The Murine Nck SH2/SH3 Adaptors Are Important for the Development of Mesoderm-Derived Embryonic Structures and for Regulating the Cellular Actin Network

Abstract

Mammalian Nck1 and Nck2 are closely related adaptor proteins that possess three SH3 domains, followed by an SH2 domain, and are implicated in coupling phosphotyrosine signals to polypeptides that regulate the actin cytoskeleton. However, the in vivo functions of Nck1 and Nck2 have not been defined. We have mutated the murine Nck1 and Nck2 genes and incorporated ␤-galactosidase reporters into the mutant loci. In mouse embryos, the two Nck genes have broad and overlapping expression patterns. They are functionally redundant in the sense that mice deficient for either Nck1 or Nck2 are viable, whereas inactivation of both Nck1 and Nck2 results in profound defects in mesoderm-derived notochord and embryonic lethality at embryonic day 9.5. Fibroblast cell lines derived from Nck1؊/؊ Nck2 ؊/؊ embryos have defects in cell motility and in the organization of the lamellipodial actin network. These data suggest that the Nck SH2/SH3 adaptors have important functions in the development of mesodermal structures during embryogenesis, potentially linked to a role in cell movement and cytoskeletal organization.Signaling by cell surface receptors commonly involves the recruitment of cytoplasmic targets into multiprotein complexes through specialized interaction domains. These domains provide a mechanism to couple different receptors to the core machinery that regulates cellular function and to provide a degree of specificity in signal transduction. Receptors with intrinsic or associated tyrosine kinase activity commonly recruit their targets through phosphotyrosine-containing motifs that bind the Src homology 2 (SH2) domains of cytoplasmic regulatory proteins (30,45). Although some of these SH2-containing polypeptides have linked catalytic domains, others have an adaptor function and are comprised exclusively of interaction domains that nucleate the assembly of protein complexes around the activated receptor. Adaptors of the Nck, Grb2, and Crk families contain a single SH2 domain and various numbers of SH3 domains that typically bind to effector proteins through proline-rich peptide motifs. Each of these adaptors preferentially binds a distinct phosphotyrosine-containing motif through its SH2 domain and a specific group of signaling proteins through its SH3 domains. Grb2, for example, links phosphorylated Tyr-X-Asn sequences on activated receptor tyrosine kinases (RTKs) to proteins such as Sos and Gab1, involved in Ras/mitogen-activated protein (MAP) kinase and phosphatidylinositol 3Ј-kinase signaling (34,36,48).Nck adaptor proteins possess three SH3 domains, followed by a C-terminal SH2 domain that binds optimally to pYDEP/

SDF1α‐induced interaction of the adapter proteins Nck and HS1 facilitates actin polymerization and migration in T cells

“…Nck1 and Nck2 display 68% identity at the amino acid level and are regarded to be functionally redundant in many aspects [2,5]. We therefore analyzed, whether HS1 would also associate with the SH2 domain of Nck2.…”

“…We detected a major band at about 80 kDa only after precipitation of HS1 from stimulated cells and not in the respective controls. By stripping and reprobing the blot with an HS1-specific antibody we clearly assigned this band to HS1.Nck1 and Nck2 display 68% identity at the amino acid level and are regarded to be functionally redundant in many aspects [2,5]. We therefore analyzed, whether HS1 would also associate with the SH2 domain of Nck2.…”

“…This regulatory network also includes the adapter proteins Nck and HS1 (reviewed in [1][2][3][4]). The Nck (noncatalytic region of tyrosine kinase) family of adapter proteins comprises two members (Nck1/Nckα and Nck2/Nckβ, also termed Grb4) that are structurally and functionally redundant in many aspects [5]. Nck1/2 are almost exclusively built of a single SH2 (Src homology) and three SH3 domains that allow for different individual and potentially simultaneous protein-protein interactions.…”

Src and FAK Kinases Cooperate to Phosphorylate Paxillin Kinase Linker, Stimulate Its Focal Adhesion Localization, and Regulate Cell Spreading and Protrusiveness

“…Normal mouse embryo fibroblasts (MEF) and paxillin null (Hagel et al, 2002), SYF null (Klinghoffer et al, 1999), and FAK null cells have been described previously. Nck1/2 null cells were provided by Tony Pawson (Samuel Lunenfeld Research Institute) (Bladt et al, 2003), Csk and p130Cas null cells were from Akira Imamoto (Imamoto and Soriano, 1993;Honda et al, 1998), Abl null cells from Jean Wang (Hardin et al, 1996) and PTP-PEST null cells from Michel Tremblay (Angers-Loustau et al, 1999). Human embryonic kidney (HEK)293A cells (AD-HEK) were from Stratagene, and FAK null cells were obtained from American Type Culture Collection (Manassas, VA).…”

“…Synchronization of adhesion formation and turnover is critical to the process of cell motility (Webb et al, 2004), and perturbation in the PKL-paxillin link results in enhanced spreading, combined with a significant inhibition of cell polarity, focal adhesion dynamics, and motility Zhao et al, 2000b;West et al, 2001;Brown et al, 2002;Manabe Ri et al, 2002;Webb et al, 2004), in part through the misregulation of Rac (West et al, 2001). Similarly, fibroblasts derived from mice with germline deletions in Src/Yes/Fyn (SYF), FAK, paxillin, and Nck exhibit profound defects in cytoskeletal organization and the capacity to efficiently migrate, providing concrete evidence for the essential nature of these associations and signaling pathways Klinghoffer et al, 1999;Hagel et al, 2002;Bladt et al, 2003;Webb et al, 2004).We have sought to further characterize the mechanism(s) by which the Nck-PAK-PIX-PKL complex becomes activated and competent to localize to focal adhesions and modulate adhesion and motility signaling. Protein phosphorylation is a common means of regulating protein function through allosteric modification as well as providing inducible protein recognition motifs (Cohen et al, 1995;Hubbard and Till, 2000).…”