Functional study of gas7 in CNS neurons and the characterization of the phosphorylation of the protein
| Autor: | Yu-Ten Ju, 朱有田 |
|---|---|
| Rok vydání: | 1999 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 87 Growth arrest-specific (gas) genes are preferentially expressed in cells that have entered a quiescent state upon contact inhibition or the absence of required growth factors. The gas7 gene was identified using a promoter trapping vector from serum-starved NIH3T3 cells. The cDNA of gas7 (2875 bp) encodes an open reading frame of 421 amino acids with a predicted molecular weight of 48 kDa. By performing mouse multiple-tissue Northern Blot analysis, we found that the transcription of gas7 was expressed abundantly in the brain. A cDNA (cb1) was cloned from mouse brain cDNA library in which the first 60 nucleotides are different from that of the gas7. This suggests that gas7 gene has different mRNA species in the brain. Using RNA in situ hybridization on adult mouse brain sections, gas7 mRNA was further localized in Purkinje and granule neurons of the cerebellum. Immunohistochemistry on brain sections showed the same pattern compared to mRNA expression. GAS7 protein is expressed abundantly in cells that also express MAP II, a neuron cell marker; but expressed less in those cells express GFAP, a glial cell marker. Using the primary culture from mouse cerebellum and the antisense oligonucleotide technique, we found that gas7 antisense oligonucleotides can reduce the neurite numbers per neuron. Together with our findings that overexpression of GAS7 induces filopodia in NIH3T3 cells and a dendrite-like structure in neuroblastoma cells, we provide evidences suggesting that GAS7 may have a role in neurite formation during terminal differentiation of neuron cells. Besides, from the in vivo 32P-orthophosphate labeling studies, GAS7 protein is phosphorylated both in primary cultures of mouse cerebellum or in neuroblastoma cells transfected with gas7. Furthermore, I found that GAS7 protein was phosphorylated at the serine residues from phosphoamino-acid analysis. On the other hand, series of clones with truncated N and C-terminal of gas7 were also transfected into neuroblastoma cells. Cells were labeled with 32P-orthophosphate in order to map the phosphorylated regions of GAS7. The result indicates GAS7 is phosphorylated at its N-terminal region. To characterize which residues are phosphorylated sites, six highly conserved phosphorylation sites of serine were chosen for site directed mutagenesis. The serine residue located in 114th amino acid was identified as the phosphorylated serine. Because this peptide sequence of the serine phosphorylation site (RKXS) is highly conserved for the cGMP dependent protein kinase and PKB (Akt) substrate. The phosphorylation of GAS7 protein was reduced after PI 3-Kinase specific inhibitor (wortmannin) treatment. Cotransfection of GAS7 with constitutive active or kinase inactive Akt, a down stream effecter of PI 3-Kinase revealed that the phosphorylation of GAS7 is regulated by PI 3-Kinase signaling cascade. The function of PI 3-kinase is involved in inhibition of program cell death and cytoskeleton rearrangement. The cGMP dependent protein kinase plays an important role in controlling plasticity of neurons. The results suggest that the physiological significance of the phosphorylation of GAS7 may involve in controlling the viability or plasticity of neurons. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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