Study On The Biological Function Of Human PTPN4, ABCF3 And PINX1 Genes

This thesis is composed of three separate parts:PTPN4, a novel Crk1-interacting protein, negatively regulates CrkI in human cell lines; The ATP-binding cassette (ABC) transporter superfamily member ABCF3 enhances the proliferation of human cancer cell lines. Telomerase inhibitor PINX1 compete with TIN2 to bind TRF1, regulate the location of TEN2 and TRF1.In the first part, PTPN4 is a widely expressed non-receptor protein tyrosine phosphatase. Although the overexpression of PTPN4 inhibits cell growth, the proteins with which PTPN4 interacts to regulate cell growth are unknown. Here we identify CrkI as a PTPN4 interacting protein via yeast two-hybrid, confirm this interaction by in vitro GST-pulldown and in vivo co-immunoprecipitation and co-localization assays. We further determine the interactional regions as the SH3 domain of CrkI and the proline-rich region between amino acids 462-468 of PTPN4. Notably, overexpression of PTPN4 inhibits Crk1-mediated proliferation and wound healing of HEK293T cells, while knockdown of PTPN4 by siRNA in Hep3B cells enhances Crkl-mediated cell growth and motility. Moreover, our data show that ectopic expression of PTPN4 reduces the phosphorylation level of CrkI in HEK293T cells. Taken together, these findings suggest that PTPN4 negatively regulates cell proliferation and motility through dephosphorylation of CrkI.In the second part, we describe a ATP-binding cassette (ABC) transporter superfamily member, named ABCF3, which encodes 709 aa and contained two ATP-binding cassette but no transmembrane segment. It is a evolutionary conserved gene and expressed in all tissues examined. Interestingly, ABCF3 inhibit p53 pathway activation and promoted proliferation of 293t cells, In contrast, knock down of ABCF3 protein expression by siRNA inhibited cell proliferation. Taken together, these findings suggest that ABCF3 is not a classic ABC transporter family member but play a significant role in the regulation of cell proliferation.In the third part, we cooperate with Lei lab, they solve the structure of the binding region of PINX1and TRF1, and find that the motif 289FTLKP293 is important for the binding, we construst several mutation and verify that F289 and L291 are charged residues. P INX1 locate on telomere rely on the binding to TRF1, it regulate the location of TRF1 and TIN2.