Biochem. J. (2011) 435, 509–518 (Printed in Great Britain) doi:10.1042/BJ20101876 509 Cytosolic lysine residues enhance anterograde transport and activation of the erythropoietin receptor Liron YOSHA, Orly RAVID, Nathalie BEN-CALIFA and Drorit NEUMANN 1 Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel Lysine residues are key residues in many cellular processes, in part due to their ability to accept a wide variety of post-translational modifications. In the present study, we identify the EPO-R [EPO (erythropoietin) receptor] cytosolic lysine residues as enhancers of receptor function. EPO-R drives survival, proliferation and differentiation of erythroid progenitor cells via binding of its ligand EPO. We mutated the five EPO-R cytosolic lysine residues to arginine residues (5KR EPO-R), eliminating putative lysine- dependent modifications. Overexpressed 5KR EPO-R displayed impaired ubiquitination and improved stability compared with wt (wild-type) EPO-R. Unexpectedly, fusion proteins consisting of VSVGtsO45 (vesicular stomatitis virus glycoprotein temperature- sensitive folding mutant) with wt or 5KR EPO-R cytosolic domains demonstrated delayed glycan maturation kinetics upon substitution of the lysine residues. Moreover, VSVG-wt EPO-R, but not VSVG-5KR EPO-R, displayed endoplasmic reticulum- associated ubiquitination. Despite similar cell-surface EPO- binding levels of both receptors and the lack of EPO-induced ubiquitination by 5KR EPO-R, the lysine-less mutant produced weaker receptor activation and signalling than the wt receptor. We thus propose that EPO-R cytosolic lysine residues enhance receptor function, most probably through ubiquitination and/or other post-translational modifications. Key words: endoplasmic reticulum, post-translational modifica- tion, signalling, trafficking, ubiquitin, vesicular stomatitis virus glycoprotein. INTRODUCTION Lysine residues are key residues in many cellular processes. Owing to their positive charge, they play a major role in protein structure and function, often stabilizing protein–protein interactions or catalytic domains through non-covalent bonds [1,2]. In addition, lysine residues have a more specific role, directed by their ability to undergo different post-translational modifications (e.g. ubiquitination, acetylation, etc.) [3–5]. Lysine-dependent post-translational modifications have been implicated in protein metabolism and function; however, the nature and extent of their role in these processes remain largely unresolved. The EPO-R [EPO (erythropoietin) receptor] is unique among cytokine receptors owing to its strong intracellular retention and short half life, the underlying molecular mechanisms of which have not yet been fully resolved. We therefore questioned the involvement of the cytosolic lysine residues of EPO-R in receptor metabolism and activation. EPO-R is a type I transmembrane protein ranging between 59 and 70 kDa [6,7] which belongs to the cytokine receptor superfamily [8]. Its ligand, EPO, is required for the survival, proliferation and differentiation of committed erythroid progenitor cells [9]. Members of the cytokine receptor family are devoid of intrinsic kinase activity and therefore rely on an associated tyrosine kinase(s) to initiate downstream signalling. EPO binding to cell-surface EPO-R results in activation of JAK2 (Janus kinase 2), the main tyrosine kinase associated with EPO- R, which undergoes autophosphorylation and then proceeds to phosphorylate the EPO-R homodimer at multiple cytoplasmic tyrosine residues [10]. Subsequently, different downstream effectors are recruited to the phosphorylated receptor, such as the transcription factor STAT5 (signal transducer and activator of transcription 5), MAPK (mitogen-activated protein kinase), and the p85 regulatory subunit of PI3K (phosphoinositide 3-kinase) [11]. Concurrently, down-regulation pathways are activated, resulting in EPO-R dephosphorylation and degradation, mainly in the lysosome, as part of a classic regulatory loop [12–16]. Unlike other cytokine receptors (e.g. [17]), a striking property of the EPO-R is its enigmatically low expression at the cell surface, whereas most of the newly synthesized EPO-R remains sequestered intracellularly [18]. These metabolic features are similar in both transfected [18] and fetal liver cells that express EPO-R endogenously [19], supporting the idea that intrinsic features of the receptor molecule regulate its surface expression. Sequences in both the extracellular and intracellular domains of EPO-R have been documented to affect anterograde transport of the receptor from the ER (endoplasmic reticulum) [20–23]. Ubiquitination is a widely studied lysine-dependent post- translational modification. First discovered as a signal for proteasomal degradation of ubiquitin-tagged proteins, it has since been found to participate in modulation of a wide range of cellular processes, including transcription, DNA repair, endocytosis, endosomal sorting and viral infection [24–26]. Ubiquitination of EPO-R has been demonstrated in different contexts [15,16,27–30]. Most of the studies link EPO-R ubiquitination to down-regulation of receptor signalling; upon activation of the receptor with EPO, the E3 ubiquitin ligase β - TrCP (β -transducin repeat-containing protein) ubiquitinates the Abbreviations used: CHX, cycloheximide; DMEM, Dulbecco’s modified Eagle’s medium; DOC, deoxycholate; Endo H, endoglycosidase H; EPO, erythropoietin; EPO-R, EPO receptor; ER, endoplasmic reticulum; FCS, fetal calf serum; FLRF, fetal liver ring finger; GFP, green fluorescent protein; HA, haemagglutinin; HEK, human embryonic kidney; IL, interleukin; JAK2, Janus kinase 2; NEM, N-ethylmaleimide; PIC, protease inhibitor cocktail; PI3K, phosphoinositide 3-kinase; rHuEPO, recombinant human EPO; STAT5, signal transducer and activator of transcription 5; TBS, Tris-buffered saline; TBS-T, TBS with Tween 20; TCL, total cell lysate; β-TrCP, β-transducin repeat-containing protein; VSVG, vesicular stomatitis virus glycoprotein; wt, wild-type; YFP, yellow fluorescent protein. 1 To whom correspondence should be addressed (email histo6@post.tau.ac.il). c  The Authors Journal compilation c  2011 Biochemical Society www.biochemj.org Biochemical Journal