Ubiquitination of EpoR and p85 in Ligand Induced EpoR Down-Regulation
| dc.contributor.advisor | Yin, Helen L. | en |
| dc.contributor.committeeMember | Huang, Lily | en |
| dc.contributor.committeeMember | Zhang, Chengcheng "Alec" | en |
| dc.contributor.committeeMember | Seemann, Joachim | en |
| dc.creator | Bulut, Gamze Betul | en |
| dc.date.accessioned | 2016-09-01T19:22:33Z | |
| dc.date.available | 2016-09-01T19:22:33Z | |
| dc.date.created | 2014-08 | |
| dc.date.issued | 2014-07-09 | |
| dc.date.submitted | August 2014 | |
| dc.date.updated | 2016-09-01T19:09:15Z | |
| dc.description.abstract | Erythropoietin (Epo) is the primary cytokine that drives red blood cell production and signals through its receptor, the EpoR, on erythroid progenitor cells. Epo binding to EpoR activates Janus kinase 2 (JAK2), which phosphorylates cytoplasmic tyrosines of the EpoR. Signaling proteins bind these phosphotyrosines through SH2 domains, leading to the survival and proliferation of erythroid progenitor cells and the differentiation of these progenitors into mature erythrocytes. Therefore, EpoR signaling is essential for red blood cell production. To maintain physiologic numbers of circulating red blood cells EpoR signaling is also subject to negative regulation. Mutations in EpoR or JAK2 that abrogate negative regulation cause erythrocytosis in hematological malignancies. Primary familial and congenital polycythemia (PFCP) is a proliferative disorder characterized by erythrocytosis and hypersensitivity of erythroid progenitors to Epo. Defects in negative regulation of EpoR signaling contribute to the etiology of PFCP. However, the underlying molecular mechanisms are poorly understood. Here we show that ubiquitination of EpoR controls internalization, lysosomal sorting, degradation and signaling of the EpoR. Ubiquitination of EpoR at K256 is necessary and sufficient for efficient Epo-induced receptor internalization, while ubiquitination at K428 promotes trafficking of activated receptors to the lysosomes for degradation. Interestingly, EpoR that cannot be ubiquitinated has reduced mitogenic activities and ability to stimulate the downstream signaling pathways. We propose that ubiquitination of the EpoR critically controls both receptor down-regulation and signaling. Secondly, we identified a novel mechanism mediating Epo-dependent EpoR internalization. Epo induces Cbl-dependent ubiquitination of the p85, which binds to phosphotyrosines on EpoR. Ubiquitination allows p85 to interact with epsin-1, thereby driving EpoR endocytosis. Knockdown of Cbl, expression of its dominant negative forms, or expression of an epsin-1 mutant all compromise Epo-induced EpoR internalization. Mutated EpoRs mimicking those from PFCP patients cannot bind p85, co-localize with epsin-1, nor internalize upon Epo stimulation and exhibit Epo hypersensitivity. Restoring p85 binding to PFCP receptors rescues Epo-induced epsin-1 co-localization, EpoR internalization, and normalizes Epo hypersensitivity. Our results uncover the role of EpoR ubiquitination and a novel Cbl/p85/epsin-1 pathway in EpoR endocytosis and show that defects in this pathway contribute to excessive Epo signaling and erythroid hyperproliferation in PFCP. | en |
| dc.format.mimetype | application/pdf | en |
| dc.identifier.oclc | 957676334 | |
| dc.identifier.uri | https://hdl.handle.net/2152.5/3581 | |
| dc.language.iso | en | en |
| dc.subject | Class Ia Phosphatidylinositol 3-Kinase | en |
| dc.subject | Endocytosis | en |
| dc.subject | Erythropoietin | en |
| dc.subject | Proto-Oncogene Proteins c-cbl | en |
| dc.subject | Receptors, Erythropoietin | en |
| dc.title | Ubiquitination of EpoR and p85 in Ligand Induced EpoR Down-Regulation | en |
| dc.type | Thesis | en |
| dc.type.material | text | en |
| thesis.degree.department | Graduate School of Biomedical Sciences | en |
| thesis.degree.discipline | Genetics and Development | en |
| thesis.degree.grantor | UT Southwestern Medical Center | en |
| thesis.degree.level | Doctoral | en |
| thesis.degree.name | Doctor of Philosophy | en |