Investigation Of The Interaction And Function Between TRAF Adaptor Protein And CKIP-1/Smurf1

Smad ubiquitination regulatory factor 1 (Smurf1) belongs to the Nedd4 (neural precursor cell-expressed developmentally downregulated gene 4) family of HECT (homologous to E6AP C-terminus)-type E3 ubiquitin ligases. It was originally identified as an E3 ubiquitin ligase for Smad1 and Smad5, the intracellular signaling mediators of BMP pathways. Smurf1 also induces ubiquitination and degradation of phosphorylated MEKK2 (but not other MEKK members) to reduce the rate of bone formation in vivo in a Smad-independent manner. CKIP-1 (casein kinase 2 interacting protein-1) is a PH domain-containing protein which is predominantly localized at the plasma membrane. Most recently, our lab showed that Smurf1 interacted with CKIP-1, resulting in an increase of the E3 ligase activity of Smurf1. Surprisingly, CKIP-1 targets specifically the linker region between the WW domains of Smurf1, thereby augmenting its affinity for and promoting ubiquitylation of the substrate. These findings provide evidence that the WW domains linker is important in complex assembly and in regulating activity of HECT-type E3s and that CKIP-1 functions as the first auxiliary factor to enhance the activation of Smurf1.The tumor necrosis factor (TNF) receptor-associated factor (TRAF) family is a group of adaptor proteins that couple the TNF and Toll-like/IL-1 receptor family to diverse kinase cascades which ultimately lead to the activation of NF-κB (nuclear factor kappa B), JNK (Jun N-terminal kinase) and other signaling pathways. TRAFs can join in the regulation of cellular processes ranging from cell proliferation, innate and required immune system, embryonic development and differentiation to apoptosis. The TRAF domain is capable of binding to the cytoplasmic regions of receptors, and other proteins which contain the TRAF-interacting motif (TIM). CKIP-1 contains several potential TIMs. We identified strong interaction of CKIP-1 with TRAF4 and TRAF1 by co-immunoprecipitation assays in 293T cells. The C-TRAF domain of TRAF1 and TRAF4 mediated the interaction with CKIP-1. Furthermore, reporter gene assays showed that CKIP-1 interfered with the functions of TRAFs in NF-κB signaling. Overexpression of CKIP-1 can also enhance the NF-κB activity.Meanwhile, to search for novel substrates of Smurf1, we performed a yeast two-hybrid screening in the human brain library using its WW domain as the bait. We identified TNF receptor associated factor 4 (TRAF4) as a candidate and the interaction of Smurf1 with TRAF4 was further validated by co-immunoprecipitation assays in mammalian cells. The PY motifs of TRAF4 and the second WW domain of Smurf1 mediated their interaction. TRAF4 protein level and in vivo ubiquitination assays suggested that TRAF4 is a new substrate of Smurf1. CKIP-1 interacted with both Smurf1 and TRAF4 resulting in an increase in Smurf1 E3 ligase activity towards TRAF4. Consequently, Smurf1 interfered with the function of TRAF4 in NF-κB signaling. Collectively, these results suggested a new role of Smurf1 and CKIP-1 in controlling the degradation of TRAF4 and implied the involvement of Smurf1 in immune response.