Abl protein-tyrosine kinase selects the Crk adapter as a substrate using SH3-binding sites.

Abstract

To understand the normal and oncogenic functions of the protein-tyrosine kinase Abl, the yeast two-hybrid system has been used for identifying proteins that interact with it. One interacting protein is Crk-I, an SH3/SH2-containing adapter protein that was originally identified as the oncogenic element in the avian sarcoma virus CT10. Direct interaction between the Crk-I SH3 and Abl at novel, -10 amino acid sites just carboxy-terminal to the Abl kinase domain occurs in vitro and in mammalian cells. There is a nearby site specific for binding another adapter, Nck, and these sites also bind Grb-2. When bound to Abl, Crk-I was phosphorylated on tyrosine. Thus, the SH3-binding sites on Abl serve as substrate recognition sites for the relatively nonspecific kinase of Abl. In Crk-I-transformed cells, Crk-I associates with endogenous c-Abl and is phosphorylated on tyrosine. The association of Crk and Abl suggests that Abl could play a role in v-Crk and Crk-I transformation and that normal Abl function may be partly mediated through bound adapter molecules.

The SH2-containing adapter protein GRB10 interacts with BCR-ABL

“…The interaction of Grb10 with Bcr-Abl in yeast was comparable in strength both by histidine autotrophy (Figure 2a) and b-galactosidase activity (data not shown). As expected, the Crk-SH3 domain which is known to bind to a proline rich sequence in the Abl C-terminus (Feller et al, 1994a;Ren et al, 1994), still showed interaction with the kinase-defective Bcr-Abl in yeast ( Figure 2a). Thus, binding of Grb10 to Bcr-Abl is mediated by an interaction between the Grb10-SH2 domain and a BcrAbl autophosphorylation site.…”

“…Among them were the SH2 domains of Grb2, PI3kinase and the SH3 domain of Crk. These signaling molecules have been previously identi®ed to interact with Bcr-Abl and to be of importance for the biological function of Bcr-Abl (Feller et al, 1994b;Pendergast et al, 1993b;Puil et al, 1994;Ren et al, 1994). This demonstrates that autophosphorylation of Bcr-Abl in yeast resembles the autophosphorylation of tyrosine residues occurring in vivo.…”

“…3610 7 colonies were screened and 62 clones were isolated which speci®cally interacted with Bcr-Abl in the yeast system. Among these clones were proteins already known to bind to Bcr-Abl both in a phosphotyrosine-dependent and -independent manner like the SH2 domains of Grb2 and p85PI3K, and the SH3 domain of Crk, respectively (Pendergast et al, 1993b;Puil et al, 1994;Ren et al, 1994). Ten of the clones obtained coded for the novel SH2-containing adapter molecule Grb10 (Ooi et al, 1995).…”

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

“…The SH3 domains are separated by a`spacer region' which contains a protein conformation-regulating tyrosine residue (Feller et al, 1994a;Rosen et al, 1995). c-Crk II and Crk-like (CRKL, a homologous but distinct gene product abundant in some hematopoietic cell lines) are substrates for tyrosine kinases, including those of the Abl family (c-Abl, Bcr-Abl and Arg) (Ren et al, 1994;Feller et al, 1994a;ten Hoeve et al, 1994;Wang et al, 1996b). Although detailed ultrastructural data are missing at this point, indirect evidence suggests that a CrkSH2-Y221 interaction somehow inhibits binding of the ®rst CrkSH3 domain to other cellular signalling proteins (Feller et al, 1994a).…”

Induced direct binding of the adapter protein Nck to the GTPase-activating protein-associated protein p62 by epidermal growth factor

“…Several potential downstream targets, which bind to SH3 domains of Nck, have been indicated. The Abl protein tyrosine kinase was the ®rst to be indicated to bind SH3 domains of Nck in vitro (Ren et al, 1994). A novel serine/threonine kinase NAK (Nck-associated kinase) was identi®ed by coimmunoprecipitation followed by in vitro kinase assay (Chou and Hanafusa, 1995).…”