Regulation, Novel Function And Crystal Structure Research Of Hect-type Ubiquitin Ligase Smurf1

Ubiquitin-proteasome pathway plays crucial roles in cellular physiological function through selective removal of misfolded and specific proteins. Protein ubiquitination process is catalyzed sequentially by ubiquitin-activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin protein ligase E3 to label substrate proteins with ubiquitin chains which promote their degradation in the 26S proteasome. Ubiquitin ligase E3s determine the substrate specificity during this process. Two main E3 families are characterized: RING (really interesting new gene) finger-type E3s and HECT domain (homologous to E6AP C-terminus)-type E3s. Knowledge of HECT type E3s is mainly obtained from research on Nedd4 family which exerts important functions in various signaling pathways including those induced by TGF-β, EGF, IGF, VEGF, SDF-1, TNF-αand other growth factors or cytokines.As a member of Nedd4 family, Smurf1 (Smad ubiquitination regulatory factor 1) was originally identified in 1999 as an E3 ligase of TGF-β/BMP pathway signaling molecules Smad1/5. RhoA, Runx2 and phosphorylated form of MEKK2 were subsequently found to be further substrates of Smurf1. Analysis of Smurf1 gene knockout and transgenic mice proved Smurf1 to be a negative regulator of bone formation, implicating that it might be a potential drug targets for bone diseases. However, whether and how its activity is regulated remains unclear.In 2005, one Science paper reported CKIP-1 (caseine kinase 2 interacting protein-1) as an interacting partner of Smurf1 in a high throughput screening for protein-protein interactions in the TGF-β/BMP signaling network. However, the biochemical and physiological functions of the interaction between CKIP-1 and Smurf1 has not been investigated. CKIP-1 was originally found to be an interacting protein of casein kinase 2. Our laboratory identified the CKIP-1 gene firstly in the establishment of human fetal liver transcriptome and secondly investigated its function and regulation for about a decade. Studies based on the molecular and cellular levels revealed that CKIP-1 is involved in regulation of cell apoptosis, differentiation, cytoskeleton reorganization and recruitment of ATM and CK2 to the plasma membrane. However, its physiological role in vivo is still unknown due to lack of research on animal levels.In this study, both the biochemical and physiological functions of the Smurf1-CKIP-1 interaction are thoroughly demonstrated. We first confirmed that CKIP-1 indeed specifically interacts with Smurf1 but not with other members of Nedd4 family, and enhances the E3 ligase activity of Smurf1. CKIP-1 functions as the first auxiliary factor to enhance the activation of Smurf1. Interestingly, CKIP-1 specifically targets the short linker region between the two WW domains of Smurf1 which mediate its interaction with substrates. The WW linker of Smurf1 consists of 15 amino acids and is distinct from that of other Nedd4 E3s. Through this manner, CKIP-1 enhances Smurf1 affinity with its substrates and promotes their ubiquitination and degradation. It is the first time to demonstrate that the short linker region between WW domains of Smurf1 could mediate the binding to its interacting protein; it is also the first time to show the importance of this short linker region for regulation of Smurf1 activity. This novel kind of regulatory mechanism is distinct from any known for HECT-type E3s. A long-standing question in understanding E3s is why closely related E3 members possess distinct substrate specificity and regulatory mechanism. Our results provide strong evidence that small differences in amino-acid sequence could accout for that specificity.We recently established the CKIP-1 gene knockout mice model. Phenotype analysis showed that CKIP-1-deficient mice undergo an age-dependent bone mass increase as a result of accelerated osteogenesis and decreased Smurf1 activity. These findings confirm the importance of the WW domains linker in complex assembly, regulating activity of HECT-type E3s and in bone mass control in vivo. CKIP-1, like Smurf1, ATF4 and Shn3, belongs to the small group of factors that regulate potential osteoblast activity. As far as we know, this is also the first time to characterize the physiological function of CKIP-1. The linker region of Smurf1 and the CKIP-1-Smurf1 interaction may thus serve as therapeutic targets for the treatment of bone diseases such as osteoporosis.In order to search for new substrates and/or regulators of Smurf1, we performed a yeast two-hybrid screening with the WW domains of Smurf1 as the bait and identified TRAF4 (TNF receptor associated factor 4) as one potential interacting partner. TRAF4 belongs to the TRAF family which functions as intracellular signaling adaptors to mediate NF-κB and JNK-AP-1 pathways and participate in bone metabolism, native and adaptive immunity and inflammation. Our further research showed that TRAF4 is a new substrate of Smurf1. When extended to other TRAF family members, we found that Smurf1 could promote their ubiquitination and reverse their negative or positive roles in NF-κB transcription activation. These results indicate novel functions of Smurf1 in immunity and inflammatory responses through targeting TRAF family members as its ubiquitination substrates.The C2 domain of Smurf1 not only affects its self-ubiquitination but also is important for its localization and function. Based on the crystal structure information of C2 We demonstrate that Smurf1-C2 belongs to classⅡtype and its molecular potential especially the regions for phospho-lipid binding is different from classical C2 domain of PKC. The knowledge of Smurf1-C2 structure will contribute to develop ment of clinical drugs that target C2 domain and affect functions of Smurf1.Taken together, we demonstrated for the first time that the short linker region between WW domains of Smurf1 exerts critical functions in complex assembly and in E3 activity regulation. CKIP-1 functions as the first auxiliary factor of Smurf1 and the interaction between CKIP-1 and Smurf1 is critical in bone mass control as indicated by gene knockout mouse models. Our research extends substrates of Smurf1 to TRAF family and indicates novel functions of Smurf1 in immunity and inflammation. Furthermore, we obtained the crystal structure of the C2 domain of Smurf1 which is beneficial for potential drug design.