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Characterization of the in vitro kinase activity of a partially purified soluble GST/JAK2 fusion protein

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Abstract

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(CΔ795)) 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.

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Authors and Affiliations

  1. Intramural Research Support Program, SAIC – Frederick, Frederick, D, USA

    Roy J. Duhé

  2. Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, MS, USA

    Roy J. Duhé

  3. Cytokine Molecular Mechanisms Section, Laboratory of Molecular Immunoregulation, National Cancer Institute – Frederick Cancer Research and Development Center, Frederick, MD, USA

    Emily A. Clark & William L. Farrar

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  1. Roy J. Duhé
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  2. Emily A. Clark
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  3. William L. Farrar
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Duhé, R.J., Clark, E.A. & Farrar, W.L. Characterization of the in vitro kinase activity of a partially purified soluble GST/JAK2 fusion protein. Mol Cell Biochem 236, 23–35 (2002). https://doi.org/10.1023/A:1016186907376

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