Abstract 1109: c-Cbl ubiquitination of p85 is essential for EpoR downregulation.
Authors: Bulut Gamze B. , Huang Lily 
Published: August 2014

Abstract
Impaired deactivation of growth factor receptors is strongly associated with carcinogenesis. Among these receptors erythropoietin receptor (EpoR) is the main regulator for red blood cell production. EpoR−/− mice die of anemia, whereas constitutive EpoR signaling is oncogenic. Binding of Epo to the EpoR activates cytosolic JAK2 kinase, which in turn phosphorylates tyrosine residues in the EpoR cytoplasmic domain, to recruit and activate signaling proteins for erythropoiesis. Following this activation, EpoR is internalized and degraded to terminate the signaling. We have shown that Epo induces clathrin-dependent EpoR internalization and p85 regulatory subunit of PI3 Kinase (PI3K) plays a key role in this process. Interestingly, this mechanism does not require PI3K activity. One disease associated with this down-regulation mechanism is Primary familial and congenital polycythemia (PFCP). PFCP patients harbor truncated EpoRs, have increased red blood cell mass and their erythroid progenitors are hypersensitive to Epo. These truncated EpoR lack the binding site for p85 and restoring p85 binding site rescues EpoR internalization and corrects Epo hypersensitivity. However, the exact mechanism of how p85 aids in EpoR internalization is unclear.
We observed that Epo induces p85 mono-ubiquitination. To test if ubiquitination of p85 regulates EpoR internalization, we generated p85 truncation mutants to identify the domains required for binding to EpoR. We found that the C-terminal SH2 domain and p110 binding domain of p85 interact with the EpoR. c-Cbl-/- mice were reported to have erythroid hyperplasia and c-Cbl was shown to bind p85. We hypothesized that upon stimulation c-Cbl ubiquitinates p85, which recruits endocytic proteins such as Epsin for EpoR down-regulation. In an in vitro kinase assay, c-Cbl can be directly phosphorylated by wild-type but not kinase-deficient JAK2. In addition, Y731 of c-Cbl became phosphorylated upon Epo stimulation which is in the pYXXM p85 binding motif.
Importantly, Epo induced EpoR internalization was abolished in cells knock down of c-Cbl by siRNA or in mouse embryonic fibroblasts (MEFs) isolated from c-Cbl knockout animals. Two ligase-deficient c-Cbl mutants,ΔY368 and ΔY371 both had a dominant negative effect on the internalization of EpoR. Together, our results show that c-Cbl, and its ligase activity, play a crucial role in Epo-induced EpoR internalization.
Secondly, we observed that upon Epo stimulation EpoR co-localizes with Epsin1 on the plasma membrane. We examined if PFCP EpoR recruits Epsin1 upon Epo stimulation. Consistent with our hypothesis that Epsin1 is recruited to the EpoR by ubiquitinated p85, PFCP EpoR does not co-localize with Epsin1 on the plasma membrane upon Epo stimulation. Studying molecular players and mechanisms in EpoR down-regulation will shed light onto our understanding of hematological malignancies.
Citation Format: Gamze B. Bulut, Lily Huang. c-Cbl ubiquitination of p85 is essential for EpoR downregulation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1109. doi:10.1158/1538-7445.AM2013-1109