CKIP-1Couples Smurf1Ubiquitin Ligase With Rpt6Subunit Of26s Proteasome To Promote Substrate Degradation

Smurf1(Smad ubiquitylation regulatory factor1) belongs to HECT (homologousto the E6-accessory protein C-terminus)-type E3ligase, which promotes theubiquitylation and degradation of target proteins, such as Smad1/5, MEKK2, RhoA,Runx2, Prickle-1and TRAF6. Smurf1is a key negative regulator of TGF-β/BMPsignaling pathway and plays important roles in cell adhesion, embryonic development,bone formation and neuron growth. Previously, we discovered that CKIP-1(caseinkinase2interacting protein-1) acts as an activator for Smurf1to promote the substratedegradation. We also showed that CKIP-1targets specifically the linker regionbetween the WW domains of Smurf1. Moreover, CKIP-1-deficient mice undergo anage-dependent increase in bone mass as a result of accelerated osteogenesis anddecreased Smurf1activity.However, how the substrates and Smurf1itself are recognized and degraded by26S proteasome is not clear. Our yeast two-hybrid screening of human adult brainlibrary using the full length CKIP-1as bait identified a potential interacting proteinRpt6. The26S proteasome consists of two basic parts: a20S proteolytic core complexand one or two19S regulatory particle(s). The19S regulatory particle can be furtherdivided into base and lid subunits. Rpt6, which contains406amino acids, belongs tobase subunits. Rpt6and other Rpt members arrange heterohexameric ring and attachat β ring which belongs to20S proteolytic core complex. The base unfolds substrateand translocates them into the proteolytic20S proteasome.First of all, we identify CKIP-1-Rpt6interaction and determine that theCoiled-coil domain of Rpt6interacts with the leucine zipper (LZ) domain of CKIP-1.We detect an interaction between Smurf1and Rpt6in HEK293T cells no matter theexogenous or endogenous expression of Smurf1and Rpt6. We also observe that theinteraction of Smurf1and Rpt6is enhanced when CKIP-1is co-expressed. When weknockdown CKIP-1，we find the CKIP-1siRNA significantly impaired the associationof endogenous Smurf1and Rpt6. Thus, we hypothesize that the relationship ofSmurf1and Rpt6might rely on CKIP-1. Rpt6can promote the degradation of Smurf1and its substrate. We find that knockdown of endogenous Rpt6results in an increasein the steady-state levels of endogenous Smurf1. It also demonstrates that thedegradation of Smurf1depends on26S proteasome. This is the first time to researchthe relationship between Nedd4E3and26S proteasome.