探討重組第一型蛋白質磷酸水解酶與磷酸化第一型抑制蛋白間之交互作用

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

吳宗憲(Tsung-Hsien Wu)

指導教授 ： 黃憲斌

國立中正大學/理學院/生命科學系分子生物研究所/碩士(2013年)

摘要

第一型蛋白質磷酸水解酶(PP1)為真核細胞內最主要的絲胺酸/酥胺酸蛋白質磷酸水解酶。第一型蛋白質磷酸水解酶的催化次單體會和很多不同的調節次單體結合，產生不同的全酶。這些調節次單體會結合把PP1標地到細胞特定區域內，藉此第一型蛋白質磷酸水解酶便可調控受質活性，因此第一型蛋白質磷酸水解酶可調控多種不同的細胞功能。 第一型蛋白質磷酸水解酶會被三種具有耐酸和熱的抑制蛋白所抑制，包括第一型抑制蛋白（inhibitor-1）、多巴胺及環磷酸腺苷調節的磷酸化蛋白（DARPP-32）和第二型抑制蛋白（inhibitor-2）。環磷酸腺苷依賴蛋白磷酸激酶( PKA) 磷酸化第一型抑制蛋白位於第35的酥胺酸後，第一型抑制蛋白才會轉變成具有強效的抑制功能的抑制蛋白。大多數的第一型蛋白質磷酸水解酶的調節蛋白和蛋白抑制劑都包含有一個相似的結合序列為(R/K)(V/I)X(F/W)，會結合到第一型蛋白質磷酸水解酶的表面。早期的研究發現，磷酸化的第一型抑制蛋白只需約10-9次方莫耳濃度，便可抑制從兔子的肌肉純化出來的第一型蛋白質磷酸水解酶50%的活性，然而，從大腸桿菌大量表達來獲得的重組第一型蛋白質磷酸水解酶不但不太會被磷酸化的第一型抑制蛋白所抑制，還會去磷酸化磷酸化的第一型抑制蛋白。近來我們用核磁共振研究發現，磷酸化的第一型抑制蛋白的結合序列R8KIQF12，不會結合到重組第一型蛋白質磷酸水解酶。因此，本研究之主要目的是來了解是否第一型抑制蛋白的結合序列RKIQF會結合到從兔子的肌肉純化出來的第一型蛋白質磷酸水解酶，但不會結合到重組第一型蛋白質磷酸水解酶上。研究結果證實了我們的假說，並且解釋了為何重組第一型蛋白質磷酸水解酶會抵抗磷酸化的第一型抑制蛋白的抑制。

關鍵字

磷酸化第一型抑制蛋白 ； 第一型蛋白質磷酸水解酶

並列摘要

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.

並列關鍵字

protein phosphatase-1 ； phospho-inhibitor-1

參考文獻

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