C3G, a guanine nucleotide-releasing protein expressed ubiquitously, binds to the Src homology 3 domains of CRK and GRB2/ASH proteins.

Abstract

CRK protein, together with GRB2/ASH and Nck proteins, belongs to the adaptor-type Src homology (SH)2-containing molecules, which transduce signals from tyrosine kinases. Here another guanine nucleotide-releasing protein (GNRP), C3G, has been identified as a CRK SH3-binding protein. The nucleotide sequence of a 4.1-kb C3G cDNA contains a 3.2-kb open reading frame encoding a 121-kDa protein, and antibodies against C3G have been shown to detect a protein of 130-140 kDa. The carboxyl terminus of C3G has a peptide sequence homologous to GNRPs for Ras, and the expression of this carboxyl terminus region suppresses the loss of CDC25 function in the yeast Saccharomyces cerevisiae. The C3G protein expressed in Escherichia coli binds to CRK and GRB2/ASH proteins. Mutational analysis of C3G assigns the SH3 binding region to a 50-amino acid region containing a proline-rich sequence. The mRNAs of both the C3G and CRK proteins are expressed ubiquitously in human adult and fetal tissues. The results of these studies suggest that the complex of CRK and C3G, or GRB2/ASH and C3G, may transduce the signals from tyrosine kinases to Ras in a number of different tissues.Growth factors elicit various responses through the activation of receptor-type and non-receptor-type tyrosine kinases (1). A group of cytoplasmic enzymes containing common amino acid sequences, designated Src homology (SH)2 and SH3 domains, play a pivotal role in transducing signals from the tyrosine kinases (2, 3). The SH2 domain responds to the signals from tyrosine kinases by binding to the tyrosinephosphorylated proteins, including the tyrosine kinases themselves. Some of the signals are also transmitted to proteins bound to the SH3 domains, but much less information is available on SH3-mediated signaling.The v-Crk protein was originally identified as an oncoprotein of a chicken retrovirus, CT10 (4). The protooncogene product v-Crk represents a newly emerging class of proteins consisting mostly of the SH2 and SH3 domains (5, 6). These proteins, now known as adaptorproteins, include Nck, GRB2/ ASH, Sem-5, and Drk (2, 7, 8). Sem-5 of Caenorhabditis elegans and Drk of Drosophila melanogaster appear to be homologues of mammalian GRB2/ASH. All of the adaptor proteins may be involved in the growth of fibroblasts. Overexpression or microinjection ofCRK, Nck, and GRB2 induces transformation of rat 3Y1 fibroblasts or DNA replication in mouse 3T3 fibroblasts (6,9,10). One common feature of the adaptor proteins may be signal transmission to Ras. The sem-5 gene of C. elegans has been mapped genetically downstream of let-23 tyrosine kinase and upstream of let-60 Ras-like protein (11). Similarly, drk of Drosophila is mapped between sevenless (sev) receptor tyrosine kinase and son of sevenless (sos), which encodes a guanine nucleotide-releasing protein (GNRP) for Rasl. Recently, the Sos protein ofDrosophila was shown to bind to the Drk protein, which contains SH2 and SH3 domains (7,8). Anti-Ras antibody also inhibited neuronal differentiation of PC12 cells induced by th...

CSF-1 stimulation induces the formation of a multiprotein complex including CSF-1 receptor, c-Cbl, PI 3-kinase, Crk-II and Grb2

“…Crk-II is an adapter protein carrying one SH2 domain and two SH3 domains that can bind to a variety of signaling proteins (Beitner-Johnson and LeRoith, 1995;Birge et al, 1992;Buday et al, 1996;Feller et al, 1995;Hempstead et al, 1994;Ribon and Saltiel, 1996;Tanaka et al, 1994). Here, we provide evidence that Crk-II interacts with c-Cbl upon CSF-1 stimulation following a time course compatible with c-Cbl phosphorylation.…”

Identification of Tyr-762 in the platelet-derived growth factor α-receptor as the binding site for Crk proteins

“…The SH3 domains have been shown to bind to e.g. the cytoplasmic tyrosine kinase c-Abl, the adaptor molecule DOCK180, and the guanine nucleotide exchange factor C3G and Sos (Feller et al, 1994;Hasegawa et al, 1996;Knudsen et al, 1995;Matsuda et al, 1996;Reedquist et al, 1996;Tanaka et al, 1994).…”

“…A guanine nucleotide exchange factor C3G forms complex with Crk proteins and the autophosphorylated PDGF a-receptor Several intracellular proteins are known to associate with the SH3 domains of the Crk proteins, including the guanine nucleotide exchange factor C3G Tanaka et al, 1994). C3G has been shown to be an activator of a Ras-related small G protein Rap 1.…”

Development of highly selective SH3 binding peptides for Crk and CRKL which disrupt Crk-complexes with DOCK180, SoS and C3G

“…Crk/CRKLSH3(1)-binding proteins currently identi®ed include the tyrosine kinases cAbl, Bcr ± Abl and Arg, the guanine nucleotide exchange factors C3G and SoS (Feller et al, 1994a,b;Knudsen et al, 1994;Tanaka et al, 1994;ten Hoeve et al, 1994;Wang et al, 1996b) as well as the possibly non-enzymatic proteins Eps15 and DOCK180 (Schumacher et al, 1995;Hasegawa et al, 1996). No speci®c protein interactions have been reported at this point for the C-terminal SH3 domains of Crk and CRKL, despite attempts of various laboratories to ®nd interacting partners or motifs.…”

“…C3G, a guanine-nucleotide exchange factor for the GTPases Rap 1 A and B (Gotoh et al, 1995), contains four potential CrkSH3 binding motifs (CB1 to CB4) clustered in the central region of the protein (Knudsen et al, 1994;Tanaka et al, 1994). A 10-amino acid peptide corresponding to the CB1-motif was found to bind the CrkSH3(1) domain with a K d of 1.9 mM , a remarkably high a nity when compared to other SH3-interacting peptides of this length measured with tryptophan¯uorescence (Lee et al, 1995).…”