The Role Of SH₂ A Gene In The PKC Signal Transduction Passway

IntroducionThe Src homology 2 (SH₂) domain is a protein domain of about 100 amino - acid residues first identified as a conserved sequence region between the onco-proteins Src and Fps. Similar sequences were later found in many other intracel-lular signal - transducing proteins. SH₂ domains function as regulatory modules of intracellular signalling cascades by interacting with high affinity to phosphoty-rosine - containing target peptides in a sequence - specific and strictly phospho-rylation - dependent manner. SH₂ family plays an important role in signal trans-duction by tyrosine protein kinase. The expressional difference between tumor and tumor neighboring tissue suggested SH₂A might relate to tumor onset or development. 8p22 of human chromosome is a region rich in disease - related genes and is also the susceptible loci of oncogenes and tumor suppressor genes. In the previous work, we cloned SH₂A gene in this region by exon trapping and exon linking, which is a novel member of SH₂ family. Also, we constructed its recombinant expression vector, and investigated its function by cell transfection, kinase assay, subcellular localization and expression analysis. SH₂ domain is considered as "Protein Recognized Code". Besides these, the exact function of SH₂ A gene has not been reported yet. So we constructed the SH₂ - domain - deleted type, named with pcDNA3.1 - SH2A - Del, and the SH₂ - domain - mu-tagenic type, named with pcDNA3.1 - SH₂A - Mut, and made a further investigation on its function by cell transfection, kinase assay, and expression analysis. And then we tried to create the stable cell lines using neomycin (Geneticin).Materials1. Bel7402 cell line and C0S7 cell line;2. Molecular Biology Reagents of Gene Cloning;3. Reagents for Kinase Assay;4. Cell Transfection Kit.MethodsBy SOE - PCR method to amplify the coding sequence of SH₂ A gene including the mutagenic points, eukaryotic recombined expression vector, pcDNA3. 1- SH₂ A - Mut was constructed; in the manner of digestion by EcoR and self -ligation by T₄ ligase of the pcDNA3. 1 - SH₂A, the most part of the SH₂ domain was knocked out of the plasmid and pcDNA3. 1 - SH₂A - Del was constructed. Kinase assay was performed to examine PKC activity in the transfected cells, Bel7402 & COS7, by the method of CaPO₄ - DNA coprecipitation. After linearizing the plasmids, the COS7 cell line was transfected by lipofection, and neomy-cin (Geneticin ) was expected to use to create stable cell lines.ResultsThe coding sequence of SH₂A gene, a fragment of 896bp was got by SOE -PCR, which contains mutagenic point. Eukaryotic recombined expression vector, pcDNA3.1 -SH₂A — Mut was constructed. Eukaryotic recombined expression vector, pcDNA3. 1 -SH₂A -Del was constructed by means of enzyme cutting and self - ligation. After transfection, in both cell lines, Bel7402 & COS7, expressing SH₂A gene, the cytoplasm PKC activity decreased 27. 3% and 27. 8% compared with the control groups, but still no apparent alteration was found in membrane PKC activity. We have expected to create stable cell lines, COS7- SH₂ A as well as the mutagenic and the domain - deleted types. After using...