The Amino-terminal Portion of the JAK2 Protein Kinase Is Necessary For Binding and Phosphorylation of the Granulocyte-Macrophage Colony-stimulating Factor Receptor β Chain

• Published in: The Journal of biological chemistry 1995-06, Vol.270 (23), p.13814-13818
• Main Authors: Zhao, Yanming, Wagner, Fred, Frank, Stuart J., Kraft, Andrew S.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.23.13814

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 βc chain of the GM-CSF receptor. To better understand how different domains of the JAK2 function to regulate association and phosphorylation of the βc receptor, the minimal portion of the βc 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 βc 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-βc 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-βc. 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-βc 458-559. When the JAK2 WT and deletions were transiently expressed along with the α and βc subunits of the GM-CSF receptor and the cells were treated with GM-CSF, the following results were obtained: 1) WT JAK2 phosphorylated the βc subunit in a GM-CSF- dependent manner, 2) the kinase-like domain deletion phosphorylated the βc subunit, and 3) both the kinase domain deletion and the amino-terminal deletion failed to stimulate phosphorylation of the βc subunit. Therefore, phosphorylation of the βc subunit requires the binding of JAK2 through its amino terminus.