Desdouits F, Cheetham J J, Huang H B, Kwon Y G, da Cruz e Silva E F, Denefle P, Ehrlich M E, Nairn A C, Greengard P, Girault J A
INSERM U114, Collège de France, Paris.
Biochem Biophys Res Commun. 1995 Jan 17;206(2):652-8. doi: 10.1006/bbrc.1995.1092.
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 approximately 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 approximately 1 microM. Surface plasmon resonance analysis showed binding of PP-1c to nonphospho- and phospho-DARPP-32-(8-38) synthetic peptides with apparent Kd values of 1.2 and 0.3 microM, 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.
研究了重组DARPP-32和合成肽对蛋白磷酸酶-1催化亚基（PP-1c）的抑制机制。使用pEt-3a质粒在大肠杆菌中表达DARPP-32作为非融合蛋白，纯化至同质，并显示其理化性质与从牛脑中纯化的蛋白相似。经cAMP依赖性蛋白激酶在苏氨酸-34位点磷酸化的重组DARPP-32抑制PP-1c的IC50约为0.5 nM，与牛DARPP-32获得的结果相当。未磷酸化的DARPP-32以及苏氨酸-34被丙氨酸或谷氨酸取代的突变形式抑制PP-1c的IC50约为1 μM。表面等离子体共振分析表明，PP-1c与非磷酸化和磷酸化的DARPP-32-(8-38)合成肽结合，表观Kd值分别为1.2和0.3 μM，支持非磷酸化的DARPP-32与PP-1c之间存在相互作用，且这种相互作用因DARPP-32在苏氨酸-34位点的磷酸化而增强。这些结果提示了一种模型，即DARPP-32通过至少两个低亲和力位点与PP-1c相互作用，这两个位点的组合导致了高亲和力（纳摩尔级）抑制。
