Functional Study Of The Protein Kinase NLK That Interacts With The Testis Highly Expressed Protein RNF138 And The Function Study Of The Testis-specific Protein RSA-14-44

1. Exploring the function of NLK, a conserved serine/threonine protein kinaseThe transforming growth factor-?(TGF-?) family members including TGF-?isoforms, activins, bone morphogenetic proteins (BMPs), and growth and differentiation factors (PDFs), are the secreted cytokines that stimulate a broad array of cellular responses such as cell proliferation, differentiation, migration and apoptosis. Misregulation of these signaling involve in various diseases including fibrosis, malignant tumors, auto-immune diseases and vascular disorders. Thus, tightly controlling TGF-?signaling is essential for maintaining normal cellular responses and tissue homeostasis.Nemo-like kinase (NLK) is an evolutionally conserved serine/threonine protein kinase. It was demonstrated that Nemo could antagonize BMP signaling through phosphorylating MAD, the BMP effector in Drosophila, which promote exporting MAD from the nucleus to the cytoplasm. Recent study also showed that Smad4 was phosphorylated by NLK, suggesting that NLK might be involved in regulating TGF-P signaling. Thus, we certainly get new insight into the mechanism of TGF-P signaling pathway by exploring the roles of NLK in the phosphoryation of Smads.NLK associates with Smad3 in vivo, which was confirmed by the co-immunoprecipitation assay. Detailed analyses further showed that the MH2 domain, located in the C terminal of Smad3, was essential for its association with NLK. Endogenous Smad3 co-localizes with transiently expressed NLK in U2OS cells in response to TGF-?stimuli.Based on these information, we investigated the possible effects of NLK on Smad3-mediated transcription by a dual-luciferase reporter assay. NLK suppresses Smad3-mediated transcription in both HEK293T and HeLa cells. Using the RNAi technology, we knocked down the expression of NLK in HaCaT, HeLa, HepG2 and HCT-116 cells and confirm the inhibition of Smad3-mediated transcription by NLK. As expected, the protein level of p21, a classical target gene of Smad3, is elevated obviously.Down-regulated cell proliferation was further observed in the NLK knockout HCT-116 cells (NLK+/- and NLK-/-), which was confirmed by the results of plate colony formation assay and the soft agar assay. However, no significant change of apoptosis was observed in the NLK+/- and NLK-/- HCT-116 cells.2. Investigating the function of RSA-14-44, a testis-specific Rho GTPaseMammalian spermatogenesis is a paradigm for development in which the genetic information from male germ stem cells is re-edited, re-organized and finally distributed into spermatozoa while a dramatic cellular metamorphosis occurs in germ cells. This intricate process is built upon a system that features distinctive patterns of structure and regulation, most of which are the results of a unique and well-coordinated program of gene expression. Thus, identification and characterization of these differentially expressed genes has provided us additional insight into the unique mechanisms of spermatogenesis.In the previous study, we identified a testis-specific Rho GTPase named RSA-14-44, which associates with PSMB5, a catalytic subunit of the 20S proteasome during spermatogenesis. Detailed analyses showed that only the precursors of PSMB5 associate with RSA-14-44, which was not a direct regulator of proteasome activity. Furthermore, RSA-14-44 C190S resulted in the disruption of association between RSA-14-44 and PSMB5.In the present study, we clarified the effect of the C190S mutant on the location of RSA-14-44 and the possible roles of RSA-14-44 in the autocleavage of PSMB5.First, we found that in transfected MCF-7 cells, RSA-14-44 partially co-localize with PSMB5 in a perinuclear region. Furthermore, we compared the localization of the RSA-14-44C190S mutant with wide-type protein by immunofluorescence. In contrast to the perinuclear centered pattern of the wild-type protein, RSA-14-44c190S is dispersed in both nuclear and cytoplasmic regions. This dramatic difference resulting from the C190S mutation was further confirmed by the immunoblot analysis of subcellular fractions.To specify the possible effects of RSA-14-44 on the autocleavage of PSMB5, we co-expressed PSMB5 with various amounts of RSA-14-44. Surprisingly, the levels of PSMB5 precursors decrease significantly as a result of the increase in the doses of RSA-14-44; nevertheless, no disturbance was observed in the level of mature PSMB5. Following this clue, we measured the stability of PSMB5 precursor under transient expression of RSA-14-44 to determine the precise role of RSA-14-44 in down-regulating its protein level. Actually, the PSMB5 precursor is unstable and its stability is down-regulated dramatically by co-expression of RSA-14-44. In contrast, mature PSMB5 has a much longer life time and is less sensitive to the expression of RSA-14-44. Disrupting the association of RSA-14-44 and PSMB5 completely reversed the effect of RSA-14-44 on the stability of the PSMB5 precursor, which suggests this association is a prerequisite for RSA-14-44 regulation of proteasome biogenesis.