| Summary: | 碩士 === 台北醫學院 === 細胞及分子生物研究所 === 89 === The Notch signaling pathway is a ubiquitous pathway that controls a variety of processes during development. Notch signaling pathway involves Notch receptors, Notch ligands, intracellular effectors and Notch modulators. The Notch family comprises a group of highly conserved proteins that function both as cell surface receptors and direct regulators of gene transcription.
The extracellular domain of Notch receptor is composed of epidermal growth factor-like repeats as well as three repeats designated LNR repeats. Further, the intracellular domain of Notch contains six copies of an ankyrin-like motif, NLS (nuclear localization signal) sequence, TAD (transcriptional activation domain), and PEST (glutamine, serine and threonine rich) domain. The full-length Notch receptor undergoes a proteolytic cleavage event upon ligand binding which potentiates the relocation of its intracellular domain (NICD) to the nucleus and interacts with the DNA binding protein CBF1 to activate transcription of genes that regulate cell differentiation. There are regulatory proteins interact with this NICD-CBF1 complex to function as activators (e.g mastermind) or repressors. The molecular mechanisms involved have been a focus of intense research and remain controversial.
We established a co-transfection assay system in order to search and study the mechanisms of novel factors that affect the CBF1-dependent Notch signaling. Four truncated human Notch1 molecules comparable to activated Notch molecules known to be functional in other systems were used in this study. The nucleotide position of these four truncated molecules in Notch1 receptor are following. (1) ANK repeats, nt 5461-6615 (amino acid 1820-2205); (2) RAM domain and ANK repeats, nt 5290-6615 (amino acid 1763-2205); (3) ANK repeats and the C-terminal TAD, nt 5461-7332 (amino acid 1820-2444); and (4) RAM+ANK+ TAD, nt 5290-7332 (amino acid 1763-2444).
The N-terminal of all truncated human Notch1 constructs were tagged with myc-tag, the C-terminal of ANK repaets and RAM+ANK were also tagged with His-tag. The cDNA fragments were then subcloned into the mammalian expression plasmid pcDNA3 to obtain four plasmids: pcDNA3/a, pcDNA3/ra, pcDNA3/ac, and pcDNA3/rac. The calcium phosphate precipitation method was used to transfcet plasmids into COS-7 and HEK293 cells. To assay for the activation of endogenous CBF1, besides plasmids of Notch1 construct, cells were cotransfected with p(FPⅢ)6 CAT reporter plasmid and the internal control plasmid pcDNA3.1/myc-his/LacZ.
The expression of truncated Notch proteins with correct molecular weights in COS7 and HEK293 cells were confirmed by Western blot analysis using anti-myc, anti-His antibodies, and anti-Notch1 C-terminal antibodies (ANK repeats≒60 kDa；RAM domain+ANK repeats≒70 kDa；ANK+C-terminal TAD≒95 kDa；RAM+ANK +TAD≒100 kDa). All transfceted cells were harvested 48 hours after DNA transfection and chloramphenicol acetyl transferase (CAT) activities were measured. Relative activation of the reporter construct was calculated as fold activation compared to empty vector (pcDNA3) transfection and normalized for the activity of b-galactosidase. Although the CBF1 protein with the human Notch1 ANK repeats failed to activate transcription, the activity was recovered by addition of either RAM domain or TAD. Relative to the control, CBF1 activated expression was increased ~3-fold by the RAM+ANK fragment, ~5-fold by the ANK +TAD, and ~12-fold by the RAM+ANK +TAD fragment. The result of CAT assay reflects the fact that the RAM domain is the primary binding domain to CBF1, ANK repaets interact weakly with CBF1, and TAD is important for transactivation activity.
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