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Roles of JAK kinase in human GM-CSF receptor signals
1Department of Molecular and Developmental Biology, University of Tokyo, Japan.
Abstract:
The IL-3 and GM-CSF (hGMR) receptors consist of two subunits, alpha and beta, both of which are members of the cytokine receptor superfamily. Phosphorylation of tyrosine residues of hGMR beta subunit and several cellular proteins are observed with hGM-CSF stimulation. We analyzed role of tyrosine residue of hGMR beta subunit and nature of tyrosine kinase, JAK2 in hGMR signals using several hGMR beta subunit mutants. In addition to box1 region, a membrane distal region (a.a. 544-589) of hGMR beta is required for c-fos activation. Only one tyrosine residue (Tyr577) exists within the region 544-589, and substitution of Tyr577 to phenylalanine in GMR beta 589 resulted in the loss of c-fos activation. In contrast, the same substitution in a wild type receptor did not affect GM-CSF-induced activities such as c-fos mRNA induction and proliferation but abolished Shc phosphorylation. These results suggest that the activation of Shc is not essential for c-fos activation and several tyrosine residues co-ordinate to activate c-fos activation. It is well documented that IL-3 or GM-CSF activates JAK2 in BA/F3 cells. However the role of JAK2 in IL-3/GM-CSF functions is largely unknown. We examined the role of JAK2 in GM-CSF-induced signaling pathways. Dominant negative JAK2 (delta JAK2) lacking the C-terminus kinase domain, suppressed IL-3/GM-CSF induced c-fos activation, c-myc activation and proliferation suggesting that JAK2 is involved in both signaling pathways. PTP1D and Shc are phosphorylated by IL-3/GM-CSF in BA/F3 cells, however these phosphorylation events were inhibited by expression of delta JAK2. Taken together, these results indicate that JAK2 is a primary kinase regulating all the known activities of GM-CSF. JAK2 mediates GM-CSF induced c-fos activation through receptor phosphorylation and Shc/PTP1D activation.
Insights
Granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling involves JAK2 kinase, which is crucial for cellular responses like c-fos activation and proliferation. JAK2 mediates GM-CSF effects through receptor phosphorylation and activation of downstream signaling proteins.
Area of Science:
- Cellular signaling pathways
- Cytokine receptor superfamily
- Protein tyrosine kinases
Background:
- Interleukin-3 (IL-3) and GM-CSF receptors (hGMR) are heterodimeric cytokine receptors.
- GM-CSF stimulation induces phosphorylation of the hGMR beta subunit and associated proteins.
- The precise role of Janus kinase 2 (JAK2) in GM-CSF signaling remains incompletely understood.
Purpose of the Study:
- To elucidate the role of specific tyrosine residues in the hGMR beta subunit in GM-CSF signaling.
- To investigate the involvement of JAK2 in mediating GM-CSF-induced cellular responses.
- To determine the relationship between JAK2 activity, receptor phosphorylation, and downstream signaling events.
Main Methods:
- Site-directed mutagenesis of the hGMR beta subunit to create specific tyrosine residue mutants.
- Analysis of GM-CSF-induced cellular responses, including c-fos and c-myc activation and cell proliferation.
- Expression of dominant-negative JAK2 (delta JAK2) to inhibit kinase activity.
- Assessment of protein phosphorylation, including Shc and PTP1D, using Western blotting.
Main Results:
- A membrane-distal region (a.a. 544-589) of the hGMR beta subunit, containing Tyr577, is critical for c-fos activation.
- Substitution of Tyr577 abolished c-fos activation but not GM-CSF-induced proliferation or c-fos mRNA induction, suggesting distinct signaling pathways.
- Dominant-negative JAK2 suppressed IL-3/GM-CSF-induced c-fos and c-myc activation and proliferation.
- JAK2 inhibition blocked GM-CSF-induced phosphorylation of Shc and PTP1D.
- JAK2 is identified as a primary kinase regulating GM-CSF signaling.
Conclusions:
- JAK2 is essential for GM-CSF-mediated cellular activities, including proliferation and gene activation.
- JAK2 mediates GM-CSF signaling through receptor phosphorylation and the activation of Shc and PTP1D.
- While Shc phosphorylation is not essential for c-fos activation, Tyr577 plays a specific role in this pathway.