title: Measuring Gli2 Phosphorylation by Selected Reaction Monitoring Mass Spectrometry.
abstract: Phosphorylation is an important mechanism by which Gli proteins are regulated. When the Hedgehog (Hh) pathway is activated, multiple serine and threonine residues of Gli2 are dephosphorylated, while at least one residue undergoes phosphorylation. These changes in phosphorylation have functional relevance for the transcriptional activity of Gli proteins, as shown by in vitro and in vivo assays on Gli mutants lacking the phosphorylated residues. Here, we describe a method of quantitatively monitoring the phosphorylation of Gli proteins by triple quadrupole mass spectrometry of Gli2 immunoprecipitated from cell lysates. This method is broadly applicable to the monitoring of phosphorylation changes of immunoprecipitated Gli proteins when the putative phosphosites are known.
authors.affiliation: Project group Quantitative Systems Analysis, Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., 44227, Dortmund, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Ahrends
authors.forename: Pawel
authors.initials: P
authors.lastname: Niewiadomski
authors.forename: Mary N
authors.initials: MN
authors.lastname: Teruel
authors.forename: Rajat
authors.initials: R
authors.lastname: Rohatgi
mesh_terms: D000818:Animals; D047468:Immunoprecipitation / Q000379:methods; D051741:Kruppel-Like Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D041681:NIH 3T3 Cells; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D059748:Proteolysis; D000075746:Zinc Finger Protein Gli2
chemical_list: C000618551:Gli2 protein, mouse; D051741:Kruppel-Like Transcription Factors; D010446:Peptide Fragments; D000075746:Zinc Finger Protein Gli2