Cbl ubiquitination of p85 is essential for Epo-induced EpoR endocytosis

Abstract

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-induced EpoR 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 allows p85 to interact with the endocytic protein epsin-1, thereby driving EpoR endocytosis. 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 internalization. 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 and EpoR internalization and normalizes Epo hypersensitivity. Our results uncover 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.

Figure 1

p85 becomes ubiquitinated upon Epo stimulation. (A) γ2A/HA-EpoR/JAK2 cells transiently expressing p85 and Flag-tagged Ub were stimulated with Epo for 15 minutes. Immunoprecipitated p85 under nondenaturing conditions was blotted with the indicated antibodies. (B) Ubiquitinated p85 was also detected by immunoprecipitation using FK2 antibody–conjugated beads under denaturing conditions and immunoblotting for p85. (C) Epo induces endogenous p85 ubiquitination in primary Ter119⁻ erythroid progenitor cells. FK2, anti-ubiquitinated proteins antibody; IP, immunoprecipitation; IB, immunoblot.

Figure 2

Epo induces p85 interaction with Cbl. (A) Cbl becomes tyrosine-phosphorylated upon Epo stimulation in γ2A/HA-EpoR cells expressing wild-type JAK2 but not kinase-deficient (KD) JAK2. Cell lysates were subjected to immunoprecipitation by Cbl antibodies and immunoblotted with 4G10 to detect phospho-tyrosine. (B) JAK2 phosphorylates Cbl in vitro. Cbl proteins immunoprecipitated from HEK293T cells were incubated with GST-tagged wild-type or kinase-deficient JAK2 kinase domain in in vitro kinase assay. J2(K), JAK2 kinase domain; J2(KD), kinase-deficient JAK2 kinase domain. (C) Epo induces p85 interaction with Cbl. γ2A/HA-EpoR/JAK2 cells transiently expressing Cbl and T7-tagged p85 were induced with Epo. p85 was immunoprecipitated with T7 antibodies, and the immunoprecipitates were probed with antibodies against Cbl. (D) Epo induces phosphorylation of Y⁷³¹ in the p85-binding motif of Cbl. (E) p85 domain structure. SH3, src-homology 3 domain; RhoGAP, RhoGAP homology domain; SH2, src-homology 2 domain; p110BD, p110 binding domain. (F-H) The C-terminal SH2 domain of p85 mediates inducible binding to EpoR upon stimulation. Biotin-tagged p85 fragments were transiently expressed in γ2A/HA-EpoR/JAK2 cells, isolated with streptavidin agarose, and probed with anti-HA to detect bound EpoR. (G) W622X, Q430X, and D327X are truncations where residues W⁶²², Q⁴³⁰, or D³²⁷ were mutated to a stop codon. IP, immunoprecipitation; IB, immunoblot. *Nonspecific bands.

Figure 3

Cbl and its E3 ligase activity are essential for Epo-induced EpoR internalization. (A) siRNAs for Cbl or control siRNAs were transfected into γ2A/HA-EpoR/JAK2 cells, and levels of surface EpoR before and after 45 minutes of Epo stimulation were analyzed by flow cytometry. For each sample, EpoR surface expression was normalized to that from samples expressing control siRNA before Epo stimulation. Knockdown efficiency of Cbl is shown. Immunoblotting of actin was used as a loading control. (B) Cbl knockdown in BaF3 cells impairs Epo-induced EpoR internalization. Cells expressing shRNA for Cbl or control shRNA, identified by GFP expression, were gated for analyses. The numbers in parentheses are median fluorescence intensity, which represents EpoR surface expression. Representative histograms from 3 independent experiments are shown. (C) Epo-induced EpoR internalization is impaired in γ2A/HA-EpoR/JAK2 cells expressing ligase-deficient Cbl mutants (ΔY368 and ΔY371). (D) Epo-induced EpoR internalization is significantly impaired by the expression of dominant-negative Cbl in Ter119^– erythroid progenitor cells. *P < .05, **P < .005 (Student t test).

Figure 4

Cbl ubiquitinates p85 in vitro. (A) T7-tagged p85 immunoprecipitated from HEK293T cells using anti-T7 antibody and Protein A beads was incubated with 200 ng of recombinant Cbl purified from BL21 in in vitro ubiquitination assay using recombinant E1, E2 (UbcH5B), and Flag-tagged wild-type Ub. After extensive washing, ubiquitinated p85 species were eluted from Protein A beads by SDS sample buffer and immunoblotted with the indicated antibodies. (B) p85 can be ubiquitinated at multiple sites. An in vitro ubiquitination assay was performed with a lysine-less ubiquitin mutant (KR) that cannot form ubiquitin chains. (C) p85 ubiquitination is lost in Cbl^−/− MEFs and is restored in Cbl^−/− MEFs reconstituted with Cbl.

Figure 5

Epo induces EpoR co-localization with epsin-1. (A) Endogenous epsin-1 becomes co-localized with wild-type EpoR upon Epo stimulation. Nonpermeabilized γ2A/HA-EpoR/JAK2 cells were stained with anti-HA antibodies to label the exofacial HA-tag of the EpoR as described previously. Cell were then treated with Epo for 12 minutes, fixed, permeabilized, immunostained for epsin-1, and visualized by confocal microscopy. The scale bar represents 5 μm. Representative co-localization areas are marked with arrows. (B) Epo induces p85 binding to epsin-1. ECFP-tagged epsin-1 and T7-tagged p85 were transiently expressed in γ2A/HA-EpoR/JAK2. Anti-T7 immunoprecipitates were immunoblotted with anti-GFP antibody to detect epsin-1. (C) EpoR-epsin-1 co-localization is impaired in cells expressing a ligase-deficient Cbl mutant. γ2A/HA-EpoR/JAK2 cells were transfected with either wild-type myc-tagged Cbl or Cbl(ΔY368). Forty-eight hours after transfection, cells were stained as described before, except Alexa 488 anti-myc antibody was used to mark transfected cells. The blue arrows indicate the transfected cells, and the white arrow shows co-localization in an untransfected cell. (D) Expression of epsin-1(ΔUIM) impaired EpoR internalization. Epo-induced EpoR internalization was determined by flow cytometry in γ2A cells co-transfected with vectors expressing HA-EpoR, JAK2, and ECFP-tagged epsin-1 (either wild-type or the ΔUIM mutant). Data were normalized to EpoR surface expression of unstimulated control cells. *P < .05 between epsin-1(WT) and epsin-1(ΔUIM). (E) BaF3 cells expressing HA-EpoR and epsin-1 (either wild-type or the ΔUIM mutant) were grown in RPMI media containing 2% fetal bovine serum (FBS) with different concentrations of Epo. Cell growth was measured using MTT assays. Cells grew similarly in WEHI media and 10% FBS. Epsin-1(ΔUIM)–expressing cells exhibit Epo hypersensitivity. **P < .005.

Figure 6

Appendage of p85-binding sites restores Epo-induced epsin-1 co-localization of a truncated EpoR that mimics those from PFCP patients in γ2A cells. (A) Schematic diagram of PFCP and rescue EpoR constructs. (B) PFCP EpoR did not co-localize with epsin-1 upon Epo stimulation. (C) Rescue EpoR appended with p85-binding sites rescued Epo-induced epsin-1 co-localization. The arrow points to a representative co-localization area.

Figure 7

Cbl knockdown results in hypersensitivity to Epo. (A) BaF3 cells expressing HA-EpoR and either Cbl shRNA or control shRNA were grown in RPMI media containing 2% FBS with different concentrations of Epo. Cell growth was measured using MTT assays. Cells grew similarly in control media containing no Epo but IL-3 (WEHI media) and 10% FBS. *P < .05, **P < .005. (B) Expression of Cbl shRNA but not control shRNA in primary Ter119^– erythroid progenitors resulted in Epo hypersensitivity in IMDM media containing 2% FBS and different concentrations of Epo. Cells grew similarly in control expansion media (2 U/mL Epo). *P < .05, **P < .005. (C) A working model of p85-mediated EpoR internalization. Epo stimulates binding of p85 to both Cbl and the EpoR through its SH2 domains, possibly forming a tripartite complex. Epo also induces Cbl ubiquitination of p85, which in turn recruits epsin-1 through its UIMs to further enlist the endocytic machinery for EpoR internalization (details are described in the Discussion).