Study Of The Molecular Mechanisms Regulating Signal Transduction And Cell Cycle In Human Lnx

LNX (Ligand of Numb-protein X) was originally isolated as a binding partner to the cell-fate determinant Numb during development, and then identified to act as a RING finger-type E3 ubiquitin ligase for the ubiquitylation and degradation of Numb. LNX protein includes domains as follows:an N-terminal RING finger domain which specifies LNX as an E3 ubiquitin ligase, the LDNPAY motif which is for Numb PTB domain-binding, and four PDZ domains which mediate protein-protein interactions. LNX protein is the first RING finger-type E3 ubiquitin ligase being found which acts as PDZ-domain-based scaffolds. Studies have approved that LNX could participate in signal transduction, such as Notch pathway, and play an important role in tumorogenesis.LNX contains 4 PDZ domains which are proved to play a central role in organizing diverse cell signaling assemblies. A yeast two-hybrid screen was used to identify LNX as a binding partner to RhoC. RhoC, a member of the Ras family, promotes reorganization of the actin cytoskeleton and regulation of cell shape, attachment, and motility. Co-immunoprecipation results testified the interaction between RhoC and LNX in mammalian cells, and clarified that the first PDZ domain of LNX was indispensable for the interaction between LNX and RhoC. The fluorescence co-localization assay showed that RhoC proteins changed its sublocalization from cytoplasm to nucleus when co-transferred with LNX. We also attentioned that co-expression of RhoC reduced the transcriptional activity of AP-1, which was up-regulated by over-expression of LNX alone. We speculate that LNX and RhoC are part of a larger protein complex that might have important functions in signaling transduction about regulating the transcriptional activities of AP-1.We demonstrate down-regulation of LNX results in GO/G1 cell cycle arrest in HEK293 cells. To elucidate the molecular mechanism of cell cycle arrest following LNX down-regulation, we employed microarray to investigate the genome-wide expression profile in LNX-inhibition cells and normal cells. Quantitative real-time PCR were used to confirm the differential expression patterns of ~30 transcripts involved in cell cycle and signaling identified by microarray. Combined with known information of online database about functions of relative genes, signal pathways and cell cycle machinery, analysis about the role of endogenous LNX involved in cell cycle was performed. The results demonstrate that down-regulation of LNX could result in cell cycle arrest in G0/G1 phase through inhibition ofβ-catenin, MAPK, c-Myc, NF-κB-dependent pathway and activation of TGF-β,p53-dependent pathway. This study provides novel insights into the pleiotropic activities of endogenous LNX protein, especially its crucial functions of E3 ubiquitin ligase and its essential role in cell proliferation, cell cycle and tumorogenesis.