The amino-terminal portion of the JAK2 protein kinase is necessary for binding and phosphorylation of the granulocyte-macrophage colony-stimulating factor receptor β_c chain

The binding of granulocyte-macrophage colony-stimulating factor (GM-CSF) to its receptor stimulates JAK2 protein kinase activation, protein phosphorylation, and JAK2 association with the β_c chain of the GM-CSF receptor. To better understand how different domains of the JAK2 function to regulate association and phosphorylation of the β_c receptor, the minimal portion of the β_c receptor necessary for JAK2 binding has been determined. Using glutathione S-transferase (GST) fusion proteins expressing different portions of the membrane-proximal domain of the β_c chain, we demonstrate that JAK2 binds to amino acids 458-495, but showed little binding to fusion proteins containing amino acids 483-559, 483-530, or 458-484. The GST-β_c 458-495 bound equally well to the wild type (WT) JAK2, a carboxyl-terminal deletion of JAK2 removing the protein kinase domain (amino acids 1000-1129), and a deletion of the kinase-like domain (amino acids 523-746). However, an amino-terminal JAK2 deletion (amino acids 2-239) markedly reduced binding to this GST-β_c. Far Western blotting demonstrated that a GST fusion protein containing amino acids 1-294 of JAK2, but not fusion proteins containing amino acids 295-522, 523-746, or 747-1127, bound GST-β_c 458-559. When the JAK2 WT and deletions were transiently expressed along with the α and β_c subunits of the GM-CSF receptor and the cells were treated with GM-CSF, the following results were obtained: 1) WT JAK2 phosphorylated the β_c subunit in a GM-CSF- dependent manner, 2) the kinase-like domain deletion phosphorylated the β_c subunit, and 3) both the kinase domain deletion and the amino-terminal deletion failed to stimulate phosphorylation of the β_c subunit. Therefore, phosphorylation of the β_c subunit requires the binding of JAK2 through its amino terminus. * This work was supported in part by National Institutes of Health Grants DK44741 (to A. S. K.) and R29-DK-46395 (to S. J. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.