JAK2 associates with the beta c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region.

Abstract

The high-affinity receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a unique a chain and a Pc subunit that is shared with the receptors for interleukin-3 (IL3) and . Two regions of the Pc chain have been defined; these include a membrane-proximal region of the cytoplasmic domain that is required for mitogenesis and a membrane-distal region that is required for activation of Ras, Raf-1, mitogen-activated protein kinase, and S6 kinase. Recent studies have implicated the cytoplasmic protein tyrosine kinase JAK2 in signalling through a number of the cytokine receptors, including the IL-3 and erythropoietin receptors. In the studies described here, we demonstrate that GM-CSF stimulation of cells induces the tyrosine phosphorylation of JAK2 and activates its in vitro kinase activity. Hematopoiesis is regulated through the interaction of one or more of a variety of cytokines with their cognate receptors. Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates the proliferation and differentiation of cells at various stages of differentiation along the myeloid lineages (1, 17). The functional, high-affinity receptor for GM-CSF consists of two subunits, each of which is a member of the cytokine receptor superfamily (19,20). The binding of GM-CSF occurs through an a subunit of 60 to 80 kDa, which alone binds GM-CSF with low affinity. Association of the a chain with a 140-kDa 1c chain results in the formation of a high-affinity binding site for GM-CSF. The 1c chain also associates with 'a subunits that specifically bind interleukin-3 (IL-3) and IL-5 and similarly contributes to the formation of high-affinity binding sites for these ligands.Stimulation of growth factor-dependent cell lines with GM-CSF has been shown to induce a variety of immediate cellular responses, including the rapid tyrosine phosphorylation of the 1c subunit and a number of cellular substrates (7, 16, 30); induction of transcription of several immediate-early genes (5); and activation of components of the Ras signalling pathway, including SHC phosphorylation, increases in GTP-bound Ras, activation of Raf-1 kinase, and activation of mitogenactivated protein (MAP) kinase (4,8,23,26,27 inducing mitogenesis, while a distal region is required for activation of Ras, Raf-1, MAP kinase, and S6 kinase.Although considerable evidence demonstrates that induction of protein tyrosine phosphorylation is critical to cytokine function, only recently have the JAK family of kinases been implicated in coupling ligand binding to tyrosine phosphorylation. The JAK family consists of JAK1 (15), JAK2 (29), and TYK2 (9) and is characterized by proteins of approximately 130 kDa that contain a carboxyl kinase domain and a second kinase-like domain and lack SH2 or SH3 domains. JAK2 has been shown to be activated in the responses to erythropoietin (Epo) (36), , growth hormone (2), G-CSF (35a), and prolactin (3). In contrast, IL-6, ciliary neurotrophic factor (CNTF), and leukemia-inhibitory factor activate JAK1, JAK2, and to some extent T...

IL-3 signaling and the role of Src kinases, JAKs and STATs: a covert liaison unveiled

“…The membrane proximal intracellular portion of the receptor till amino-acid number 589, that includes the Box 1 region, was shown to be necessary and su cient for DNA synthesis mediated by several of these cytokines including IL-3 (Sato et al, 1993;Tian et al, 1996). This region was also found to be required for the induction of proliferation-associated genes such as c-myc, pim-1 and oncostatin M. Following cytokineinduced hetero-dimerization, this region of the receptor was found to bind to multiple signal-transducing proteins, which include JAKs, STATs, c-Src, phosphotidylinositol-3 kinase as well as Vav (Quelle et al, 1994;Jaster et al, 1997;Yoshimura et al, 1995Yoshimura et al, , 1998Rao and Mufson, 1995;Matsuguchi et al, 1995). The membrane distal domain of the receptor, spanning amino acids 589 ± 881, was shown to be required for growth inhibition that can be induced by some of the cytokines.…”

Association of RACK1 and PKCβ with the common β-chain of the IL-5/IL-3/GM-CSF receptor

“…Phosphorylated tyrosine residues on bc then serve as docking sites for intracellular signalling molecules, for example, STAT transcription factors and adapter molecules such as Shc (Chin et al, 1996;Lanfrancone et al, 1995). Non-phosphotyrosine mediated interactions have been reported for Jak2 (Quelle et al, 1994) and members of the Src family of kinases (Al-Shami et al, 1997). We have performed a two-hybrid screen with a HeLa cell library, utilizing the cytoplasmic domain of human bc lacking the box1 region (bc cyt Dbox1) to assess the occurrence of other tyrosine phosphorylation independent interactions.…”

“…Although bc contains no intrinsic kinase domain, upon ligand binding, rapid tyrosine phosphorylation of many cellular proteins, including the receptor itself is observed. (Quelle et al, 1994;Eder et al, 1993) Recent studies have demonstrated that the tyrosine kinase Jak2 is associated with the bc in a region containing a peptide stretch known as the box1 homology domain (Quelle et al, 1994) which is conserved among many cytokine receptors (Murakami et al, 1991). Its association with bc has been shown to be critical for receptor phosphorylation and downstream signalling.…”

Analysis of mechanisms involved in the prevention of γ irradiation-induced apoptosis by hGM-CSF

“…There was a report showing an essential and su cient role of JAK2 activation through the plasma membrane proximal domain of erythropoietin receptor (EPOR) for anti g irradiation-induced apoptosis in 32D cells (Quelle et al, 1998). The observation that the box1 region of EPOR and bc exerts activity through interaction with JAK2 (Quelle et al, 1994;Witthuhn et al, 1993), also means that only JAK2 is essential and su cient for prevention of the apoptosis induced by g irradiation. Our attempt to con®rm this point using chimeric bc/JAK2 constructs (Liu et al, 1999a) revealed that BA/F3 cells expressing b-JAK2 failed to suppress the DNA fragmentation induced by g irradiation.…”

“…JAK2 plays an essential role for various hGM-CSF activities and that JAK2 activation depends on the box1 region of bc (Quelle et al, 1994;Watanabe et al, 1996). Since deletion of the box1 region abrogated the potential for hGM-CSFR to maintain cell survival, JAK2 is likely to play an essential role in transducing signals for anti g irradiation-induced apoptosis.…”