Development of highly selective SH3 binding peptides for Crk and CRKL which disrupt Crk-complexes with DOCK180, SoS and C3G.
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 first SH3 domains of the adapters Crk and CRKL were selectively mutagenised to generate Crk/CRKL SH3-binding peptides of very high affinity and selectivity. Affinities 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 affinity 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 efficiently to the HACBPs. A GST-HACBP fusion protein precipitated Crk and CRKL proteins out of 35S-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.
Posern
G
G
Laboratory of Molecular Oncology, Institute for Medical Radiation and Cell Research (MSZ), Bavarian Julius-Maximilians University, Würzburg, Germany.
Zheng
J
J
Knudsen
B S
BS
Kardinal
C
C
Müller
K B
KB
Voss
J
J
Shishido
T
T
Cowburn
D
D
Cheng
G
G
Wang
B
B
Kruh
G D
GD
Burrell
S K
SK
Jacobson
C A
CA
Lenz
D M
DM
Zamborelli
T J
TJ
Adermann
K
K
Hanafusa
H
H
Feller
S M
SM
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050737:Proto-Oncogene Proteins c-crk; D020664:Son of Sevenless Proteins; D020744:rac GTP-Binding Proteins; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D002352:Carrier Proteins; C099818:DOCK1 protein, human; D020662:Guanine Nucleotide Exchange Factors; D008565:Membrane Proteins; D009687:Nuclear Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D020664:Son of Sevenless Proteins; D020744:rac GTP-Binding Proteins