Rnf 11 sequestration of Smurf 2 on membranes antagonizes Smad 7 down-regulation of TGFβ signaling

The activity of the E3 ligase, Smurf2, is antagonized by an intra-molecular, auto-inhibitory interaction between its C2 and Hect domains. Relief of Smurf2 auto-inhibition is induced by TGFβ and is mediated by the inhibitory SMAD, Smad7. In a proteomic screen for endo-membrane 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 over-expressed in certain tumors, and, importantly, we find that depletion of this protein down-regulated gene expression of several TGFβresponsive genes, dampened cell proliferation, and dramatically reduced cell migration in response to TGFβ. Our data suggest for the first time that choice of binding partners for Smurf2 can sustain or repress TGFβ signaling and Rnf11 may promote TGFβ-induced cell migration. INTRODUCTION Endocytosis of plasma membrane-associated growth factor receptors can either attenuate or propagate and sustain receptor-mediated key signaling pathways. For example, downregulation of the Epidermal Growth Factor Receptor (EGFR) occurs through clathrindependent endocytosis. Here, cargo destined for degradation traffics first to the early endosome (also known as the sorting endosome) and is then sorted to the lysosome. Alternatively, activated EGF receptor internalized through clathrindependent endocytosis can be routed to the sorting endosome, where it continues to signal, or it can be recycled back to the plasma membrane directly or indirectly via recycling endosomes (1). Thus, clathrin dependent endocytosis of the EGFR functions both in the transduction as well as the termination of growth factor signaling. In contrast, the regulation of TGFβ signaling is http://www.jbc.org/cgi/doi/10.1074/jbc.M117.783662 The latest version is at JBC Papers in Press. Published on March 14, 2017 as Manuscript M117.783662 Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc. at H A C E T T E PE U N IV E R IT Y on M arch 3, 2017 hp://w w w .jb.org/ D ow nladed from Ring finger protein 11 sequesters Smurf2 on membranes 2 compartmentalized from its initial activation at the plasma membrane (2). At the plasma membrane, the receptor (TGFβR) is uniformly distributed between lipid rich micro-domains, termed lipid rafts, and non-raft regions. Following ligand engagement, the TGFβR-ligand complex can be endocytosed using two distinct mechanisms. In one pathway, ligand-receptor complexes internalized via clathrin-dependent endocytosis continue to actively signal on early endosomes through receptor-mediated phosphorylation of SMAD proteins. In a second pathway, internalization of ligand-receptor complexes residing in lipid rafts occurs via clathrinindependent/caveolin-dependent endocytosis, which is driven by ubiquitylation of the TGFβR by the Smurf2/Smad7 E3 ligase complex and which targets the receptor for degradation by the lysosome or the proteasome (2). Thus, in contrast with EGFR signaling, there is physical segregation of the molecular machineries that mediate the propagation and termination of TGFβ signaling (2), since endosomes generated by caveolindependent endocytosis of the activated TGFβR and the Smurf2/Smad7 complex are thought to bypass the sorting endosome en route to the lysosome. Mechanistically, compartmentalized activation of Smurf2 ligase in response to TGFβ stimulation is achieved by regulated assembly of a stable complex with Smad7 in the nucleus and subsequent recruitment to activated TGFβ receptors in lipid raft domains in the plasma membrane. This complex is stabilized by a protein-protein interaction between WW domains in Smurf2 and a PPXY motif in Smad7, generating an active E3 ligase that is essential for the termination of TGFβ signaling (3, 4). The modularity of PPXY and WW domains prompts speculation that an additional layer of regulation, such as the sequestration of Smurf2, could be achieved by other proteins harboring proline-rich motifs that reside outside of the nucleus. Under these circumstances, the net effect of this type of regulation would be a prolonged or sustained level of responsiveness to TGFβ, since less Smurf2 would be available for recruitment to the nucleus for complex formation with Smad7. Such a scenario could contribute to the deregulation of TGFβ signaling observed in disease (5). A strong candidate for this novel type of regulation is RING Finger protein 11 (Rnf11), a gene identified in screens for cDNAs encoding RING finger proteins and genes highly expressed in breast cancer (6,7). Rnf11 encodes a protein of 154 amino acids with several potential interaction domains, including a PPXY motif, a RING domain, ubiquitin interaction motif (UIM), and two acidic di-leucine or DXXLL motifs. In growing cells, Rnf11 displays a complex intracellular vesicular distribution, and it is thought to localize at steady state to the early endosome and recycling endosomes (8-11). Entry into these compartments is strictly regulated by multiple sorting determinants, which regulate its association with membranes and trafficking within the cell. First, acylation of Rnf11 was shown to be essential for association with endosomes, as mutation of a putative myristoylation site prevented its trafficking to cellular membranes (8, 9, 10). Analysis of both acidic di-leucine motifs also provided critical insight into how nascent Rnf11 enters the endosomal compartment. A mutation (Lc) in the carboxy-terminal DXXLL motif of Rnf11 (amino acids 144-148) led to Golgi accumulation, suggesting a defect in export from this organelle, whereas a mutation (Ln) in the amino-terminal DXXLL motif (amino acids 1216) and the double mutation (Lc/Ln) caused the protein to be restricted to the plasma membrane (11). Based on these and other data, it has been proposed that newly synthesized Rnf11 is directed to the plasma membrane from the sorting endosome, following its export from the Golgi, and re-entry into the endosomal compartment from the plasma membrane is thought to occur through clathrin-dependent endocytosis (CDE)(10, 11). There is also accumulating evidence that at H A C E T T E PE U N IV E R IT Y on M arch 3, 2017 hp://w w w .jb.org/ D ow nladed from Ring finger protein 11 sequesters Smurf2 on membranes 3 ubiquitylation of Rnf11 and/or its trafficking machinery could also regulate the subcellular localization of Rnf11 (8, 9, 10, 11). The intricate and strict controls that the cell has developed to establish the steady state localization of Rnf11 led us to test whether this might impact its function in the cell. In this report, we show for the first time that Rnf11 is able to block Smurf2-mediated repression of TGFβ signaling by sequestering Smurf2 in an endosomal compartment. We further show a key element of this regulation entails the proper sorting of Rnf11 to antagonize Smurf2 function. Moreover, we find for the first time that the Smurf2/Rnf11 complex is a functional E3 ligase. Most importantly, while Rnf11, like Smad7, can stimulate the enzymatic activity of Smurf2, these proteins exhibit antagonistic effects on silencing of TGFβ activity in vivo, due to sequestration of Smurf2 to different compartments. Furthermore, depletion of Rnf11 in the metastatic tumor cell line, 4T1 significantly mitigated TGFβ-responsive gene expression and cell migration. We propose that up-regulation of Rnf11, which has been observed in multiple types of cancer, could inappropriately sustain TGFβ signaling by sequestering Smurf2 in an endosomal compartment, thereby pre-emptively antagonizing the function of Smurf2/Smad7 complex in suppressing cellular TGFβ responsiveness, enhancing cell migration, and promoting the progression of these cancers.