Screening For The Signal Transduction Molecule-PAK4 Binding Proteins By Yeast Two Hybridization

IntroductionTumor is among the most severe human diseases around the world threatening public health with dead rate keeping on increasing. Therefore, the study on the pathogenesis , diagnosis and therapy of tumor is a major subject in our county. By exploring the molecule mechanism of the key target proteins in cell signal transduction pathways , we know more about the genesis and development of the malignant tumor. Now most researchers are searching for the target proteins which are essential in the tumor therapy. So we may cure patients with cancer by targeting the proteins.P21 - activated kinase, one of the evolutionarily conserved families of ser-ine/threonine protin kinases , is a down - stream effector of small GTPase Cdc42/Rac, which have been implicated in a wide range of biological activities. Until now, PAK familie has 6 members which contain an N - terminal regulatory domain and a c - terminal catalytic domain. PAKs are now categorized into two subgroups based on architectrual similarities. Group I contains PAK1, PAK2, PAK3, Group II contains PAK4,PAK5 ,PAK6. There are many differences between the two PAK subgroups on the structrue, regulation of activity, biological function and PAKs may have different down - stream target proteins. We have known that PAK1 is effect on the genesis and development of tumor through the research on the down - stream target proteins. Meanwhile, many researchers reported several target proteins that can be interacted with PAK4,which have different interaction with Group I on the development of tumor. In order to explorethe biological character and function of PAK4, we screened PAK4 binding proteins from the human fetal brain cDNA library by using yeast two hybrid essay in this subjuct.Materials and methodsMaterialsReagents for gene cloningReagents for yeast two hybrid and extracts for yeast proteins and yeast plas-mids.MethodsConstructing the Bait Plasmids pGBKT7 - PAK4B/K Verifying Fusion Protein Expression in Yeast Strain Y187/AH109 Human Fetal Brain cDNA Library Amplification, Large - scale Transforming Yeast Strain AH109 with cDNA LibraryScreening PAK4 Binding Proteins by Yeast two — Hybrid Mating Screening for Ade+/His+/LacZ+ positive colonies and analyzing the se-quencResultsSDS - PAGE and Western blotting showed that fusion genes pGBKT7 -PAK4B/K can express fusion proteins in yeast strain Y187/AH109. Screening several Ade + /His + /LacZ + positive colonies which can be interacted with PAK4 by using yeast two - hybrid mating. Comparing the sequences with that of other proteins in NCBI, we got P24,P34,P18,P48 and P28.ConclusionsWe screened several proteins which can be interacted with PAK4 by using yeast two - hybrid system. They are close to cytoskeletal reorganization, cancer diversion, and protein ubiquitination.