Proteomic analysis of Src family kinases signaling complexes in Golgi/endosomal fractions using a site-selective anti-phosphotyrosine antibody: identification of LRP1-insulin receptor complexes

Journal of Proteome Research
Nicolas BilodeauRobert L Faure

Abstract

A role for Src Family Kinases (SFKs) in the dynamics of endocytic and secretory pathways has previously been reported. Identification of low-abundance compartmentalized complexes still remains challenging, highlighting the need for novel tools. Here we describe analysis of SFK-signaling complexes of hepatic Golgi/endosomes (G/E) fractions by sequential affinity enrichment of proteins. Mouse G/E permeabilized membranes were first validated in terms of electron microscopy, 1-D electrophoresis (1-DE), insulin-mediated endocytosis and protein content. With the use of quantitative N-terminal labeling of tryptic peptides (iTRAQ), 1-DE and IEF tryptic peptides separation methods, a total of 666 proteins were identified, including the SFK Lyn. Following insulin injection, a series of proteins were recognized by an anti-phosphotyrosine antibody (alpha P42-2) raised against the residue most frequently phosphorylated by SFK on the adenoviral protein E4orf4 and that cross-reacts with endosomal SFK targets. By using affinity chromatography coupled with mass spectrometry, we identified 16 proteins classified as (1) recycling receptors, (2) vesicular trafficking proteins, (3) actin network proteins, (4) metabolism proteins, or (5) signaling p...Continue Reading

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Citations

Sep 6, 2014·Biologie aujourd'hui·Bernard Desbuquois, François Authier
Mar 18, 2015·FEBS Letters·Danielle CaronRobert L Faure
Apr 2, 2019·Frontiers in Cell and Developmental Biology·Ewa E Bres, Andreas Faissner
Jun 7, 2017·Journal of Diabetes Research·Dianaly T AuSelen C Muratoglu
Jun 15, 2021·Frontiers in Cardiovascular Medicine·Jiefang ChenLing Mao

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