Screening For Proteins Interacting With SH2A, A New Signaling Molecule

ObjectiveSignal transduction is a complicated network which consists of interactions of protein. It researches into the molecular mechanism, which cells respond to outer stimuli by 'Signal transduction network'. As corpuscular nervous system, the complicated network of signal transduction controls corpuscular growth, differentiation and apoptosis. The dysfunction of the cellular signal transduction could result in many human diseases. The research of the cellular signal transduction has been a very important field to the future pharmaprojects. Therefore, it has been general to study protein interactions which are the main content of the signal transduction. Many methods of studying interactions of protein come into being because of the demand. Among them, the yeast two-hybrid system is currently most extensively used in the study of protein interactions, protein function domains, interaction with unknown protein and small molecules, as well as in the screening and designing targets of pharmacological therapy.In the previous work, we have cloned a new gene, SH2A, in the 8p22 of human disease genes region by exon trapping and exon linking. The expression product of this new gene was deduced to be is a novel member of SH2 signal protein family using SMART software. The Src homology 2 domain (or SH2 domain) is a protein domain of about 100 amino acid residues, typically bind to a phosphorylated tyrosine residue in the context of a longer peptide motif within a target protein, and SH2 domains represent the largest class of known pTyr-recognition domains. Proteins having SH2 domain play an important role in signal transduction of tyrosine protein kinase. SH2 domains mediate interactions of protein as 'recognition codes', which regulate the cells apoptosis, differentiation, proliferation and functions, and have closely relationship with the occurrence of some diseases. Considerable evidence indicated that the disorder of SH2 domains contributed to the development of many diseases. For this reason, elucidating the mechanism of interactions between proteins containing SH2 domains and other proteins will be very important for clinical parmaprojects. We have sub-located the SH2A protein in cytoplasm and we also found the wild type SH2A could strongly inhibit the activity of protein kinase C following transiently transfection. This suggested that SH2A might block the signal transduction of PKC pathway. To data, the exact function of SH2A gene was still not understood.To investigate the biological function of SH2A, determine the interacting protein in the cell signal pathway and elucidate the function of SH2A, in this work, we screened the human kidney cDNA library with baiting expressing vector pGBKT7-SH2A by yeast two-hybrid system and found some SH2A interacting protein. Thus provide further information on the biological function of SH2A in the cell signal transudation pathway.Methods1. Construct the pGBKT7-SH2A plasmid.2. Amplification of human kidney cDNA library.3. Co-transform yeast competent cell and screen for interacting protein of SH2A by yeast two-hybrid system.4. Select positive clones to sequence and BLAST analysis.5. Predict the sites of tyrosine phosphorylation of the five proteins.Results1. Cloned SH2A into pGBKT7 vector containing BD to construct pGBKT7- SH2A bait protein expression vector.2. Amplified the human kidney cDNA library.3. Extracted the human kidney cDNA library and pGBKT7-SH2A plasmids, co-transformed yeast competent cell, selected 61 clones following the culturing in SD/-Ade/-His/-Leu/-Trp and obtained 46 positive clones by the SD/-Ade/-His/-Leu/-Trp/X-α-Gal detection.4. The positive clones were sequenced and underwent BLAST analysis. Five SH2A interacting proteins (alpha-2- glycoprotein 1, zinc,AZGP1; defender against cell death 1, DAD1, hydroxysteroid 17-beta, HSD17B10; HIV-1 Tat interactive protein, 60kDa, HTATIP and pyruvate kinase, muscle, PKM2) and two non-coding sequences (CARD14 and RAS-like, estrogen-regulated, growth inhibitor) were identified.5. Predict the sites of tyrosine phosphorylation of the five proteins screened by yeast two-hybrid system. Except the PKM2 without the sites of tyrosine phosphorylation, the others, AZGP 1,DAD1, HSD 17B 10 and HTATIP, all have the sites of tyrosine phosphorylation.Conclusions1. Applied the yeast two-Hybrid system in protein interactions study.2. Screened five SH2A interacting proteins.