Investigation of the Role of JAK2 in SOCS1-mediated Granulocyte-macrophage colony-stimulating Factor Receptor Turnover

Our lab has recently shown that SOCS1 binds and degrades the granulocyte-colony stimulating factor receptor beta common subunit (GMRBc) in a proteasome-dependent manner. The focus of this project was to determine the role of JAK2 in SOCS1-mediated GMRBc turnover. Results: The binding of SOCS1 to GMRBc was observed in the JAK2-null gamma2A fibrosarcoma cell line. In TF1s, an erythroleukemic cell line that endogenously expresses GMRBc, transient knockdown of JAK2 resulted in the marked stabilization of GMRBc levels by thirty minutes. Using the kinase-dead K882E JAK2 mutant and the Y1007F JAK2 mutant, which abrogates SOCS1-binding as well as JAK2 activation because it is a key phosphorylation site, it was determined that the kinase activity of JAK2 is required SOCS1-mediated degradation of GMRBc. Lastly, SOCS1 has been predicted to bind Y468 on GMRBc.Conclusions: JAK2 is not necessary for SOCS1 to bind GMRBc; however, active JAK2 is necessary for substantial GMRBc degradation.M.Sc

SOCS-1 Mediates Ubiquitylation and Degradation of GM-CSF Receptor

<div><p>Granulocyte-macrophage colony-stimulating factor (GM-CSF) and the related cytokines interleukin (IL)-3 and IL-5 regulate the production and functional activation of hematopoietic cells. GM-CSF acts on monocytes/macrophages and granulocytes, and several chronic inflammatory diseases and a number of haematological malignancies such as Juvenile myelomonocytic leukaemia (JMML) are associated with deregulated GM-CSF receptor (GMR) signaling. The downregulation of GMR downstream signaling is mediated in part by the clearance of activated GMR via the proteasome, which is dependent on the ubiquitylation of βc signaling subunit of GMR via an unknown E3 ubiquitin ligase. Here, we show that suppressor of cytokine signaling 1 (SOCS-1), best known for its ability to promote ubiquitin-mediated degradation of the non-receptor tyrosine kinase Janus kinase 2 (JAK2), also targets GMRβc for ubiquitin-mediated degradation and attenuates GM-CSF-induced downstream signaling.</p></div

Knockdown of endogenous SOCS-1 in TF-1 cells promotes GMRβc stabilization and GM-CSF-induced downstream signaling.

<p>(A) Serum and cytokine starved TF-1 cells were treated with GM-CSF for the indicated times, lysed, immunoprecipitated (IP) using anti-GMRβc antibody, and immunoblotted with the indicated antibodies. (B) TF-1 cells transduced with lentivirus-shSOCS-1 or non-targeting scrambled shRNA (shScr) were lysed and immunoblotted with the indicated antibodies. (C) TF-1-shSOCS-1 and TF-1-shScr cells were treated with (+) MG132 or (–) DMSO for 4 h prior to immunoprecipitation (IP) with anti-GMRβc antibody and subsequent immunoblot analysis using the indicated antibodies. (D) Serum and cytokine starved TF-1-ShScr or TF-1-ShSOCS-1 cells were treated with GM-CSF for the indicated times, lysed and immunoblotted with the indicated antibodies. WCE: whole cell extract.</p

SOCS-1 preferentially binds to and ubiquitylates GMRβc.

<p>(A) HEK293 cells transfected with plasmids encoding GMRα and βc in combination with Flag-SOCS-1, -2, -3 or an empty plasmid (mock) were lysed, immunoprecipitated (IP) using anti-GMRβc antibody, and immunoblotted with the indicated antibodies. (B) HEK293 cells transfected with plasmids encoding GMRα, βc and HA-ubiquitin (HA-Ub) in combination with Flag-SOCS-1, -3 or an empty plasmid (mock) were treated for 4 h with (+) MG132 or (−) DMSO then lysed, immunoprecipiated (IP) using anti-GMRβc antibody, and immunoblotted with the indicated antibodies. (C) HEK293 cells transfected with plasmids encoding GMRα and βc in combination with Flag-SOCS-1, -SOCS-1▵SOCS-Box mutant, or empty plasmid (mock) were treated for 4 h with (+) MG132 or (–) DMSO then lysed, immunoprecipiated (IP) using anti-GMRβc antibody, and immunoblotted with the indicated antibodies. WCE: whole cell extract.</p