IDENTIFICATION OF SIGNALING COMPONENTS IN TYROSINE KINASE CASCADES USING PHOSPHOPEPTIDE AFFINITY-CHROMATOGRAPHY

Various methods are now available to identify the molecular partners o f the component of a signal transduction pathway. Some interactions, h owever, can be technically difficult to detect because they depend upo n transient tyrosine phosphorylation. Here, we present a simple affini ty chromatography approach based on synthetic phosphopeptides to purif y potential partners of phosphotyrosine-containing proteins. With this approach, we confirm the previously characterized interaction between Grb2 and the EGF receptor, and we identify novel partners of the IGF- 1 receptor and of the JAK proteins. Methenyltetrahydrofolate synthetas e (MTHFS) was identified as a potential mediator of IGF-1R dependent t ransformation. P85 alpha, the regulatory subunit of PI3 kinase, was id entified as one of four proteins recruited by a phosphopeptide mimicki ng a motif conserved in all JAK family members. (C) 1997 Academic Pres s.