Functional Investigation Of SH₂A Gene

Introducion:Functional Genomics is to analyze the function of genes systematically by using the information of structural genomics, developing and utilizing new experimental tools. The most effective methods to identify gene function is to examine the phenotypic alteration of cells or the whole body after gene expression is increased or blocked.Another important aspect of functional genomics is the research on human disease - related genes. 8p22 of human chromosome is an 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. SH2 domain is considered as " Protein Recognized Code". SH2 family plays an important role in signal transduction by tyrosine protein kinase. The expressional difference between tumor and tumor neighboring tissue suggested SH2A may relate to tumor onset or development. Besides these, the exact function of SH2A gene has not been reported yet. To study the biological function of SH2A gene, we constructed its recombinant expression vec-tor, and investigated its function by cell transfection, kinase assay, subcellular localization and expression analysis.Materials and Methods1. Materials1) Bel7402 cell line and COS? ceU line2) Molecular Biology Reagents of Gene Cloning 3) Reagents for Northern Blot and RT - PCR4) Cell Transfection Kit5) Reagents for Kinase Assay2. Methods;RT - PCR method was used to amplify the coding sequence of SH2A gene. Eukaryotic recombined expression vector, pcDNAS. 1 -SH2A was constructed and then transfected Bel7402 cell and COS? cell by lipofetion. Multiple kinase assay was performed to examine PKC^MAPK.TPK activity in the transfected cells. Meantime, pEGFP - SH2A vector was also constructed and the cells transfected with it were examined by fluorescent microscopy. The expression of SH2A gene was examined under different concentration and time of bFGF as a stimulating factor.Results:A fragment of 896bp was got by RT - PCR, which contains the coding sequence of SH2 A gene. ( Genbank submission No. AY190323) Eukaryotic recombined expression vector, pcDNA3. 1 -SH2A and pEGFP - SH2A were constructed. In both cell lines ex-pressing SH2A gene, the cytoplasm PKC activity decreased 40% , but no apparent alteration was found in MAPK, TPK and membrane PKC activity. The proliferation index of transfected cells exhibited an decline in cytometry detection. SH2A gene was localized in the cytoplasm of transfected cells under the examination of fluorescent microscope. The expression of SH2 A gene was up - regulated under the stimulation of 50ng/ml bFGF at 24hours.Discussion:1. The effect of SH2A gene in cell signal transduction:SH2A is a novel member of SH2 signaling protein family. SH2 domain can specifically recognize phosphated tyrosine residue, and mediate receptor tyrosine protein kinase signal transduction. To investigate the role of SH2A in signal transduction, we constructed eukaryot-ic recombined expression vector. By cell transfection and kinase assay, we found that SH2A may be an inhibitor and play an important role in some key step of PKC signal transduction.PKC, an enzyme that is activated by the receptor - mediated hydrolysis of inositol phospholipids, can lead to the increase or disturbance of the transcription of some genes when stimulated persistently. Our previous work has show that SH2A may relate to tumor onset or development, and our current research suggests that SH2A gene participate tumor onset or development by altering the PKC activity in cytoplasm. The decline of proliferation index of transfected cells in cytometry detection also demonstrated that SH2A gene has an inhibitory effect in cell proliferation.During the stimulation of PKC, it is very important that PLCr isactivated first. SH2A protein expressed in the transfected cells may cause an inhibitory competition with PLCr for the S...