Simultaneous assay of Src SH3 and SH2 domain binding using different wavelength fluorescence polarization probes.
pp60(c-src) is a prototypical nonreceptor tyrosine kinase and may play a role in diseases as diverse as cancer and osteoporosis. In Src, the SH3 domain (Src homology 3) binds proteins at specific, proline-rich sequences, while the SH2 domain (Src homology 2) binds phosphotyrosine-containing sequences. Inhibition of Src SH3 and SH2 domain function is of potential therapeutic value because of their importance in signaling pathways involved in disease states. We have developed dual-wavelength fluorescent peptide probes for both the Src SH3 and the Src SH2 domains, which allow the simultaneous measurement of compounds binding to each domain in assays based on the technique of fluorescence polarization. We demonstrate the utility of these probes in a dual-binding assay (suitable for high-throughput screening) to study the interactions of various peptides with these domains, including a sequence from the rat protein p130(CAS) which has been reported to bind simultaneously to both Src SH3 and SH2 domains. Utilizing this dual-binding assay, we confirm that sequences from p130(CAS) can simultaneously bind Src via both its SH3 and its SH2 domains. We also use the dual-binding assay as an internal control to identify substances which inhibit SH3 and SH2 binding via nonspecific mechanisms.
Lynch
B A
BA
ARIAD Pharmaceuticals Inc., 26 Landsdowne Street, Cambridge, Massachusetts 02139, USA. berkley.lynch@ucb-group.com
Minor
C
C
Loiacono
K A
KA
van Schravendijk
M R
MR
Ram
M K
MK
Sundaramoorthi
R
R
Adams
S E
SE
Phillips
T
T
Holt
D
D
Rickles
R J
RJ
MacNeil
I A
IA
D000818:Animals; D050719:Crk-Associated Substrate Protein; D004926:Escherichia coli; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000138:chemical synthesis; D006801:Humans; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D050717:Retinoblastoma-Like Protein p130; D018909:src Homology Domains
C493841:BCAR1 protein, human; C493843:Bcar1 protein, rat; D050719:Crk-Associated Substrate Protein; D005456:Fluorescent Dyes; D010750:Phosphoproteins; D011506:Proteins; D011994:Recombinant Proteins; D050717:Retinoblastoma-Like Protein p130; D015688:Oncogene Protein pp60(v-src)