Dominant-negative mutants of the SH2/SH3 adapters Nck and Grb2 inhibit MAP kinase activation and mesoderm-specific gene induction by eFGF in Xenopus

Abstract

The SH2/SH3 adapters Nck, Grb2 and Crk promote the assembly of signaling complexes by binding to tyrosine phosphorylated proteins using their SH2 domains and to proline-rich sequences on e ector molecules using their SH3 domains. FGF, which activates a receptor tyrosine kinase, induces mesoderm formation in Xenopus embryos through activation of the Ras/Raf/MAPK signaling pathway. We present evidence that dominant-negative mutants of Nck and Grb2, but not Crk1, can inhibit mesoderm-speci®c gene induction by eFGF in Xenopus animal cap explants. We also show that dominantnegative mutants of Grb2 and Nck can inhibit eFGFinduced Erk1 activation in Xenopus animal caps, and that targeting the ®rst two SH3 domains of Nck to the membrane can activate Erk1 in the absence of eFGF. Furthermore, combinations of the dominant-negative Grb2 mutants with the inhibitory Nck mutant synergistically inhibited Erk1 activation by eFGF in Xenopus animal caps, suggesting that the dominant-negative Nck and Grb2 mutants inhibit Erk1 activation by binding to di erent proteins. By contrast only Grb2 mutants could inhibit eFGF-induced Erk1 activation in human 293 cells, demonstrating diversity in the speci®c mechanisms of signaling from FGF to MAP kinases in di erent cells.

Vertebrate Ctr1 coordinates morphogenesis and progenitor cell fate and regulates embryonic stem cell differentiation

“…A dramatic example of this phenomenon occurs during early vertebrate embryogenesis, when fibroblast growth factor (FGF) mediates multiple developmental processes. During mesoderm induction, FGF triggers activation of a Ras-MAPK cascade (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). In contrast, subsequent morphogenesis of the dorsal mesoderm is regulated by a noncanonical FGF pathway that is independent of Ras-MAPK signaling and may involve stimulation of Ca 2ϩ release and/or activation of phospholipase C␥ (PLC␥) (13,14).…”

Oncogenic Met receptor induces ectopic structures in Xenopus embryos

“…Direct Grb2 binding is not required, but Grb2 function is necessary for induction of ectopic structures Although, direct binding of Shc or Grb2 to Tpr-Met is not required for the induction of ectopic structures, we have established that in the absence of direct Grb2 binding, a direct interaction between Tpr-Met and Shc is required. Since Grb2 is required for the morphogenesis associated with gastrulation in vertebrates (Cheng et al, 1998;Gupta and Mayer, 1998;Dupont and Blancq, 1999), we tested whether Grb2 function (either direct or indirect) is critical to the morphogenesis of the ectopic protrusions. Co-expression of dominant-negative Grb2 (DnGrb2) along with Tpr-Met blocked the formation of ectopic structures, and resulted in a phenotypically normal embryo (Figure 7b, c, f).…”

SLP-76 Coordinates Nck-Dependent Wiskott-Aldrich Syndrome Protein Recruitment with Vav-1/Cdc42-Dependent Wiskott-Aldrich Syndrome Protein Activation at the T Cell-APC Contact Site

“…Although this domain of WASP has been shown to interact in vitro with the SH3 domains of numerous proteins (23-29), we focused on Nck and Grb2, which have been shown to recruit WASP or N-WASP to the comet tails of vaccinia virus and the pedestals formed by enteropathogenic Escherichia coli (EPEC) (39 -43). To test the role of Nck and Grb2 in recruiting WASP in T cells, we used a panel of point mutants that perturb protein interactions with the SH3 domains of these proteins (39,44,45). Wild-type Jurkat T cells were transiently transfected with clones expressing wild-type or mutant versions of Grb2 or Nck tagged with GFP, and relative levels of expression were assessed by flow cytometry.…”