Reciprocal cross-regulation between RNF41 and USP8 controls cytokine receptor sorting and processing

Abstract

SummaryThe mechanisms controlling the steady-state cell surface levels of cytokine receptors, and consequently the cellular response to cytokines, remain poorly understood. The number of surface-exposed receptors is a dynamic balance of de novo synthesis, transport to the plasma membrane, internalization, recycling, degradation and ectodomain shedding. We previously reported that the E3 ubiquitin ligase RING finger protein 41 (RNF41) inhibits basal lysosomal degradation and enhances ectodomain shedding of JAK2-associated cytokine receptors. Ubiquitin-specific protease 8 (USP8), an RNF41-interacting deubiquitylating enzyme (DUB) stabilizes RNF41 and is involved in trafficking of various transmembrane proteins. The present study identifies USP8 as a substrate of RNF41 and reveals that loss of USP8 explains the aforementioned RNF41 effects. RNF41 redistributes and ubiquitylates USP8, and reduces USP8 levels. In addition, USP8 knockdown functionally matches the effects of RNF41 ectopic expression on the model leptin and leukemia inhibitory factor (LIF) receptors. Moreover, RNF41 indirectly destabilizes the ESCRT-0 complex through suppression of USP8. Collectively, our findings demonstrate that RNF41 controls JAK2-associated cytokine receptor trafficking by acting as a key regulator of USP8 and ESCRT-0 stability. Balanced reciprocal cross-regulation of RNF41 and USP8 thus determines whether receptors are sorted for lysosomal degradation or recycling, this way regulating basal cytokine receptor levels.

Proteome-scale Binary Interactomics in Human Cells

“…The pXP2d2-rPAPImKate2 reporter gene plasmid was generated by replacing the firefly luciferase sequence in pXP2d2-rPAPI-luciferase (4) with the mKate2 encoding sequence (23). The pMet7-Flag-VPS52, pMet7-VPS52-Etag, pMet7-Flag-ASB6 and pMet7-Etag-ASB6 constructs were generated through an LR reaction (Invitrogen, Carlsbad, CA) to transfer the VPS52 or ASB6 ORF from a Gateway entry clone of the human ORFeome collection to a previously described pMet7-Flag or pMet7-Etag destination vector, respectively (21). pMet7-RNF41-Flag was generated by replacing the Etag coding sequence in pMet7-RNF41-Etag (20) by the Flag-tag sequence.…”

“…Through this regulatory mechanism it has been implicated in various signaling pathways, including the EGFR and TLR signal transduction cascades (29). Our group previously established that RNF41 also controls the sorting and processing of a number of JAK2-associated cytokine receptors, including the leptin, LIF and IL-6 receptors (20,21). It does so by modulating the ubiquitination status of USP8, which in turn regulates ubiquitination and stability of the ESCRT-0 complex involved in transport of internalized cytokine receptors toward multivesicular bodies for subsequent degradation in lysosomes.…”

“…RNF41-dependent ubiquitination and ensuing destabilization of USP8 results in ESCRT-0 ubiquitination and degradation, abolishing receptor sorting to the lysosomes. Instead, receptors are recycled to the plasma membrane leading to enhanced ectodomain shedding by ADAM proteases (21).…”

Endospanin 1 Determines the Balance of Leptin-Regulated Hypothalamic Functions

“…The interaction of ubiquitin ligase RNF41 with ObR and with deubiquitinase USP8 promotes the export of ObR to the plasma membrane, via the destabilization of the ESCRT-O endocytic machinery complex required for lysosomal degradation of cargos [68, 69]. More recently, the identification of other regulators of ObR trafficking has been extended to necdin and MAGEL2 proteins, which assemble into ubiquitination complexes and act jointly with RNF41 to regulate ObR levels at the plasma membrane (Fig.…”

Expression array analysis of the hepatocyte growth factor invasive program

“…RNF41 reportedly negatively regulates ErbB3 expression by the androgen receptor in androgen-dependent, but not androgen-independent, PCa cells [65]. RNF41 also negatively regulates the stability of the ubiquitin isopeptidase USP8, a regulator of JAK2-associated cytokine receptor sorting and processing: elevated RNF41 destabilizes the endosomal-sorting-complexes-required-for-transport (ESCRT)-0 complex (Hrs and signal-transducing-and adapter-molecule (STAM)-1 or STAM2), which results in cargo arriving at sorting endosomes being routed to recycling endosomes rather than to lysosomes [66]. By the same mechanism, USP8 also directs the trafficking and lysosomal degradation of CXCR4 [67], MET and epidermal growth factor receptor [68,69], implying that its loss consequent to increased RNF41 abundance would prolong and potentially amplify invasion signaling by these receptors.…”