The Prokaryotic Expression, Purification And Structural Identification Of Type 2C Protein Phosphatase Wip1

The Wild-type p53-induced phosphatase 1,Wipl(or PPMID),is unusual in that it is a serine/threonine phosphatase with oncogenic activity.A member of the type 2C phosphatases(PP2Cδ),Wip1 has been shown to be amplified and overexpressed in multiple human cancer types.Wip1 acts as a homeostatic regulator of the DNA damage response by dephosphory lating proteins that are substrates of both ATM and ATR,important DNA damage sensor kinases.Wip1 also suppresses the activity of multiple tumor suppressors,including p53,ATM and ARF.We present evidence that the suppression of p53,p38 MAP kinase,and ATM/ATR signaling pathways by Wip1 are important components of its oncogenicity when it is amplified and overexpressed in human cancers.In order to study the oncogenicity of Wip1,we designed a point mutation protein-D314A,which turned the 314th amino acid of Wip1 Asp to Ala,which probably be the active site.We constructed prokaryotic expression vectors for the two proteins wild and point mutation type,and finally collected the purified proteins.We researched the binding capacity of Wip1 with ATP,and also indentified the secondary structure by circular dichroism spectrum to try to find the difference between the two proteins.All the researchs were to confirm the active site of Wip1,and laid a foundation for the further research,including crystallization for steric configuration study and the mechanism of oneogenicity.