Identification of signalling components in tyrosine kinase cascades using phosphopeptide affinity chromatography.
Various methods are now available to identify the molecular partners of the component of a signal transduction pathway. Some interactions, however, can be technically difficult to detect because they depend upon transient tyrosine phosphorylation. Here, we present a simple affinity chromatography approach based on synthetic phosphopeptides to purify 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 synthetase (MTHFS) was identified as a potential mediator of IGF-1R dependent transformation. P85alpha, the regulatory subunit of PI3 kinase, was identified as one of four proteins recruited by a phosphopeptide mimicking a motif conserved in all JAK family members.
Duménil
G
G
Unité INSERM 276, Institut Pasteur, Paris, France. gdumenil@Pasteur.fr
Rubini
M
M
Dubois
G
G
Baserga
R
R
Fellous
M
M
Pellegrini
S
S
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D066246:ErbB Receptors / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D018922:HL-60 Cells; D006801:Humans; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D010748:Phosphopeptides; D011506:Proteins; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D017526:Receptor, IGF Type 1