PDGF-BB stimulation in the rescue cell lines.To confirm the need for Nck adapters in the formation of membrane ruffles, we next treated WT cells with siRNA specific for Nck1 or Nck2 and stimulated the cells with PDGF-BB (the efficiency of Nck knock down is shown in supplemental Fig. 1B). Cells transfected with control siRNA behaved as normal WT MEFs, and 82% of the cells had developed membrane ruffles, predominantly dorsal ruffles, after 10 min of stimulation (Fig. 2, A and C). In contrast, treatment of the WT MEFs with Nck1-specific siRNA almost completely abolished the PDGF-BB responsiveness, and only 8% of the cells displayed membrane ruffles. The Nck2-specific siRNA was less effective in suppressing the PDGF-BB-induced membrane ruffles, and 38% of the cells formed membrane ruffles. Taken together, these observations suggest that the Nck adapters are critical for the formation of dorsal ruffles. Although the individual Nck adapters can partially rescue the membrane ruffling activity, the presence of both Nck paralogues is needed for a full ruffling response.FIGURE 2Knock-down of Nck adapters affect the formation of membrane ruffles. A, WT cells transfected with control siRNA were stimulated with 100 ng/ml PDGF-BB for 10 min, and the actin filament system was visualized with AlexaFluor 488-conjugated phalloidin. The bar represents 20 μm. B, WT cells transfected with siRNA specific for Nck1 or Nck2 were stimulated with 100 ng/ml PDGF-BB for 10 min, and the actin filament system was visualized as in A. Bar represents 20 μm. C, quantification of the membrane ruffling activity was performed by microscopy analysis. The values represent analyses of at least 100 cells from three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Nck Adapters Are Needed for Efficient PDGF-BB-induced Chemotaxis—We next tested the requirement for the Nck adapter proteins for PDGF-induced chemotaxis. To this end, cells were seeded in the upper chamber in the ChemoTx chemotaxis system, and PDGF-BB in concentrations ranging from 0 to 100 ng/ml was added to the lower chamber. This triggered the migration of the cells from the upper part of the filter chamber toward the gradient of growth factor in the lower chamber. The amount of cells that had migrated from the upper to the lower part of the filter after 4 h was determined by a CCD camera, as described under "Experimental Procedures." WT cells migrated efficiently to PDGF-BB; concentrations of PDGF-BB up to 50 ng/ml were increasingly efficient in inducing chemotaxis (Fig. 3A). In contrast, KO cells migrated with a reduced efficiency compared with the WT cells (Fig. 3A). The KO/Nck1 rescue cells also had a reduced chemotactic response, in particular at higher concentrations of PDGF-BB (Fig. 3A). The KO/Nck2 rescue cells displayed a normal migratory ability at lower concentrations of PDGF-BB, but at 25, 50, and 100 mg/ml PDGF-BB they migrated with significantly reduced capacity compared with the WT cells (Fig. 3A). We also tested the ability of the WT and KO cells to migrate in a tissue culture wound healing assay. The KO cells had a markedly reduced ability to close the wound compared with wild-type cells (data not shown and Ref. 19Bladt F. Aippersbach E. Gelkop S. Strasser G.A. Nash P. Tafuri A. Gertler F.B. Pawson T. Mol. Cell. Biol. 2003; 23: 4586-4597Crossref PubMed Scopus (142) Google Scholar). These observations implicate that Nck adapters are needed for an efficient PDGF-BB-dependent chemotaxis; however, the chemotactic response is not entirely ablated by the absence of Nck. Additional signaling pathways, such as the PI3K pathway, are also known to participate in the PDGF-BB-induced chemotaxis. The PI3K/Akt pathway downstream of PDGFRβ is not affected by the genetic inactivation of the Nck adaptors, as PDGF-BB stimulates the phosphorylation of Akt with the same timing and efficiency in WT and KO cells (supplemental Fig. 2A). Importantly, PAE cells stably transfected with the PDGFRβY751F had an intact ability to phosphorylate Akt in response to PDGF-BB, whereas the PDGF-BB-dependent Akt phosphorylation was entirely ablated in PAE/PDGFRβ-Y740F mutant cells (supplemental Fig. 2B). This is in agreement with the notion that Tyr-740 is the preferred binding site for the p85 subunit of PI3K (although it binds both Tyr-740 and Tyr-751), whereas Nck1 binds only to Tyr-751 (8Kazlauskas A. Kishishian A. Cooper J.A. Valius M. Mol. Cell. Biol. 1992; 12: 2534-2544Crossref PubMed Google Scholar).FIGURE 3KO cells have a reduced chemotactic ability. A, WT, KO, KO/Nck1 rescue, or KO/Nck2 rescue cells were seeded into filter chambers of the Boyden type. Chemotaxis was triggered by the addition of different amounts of PDGF-BB or 10% FBS. The reaction was terminated after 4 h, and the amount of cells that had passed across the membrane from the upper to the lower phase of the filter was determined in a plate-reader. B, KO cells were seeded into filter chambers of the Boyden type. Chemotaxis was triggered by the addition of various combinations of PDGF-BB, PDGF-AA, or FBS as depicted in the figure. The reaction was terminated after 4 h, and the amount of cells that had passed across the membrane from the upper to the lower phase of the filter was determined. Note that the arbitrary units (AU) can vary between different experiments due to differences in the color reaction induced by the incubation with Giemsa solution. Therefore, we took care to set up all conditions to be studied on the same filter to allow a true comparison of the chemotactic abilities in the different conditions. This kind of difference are normally avoided by normalizing the chemotactic against the migration in 10% FBS. This could not be done in the present study, as serum strongly interfered with the chemotaxis of the KO and rescue cell lines.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Serum Abolishes Residual PDGF-induced Chemotaxis in Cells Lacking Nck Adapters—Interestingly, although the addition of 10% serum efficiently induced chemotaxis of WT cells, neither the KO, the KO/Nck1 rescue, nor the KO/Nck2 rescue cells moved at all in the presence of 10% FBS (Fig. 3A). This was a surprising observation, as FBS normally constitutes an efficient chemotactic cue. We next tested if the inability of the KO cells to move in the presence of serum was caused by an inability of serum constituents to trigger migration of the KO cells or if factors in the serum actively blocked migration. To this end increasing amounts of FBS were added in the presence of 50 ng/ml PDGF-BB. Whereas 1% of serum had a mild effect on the PDGF-BB-induced chemotaxis, 5 and 10% serum efficiently blocked the PDGF-BB-induced migration of the KO cells. PDGF-AA did not induce chemotaxis in the KO cells or in the WT cells, which is consistent with the described inability of the PDGFRα to induce chemotaxis (Fig. 3B) (1Rönnstrand L. Heldin C.-H. Int. J. Cancer. 2001; 91: 757-762Crossref PubMed Scopus (130) Google Scholar). No blocking effect of FBS could be seen in WT cells (data not shown). These findings strongly suggest that serum constituents actively block migration of the KO cells.Cells Lacking Nck Adapters Have a Reduced Ability to Form Focal Adhesions—To gain insights into why the addition of FBS inhibited the chemotactic and migratory properties of the KO cells, we stimulated cells with 10% FBS. The wild-type cells had not entirely lost the stress fibers and focal adhesions upon serum starvation; however, a marked increase in the amount of stress-fibers and focal adhesions could be noticed after 10 min of stimulation, and the size of the focal adhesions increased from 1.4 to 1.9 and 1.8 μm2 after 10 and 20 min, respectively (Fig. 4, A and B). In contrast, although the KO cells had some focal adhesions, they were fewer and significantly smaller than in the WT cells (1.1 μm2), which did increase to 1.3 μm2 after 20 min, but the size was still significantly smaller than the WT focal adhesions (Fig. 4, A and B). The response in KO/Nck1 rescue cells very much resembled the one in KO cells with a reduced formation of focal adhesions (Fig. 4, A and B). In contrast, the focal adhesions in the KO/Nck2 rescue cells were not smaller than the WT cells, but they did not increase in size upon FBS treatment (Fig. 4, A and B).FIGURE 4Nck adapters are needed for the assembly of focal adhesions. A, WT, KO, KO/Nck1 rescue and KO/Nck2 rescue cells were serum-starved for 16 h and then stimulated with 10% FBS for different time periods. Filamentous actin was detected by fluorescein isothiocyanate-conjugated phalloidin. Vinculin was detected by a mouse anti-vinculin antibody followed by a TRITC-conjugated anti-mouse antibody. The bar represents 20 μm. B, quantification of the area of the focal adhesions. Fifteen micrographs of each condition were generated by microscopy analysis. The pictures were taken of randomly chosen areas of the slides. The mean area of the focal adhesions on each micrograph was determined using the ImageJ software and the Analyze Particles function. The statistics analysis was carried out using Student's t test. The *, p < 0.05; **, p < 0.01; ***, represent p < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To confirm the need for Nck adapters in the formation of focal adhesions, we treated WT cells with siRNA specific for Nck1 or Nck2 and stimulated the cells with 10% FBS for 20 min. The focal adhesions were again visualized with a vinculin-specific antibody, and mean area of the focal adhesions was measured before and after FBS treatment (Fig. 5A). In control siRNA-treated cells the mean area increased from 0.9 to 1.0 μm2, which is a significant increase in the surface area (Fig. 5, A and C). The apparent size of the focal adhesions are generally smaller under this experimental conditions, as the cells are allowed to attached for shorter periods and are kept in the absence of serum for most of the transfection procedure. The surface area of the focal adhesions in the Nck1- and Nck2-specific siRNA-treated cells were in the same range as in the control siRNA-treated cells (0.9 and 0.8 μm2, respectively), but importantly, the surface area of the focal adhesions did not