Self-regulatory Mechanism Of HECT-Type Ligase Smurf1

Ubiquitin-proteasome pathway plays crucial roles in biological processes such as transcriptional regulation, signal transduction, cell stress, protein degradation. Ubiquitin-proteasome pathway is a cascade of reaction catalyzed by ubiquitin-activation enzyme (E1), ubiquitin-conjugation enzyme (E2) and ubiquitin-protein liagse (E3). The roles of E3s are related with the selection of specific substrates and E2s, which determines specificity of the whole ubiquitin-proteasome pathway. E3s can be classified as HECT-Type and RING-Type. Previous reports indicate there are more RING-type E3s and less HECT-Type E3s. We are interested in Nedd4 family members which belong to HECT-Type E3s.Smurf1 (Smad ubiquitin regulatory factor 1) is a HECT domain ubiquitin ligase, which contains an amino-terminal phospholipids/calcium-binding C2 domain, two WW domains and a carboxy-terminal HECT domain. The main function of Smurf1 is to negatively regulate the bone formation. In C2C12 myoblasts, overexpression of Smurf1 can block the osteogenic conversion induced by BMP but has little impact on the ability of TGF-βto inhibit the myogenic differentiation through the ubiquitin-dependent degradation of Smad5; In Smurf1 ?/?mice, ubiquitin-dependent degradation phosphorylated MEKK2 by Smurf1 inhibits bone formation which depends on age ;Smurf1 functions in regulation of cell polarity and protrusion through the degradation of RhoA.We have reported that CKIP-1 can enhance not only the affinity of Smurf1 to substrates but also atuoubiquitination of Smurf1.The mechanism of atuoubiquitination of Smurf1 remain undiscovered.We demonstrated that the intermolecular interaction of Smurf1 which depended on activity of HECT-Type ubiqutin ligase can occur through different experiments such as degradation experiment, in vivo ubiquitination assay and half life period experiment. Smurf1 was subject to intermolecular interaction and intermolecular degradation both of which were mediated between the C2 and the WW domains. Furthermore, WW domain and C2 domain degraded by Smurf1 can be prevented by MG132. One paper reported the loss of C2 domain of Smurf1can not degrade Smad7 and Smad1.Our lab found C2 domain can enhance autoubiquitination of Smurf1.And the activity of the loss of C2 domain of Smurf1 did not disappear, but autoubiquitination of Smurf1 which lost C2 domain was lower. Taken together, we demonstrated a new activity regulation mechanism of Smurf1 which was fully different from Smurf2. The intermolecular regulatory mode of Smurf1 might represent a novel model of E3 activity control.