Regular Article RED CELLS, IRON, AND ERYTHROPOIESIS Cbl ubiquitination of p85 is essential for Epo-induced EpoR endocytosis

internalization is mediated through a novel Cbl/p85/ epsin-1 pathway. Mutated EpoR in primary familial and congenital polycythemia patients cannot activate this pathway, exhibiting excessive Epo signaling. Erythropoietin (Epo) binding to the Epo receptor (EpoR) elicits downstream signaling that is essential for red blood cell production. One important negative regulatory mechanism to terminate Epo signaling is Epo-inducedEpoR endocytosis and degradation. Defects in this mechanism play a key role in the overproduction of erythrocytes in primary familial and congenital polycythemia (PFCP). Here we have identified a novel mechanism mediating Epo-dependent EpoR internalization. Epo induces Cbl-dependent ubiquitination of the p85 regulatory subunit of PI3K, which binds to phosphotyrosines on EpoR. Ubiquitination allowsp85 to interactwith the endocytic protein epsin-1, therebydrivingEpoRendocytosis. Knockdown of Cbl, expression of its dominant negative forms, or expression of an epsin-1 mutant devoid of ubiquitin-interacting motifs all compromise Epo-induced EpoR in-ternalization. Mutated EpoRs mimicking those from PFCP patients cannot bind p85, co-localize with epsin-1, or internalize on Epo stimulation and exhibit Epo hypersensitivity. Similarly, knockdown of Cbl also causes Epo hypersensitivity in primary erythroid progenitors. Restoring p85 binding to PFCP receptors rescues Epo-induced epsin-1 co-localization andEpoR internalization and normalizes Epohypersensitivity.Our results uncover a novelCbl/p85/epsin-1 pathway in EpoR endocytosis and show that defects in this pathway contribute to excessive Epo signaling and erythroid hyperproliferation in PFCP. (Blood. 2013;122(24):3964-3972