The Distribution Of SPAP2 In Mice And The Expression In Hela Cells

In the quantities of regulation mechanisms of cell signal transduction, thephosphorylation and dephosphorylation of proteins are of importance.Especially, phosphorylation of specific cellular proteins on tyrosyl residues isone of the most fundamental regulatory mechanisms in signal transduction thatdictates cell-cell communication, cell growth and proliferation, regulation ofcell cycle and differentiation, tumorogenesis and tumor metastasis, nerotranmission, angiogenesis, embryogeny, and kinds of hereditary andnon-hereditary diseases. The research of protein tyrosine phophorylation hasbeen hot spot in the field of biological science.SPAP2 is an intramembrane protein of immune receptor familiescontaining both ITIMs and ITAMs. The extracellular portion of SPAP2contains six immunoglobulin-like domains and its intracellular segment hastwo immunoreceptor tyrosine-based activation motifs (ITAMs) and twoimmunoreceptor tyrosine-based inhibition motifs (ITIMs). The SPAP2 genecontains 16 exons and is localized at human chromosome 1q21. A full-lengthcDNA of SPAP2 encodes a protein of 734 amino acid residues, intracellularsegment of which has 137 amino acid residues.SH2-domain containing PTP is a PTP sub-family with high conservativesequence,and this sub-family mostly includes three PTPs: SHP1,SHP2 anddrosophila generic Csw. SHP1 and SHP2 are highly homologous cytosolicprotein tyrosine phosphorylatase (sharing 60% overall sequence identity)consisting of two tandem SH2 domain, followed by a catalytic domain (PTPdomain).In spite of having highly homologous structures, they displayed adistinct tissue distributions and biological fuctions. SHP1 negatively regulatesa larger number of different signaling pathways. SHP2 positively regulatessignaling events from the activated receptor protein tyrosine kinases.The functions of SHP1 in cell signal transduction mostly contain:(1)The negative regulation in antigen receptor signal transductionprocess.(2)The positive regulation in apoptosis process mediated by as.(3)Both positive and negative regulation in JAK and STAT signaltransduction.(4)The positive regulation in MAP kinase activation pathway.The functions of SHP2 in cell signal transduction mostly contain:(1)The positive regulation transducer of MAP kinase activationpathway induced by factors.(2)The positive regulation in JAK and STAT signal transduction.(3)The positive regulator in synthesizing DNA and cell proliferation.(4)The negative regulator in K/p38 MAP kinase activation pathway.(5)The positive regulator in cell migration process.When stimulated and activated by signals fome outside , the tyrosylresidues of intracellular segment embedded in the two ITIM and twoITAM are phosphorylated, which induce SH2-domain containing PTPand PTK are translocalized from cytoplasm to membrane and bound to thephosphorylized ITIM and ITAM, followed by the signal transduction cascades.So The research of SPAP2 is necessary for representing the biophysicalfunctions of SH2-domain containing PTP and searching unkown SPAP2binding PTP and PTK.1. The functional research of SPAP2The expression changes were analysed both varieties of tissues in miceand development stages of peticular organs by Immunoprecipitition andWestern-blot, the result indicates the high expression in kidney , adrenal gland,lung ,pancreas and liver which implicate the high expression of SPAP2 may berelated to the regulation of glands' secretion. The intraperitoneal injection ofperoxovanadate into mice resulted within minutes in the appearance ofnumerous tyrosine-phosphorylated proteins in tissues. Then we find thebinding of SPAP2 and SHP1 in mice by Immunoprecipitition andWestern-blot. Then the level of tyrosine-phosphorylated is improved.2. The cloning of SPAP2 geneThe SPAP2 was cloned by means of gene engineering and constructedinto cloning vector pBluescript II KS, which was then sequenced and verified.Then we cut it and constructed into expression vector pcDNA3-SPAP2.It isthe most of all .At last we transfecte the Hela cells.3. The research about the binding proteins of SPAP2 in cell levelThe plasmid pcDNA3-SPAP2 was transfected into Hela cells. Then thebinding of SPAP2 and SHP1 was found in Hela cells by Immunoprecipititionand Western-blot. In order to build a stable cell lines that can express SPAP2stably, We definite the screening conditions of G418. It has establishedsubstructure for building the stable cell lines and other researchs about thebinding proteins of SPAP2.