No abstract

A framework for open discourse on the use of CRISPR-Cas9 technology to manipulate the human genome is urgently needed

The Ras protein signals to a number of distinct pathways by interacting with diverse downstream effectors. Among the effectors of Ras are the Raf kinase and RalGDS, a guanine nucleotide dissociation stimulator specific for Ral. Despite the absence of significant sequence similarities, both effectors bind directly to Ras, but with different specificities. We report here the 2.1 A crystal structure of the complex between Ras and the Ras-interacting domain (RID) of RalGDS. This structure reveals that the beta-sheet of the RID joins the switch I region of Ras to form an extended beta-sheet with a topology similar to that found in the Rap-Raf complex. However, the side chain interactions at the joining junctions of the two interacting systems and the relative orientation of the two binding domains are distinctly different. Furthermore, in the case of the Ras-RID complex a second RID molecule also interacts with a different part of the Ras molecule, the switch II region. These findings account for the cross-talk between the Ras and Ral pathways and the specificity with which Ras distinguishes the two effectors.

Cytoplasmic proteins that regulate signal transduction or induce cellular transformation, including cytoplasmic protein-tyrosine kinases, p21w GTPase-activating protein (GAP), phospholipase Cy, and the v-crk oncoprotein, possess one or two copies of a conserved noncatalytic domain, Src homology region 2 (SH2). Here we provide direct evidence that SH2 domains can mediate the interactions of these diverse signaling proteins with a related set ofphosphotyrosine ligands, including the epidermal growth factor (EGF) receptor. In src-transformed cells GAP forms heteromeric complexes, notably with a highly tyrosine phosphorylated 62-kDa protein (p62). The stable association between GAP and p62 can be specifically reconstituted in vitro by using a bacterial polypeptide containing only the N-terminal GAP SH2 domain. The efficient phosphorylation of p62 by the v-Src or v-Fps tyrosine kinases depends, in turn, on their SH2 domains and correlates with their transforming activity. In lysates of EGF-stimulated cells, the N-terminal GAP SH2 domain binds to both the EGF receptor and p62. Fusion proteins containing GAP or v-Crk SH2 domains complex with similar phosphotyrosine proteins from src-transformed or EGF-stimulated cells but with different efficiencies. SH2 sequences, therefore, form autonomous domains that direct signaling proteins, such as GAP, to bind specific phosphotyrosine-containing polypeptides. By promoting the formation of these complexes, SH2 domains are ideally suited to regulate the activation of intracellular signaling pathways by growth factors.