Abstract
The activity of the E3 ligase, SMURF2, is antagonized by an intramolecular, autoinhibitory interaction between its C2 and Hect domains. Relief of SMURF2 autoinhibition is induced by TGFβ and is mediated by the inhibitory SMAD, SMAD7. In a proteomic screen for endomembrane interactants of the RING-domain E3 ligase, RNF11, we identified SMURF2, among a cohort of Hect E3 ligases previously implicated in TGFβ signaling. Reconstitution of the SMURF2·RNF11 complex in vitro unexpectedly revealed robust SMURF2 E3 ligase activity, with biochemical properties previously restricted to the SMURF2·SMAD7 complex. Using in vitro binding assays, we find that RNF11 can directly compete with SMAD7 for SMURF2 and that binding is mutually exclusive and dependent on a proline-rich domain. Moreover, we found that co-expression of RNF11 and SMURF2 dramatically reduced SMURF2 ubiquitylation in the cell. This effect is strictly dependent on complex formation and sorting determinants that regulate the association of RNF11 with membranes. RNF11 is overexpressed in certain tumors, and, importantly, we found that depletion of this protein down-regulated gene expression of several TGFβ-responsive genes, dampened cell proliferation, and dramatically reduced cell migration in response to TGFβ. Our data suggest for the first time that the choice of binding partners for SMURF2 can sustain or repress TGFβ signaling, and RNF11 may promote TGFβ-induced cell migration.
Highlights
Endocytosis of plasma membrane-associated growth factor receptors can either attenuate or propagate and sustain receptor-mediated key signaling pathways
Internalization of ligand1⁄7receptor complexes residing in lipid rafts occurs via clathrin-independent/caveolin-dependent endocytosis, which is driven by ubiquitylation of the TGFR by the SMURF21⁄7SMAD7 E3 ligase complex and which targets the receptor for degradation by the lysosome or the proteasome [2]
We propose that up-regulation of RNF11, which has been observed in multiple types of cancer, could inappropriately sustain TGF signaling by sequestering SMURF2 in an endosomal compartment, thereby preemptively antagonizing the function of SMURF21⁄7SMAD7 complex in suppressing cellular TGF responsiveness, enhancing cell migration, and promoting the progression of these cancers
Summary
Because all previous screens for RNF11-interacting proteins were yeast two-hybrid screens, and it was subsequently shown that RNF11 resides in an endosomal compartment, we initiated a proteomic screen in HEK293T cells to determine whether any known or novel interactions were localized to endomembranes. Cells transiently expressing C-terminally tagged RNF11 were lysed in a detergent-free isosmotic buffer to preserve protein interactions associated with membranes, and the resulting crude cytosol (S800) was fractionated by lowspeed centrifugation (20,000 ϫ g) to enrich for endosomal proteins (Fig. 2A). A striking feature of the set of Hect E3 ligases identified in our RNF11 endosomal proteomic screen is the presence of all known members of the NEDD4 family (Table 1). This group of enzymes shares a common set of motifs, including an N-terminal C2 domain, several WW domains, and a catalytic C-terminal Hect domain [13]. Depicted are the results of two proteomic screens, P20K pellet and crude cytosol (S800)
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