Characterization of The Interaction Between The Recombinant Protein Phosphatase-1 and Phospho-inhibitor-1

• Main Authors: Tsung-Hsien Wu, 吳宗憲
• Other Authors: Hsien-Bin Huang
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/20897158884981303329

碩士 === 國立中正大學 === 分子生物研究所 === 101 === Protein phosphatase 1 (PP1) is one of the major serine/threonine eukaryotic protein phosphatases. The catalytic subunit of PP1 is associated with different regulatory subunits to form a variety of holoenzymes. These regulatory subunits target the enzyme to specific subcellular compartments in which PP1 regulates the functions of its substrates, accounting for PP1 that can modulate the diverse cellular functions. PP1 is also specifically inhibited by three acid- and heat-stable protein inhibitors, including inhibitor-1, DARPP-32 and inhibitor-2. Phosphorylation of inhibitor-1 at Thr-35 by PKA converts the protein into a potent inhibitor of PP1. Most of PP1-regulatory proteins and protein inhibitors contain one consensus PP1-binding motif, (R/K)(V/I)X(F/W) that binds to surface of PP1. The early studies indicated that PP1 purified from rabbit muscles is inhibited by the phospho-inhibitor-1 with the IC50 at the nano-molar levels. However, the recombinant PP1 obtained from the E. coli overexpression cannot be inhibited by phospho-inhibitor-1, but dephosphorylates the phospho-inhibitor-1. Recently, our structural studies of NMR identified that the consensus PP1-binding motif, R8KIQF12, of inhibitor-1 cannot bind to the recombinant PP1. Thus, my studies carried out the experiments to examine whether RKIQF of inhibitor-1 binds to the native PP1, purified from the rabbit muscles, but not to the recombinant PP1. Our findings confirmed our suggestion and explained why the recombinant PP1 resists to the inhibition by phospho-inhibitor-1.