Requirement of Nck adaptors for actin dynamics and cell migration stimulated by platelet-derived growth factor B

G. M. Rivera*, S. Antoku*, S. Gelkop†, N. Y. Shin‡, S. K. Hanks‡, T. Pawson†§, and B. J. Mayer*§

*Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Developmental Biology and Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, CT 06030; †Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5; and ‡Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232

Contributed by T. Pawson, May 8, 2006 The Nck family of Src homology (SH) 2͞SH3 domain adaptors been shown to play an important role during embryogenesis functions to link tyrosine phosphorylation induced by extracellular potentially linked to cell motility and cytoskeletal organization (7). signals with downstream regulators of actin dynamics. We inves- Although circumstantial evidence suggests that the Nck adaptors tigated the role of mammalian Nck adaptors in signaling from the can interact with the activated PDGF␤R (8–10), their role in activated platelet-derived growth factor (PDGF) receptor (PDGF␤R) signaling to the actin cytoskeleton downstream of this receptor to the actin cytoskeleton. We report here that Nck adaptors are remains largely unknown. Here we report that Nck adaptors are required for cytoskeletal reorganization and chemotaxis stimu- strictly required for cytoskeletal reorganization and chemotaxis lated by PDGF-B. Analysis of tyrosine-phosphorylated proteins stimulated by PDGF-B. Furthermore, we provide mechanistic demonstrated that Crk-associated substrate (p130Cas), not the insights suggesting that Nck adaptors transduce signals downstream activated PDGF␤R itself, is the major Nck SH2 domain-binding of the activated PDGF␤R by an indirect mechanism involving the protein in PDGF-B-stimulated cells. Both Nck- and p130Cas-deficient scaffolding protein p130Cas. cells fail to display cytoskeletal rearrangements, including the formation of membrane ruffles and the disassembly of actin Results bundles, typically shown by their WT counterparts in response to Nck Adaptors Are Required for Cytoskeletal Changes Induced by PDGF-B. Furthermore, Nck and p130Cas colocalize in phosphoty- PDGF-B. To determine the significance of Nck adaptors in cytoskel- rosine-enriched membrane ruffles induced by PDGF-B in NIH 3T3 etal changes induced by PDGF-B, serum-starved, Nck-deficient cells. These results suggest that Nck adaptors play an essential role (both Nck genes inactivated; dKO), and WT mouse embryonic in linking the activated PDGF␤R with actin dynamics through a fibroblasts (MEFs) were left untreated or were stimulated with pathway that involves p130Cas. PDGF-B. Profound remodeling of the cytoskeleton, including the disassembly of actin bundles and the formation of various types of Crk-associated substrate ͉ actin cytoskeleton ͉ SH2 domain ͉ SH3 domain ͉ membrane protrusions, occurred in WT but not dKO cells after tyrosine phosphorylation PDGF-B stimulation (Fig. 1A and Fig. 7A, which is published as supporting information on the PNAS web site). Occurrence of ell motility is critically important in developmental processes ‘‘peripheral’’ or ‘‘dorsal’’ ruffles (11) was compared between the Cand in the pathogenesis of a variety of diseases. Remodeling of genotypes. Whereas dKO cells remained mostly in a ‘‘quiescent’’ the actin cytoskeleton, i.e., the dynamic assembly and disassembly state, the WT cells, in contrast, showed a dramatic increase in of filamentous actin, governs essential aspects of cell motility such peripheral and dorsal ruffling after PDGF-B stimulation (Fig. 7B). These observations suggested a critical role for Nck adaptors in as the formation of membrane protrusions and cell adhesion to ␤ other cells or to the substrate (1). Extracellular signals can induce transducing signals from the activated PDGF R to the actin remodeling of the actin cytoskeleton through changes in tyrosine cytoskeleton. phosphorylation. For example, ligand-induced dimerization of platelet-derived growth factor (PDGF) receptors (PDGF␤R) stim- Expression of Nck Rescues the Response to PDGF-B in Nck-Deficient ulates their intrinsic kinase activity, leading to the autophosphor- Cells. Given the striking contrast in the response to PDGF-B stimulation of WT vs. dKO cells, we next tested whether the ectopic ylation in trans of multiple intracellular tyrosine residues on the expression of Nck1 or Nck2 in dKO cells could rescue the respon- receptor (2, 3). This event results in the creation of phosphotyrosine siveness to PDGF-B. Cells were cotransfected with actin-GFP to docking sites for Src homology (SH) 2 domain-containing signaling visualize cytoskeletal changes and a vector expressing Nck1, Nck2, molecules (4). Cellular effects mediated by signaling pathways a loss-of-function mutant Nck1 (with inactivating mutation of all activated by PDGF␤R involve proliferation, survival, actin reorga- three SH3 domains; Kall), or empty vector (EBB). No major nization, and migration (3). Despite extensive efforts aimed at cytoskeletal changes were observed after PDGF-B stimulation in characterizing effectors that associate with PDGF␤R (2), the serum-starved dKO cells cotransfected with a loss-of-function dynamic assembly of signaling complexes induced by the activation mutant Nck1 (Fig. 1B,Kall) or with the empty vector (EBB in Fig. of this receptor and their relation to specific cellular responses are 8A, which is published as supporting information on the PNAS web still poorly understood. site). In contrast, actin bundles disassembled and peripheral and Nck, a two-gene family in mammals, is an important link in transducing signals from tyrosine phosphorylation to the cytoskel- ␣ ␤ eton (5, 6). Nck proteins (termed Nck, Nck or Nck1, and Nck or Conflict of interest statement: No conflicts declared. Nck2) consist of three N-terminal SH3 domains followed by a single Freely available online through the PNAS open access option. C-terminal SH2 domain. The SH2 domain of Nck can bind a Abbreviations: PDGF, platelet-derived growth factor; PDGF␤R, PDGF receptor; SH, Src number of activated receptor tyrosine kinases and tyrosine- homology; MEF, mouse embryonic fibroblast; PI3K, phosphatidylinositol 3-kinase; Fak, phosphorylated docking proteins; on the other hand, the Nck SH3 focal adhesion kinase. domains engage proline-rich binding sites on a host of effectors §To whom correspondence may be addressed. E-mail: [email protected] or bmayer@ implicated in cytoskeletal regulation, including members of the neuron.uchc.edu. WASp͞Scar family (reviewed in refs. 5 and 6). Nck adaptors have © 2006 by The National Academy of Sciences of the USA

9536–9541 ͉ PNAS ͉ June 20, 2006 ͉ vol. 103 ͉ no. 25 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603786103 Downloaded by guest on September 28, 2021 Fig. 2. Nck deficiency impairs PDGF-B- and fibronectin-stimulated cell mi- gration. Data show the number of migrating cells per microscopic field. Bars represent means Ϯ SD from eight pictures taken at random from each treat- ment combination in each of two independent experiments. Genotypes com- pared are WT, Nck1Ϫ/Ϫ Nck2Ϫ/ϩ (Nck2), Nck1Ϫ/ϩ Nck2Ϫ/Ϫ (Nck1), NckϪ/Ϫ Nck2Ϫ/Ϫ (dKO), and Nck-deficient cells rescued with an empty retroviral vector (IRES) or a retroviral vector expressing Nck (IRES͞Nck1). Within each genotype, means with a different letter over the bar are different (P Ͻ 0.01).

tional transwell migration assay. Nck-deficient (dKO) cells had a readily apparent migration disadvantage compared with their WT counterparts (Fig. 9, which is published as supporting information on the PNAS web site). As shown in Fig. 2, more WT MEFs migrated in the presence of PDGF-B, fibronectin, or both than in starvation medium alone (P Ͻ 0.001). In contrast, the lack of either Nck1 or Nck2 abrogated the chemotactic response to PDGF (P Ͼ 0.05). The haptotactic response to fibronectin was severely com- promised in MEFs with inactivation of both Nck genes (dKO and IRES). However, more (P Ͻ 0.001) MEFs with only one of the two Nck genes migrated in the presence of fibronectin than in the

presence of medium alone. Interestingly, Nck-deficient MEFs CELL BIOLOGY rescued with a retrovirus expressing Nck (expression levels Ϸ3-fold higher than in WT cells) showed a slight, but significant (P Ͻ 0.01), Fig. 1. Requirement of Nck in actin rearrangements induced by PDGF-B. increase in migration in response to PDGF and͞or fibronectin Shown are confocal images of serum-starved MEFs left untreated (time 0) or compared with cells exposed to medium alone. These results stimulated with PDGF-B for 10 min. (A) Actin rearrangements in Nck-deficient strongly suggest that both Nck genes are required for normal (dKO) and WT cells fixed and stained with Texas red phalloidin and Hoechst directed cell motility and that Nck1 and Nck2 are at least partially 33342 to visualize the actin cytoskeleton and nuclei, respectively. (B) Cytoskel- redundant. etal rearrangements in Nck-deficient cells coexpressing actin-GFP and WT (Nck1) or a loss-of-function mutant Nck (Kall). Arrows and arrowheads indi- cate dorsal and peripheral ruffles, respectively. (Scale bars: 25 ␮m.) Profile of Tyrosine Phosphorylation in Response to Activation of PDGF␤R. To begin to understand the mechanism by which Nck adaptors link signaling from the activated PDGF␤R to the cytoskel- dorsal ruffles formed in cells transfected with either Nck1 (Fig. 1B) eton, we examined tyrosine-phosphorylated proteins in cell lysates or Nck2 (Fig. 8 A and B) after PDGF-B stimulation. To further evaluate the role of Nck in cytoskeletal dynamics induced by obtained from serum-starved NIH 3T3 cells left untreated or PDGF-B, we performed live-cell imaging of Nck-deficient cells stimulated with PDGF-B. Western blot analysis using an antibody cotransfected with actin-GFP and either empty vector or Nck1. As against phosphotyrosine (Fig. 3, pY) showed that three major shown in Movie 1, which is published as supporting information on proteins of Ϸ185, 130, and 65 kDa underwent changes in their the PNAS web site, dKO cotransfected with the empty vector phosphotyrosine levels in association with PDGF-B stimulation. (EBB) showed a very stable cytoskeleton. Conversely, cells cotrans- We also determined the pattern of SH2-binding proteins by far- fected with Nck1 exhibited formation of very dynamic peripheral Western blot analysis using GST fusions of the isolated SH2 domain (Movie 2, which is published as supporting information on the from Nck1, Nck2, or phosphatidylinositol 3-kinase (PI3K) or GST PNAS web site) and dorsal (Movie 3, which is published as alone as a control (Fig. 3). A band of Ϸ185 kDa, the PDGF␤R, was supporting information on the PNAS web site) membrane ruffles clearly observed in pY and PI3K blots soon after PDGF-B stimu- after PDGF-B stimulation. Thus, these results demonstrate that lation but was undetectable in lysates of cells left untreated. either Nck1 or Nck2 is required for PDGF-B-stimulated actin ␤ Noticeably, this band was absent in filters probed with SH2 domains rearrangements. Importantly, phosphorylation of the PDGF R from Nck1 and Nck2, indicating that the activated PDGF␤Risnot and activation of p42͞44Erk, cell proliferation-related signaling efficiently bound directly by Nck adaptors. In contrast, a single molecules, was not affected in the absence of Nck adaptors (see Ϸ below). Actin dynamics is the cellular function most profoundly major band of 130 kDa was consistently detected in membranes altered in the absence of Nck adaptors. probed with Nck1 and Nck2 SH2 domains; this band was not seen in the PI3K SH2 blot. The Ϸ130-kDa band showed low but Lack of Nck Adaptors Leads to Impaired Chemotactic and Haptotactic detectable levels of tyrosine phosphorylation in lysates from serum- Responses. We took advantage of the availability of MEFs of various starved, unstimulated cells and a modest (Ϸ3- to 5-fold) increase in genotypes and compared their ability to migrate using a conven- lysates obtained soon after PDGF-B stimulation. Interestingly, the

Rivera et al. PNAS ͉ June 20, 2006 ͉ vol. 103 ͉ no. 25 ͉ 9537 Downloaded by guest on September 28, 2021 Fig. 3. Pattern of tyrosine phosphorylation in response to activation of PDGF␤R. Lysates from NIH 3T3 cells left untreated (time 0) or stimulated with PDGF-B for various intervals were transferred to membranes and probed with anti-pTyr antibody (pY) or subjected to far-Western blot analysis using GST fusions of the Ϫ3 isolated SH2 domain from Nck1 (Nck1), Nck2 (Nck2), or PI3K p85 subunit and GST alone as a control. Apparent molecular weights (Mr ϫ 10 ) of standards are indicated. Arrows indicate a band corresponding to the activated PDGF␤R, and arrowheads indicate a band of Ϸ130 kDa in size that binds Nck SH2 domains. Similar kinetics of tyrosine phosphorylation were observed in three independent experiments.

Ϸ130-kDa band exhibited a dynamic, biphasic pattern of phosphor- on their molecular weights and previous work suggesting possible ylation, with a rapid increase soon after PDGF-B stimulation (2–5 association with Nck (12, 13). Lysates from serum-starved NIH 3T3 min) followed by an equally rapid and sustained decrease thereafter cells were subjected to immunoprecipitation with polyclonal anti- to a level lower than that in starved cells (Figs. 3 and 4 and Figs. 10 p130Cas (Fig. 4) or anti-Fak antibodies (Fig. 10). Immunoprecipi- and 11, which are published as supporting information on the PNAS tation with anti-Cas almost completely cleared the Ϸ130-kDa Nck web site). Because the Ϸ130-kDa Nck-interacting protein is likely SH2-binding protein from the lysates. In contrast, only a minor Ϸ to mediate Nck recruitment to sites of actin rearrangements, we fraction of the 130-kDa protein was cleared from the lysates by sought to determine its identity in subsequent experiments. immunoprecipitation with anti-Fak, and this fraction did not bind Nck SH2 domains (Fig. 10). Western blot analysis of the same p130Cas Is the Major Tyrosine-Phosphorylated Protein That Binds Nck IP:Cas (Fig. 4) and IP:Fak (Fig. 10) filters (IP, immunoprecipita- Cas in PDGF-B-Stimulated Cells. We investigated the identity of the tion) stripped and reprobed with specific antibodies against p130 Ϸ130-kDa Nck SH2-binding protein by immunoprecipitation. Fo- (Cas) and Fak (Fak), respectively, demonstrated the specificity of the immunoprecipitation. cal adhesion kinase (Fak) and p130Cas were likely candidates based The identity of the Ϸ130-kDa Nck SH2 domain-binding protein as p130Cas was confirmed by comparing the pattern of tyrosine- phosphorylated proteins in Cas-deficient and WT MEFs or NIH 3T3 cells. As shown in Fig. 11, tyrosine phosphorylation of the Ϸ130-kDa Nck SH2 domain-binding protein was absent in samples from Cas-deficient cells, whereas it increased after PDGF-B stim- ulation in cells containing p130Cas. Taken together, these results show unambiguously that p130Cas is the major tyrosine- phosphorylated protein that can bind Nck SH2 domains in cells stimulated with PDGF-B.

p130Cas Is Required for Cytoskeletal Changes Induced by PDGF-B. The striking finding that p130Cas is the major Nck SH2 domain-binding protein in fibroblasts stimulated with PDGF-B led us to test the hypothesis that cells lacking p130Cas, or expressing p130Cas lacking the Nck SH2 binding sites, would fail to undergo cytoskeletal rearrangements in response to PDGF-B. Accordingly, we com- pared the PDGF-responsiveness of WT vs. p130Cas-deficient MEFs or p130Cas-deficient MEFs rescued with a retroviral vector express- ing either WT p130Cas or a phosphorylation-defective mutant (Y3F substitutions of the 15 YXXP tyrosine residues of the Fig. 4. p130Cas is the major tyrosine-phosphorylated protein that binds Nck substrate domain, which include the Nck SH2 binding sites; ref. 14). in cells stimulated with PDGF-B. Lysates obtained from NIH 3T3 cells left After PDGF-B stimulation, major cytoskeletal changes, including untreated (time 0) or stimulated with PDGF-B for various intervals were the formation of membrane protrusions, were readily apparent in immunoprecipitated with polyclonal antibodies against p130Cas. Whole-cell WT and p130Cas-deficient MEFs reexpressing WT p130Cas (Fig. 5). lysates (W) and supernatant (S) and pellet (P) fractions were subjected to In contrast, the formation of membrane protrusions induced by SDS͞PAGE, and proteins were transferred to nitrocellulose filters. Pellet frac- PDGF-B was severely compromised in p130Cas-deficient and tions represent five times more lysate than whole-cell lysate or supernatant p130Cas-deficient cells reexpressing the phosphorylation-defective fractions. Membranes were probed with GST fusions of the isolated SH2 p130Cas mutant, although a few cells displayed small foci of actin on domain from Nck1 (SH2) in a far-Western blot or with monoclonal anti- phosphotyrosine (pY) or anti-p130Cas (Cas) antibodies in Western blot analysis. the dorsal surface resembling disorganized dorsal ruffles. Quanti- Cas Ϫ3 Ϸ Apparent molecular weights (Mr ϫ 10 ) are indicated. Arrowheads indicate tative analysis showed that 50% of the WT and p130 -deficient Cas a band of Ϸ130 kDa in size corresponding to p130Cas, and the arrow indicates MEFs reexpressing WT p130 formed readily distinguishable the PDGF␤R. dorsal ruffles in response to PDGF-B stimulation whereas only

9538 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603786103 Rivera et al. Downloaded by guest on September 28, 2021 CELL BIOLOGY

Fig. 6. Recruitment of endogenous Nck and p130Cas to membrane ruffles Fig. 5. p130Cas is required for membrane ruffling induced by PDGF-B stim- induced by PDGF-B. Shown are confocal images of serum-starved NIH 3T3 ulation. Shown are confocal images of WT, p130Cas-deficient (Ϫ͞Ϫ), and fibroblasts left untreated (Starvation) or stimulated with PDGF for 10 min p130Cas-deficient MEFs reexpressing either WT (Ϫ͞Ϫ WT) or a phosphoryla- (PDGF-B). Fixed cells were stained with Texas red phalloidin and anti-Nck and tion-deficient mutant (Ϫ͞Ϫ F1–15) p130Cas. Cells were left untreated (Starva- anti-p130Cas antibodies. In the merged images, filamentous actin, Nck, and tion) or were stimulated with 30 ng͞ml PDGF-B. Arrows indicate dorsal ruffles. p130Cas are colored red, green, and blue, respectively. Arrows indicate dorsal (Scale bar: 25 ␮m.) ruffles. (Scale bar: 25 ␮m.)

Ϸ10–15% of the p130Cas-deficient and p130Cas-deficient cells re- Cas bution of tyrosine-phosphorylated proteins and Nck in relation to expressing the phosphorylation-defective p130 mutant showed F-actin structures in NIH 3T3 cells. We found that both endogenous actin foci on the dorsal surface (Fig. 12, which is published as p130Cas and Nck colocalized to actin structures induced by PDGF-B supporting information on the PNAS web site). stimulation in NIH 3T3 fibroblasts (Fig. 6). In Nck-deficient and NIH 3T3 cells, respectively, ectopically expressed (Fig. 13C) and Nck and p130Cas Colocalize in Phosphotyrosine-Enriched Membrane endogenous (Fig. 14B, which is published as supporting information Ruffles Induced by PDGF-B. We first investigated the effects of on the PNAS web site) Nck displayed a diffuse cytoplasmic PDGF-B stimulation on the subcellular distribution of GFP fusions distribution under serum starvation and localized strongly to of either full-length Nck1 or its isolated SH2 domain in Nck- PDGF-B-induced membrane ruffles. In serum-starved NIH 3T3 deficient MEFs or NIH 3T3 cells. In Nck-deficient cells, full-length cells, tyrosine-phosphorylated proteins accumulated in foci at the Nck1-GFP was recruited to structures resembling peripheral and cell periphery, presumably sites of cell-substrate adhesion that dorsal ruffles (Fig. 13A Upper, which is published as supporting coincided with tips of actin bundles. In sharp contrast, tyrosine- information on the PNAS web site). Consistent with the failure of phosphorylated proteins accumulated at the edges of membrane the Nck SH3 domain mutant (Fig. 1B,Kall) to rescue the response ruffles in PDGF-B-stimulated cells, and, consistent with the dis- to PDGF-B, no structures resembling peripheral or dorsal ruffles assembly of actin bundles, the foci of phosphotyrosine proteins at the cell periphery were no longer observed (Fig. 14A). Taken were detected in dKO cells transfected with Nck1 SH2-GFP after together, these results suggest that PDGF-stimulated accumulation PDGF-B stimulation (Fig. 13A Lower). In contrast, the GFP-Nck1 of tyrosine-phosphorylated proteins recruits Nck to the membrane SH2 domain fusion localized strongly to membrane ruffles induced through SH2 domain-mediated interactions, where it colocalizes by PDGF-B in NIH 3T3 cells where endogenous Nck is present with p130Cas at sites of active actin polymerization. (Fig. 13B). This observation suggested that Nck recruitment de- pends on SH2-domain-mediated interactions with tyrosine- Actin Dynamics Is the Cellular Function Most Profoundly Altered in the phosphorylated proteins enriched at sites of actin polymerization. Absence of Nck Adaptors. To analyze whether other aspects of Next we analyzed by immunofluorescence the subcellular distri- PDGF␤R signaling were affected in the absence of Nck adaptors,

Rivera et al. PNAS ͉ June 20, 2006 ͉ vol. 103 ͉ no. 25 ͉ 9539 Downloaded by guest on September 28, 2021 we compared the overall patterns of tyrosine phosphorylation, GST-Nck pull-down fractions was not investigated, and thus the p42͞44Erk activation, and p130Cas phosphorylation of Nck- possibility of an indirect interaction was not ruled out. deficient vs. Nck-deficient cells rescued with Nck1 (Fig. 15, which Crk-associated substrate (p130Cas) is implicated in cytoskeletal is published as supporting information on the PNAS web site). The regulation and cell migration, forms a complex with cell-matrix overall pattern of tyrosine phosphorylation did not differ signifi- adhesion-associated proteins, and is phosphorylated upon integrin cantly between dKO and IRES͞Nck1 cells. Importantly, the ex- receptor engagement (17). Integrin receptor clustering by fibronec- pression and functionality of PDGF␤R are not affected in cells tin promoted p130Cas phosphorylation via Fak͞Src and the forma- lacking Nck adaptors, as evidenced by similar patterns of PDGF␤R tion of a complex between phosphorylated p130Cas and Nck (12). autophosphorylation in dKO and IRES͞Nck1 cells (pY blot). Consistent with the PDGF-B-stimulated phosphorylation of Furthermore, PDGF-B-dependent activation of the cell prolifera- p130Cas previously reported (18), our results point to a complex tion-related signaling molecules p42͞44Erk did not differ between between p130Cas and Nck as a critical node in the signaling network dKO and IRES͞Nck1 cells. linking the activated PDGF␤R with the actin cytoskeleton. Several Tyrosine phosphorylation of proteins in the Ϸ120- to 130-kDa converging observations lend support to this notion: (i) the rapid range showed similar basal levels under starvation conditions and increase (3- to 5-fold) in phosphorylation of p130Cas after stimu- an Ϸ3- to 5-fold increase after PDGF-B stimulation (Fig. 15). In lation with PDGF-B; (ii) the change in subcellular distribution of IRES͞Nck1 cells, tyrosine phosphorylation of these proteins fol- endogenous p130Cas and Nck from cytoplasmic͞focal adhesion- lowed a biphasic pattern that resembled that observed in WT NIH associated in quiescent cells to the phosphotyrosine-enriched mem- 3T3 cells. Interestingly, in cells lacking Nck adaptors (dKO cells), brane ruffles induced by PDGF-B; (iii) the preferential binding of phosphorylation of proteins in the Ϸ120- to 130-kDa range ap- Nck SH2 domain to p130Cas in far-Western blot analysis; (iv) the peared to increase linearly after PDGF-B stimulation (i.e., down- strikingly similar cytoskeletal phenotypes, i.e., the absence of actin regulation was not apparent in this time scale). Last, the phosphor- rearrangements after PDGF-B stimulation, in Nck- and p130Cas- ylation of p130Cas in response to PDGF-B increased to the same deficient cells; and (v) the inability of a phosphorylation-defective extent, as detected by far-Western blotting with SH2 domains from mutant of p130Cas (which cannot bind Nck SH2 domains) to rescue Nck, in dKO and IRES͞Nck1 cells (Fig. 15, SH2 blot). Taken the response to PDGF-B in p130Cas-deficient cells. Interestingly, together, these observations suggest that actin dynamics, but not unpublished work from our laboratory shows that specific down- other aspects of PDGF signaling, is the cellular function most regulation by small interfering RNA interference of Crk adaptors profoundly altered in the absence of Nck adaptors. (Crk and Crk L), strong binding partners of p130Cas, does not alter membrane ruffling induced by PDGF-B stimulation. This finding is Discussion consistent with our observation of increased phosphorylation of Using a combination of genetics, cell biology, and biochemistry we Crk II and L, which renders them in an inactive conformation, after have uncovered an essential role of Nck adaptors in linking tyrosine PDGF-B stimulation. Thus, the above observations suggest that a phosphorylation induced by activation of the PDGF␤R, remodel- molecular complex involving p130Cas and Nck, but not Crk adap- ing of the actin cytoskeleton, and directed cell motility. Further- tors, is a critical link in signaling from the activated PDGF␤Rtothe more, our studies provide mechanistic insights suggesting that Nck actin cytoskeleton. Future studies will be necessary to elucidate adaptors transduce signals to the cytoskeleton downstream of the the molecular mechanism linking the activated PDGF␤Rwiththe PDGF␤R through a signaling pathway that involves p130Cas. formation of a complex between p130Cas and Nck, including the Inactivation of both Nck1 and Nck2 results in profound defects mechanisms of p130Cas phosphorylation and relocalization. in development of mesoderm-derived tissues and the formation of p130Cas exhibited a biphasic pattern of phosphorylation, with a the lamellipodial actin meshwork leading to embryonic lethality (7). rapid increase soon after PDGF-B stimulation (2–5 min) followed Here we report that MEFs lacking functional Nck proteins fail to by an equally rapid and sustained decrease thereafter. This obser- show the typical cytoskeletal changes observed in their WT coun- vation suggests the existence of a limiting or ‘‘off’’ mechanism, terparts in response to PDGF-B stimulation and have seriously presumably mediated by a protein tyrosine phosphatase, that could compromised directional motility. Importantly, the responsiveness be important in fine-tuning actin rearrangements induced by to PDGF-B in Nck-deficient cells was rescued by ectopic expression PDGF-B. Further studies will be necessary to uncover the molec- of WT Nck1 or Nck2 but not loss-of-function mutants. These results ular mechanisms underlying the dynamics of p130Cas phosphory- are entirely consistent with an essential role of Nck adaptors in lation and its physiological consequences in the context of promoting the dynamic assembly of actin in response to tyrosine PDGF␤R activation. Using a very sensitive probe, such as the SH2 kinase signals and are in agreement with previous studies demon- domains of the regulatory subunit of PI3K, we could detect strating that the Nck͞Dock adapter protein is required in photo- significant autophosphorylation of the PDGF␤R within 1 min after receptor axon guidance and target recognition in the Drosophila PDGF-B stimulation (Fig. 3). We recently found a similarly rapid visual system (reviewed in ref. 15). increase in the GTP loading of Rac (G.M.R. and B.J.M, unpub- One of the potential mechanisms by which Nck adaptors could lished data), and major cytoskeletal rearrangements were seen soon couple to the PDGF␤R is by direct binding to autophosphorylated after (3.5 min) PDGF-B stimulation (Figs. 7A and 8A). During T docking sites induced on the receptor upon ligand-mediated acti- cell receptor activation, a rapid increase in tyrosine phosphoryla- vation. Indeed, it has been shown that Nck1 (8) and Nck 2 (10) can tion recruits Nck into a signaling complex that subsequently mi- bind in vitro, through SH2 domain-mediated interactions, to phos- grates peripherally and accumulates at a ring-shaped actin-rich photyrosine residues (Tyr-751 and Tyr-1009, respectively) in the structure (19). It is likely that highly dynamic processes similar to ligand-activated PDGF␤R. Our data, however, support a model in those occurring during T cell receptor engagement also occur which Nck proteins could couple to PDGF␤R through an indirect during activation of the PDGF␤R. Our data are consistent with a mechanism. We used an SH2 domain-based far-Western blot model in which p130Cas serves to recruit Nck to the membrane to analysis (16) to determine Nck SH2 domain-binding partners in initiate actin polymerization and that these complexes are remod- lysates obtained from serum-starved and PDGF-B-stimulated cells. eled over time as additional proteins are recruited and͞or phos- SH2 domains from both Nck1 and Nck2 consistently detected a phorylated. single band of Ϸ130 kDa that was unambiguously identified as Nck can signal to the actin cytoskeleton by interaction of its SH3 p130Cas.Incontrast,anϷ190-kDa species, presumably the activated domains with a variety of downstream effectors. PAK-1 (p21- PDGF␤R, was readily detected by anti-pY and PI3K SH2 domains activated kinase 1) is recruited by the middle SH3 domain of Nck but not Nck SH2 domains. In previous studies (8) the presence of (20, 21), and Nck-mediated targeting of PAK to the plasma tyrosine-phosphorylated proteins in addition to the PDGF␤Rin membrane is sufficient for its activation by members of the Rho

9540 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603786103 Rivera et al. Downloaded by guest on September 28, 2021 family of GTPases (22). Under these conditions PAK may also assay (Boyden chamber) as detailed in Supporting Text, which is serve as an adaptor to recruit guanine nucleotide exchange factors published as supporting information on the PNAS web site. for Rho GTPases (23) Importantly, activation of PDGF␤R leads to Nck-mediated recruitment of PAK-1 to dorsal ruffles and to the Immunofluorescence and Confocal Microscopy. Immunofluorescence edges of lamellipodia (24). Alternatively, Nck adaptors could was performed as previously described (25), and further details are stimulate actin polymerization through interactions with members provided in Supporting Text. The affinity-purified polyclonal anti- of the WASp͞WAVE family and their binding partners. We have Nck antibody was raised in rabbits immunized with a GST fusion shown recently that an increased local concentration of membrane- of full-length human Nck1. The anti-phosphotyrosine antibody was from Cell Signaling Technology (P-Tyr-100, catalog no. 9411), and targeted Nck SH3 domains is sufficient to trigger localized actin the polyclonal anti-Fak antibody was from Santa Cruz Biotechnol- polymerization through a pathway that requires N-WASp (25). ogy (C-20, sc-558). The monoclonal anti-Cas (6G11) antibody was N-WASp localizes to several actin structures, including podosomes, described previously (30). In some experiments, cells were stained invadopodia, and circular dorsal ruffles (26). Interestingly, inva- with Texas red phalloidin (Molecular Probes) and Hoechst 33342 dopodium formation in metastatic carcinoma cells downstream of dye to visualize the actin cytoskeleton and nuclei, respectively. epidermal growth factor receptor requires Nck-mediated recruit- Fluorescent images were collected on a Zeiss LSM 510 confocal ment of the N-WASp-Arp2͞3 complex (27). Also, Nck adaptors microscope with a ϫ63 NA 1.25 Plan-NEOFLUAR oil-immersion have been implicated in activation of WAVE proteins (28). Further objective. Live images were obtained on a Nikon TE2000 inverted studies will be necessary to clarify the role of Nck adaptors in microscope with a 60 ϫ 1.4 NA oil-immersion objective. Additional activation and targeting of the various downstream effectors upon details are provided in Supporting Text. PDGF stimulation. In summary, our results demonstrate that Nck adaptors are Pull-Down Assay, Immunoprecipitation, Western Immunoblotting, and strictly required for cytoskeletal reorganization and chemotaxis Far-Western Blot Analysis. Serum-starved or PDGF-B-treated cells stimulated by PDGF-B. Furthermore, these studies provide mech- were harvested, and lysates were subjected to immunoprecipitation anistic insights suggesting that Nck adaptors transduce signals to the with polyclonal anti-Fak or anti-Cas (Cas-B) antibodies (30). Pro- cytoskeleton downstream of the PDGF␤R through a signaling cedural details are described in Supporting Text. Pull-down assays pathway that involves p130Cas. The Nck family of SH2͞SH3 domain were performed with glutathione-Sepharose beads precomplexed with GST-Nck SH2 domains. Western immunoblotting was carried adaptors may constitute an important target of intervention in ␤ out by using a monoclonal anti-phosphotyrosine antibody (dilution diseases concurring with dysregulated signaling from the PDGF R, 1:5,000), a monoclonal anti-Cas antibody (dilution 1:1,000), or a altered actin dynamics, and aberrant cell migration. polyclonal anti-Fak antibody (dilution 1:1,000). We used GST fusions of Nck1, Nck2, and PI3K SH2 domains in a far-Western blot Materials and Methods analysis as previously described (16). A detailed description of these Cell Culture, Transfections, PDGF-B Stimulation, and Migration Assay. procedures is provided in Supporting Text. NIH 3T3 cells and MEF lacking Nck (7) or p130Cas (29) were cultured in DMEM supplemented with antibiotics and 10% calf We thank Amy Bouton (University of Virginia School of Medicine, serum or 10% FBS, respectively. Transient transfections were Charlottesville) for reagents including anti-Cas antibodies and for crit- carried out by using the Lipofectamine reagent according to ically reading the manuscript. We are grateful to Drs. A. Cowan and W. CELL BIOLOGY instructions provided by the manufacturer. Cells were serum- Mohler for expert advice in imaging techniques and Dr. Kazuya Machida for helping with the far-Western blot analysis. This work was supported starved (DMEM plus 0.1% FBS) for 24 h before stimulation with by National Institutes of Health Grant CA82258 (to B.J.M.) and a 30 ng͞ml PDGF-BB (Upstate Biotechnology). Migration assay of postdoctoral fellowship from the American Heart Association (to serum-starved MEFs was performed by using a transwell migration G.M.R.).

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