Abstract
Ubiquitin modification of the TGF-β pathway components is emerging as a key mechanism of TGF-β pathway regulation. To limit TGF-β responses, TGF-β signaling is regulated through a negative feedback loop whereby the E3 ligase SMURF2 targets the TGF-β receptor (TβR) complex for ubiquitin-mediated degradation. Counteracting this process, a number of deubiquitinating (DUBs) enzymes have recently been identified that deubiquitinate and stabilize the TβR. However the precise mechanism by which these DUBs act on TβR function remains poorly defined. Here, we demonstrate that apart from targeting the TβR complex directly, USP15 also deubiquitinates SMURF2 resulting in enhanced TβR stability and downstream pathway activation. Through proteomic analysis, we show that USP15 modulates the ubiquitination of Lys734, a residue required for SMURF2 catalytic activity. Our results show that SMURF2 is a critical target of USP15 in the TGF-β pathway and may also explain how USP15 and SMURF2 target multiple complementary protein complexes in other pathways.
Highlights
The phosphorylation of R-SMADs in their terminus creates an interaction interface that permits them to oligomerize with the co-SMAD, SMAD44
As deubiquitinating enzymes (DUBs) USP11, USP15, and UCH37 all form a complex with SMAD7, we first assessed if DUB function is dependent on the ubiquitination of Tβ R-I by SMURF2, the E3 ligase, which binds to SMAD7 and targets the Tβ R complex for degradation
Co-expression of catalytically inactive SMURF2 completely abolished the ability of USP15 to activate this reporter (Fig. 1C)
Summary
The phosphorylation of R-SMADs in their terminus creates an interaction interface that permits them to oligomerize with the co-SMAD, SMAD44. The binding of SMAD7 to the HECT domain of SMURF2 abrogates the inhibitory intramolecular interactions between these domains, facilitating SMURF2 ubiquitin ligase activity[7]. Using a functional genetic screen we have previously found that USP15 forms a complex with SMAD7 and SMURF2 and is recruited to the TGF-β receptor complex, where it deubiquitinates and stabilizes Tβ RI9. In this scenario SMAD7 acts as an adaptor protein, interacting with two enzymes with opposing activities resulting in a constant balancing act between the ligase and DUB in regulating TGF-β output. We demonstrate that USP15 regulates ubiquitination of a number of lysine residues on SMURF2 including a key residue in the C-Lobe of SMURF2 which has previously been suggested to be required for ubiquitin transfer from the E2 to the catalytic cysteine in SMURF213
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