title: Analysis of Janus tyrosine kinase phosphorylation and activation.
abstract: Activation of Janus kinases (Jaks) occurs through autophosphorylation of key tyrosine residues located primarily within their catalytic domain. Phosphorylation of these tyrosine residues facilitates access of substrates to the active site and serves as an intrinsic indicator of Jak activation. Here, we describe the methods and strategies used for analyzing Jak phosphorylation and activation. Tyrosine-phosphorylated (active) Jaks are primarily detected from cell extracts using anti-phosphotyrosine-directed Western blot analysis of Jak-specific immunoprecipitates. Additionally, receptor pull-down and in vitro kinase assays can also be utilized to measure cellular Jak catalytic activity. In addition to tyrosine phosphorylation, recent evidence indicates Jaks can be serine phosphorylated upon cytokine stimulation, however the lack of commercially available antibodies to detect these sites has hindered their analysis by Western blot. However, phosphoamino acid analysis (PAA) has been employed to monitor Jak serine and threonine phosphorylation. Over the past decade, remarkable advances have been made in our understanding of Jak function and dysfunction, however much remains to be learned about their complex regulatory mechanisms.
authors.affiliation: Department of Biological Sciences, The University of Texas at El Paso, El Paso, TX, USA.
authors.forename: Jeremy A
authors.initials: JA
authors.lastname: Ross
authors.forename: Georgialina
authors.initials: G
authors.lastname: Rodriguez
authors.forename: Robert A
authors.initials: RA
authors.lastname: Kirken
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D053612:Janus Kinases / Q000302:isolation & purification / Q000378:metabolism*; D019606:Phosphoamino Acids / Q000378:metabolism; D010766:Phosphorylation; D013194:Staining and Labeling; D013696:Temperature
chemical_list: D019606:Phosphoamino Acids; D053612:Janus Kinases