Autoinhibition of the HECT-Type Ubiquitin Ligase Smurf2 through Its C2 Domain

Abstract

Ubiquitination of proteins is an abundant modification that controls numerous cellular processes. Many Ubiquitin (Ub) protein ligases (E3s) target both their substrates and themselves for degradation. However, the mechanisms regulating their catalytic activity are largely unknown. The C2-WW-HECT-domain E3 Smurf2 downregulates transforming growth factor-beta (TGF-beta) signaling by targeting itself, the adaptor protein Smad7, and TGF-beta receptor kinases for degradation. Here, we demonstrate that an intramolecular interaction between the C2 and HECT domains inhibits Smurf2 activity, stabilizes Smurf2 levels in cells, and similarly inhibits certain other C2-WW-HECT-domain E3s. Using NMR analysis the C2 domain was shown to bind in the vicinity of the catalytic cysteine, where it interferes with Ub thioester formation. The HECT-binding domain of Smad7, which activates Smurf2, antagonizes this inhibitory interaction. Thus, interactions between C2 and HECT domains autoinhibit a subset of HECT-type E3s to protect them and their substrates from futile degradation in cells.

Structural Basis for Interactions of thePhytophthora sojaeRxLR Effector Avh5 with Phosphatidylinositol 3-Phosphate and for Host Cell Entry

“…In plants, PtdIns(5)P is generated either by the dephosphorylation of PtdIns(3,5)P 2 or PtdIns(4,5)P 2 (46). Just like other phosphoinositides, PtdIns(5)P also has its own subcellular locations.…”

“…Phosphatidylinositol 4-phosphate (PtdIns(4)P), the precursor of PtdIns(4,5)P 2 , is the most abundant form of phosphoinositide in plant cells (46). It is localized in the plasma membrane as well as in the membranes of trans-Golgi networks (47).…”

“…In plants, Luna and colleagues have found that proper dephosphorylation of PtdIns(4)P is crucial for normal root hair growth (52). In addition, it has been reported that PtdIns(4)P may play an essential role in cytoskeletal networking in plants (46). Furthermore, Clark and colleagues have reported that POL, a phosphatase recruited to the plasma membrane via acylation as well as the essential protein for stem cell maintenance in Arabidopsis, can be activated by PtdIns(4)P in vitro, which tentatively suggests the important role of this phosphoinositide, together with other phospholipids, in regulating important developmental pathways in planta (53).…”

Ubiquitination of E3 ligases: self-regulation of the ubiquitin system via proteolytic and non-proteolytic mechanisms

“…16 Consistent with the concept of intramolecular modification, it appears that the self-ubiquitinating activity of ITCH and other HECT ligases like NEDD4-1, NEDD4-2, SMURF2, and WWP1, is regulated through intramolecular interactions that are modulated by modifications such as phosphorylation, and that involve the HECT domain. [17][18][19] In an attempt to decipher the biological role of self-ubiquitination, it was proposed that it serves to target the ligase for degradation, 11 which has been observed indeed as a means of negative feedback for Mdm2, 6,20 E6-AP, 21 CBL ligases, 22 and the substrate receptor subunits of CRL complexes. 23 Self-ubiquitination can occur in substrateindependent 24 (Figure 2a) and -dependent modes 22 ( Figure 2b).…”

To (TGF)β or not to (TGF)β: Fine-tuning of Smad signaling via post-translational modifications

“…Smad7 serves as an adaptor for Smurf2 ubiquitin ligase activity by presenting the ubiquitin E2 conjugating enzyme UbcH7 to the HECT domain of Smurf2 [99]. Recently, Wiesner et al presented impressive data suggesting that Smad7-Smurf2 binding also relieves an intramolecular interaction between Smurf2s HECT and C2 domains which normally serves to protect steady state levels of this ubiquitin E3 ligase and its substrates in cells [100]. The association of the Smad7-Smurf complex with active TGF-β receptors results in the ubiquitination and degradation of Smad7, the receptor, and Smurf itself, via proteasomal and lysosomal pathways [101][102][103].…”