Nckβ Adapter Regulates Actin Polymerization in NIH 3T3 Fibroblasts in Response to Platelet-Derived Growth Factor bb

Src homology (SH) domains, including SH2 and SH3 domains, are protein modules found in many otherwise functionally distinct molecules (25). The ligands for SH2 and SH3 domains are phosphotyrosine-containing peptides (pY-X-X-X) and proline-rich peptides (P-X-X-P), respectively. The C-terminal amino acid residues of the phosphotyrosines and the flanking amino acid residues of proline-rich segments determine binding affinity and specificity (26, 35). A family of SH2 and SH3 domain-containing proteins, including Crk, Grb2, and Nck, contain only SH2 and SH3 domains and have no other functional motifs (2, 19). They are therefore regarded as adapters. They act by binding to tyrosine-phosphorylated proteins via SH2 domains and associating with P-X-X-P motif-containing proteins through SH3 domains. SH3-associated proteins often get translocated to the proximity of phosphotyrosine proteins (32). Thus, SH2 and SH3 domains act as a second messenger connecting protein tyrosine phosphorylation to a variety of intracellular signaling networks. The best-characterized adapter is the SH3-SH2-SH3 protein Grb2. Grb2 binds to two separate pY-X-N-V motifs in the epidermal growth factor receptor (EGFR) via its SH2 domain and associates through its two SH3 domains to the PPPVPPRRR motifs in Sos, a guanine nucleotide exchange factor for Ras. As a result, Sos is translocated to the plasma membrane and activates Ras (32). Nck contains three consecutive SH3 domains and one SH2 domain, which together occupy more than 70% of Nck's 377 amino acids (16). Similar to Grb2, Nck is widely expressed in various types of cells and acts as an adapter by linking receptor tyrosine kinases to downstream signaling networks. It has also been reported that there is a fraction of Nck that is associated with Sam68 in the nucleus (14), although its function remains unknown. The SH2 domain of Nck has been shown to bind either directly or indirectly to EGFR, platelet-derived growth factor receptor (PDGFR), Eph receptor, insulin receptor substrate 1, p130cas, and p62Dok (16, 20). For example, tyrosine 751 (Y751) in human PDGFR-β was identified as the binding site for Nckα. Since Y751 is also one of the two binding sites in the PDGFR for the p85 subunit of phosphatidylinositol 3-kinase (PI3-K) (the p85 subunit has two SH2 domains), Nckα and PI3-K may either compete with each other for binding to the PDGFR and thereby antagonize each other's function or bind to different pools of the cell surface PDGFR (22). There has been no evidence so far for or against either of these hypotheses. Stein et al. identified a binding site for Nckα in the Eph family receptor, Eph1 (ELK). They showed that Y-594 in the juxtamembrane region of Eph1 recruits Nck to the plasma membrane. The membrane-bound Nck in turn causes, apparently via Nck-interacting kinase (NIK), activation of the JNK/SAPK pathway (1, 36, 37). The current list of Nck (α, β, or both) SH3-binding molecules includes the Abl protein tyrosine kinase, Sos, Nck-associated kinase (NAK), p21cdc42/rac-activated kinases (PAK), Rho effector PKN-related kinase PRK2, protooncogene c-cbl, human Wiskott-Aldrich syndrome protein (WASp), the novel serine threonine kinase NIK, casein kinase 1 gamma-2, Sam-68, Nap1 (Nck-associated protein 1), and NAP4 (Nck-, Ash- and PLCγ-binding protein 4) (16). We and others recently reported that Nck represents a family of genes including two human (Nck/hNckα and hNckβ/Nck2) and two mouse (mNckα and mNckβ/Grb4) Nck genes (3, 4, 25, 40). hNckα and hNckβ reside in different chromosomes (4, 10, 41) and are coexpressed in most but not all cells (3). The newly identified Nckβ binds significantly better than Nck (Nckα) to both receptor and nonreceptor tyrosine kinases (3, 4, 40). Moreover, Nckα and Nckβ appear to have distinct functional assignments in the same cells (4). Recently, a growing number of studies have suggested that Nck plays an important role in mediating receptor tyrosine kinase signaling to the actin cytoskeleton. Rockow et al. showed that overexpression of Nckα blocks nerve growth factor- and basic fibroblast growth factor-induced neurite outgrowth, a Rac1/Cdc42 GTPase-dependent actin cytoskeletal change, in rat adrenal pheochromocytoma cells PC12, through an extracellular signal-regulated kinase-independent mechanism (29). Two Nck-SH3-binding proteins, WASp and PAK1, have clear roles in regulation of the actin cytoskeleton through either Cdc42- and Rac-dependent or -independent mechanisms. Symons et al. showed that WASp binds to GTP-bound Cdc42 and clusters in polymerized actin (38). N-WASp (richest in neural tissues) is also involved in Cdc42 signaling to the actin cytoskeleton (21). Sells et al. reported that PAK1-induced actin organization depends upon binding to Nck but not upon PAK1 kinase activity or binding to Rac1 and Cdc42 (33). They showed that a kinase-dead PAK1 could mimic the effect of Rac and induce lamellipodia formation (33). Consistent with their observations, Obermeier et al. showed that brain-specific PAK (γPAK/PAK3) induces cell spreading, membrane ruffling, and increased lamellipodia formation (24). The strongest support of the notion that Nck links tyrosine kinases to the actin cytoskeleton comes from a genetic study of Drosophila melanogaster. Each of the eight R cells (R1 to R8) of the Drosophila compound eye is a distinct neuron and acts as a photoreceptor. Guidance and target recognition of these R cells toward axons are believed to be regulated by receptors at the surface of the growth cone, which resides at the leading edge of the axon. The growth cones receive extracellular cues and in turn control the intracellular actin cytoskeletol rearrangement. Zipursky and his colleagues found that the gene called Dreadlocks, or Dock, was concentrated in the R-cell growth cone and essential for R-cell guidance and target recognition. Mutations in the Dock gene disrupted signaling from the surface of the growth cone to the intracellular actin cytoskeleton, resulting in defects in R-cell fasciculation, targeting, and retinotopy (5). Dock is structurally related to and has an overall 40% amino acid identity with the mammalian Nck genes (4). Rao and Zipursky showed that the first and third SH3 domains and the SH2 domain are functionally redundant, whereas the middle SH3 domain is always required (27). Depending upon the specific neuron type, the middle SH3 domain could act with the SH2 domain or with the first and third SH3 domains. The critical downstream pathways for Dock include the Cdc42-Pak1 pathway (9) and the Ste20-like kinase misshapen pathway (30). In contrast, important questions regarding the function of the mammalian Nck genes remain unanswered. Does Nck play roles in mammals similar to that of Dock in Drosophila? Are both Nckα and Nckβ involved in regulation of the actin cytoskeleton? It has previously been demonstrated that PDGF induces actin polymerization at the plasma membrane of fibroblast cells to produce edge ruffles and lamellipodia (6, 28). This signaling event appears to involve the Rho family GTPase Rac1 (23, 28) and PI3-K (23, 42, 43). PI3-K appears to act upstream of Rac1 (8). Because Nck is a direct target for PDGFR, we set out to compare the roles of Nckα and Nckβ in PDGF-stimulated actin cytoskeletal rearrangement in NIH 3T3 cells. We found that Nckβ is specifically involved in PDGF-induced membrane ruffling and formation of lamellipodia. Our results suggest that Nckβ acts either parallel to or downstream of Rac1, a mediator between PDGFR and the actin cytoskeleton.