LOSS OF JAK2 REGULATION VIA VHL-SOCS1 E3 UBIQUITIN HETEROCOMPLEX UNDERLIES CHUVASH POLYCYTHEMIA

Chuvash polycythemia (CP) is a rare congenital form of polycythemia caused by homozygous R200W and H191D mutations in the von Hippel-Lindau (VHL) gene whose gene product is the principal negative regulator of hypoxia-inducible factor. However, the molecular mechanisms underlying some of the hallmark features of CP such as hypersensitivity to erythropoietin are unclear. Here, we show that VHL directly binds suppressor of cytokine signalling 1 (SOCS1) to form a heterodimeric E3 ligase that targets phosphorylated (p)JAK2 for ubiquitin-mediated destruction. In contrast, CP-associated VHL mutants have altered affinity for SOCS1 and fail to engage and degrade pJAK2. Systemic administration of a highly selective JAK2 inhibitor, TG101209, reverses the disease phenotype in vhlR200W/R200W knock-in mice, a model that faithfully recapitulates human CP. These results reveal VHL as a SOCS1-cooperative negative regulator of JAK2 and provide compelling biochemical and preclinical evidence for JAK2- targeted therapy in CP patients

Regulation of Cellular Oxygen Sensing Pathways by VHL

Erythropoiesis represents a vital physiologic process that can be adjusted to combat compromised oxygen availability, otherwise known as hypoxia. The canonical response to adapt to hypoxia is mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1). When oxygen is available HIFalpha subunits are hydroxylated by prolyl hydroxylase enzymes (PHD1-3) to allow for binding to the von Hippel-Lindau (VHL) tumour suppressor protein, which serves as the substrate recognition subunit of an E3-ubiquitin ligase complex that ultimately targets proteins for proteasome-mediated degradation. The work presented in this thesis demonstrates a novel role of VHL in recruiting DCNL1 to initiate E3-ubiquitin ligase enzymatic activity which, in essence, allows for the degradation of HIFalpha to commence. In hypoxia, hydroxylation does not take place and as a result HIFalpha can promote the transactivation of a repertoire of genes that aid in adaptation to hypoxia including one which encodes the glycoprotein hormone erythropoietin (EPO). EPO binds to EPO receptor (EPOR) expressed on erythroid progenitor cells to promote their proliferation and differentiation into red blood cells. An erythroid specific oxygen sensing pathway is elucidated in this thesis wherein EPOR is hydroxylated by PHD3 to promote VHL-mediated degradation in a manner analogous to HIFalpha. Combined, these findings build upon our fundamental understanding of how cells detect and counter hypoxic stress.Ph.D.2017-03-31 00:00:0

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

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