Src homology region 2 domains direct protein-protein interactions in signal transduction.

Abstract

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.

Epidermal Growth Factor Modulates Tyrosine Phosphorylation of p130Cas

“…Unless otherwise indicated, the results shown in the figures are those obtained with RatER cells (2.5 ϫ 10 5 receptors/cell; Ref. 29). In addition, epidermoid carcinoma A431 cells (Ͼ10 6 receptors/cell; Ref.…”

“…29), A431 cells, and wild-type Rat-1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 50 units/ml penicillin, and 50 g/ml streptomycin. Cells were grown into 90% confluency as monolayers and serum-starved for 36 h prior to stimulation with or without EGF in the presence or absence of various inhibitors as indicated in the figure legends.…”

New insights into protein‐tyrosine kinase receptor signaling complexes

“…This observation led to the suggestion that the function of the SH2 domain was to recognize phosphotyrosine residues within a particular peptide sequence context. This novel role for protein phosphorylation in the regulation of intracellular events has since been shown to function in diverse systems Moran et al, 1990;Escobedo et al, 1991a), and this overall concept has revolutionized our thinking about the range of possible regulatory and interactive functions of protein phosphorylation within the cell.…”

Polyamines are essential for cell transformation by pp60v-src: delineation of molecular events relevant for the transformed phenotype

“…All these signal transducers, ex-cept for c-Raf, share sequences called SH2 (src homology region 2), which are involved in the association of the proteins with the receptors or each other (1). Likewise, the SH2 domain of pp60 v-s'c appears to be critical for the binding of its substrates (29,38,45). Regarding the signal transducers, it is known that PI 3-kinase associates with pp60 '~s'~ (19), and that this binding most likely occurs through the SH2 domain of pp60 vs'~ (10).…”