Characterization of the in vitro kinase activity of a partially purified soluble GST/JAK2 fusion protein.
The biochemical and biophysical characteristics of Janus protein-tyrosine kinases (JAKs), which are essential early mediators of cytokine-initiated signal propagation, are virtually undefined. To facilitate the in vitro analysis of JAK-mediated catalysis, we substantially purified a soluble recombinant JAK2 and developed a novel means of quantifying JAK-catalyzed product formation. Glutathione-S-transferase fusion proteins containing active and inactive forms of rat Janus kinase 2 (GST:rJAK2 and GST:rJAK2(CA795)) were highly purified via affinity chromatography. A microtiterplate-based ELISA was used to measure tyrosine phosphorylation of a streptavidin-immobilized biotinylated STAT1-derived peptide. The ELISA data indicated that only about 1% of the enzyme was involved in exogenous substrate phosphorylation. Other immobilized peptides served as apparent substrates with varying efficacy. Traditional radioisotopic autokinase assays demonstrated that the activity of the purified fusion protein was inhibited by a variety of tyrphostin inhibitors. Non-radiolabeled adenine nucleotides, but not guanine nucleotides, inhibited the radioisotopic autokinase assay. These observations verify that the catalytic activity of JAK2 is highly regulated, and are consistent with the suggestion that JAK2 may require additional accessory proteins, such as a potential upstream regulatory kinase, for full catalytic activity.
Duhé
Roy J
RJ
Intramural Research Support Program, SAIC-Frederick, MD, USA. RDUHE@pharmacology.umsmed.edu
Clark
Emily A
EA
Farrar
William L
WL
D000225:Adenine / Q000737:chemistry; D000818:Animals; D019921:Biotinylation; D002384:Catalysis; D002460:Cell Line; D002846:Chromatography, Affinity; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005982:Glutathione Transferase / Q000378:metabolism*; D006147:Guanine / Q000737:chemistry; D007313:Insecta; D053614:Janus Kinase 2; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D050794:STAT1 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D020032:Tyrphostins / Q000378:metabolism / Q000494:pharmacology
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D010455:Peptides; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D050794:STAT1 Transcription Factor; C494081:Stat1 protein, rat; D015534:Trans-Activators; D020032:Tyrphostins; D014443:Tyrosine; D006147:Guanine; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; C507927:Jak2 protein, rat; D053614:Janus Kinase 2; D000225:Adenine