The Roles Of NEDD4 In CD40 Signaling

NEDD4 (Neuronal precursor cell Expressed Developmentally Downregulated 4) is an E3 ubiquitin ligase which contains the HECT (Homologous to E6-AP C-Terminus) domain and conserves from yeast to human. NEDD4 consists of an N-terminal C2 domain, 2-4 WW domains, and a C-termianl HECT-type ligase domain and binds to PY (including LPxY and PPxY) modifs in substrate proteins including PTEN, Rap2A, Cbl-b, Gag and NEaC. NEDD4 regulates growth factor receptor, T cell function, neural development, virul budding and so on. Base on finding that NEDD4 is in CD40 signaling complex which is used with moden organic mass spectrometry in A20 cell but there were no report of NEDD4 function in CD40 signaling. We further investigate the roles of NEDD4 in CD40 pathway. With co- immunoprecipitation, we find that NEDD4 can interact with TRAF3 which is one of most important adaptor in CD40 signaling but not TRAF2 in HEK293 and A20 cells. Overexpression of NEDD4 in HEK293 cells promotes the polyubiquitination of TRAF3 but not TRAF2 indicating that NEDD4 is the ubiquitin E3 ligase of TRAF3. Assessment of p-P38 levels between A20/control vector and A20/NEDD4, A20/control siRNA and A20/NEDD4 siRNA and NEDD4+/+ and NEDD4-/- na?ve B cell showed that NEDD4 affected p38 activity. Because of perinatal lethality of NEDD4 KO mice, to study how the loss of NEDD4 affects B cell function, we generated fetal liver chimeras by transferring NEDD4+/+ and NEDD4-/- fetel liver cells into lethally irradiated C57BL/6 recipient mice. We assessed the survival and proliferation of na?ve B cells which were isolated from spleens by CD43(Ly-48)MicroBead with PI and CFSE. But there are no distinction between NEDD4+/+ and NEDD4-/- na?ve B cells. To our surprise, the percentage of na?ve B cells of NEDD4-/- is sharply increased in spleen lymph-cells. These findings indicate that NEDD4 promotes the ubiquitination of TRAF3 and affects the CD40 dependent MAPK signaling.