ABSTRACT
In this chapter it is described a general method that has been used successfully by more than one laboratory interested in detecting O-GlcNAc in myofilament proteins. Alternative reagents for chemo-enzymatic or metabolic labeling will be indicated, as well as references for more details in alternative methods. The outline is divided into (1) Enrichment of O-GlcNAc Stoichiometry, (2) Cardiac Myofilament Protein Isolation, (3) SDS-PAGE, (4) "Reduction and Alkylation," (5) In-Gel Protein Digestion, (6) Chemo-enzymatic Labeling of O-GlcNAc Moieties (Click Chemistry), (7) Biotin Alkyne Tagging, (8) Strong Cation Exchange (SCX) and Streptavidin, and (9) ß-Elimination and Michael Addition (BEMAD) for O-GlcNAc Site-Mapping.
Subject(s)
Acetylglucosamine/metabolism , Myocardium/chemistry , Myofibrils/chemistry , Proteins/chemistry , Staining and Labeling/methods , Acetylglucosamine/chemistry , Animals , Biotin , Click Chemistry , Electrophoresis, Polyacrylamide Gel , Glycosylation , Mass Spectrometry , Mice , Myocardium/metabolism , Myofibrils/metabolism , Peptide Mapping , Proteins/metabolism , Rats , Streptavidin , beta-N-Acetylhexosaminidases/antagonists & inhibitors , beta-N-Acetylhexosaminidases/metabolismABSTRACT
The cellular proto-oncogene c-Src is a nonreceptor tyrosine kinase involved in cell growth and cytoskeletal regulation. Despite being dysregulated in a variety of human cancers, its precise functions are not fully understood. Identification of the substrates of c-Src remains a major challenge, because there is no simple way to directly stimulate its activity. Here we combine the chemical rescue of mutant c-Src and global quantitative phosphoproteomics to obtain the first high resolution snapshot of the range of tyrosine phosphorylation events that occur in the cell immediately after specific c-Src stimulation. After enrichment by anti-phosphotyrosine antibodies, we identified 29 potential novel c-Src substrate proteins. Tyrosine phosphopeptide mapping allowed the identification of 382 nonredundant tyrosine phosphopeptides on 213 phosphoproteins. Stable isotope labeling of amino acids in cell culture-based quantitation allowed the detection of 97 nonredundant tyrosine phosphopeptides whose level of phosphorylation is increased by c-Src. A large number of previously uncharacterized c-Src putative protein targets and phosphorylation sites are presented here, a majority of which play key roles in signaling and cytoskeletal networks, particularly in cell adhesion. Integrin signaling and focal adhesion kinase signaling pathway are two of the most altered pathways upon c-Src activation through chemical rescue. In this context, our study revealed the temporal connection between c-Src activation and the GTPase Rap1, known to stimulate integrin-dependent adhesion. Chemical rescue of c-Src provided a tool to dissect the spatiotemporal mechanism of activation of the Rap1 guanine exchange factor, C3G, one of the identified potential c-Src substrates that plays a role in focal adhesion signaling. In addition to unveiling the role of c-Src in the cell and, specifically, in the Crk-C3G-Rap1 pathway, these results exemplify a strategy for obtaining a comprehensive understanding of the functions of nonreceptor tyrosine kinases with high specificity and kinetic resolution.
