The amino-terminal portion of the JAK2 protein kinase is necessary for binding and phosphorylation of the granulocyte-macrophage colony-stimulating factor receptor beta 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 beta c chain of the GM-CSF receptor. To better understand how different domains of the JAK2 function to regulate association and phosphorylation of the beta c receptor, the minimal portion of the beta 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 beta 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-beta 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-beta 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-beta c 458-559. When the JAK2 WT and deletions were transiently expressed along with the alpha and beta 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 beta c subunit in a GM-CSF-dependent manner, 2) the kinase-like domain deletion phosphorylated the beta c subunit, and 3) both the kinase domain deletion and the amino-terminal deletion failed to stimulate phosphorylation of the beta c subunit. Therefore, phosphorylation of the beta c subunit requires the binding of JAK2 through its amino terminus.
Zhao
Y
Y
Division of Hematology/Oncology, Veterans Administration Medical Center, Birmingham, Alabama 35294, USA.
Wagner
F
F
Frank
S J
SJ
Kraft
A S
AS
D001665:Binding Sites; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D053614:Janus Kinase 2; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D011518:Proto-Oncogene Proteins; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D014443:Tyrosine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011505:Protein-Tyrosine Kinases; D053614:Janus Kinase 2