EPO/EPOR signalling pathway is essential for the proliferation, differentiation, and survival of erythroid progenitors. Defects in EPO/EPOR signalling regulation cause myeloproliferative disorders, such as erythrocytosis, or polycythemia vera. Erythrocytosis is a disorder associated with truncations of the human EPOR resulting in deletions of 59-110 amino acids from the cytoplasmic tail of EPOR. The transformation capacity of JAK2V617F, a mutation responsible for myeloproliferative neoplasias is dependent on the presence of EPOR. Therefore, the elucidation of the mechanisms controlling EPO/EPOR signalling and proliferation/differentiation of erytlu'oid progenitors attracts interest. Ubiquitination and degradation is a common regulatory mechanisms affecting signalling from a variety of receptors. SOCS proteins regulate receptor signalling partly via their ubiquitin ligase (E3)-recruiting SOCS box domain. The aim of this study is to clarify the role of lysines important for EPOR ubiquitination in regulating EPO/EPOR f . signalling, erythroid cell proliferation and role of SOCS3 as ubiquitin ligase. Here, it is shown that K428 plays a key role in EPOR routing and demonstrate that the effects of SOCS3 on EPO/EPOR signalling depend on K428. Mutating K428 to arginine (K428R) blocked EPOR ubiquitination and enhanced stabilisation of the mature form of EPOR K428R in comparison to the wild-type receptor. This also leads to accumulation of EPOR in late endosomes and leads to sustained activation of ST AT5 and hypersensitivity to EPO leading to increased proliferation. SOCS3 was observed to ubiquitinate EPOR on this lysine and is important for trafficking the receptor from late endosomes to lysosomes. These findings clearly show that K428 is crucial for EPOR ubiquitination and attenuation of EPO responses. It was also observed JAK2V617F escapes SOCS3 inhibition by accumulating EPOR in late endosomes and utilising it for the constitutive signalling for transformation of the cells. This is the first demonstration of SOCS-mediated ubiquitination by targeting K428 and routing of EPOR to lysosomes.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:602404 |
| Date | January 2013 |
| Creators | Ahlawat, Anju |
| Publisher | Queen's University Belfast |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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