DEVELOPMENT OF HIGHLY SELECTIVE SH3 BINDING PEPTIDES FOR CRK AND CRKLWHICH DISRUPT CRK-COMPLEXES WITH DOCK180, SOS AND C3G

Many Src Homology 3 (SH3) domains function as molecular adhesives in i ntracellular signal transduction, Based on previous ultrastructural st udies, short motifs which bind to the first SH3 domains of the adapter s Crk and CRKL were selectively mutagenised to generate Crk/ CRKL SH3- binding peptides of very high affinity and selectivity, Affinities wer e increased up to 20-fold compared to the best wildtype sequences, whi le the selectivity against a similar SH3 domain [Grb2SH3(N)] was not o nly retained, but sometimes increased. Blot techniques with CST-fusion peptides and in solution precipitation assays with biotinylated high affinity Crk binding peptides (HACBPs) were subsequently used to analy se the binding of these sequences to a large panel of SH3 domain-conta ining fusion proteins. Only those proteins which contained the CrkSH3( 1) or CRKLSH3(1) domains bound efficiently to the HACBPs. A GST-HACBP fusion protein precipitated Crk and CRKL proteins out of S-35-labelled and unlabelled cell lysates, Very little binding of other cellular pr oteins to HACBP was detectable, indicative of a great preference for C rk and CRKL when compared to the wide variety of other endogenous cell ular proteins. Moreover, HACBP disrupted in vitro preexisting Crk-comp lexes with DOCK180 and the exchange factors SoS and C3G, which are kno wn targets of Crk adapters, in a concentration dependent manner. HACBP -based molecules should therefore be useful as highly selective inhibi tors of intracellular signalling processes involving Crk and CRKL.