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

Abstract

Many Src Homology 3 (SH3) domains function as molecular adhesives in intracellular signal transduction. Based on previous ultrastructural studies, short motifs which bind to the ®rst SH3 domains of the adapters Crk and CRKL were selectively mutagenised to generate Crk/ CRKL SH3-binding peptides of very high a nity and selectivity. A nities were increased up to 20-fold compared to the best wildtype sequences, while the selectivity against a similar SH3 domain [Grb2SH3(N)] was not only retained, but sometimes increased. Blot techniques with GST-fusion peptides and in solution precipitation assays with biotinylated high a nity Crk binding peptides (HACBPs) were subsequently used to analyse the binding of these sequences to a large panel of SH3 domain-containing fusion proteins. Only those proteins which contained the CrkSH3(1) or CRKLSH3(1) domains bound e ciently to the HACBPs. A GST-HACBP fusion protein precipitated Crk and CRKL proteins out of 35 S-labelled and unlabelled cell lysates. Very little binding of other cellular proteins to HACBP was detectable, indicative of a great preference for Crk and CRKL when compared to the wide variety of other endogenous cellular proteins. Moreover, HACBP disrupted in vitro preexisting Crk-complexes with DOCK180 and the exchange factors SoS and C3G, which are known targets of Crk adapters, in a concentration dependent manner. HACBP-based molecules should therefore be useful as highly selective inhibitors of intracellular signalling processes involving Crk and CRKL.

The Crk signaling pathway contributes to the bombesin-induced activation of the small GTPase Rap1 in Swiss 3T3 cells

“…Because the expression levels of HA-Rap1 decrease slightly with expression of CBR, blots were densitometrically analysed and the ratio was determined. The relative activation and its inhibition by CBR in the above experiment is shown in the bar diagram CrkSH3(1) domain and the Ras exchange factor SoS, which is presumably also abolished following CBR expression, apparently does not play an important role, which is in accordance with the low a nity of SoSderived peptides for the CrkSH3(1) domain (Posern et al, 1998b).…”

“…The Crk-binding region of C3G (CBR) is expected to act as a suppressor of Crk signaling by blocking the protein interactions mediated via the ®rst SH3 domain of Crk adapters (York et al, 1998). This block is likely to be very speci®c, because C3G-derived motifs bind to c-Crk and CRKL with an unusually high selectivity (Posern et al, 1998b). Cotransfection of hemagglutinin-tagged Rap1 (de Rooij et al, 1998) allowed to analyse its GTP loading speci®cally in the transfected cells.…”

Alterations in the focal adhesion kinase/Src signal transduction pathway correlate with increased migratory capacity of prostate carcinoma cells

“…In addition, substrates of the FAK/Src bipartite kinase complex have been identi®ed as regulators of migration (Cary et al, 1998;Klemke et al, 1998;Petit et al, 2000). Speci®cally, FAK/Srcmediated phosphorylation of Cas or paxillin creates binding sites for the adapter protein Crk (Hamasaki et al, 1996;Harte et al, 1996;Schaller and Parsons, 1995;Tachibana et al, 1997), which binds to DOCK180 (Kiyokawa et al, 1998b;Matsuda et al, 1996;Posern et al, 1998), an activator of Rac (Kiyokawa et al, 1998a;Nolan et al, 1998), thereby directing Rac activity to sites of integrin engagement with the ECM to stimulate membrane ru ing and cell migration (Cheresh et al, 1999;Hasegawa et al, 1996;Reddien and Horvitz, 2000;Wu and Horvitz, 1998). Indeed, Cas over expression in FG carcinoma cells and Chinese hamster ovary cells stimulates motility (Cary et al, 1998;Klemke et al, 1998).…”

MUC1 Initiates Src-CrkL-Rac1/Cdc42–Mediated Actin Cytoskeletal Protrusive Motility after Ligating Intercellular Adhesion Molecule-1

“…Once activated in the GTP-bound forms, Rac1 and Cdc42 can consequently interact with the downstream effectors, Arp2/3 activators of WAVE/WASP family, to initiate membrane ruffling and formation of membrane lamellipodial/filopodial protrusions (26), whereas RhoA is frequently involved in cellular contraction and focal adhesion dynamics (27). Acting upstream of Rho GTPases, the adaptor protein CrkL has been identified as a crucial mediator of cell migration through the association with guanine nucleotide exchange factors, such as Dock180, for catalyzing the GDP/GTP exchange and Rho GTPase activation (28,29). In response to migratory cues, like integrin engagement, CrkL can be recruited to focal contacts where Src forms a signaling complex with focal adhesion kinase (FAK) and phosphorylates FAK-associated p130CAS or paxillin on the conserved Y-x-x-P binding motifs for the CrkL-SH2 domain (23,30), thereby facilitating Rho GTPasemediated cytoskeletal rearrangements and cell migration (30).…”