Direct analysis of the binding of the abl Src homology 2 domain to the activated epidermal growth factor receptor
Src homology regions 2 (SH2) and 3 (SH3) are noncatalytic domains that are conserved among several proteins implicated in the regulation of cell proliferation. Using bacterially expressed fusion proteins containing the SH2 domain of the abl tyrosine kinase, we have quantitated the binding of these d...
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Veröffentlicht in: | The Journal of biological chemistry 1993-01, Vol.268 (3), p.1775-1779 |
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Zusammenfassung: | Src homology regions 2 (SH2) and 3 (SH3) are noncatalytic domains that are conserved among several proteins implicated in
the regulation of cell proliferation. Using bacterially expressed fusion proteins containing the SH2 domain of the abl tyrosine
kinase, we have quantitated the binding of these domains to the activated epidermal growth factor (EGF) receptor (EGFR). A
35S-labeled abl SH2 fusion protein binds to the human EGFR immunoprecipitated from EGF-treated NIH3T3 cells that overexpress
the receptor. This binding is totally dependent on the pretreatment of cells with EGF. The interaction is rapid, reaching
50% of maximum within 1 min, and attaining apparent equilibrium by 10 min. Dissociation of the complex is biphasic with a
rapidly dissociating component (t1/2 of less than 1 min), as well as a slowly dissociable component. The 35S-labeled abl SH2
fusion protein specifically binds to the EGFR in a saturable manner and is differentially inhibited by unlabeled fusion proteins
containing SH2 domains from phospholipase C, the p85 subunit of phosphatidylinositol-3 kinase, and the GTPase activation protein
of ras. To identify residues critical for abl SH2-EGFR binding, six point mutants were constructed in the highly conserved
FLVRES motif. Three mutants (V170L, E172Q, and E174Q) display binding affinities similar to that of wild type. However, three
other mutants (R171K, S173C, and S175C) have greatly reduced affinity. Interestingly, the binding affinity to the EGFR determined
by the in vitro assay directly correlates with the transforming ability of the corresponding v-abl constructs in vivo (Mayer,
B. J., Jackson, P. K., Etten, R. A. V., and Baltimore, D. (1992) Mol. Cell. Biol. 12, 609-618). These data indicate that the
Arg-171, Ser-173, and Ser-175 are critical for both transformation and abl SH2 domain binding to phosphotyrosine-containing
proteins. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(18)53920-X |