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

Abstract

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.

Two Distinct Domains Within the N-Terminal Region of Janus Kinase 1 Interact with Cytokine Receptors

“…Therefore, it is critical to define the domains in Jaks and cytokine receptors responsible for this interaction to mimic or abrogate the activation of cytokine systems with specific drugs. Some reports have indicated that the N-terminal region, containing the JH7-6 domains, of Jak2, Jak3, and Tyk2 is critical for the association with receptors (42)(43)(44)(45)(46)(47)(48)(49). Our data (Fig.…”

“…JH1 and JH2 domains correspond to a tyrosine kinase and kinase-like domain, respectively. The N terminus of Jak2, which contains the JH7-6 domains, is required for growth hormone receptor and GM-CSFR␤ common chain association (42)(43)(44). Similarly, deletion of the JH7-6 domains or mutation of tyrosine 100 to cysteine of Jak3 abrogates the interaction with the common ␥ chain and results in a SCID syndrome (44 -46).…”

Regulation of Proliferation, Differentiation and Survival by the IL-3/IL-5/GM-CSF Receptor Family

“…Janus kinases (JAKs) are to a membrane proximal region of ␤c containing Box I (Fig. 2) a family of cytoplasmic tyrosine kinases that are associated [10,12,[37][38][39]. Deletion of this region renders the ␤c chain with cytokine receptors and play a major role in cytokine unable to activate JAK2 [10].…”

Janus kinases: components of multiple signaling pathways

“…Furthermore, the authors demonstrated that a region encompassing the JH6 and JH7 domains of JAK3 was su cient for interaction of the kinase with the proline-rich Box1 region of the IL-2 receptor and was su cient in reconstituting the IL-2 dependent response. Zhao et al (1995) using a mutational analysis approach demonstrated that truncation of 239 amino terminal residues of JAK2, but not the JH1 and JH2 domains, resulted in abrogation of the interaction of JAK2 with the membrane proximal region of the beta-chain of the GM-CSF receptor. A similar N-terminal truncation of JAK2 also abolished its interaction with the growth hormone receptor .…”