Interaction Of Tfpi-2 With Rassf1c And Role Of Sok1 On Breast Cancer Cell Migration

Tissue factor pathway inhibitor-2 (TFPI-2), also named as placenta protein-5 (PP5) and matrix associated serine protease inhibitor (MSPI), is a protease inhibitor with Kunitz domains and belongs to serine protease inhibitor superfamily. The mature protein contains a short acidic amino-terminal region, three tandem Kunitz domains and a carboxyl-terminal tail highly enriched in basic amino acids. Previous studies showed that TFPI-2 could inhibit several proteases activity including trypsin, plasmin, chymotrypsin, plasma kallikrein, cathepsin G and a wide variety of matrix metalloproteases(MMPs). TFPI-2, with the similar structure and function of tissue factor pathway inhibitor-1 (TFPI-1), is also be recognized as an important endogenous anticoagulant factor and shows the inhibition to FVIIa/TF complex and FXa. But the biological functions of TFPI-2 are still unclear. Especially, the interactions of TFPI-2 with other proteins haven't been revealed well.We constructed the bait vector pGBKT7/TFPI-2 and screened the candidate proteins from hFB-cDNA library by use of Matchmaker two hybrid system3 and analysis method of bioinformatics. We fished 95 proteins which displayed different roles in the different biologic processes from hFB-cDNA library by use of pGBKT7/TFPI-2 as bait vector. Such proteins included 32 metabolism associated proteins (8 proteometabolism proteins,13 nucleic acid metabolism proteins and 11 glycometabolism proteins),21 signal transduction associated proteins, five Cell growth and/or maintenance associated proteins, seven transport associated proteins, two immune response or apoptosis associated proteins and 28 proteins with unknown function. By advanced analysis from cell location, known protein function and the possibility to express the inserted protein correctly under such yeast two hybrid system, we finally harvested eight proteins associated with the potential interaction with TFPI-2.We choose two proteins RASSF1C and the ASB in order to determine the interactions with TFPI-2. We success at copying the gene of ASB, but while using PCR detected 36 positive clones,there were no target strips at all. Therefore, the purpose of this experiment is just to determine whether TFPI-2 interact with RASSF1Conly.We use different colors of fluorescent expression vector, which were constructed in the form of (TFPI-2 or RASSF1C) fusion protein, Observed by confocal microscope found the two proteins co-localization. We used co-immunoprecipitation and determined TFPI-2 could interact with RASSF1C.The Ste20 family of serine/threonine protein kinases is implicated in a variety of signaling pathways including those involved in the control of cell migration and polarity. In mammals, over 30 Ste20 kinases exist classified into two subgroups. These are the p21-activated kinases and the germinal center kinases. Subgroups II and III contain the mammalian Ste20 (MST) kinases, MST1, MST2, MST3, MST4, and yeast Spsl/Ste20-related kinase 1 (YSK1). YSK1, also known as Ste20/oxidant stress response kinase 1, is weakly activated by reactive oxygen intermediates but not by any other environmental stresses, or by growth factors. However, this kinase does not participate in any of the known MAPK pathways and a physiological function for YSK1 remains unknown. Like YSK1, MST4 overexpression fails to activate the JNK and p38 MAPK pathways, although it promotes anchorage-independent growth and tumor formation, and has been implicated in prostate cancer progression. Investigating the MST family kinases YSK1 and MST4 and uncover a signaling function linked with the Golgi matrix protein GM130 that play a role in the control of cell migration and polarization have some results:YSK1 and MST4 localize to the Golgi apparatus, YSK1 and MST4 interact with the Golgi matrix protein GM130. We have detected some relations of YSK1 and MST3, and MST3 relative to cancer cell migration, We think that YSK1 is a important downstream protein. Here we showed that YSK1 involved in cell migration with the method of gene knocking down in breast cancer MCF-7 cell line.