MECHANISM OF INHIBITION OF PROTEIN PHOSPHATASE-1 BY DARPP-32 - STUDIES WITH RECOMBINANT DARPP-32 AND SYNTHETIC PEPTIDES
MECHANISM OF INHIBITION OF PROTEIN PHOSPHATASE-1 BY DARPP-32 - STUDIES WITH RECOMBINANT DARPP-32 AND SYNTHETIC PEPTIDES
复制标题
DOI：
10.1006/bbrc.1995.1092
复制
发表时间：
1995-01-17
影响因子：
3.1
通讯作者：
GIRAULT, JA
中科院分区：
文献类型：
--
作者：
DESDOUITS, F;CHEETHAM, JJ;GIRAULT, JA
研究了重组DARPP-32和合成肽抑制蛋白磷酸酶-1催化亚基（PP-1c）的作用机制。DARPP-32在大肠杆菌中用pEt-3a质粒作为非融合蛋白表达，纯化到均匀性，并显示出与从牛脑中纯化的蛋白相似的物理化学性质。通过camp依赖性蛋白激酶磷酸化苏氨酸-34的重组DARPP-32抑制PP-1c的IC50约为0.5 nM，与牛DARPP-32的IC50相当。非磷酸化的DARPP-32和苏氨酸-34被丙氨酸或谷氨酸取代的突变形式抑制PP-1c的IC50接近1 μ M。表面等离子体共振分析显示，PP-1c与非磷酸化和磷酸化的DARPP-32（8-38）合成肽结合，表观K-d值分别为1.2和0.3 μ M。支持非磷酸化的DARPP-32和PP-1c之间存在相互作用，这种相互作用通过DARPP-32在苏氨酸-34位点的磷酸化而增加。这些结果表明，DARPP-32通过至少两个低亲和力位点与PP-1c相互作用，这两个位点的结合导致了高亲和力（nM）抑制。(C) 1995学术出版社，Inc。
The mechanism of inhibition of protein phosphatase-1 catalytic subunit (PP-1c) by recombinant DARPP-32 and synthetic peptides was studied. DARPP-32 was expressed in Escherichia coli as a non-fusion protein using a pEt-3a plasmid, purified to homogeneity and shown to have physicochemical properties similar to those of the protein purified from bovine brain. Recombinant DARPP-32 phosphorylated on threonine-34 by cAMP-dependent protein kinase inhibited PP-1c with an IC50 approximate to 0.5 nM, comparable to that obtained with bovine DARPP-32. Non-phosphorylated DARPP-32, and mutated forms in which threonine-34 was replaced by an alanine or a glutamic acid, inhibited PP-1c with an IC50 approximate to 1 mu M Surface plasmon resonance analysis showed binding of PP-1c to nonphospho- and phospho-DARPP-32-(8-38) synthetic peptides with apparent K-d values of 1.2 and 0.3 mu M, respectively, supporting the existence of an interaction between non-phosphorylated DARPP-32 and PP-1c that is increased by phosphorylation of DARPP-32 at threonine-34. These results suggest a model in which DARPP-32 interacts with PP-1c by at least two low affinity sites, the combination of which is responsible for the high affinity (nM) inhibition. (C) 1995 Academic Press, Inc.