Watanabe T, Huang H B, Horiuchi A, da Cruze Silva E F, Hsieh-Wilson L, Allen P B, Shenolikar S, Greengard P, Nairn A C
Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
Proc Natl Acad Sci U S A. 2001 Mar 13;98(6):3080-5. doi: 10.1073/pnas.051003898.
Regulation of protein phosphatase 1 (PP1) by protein inhibitors and targeting subunits has been previously studied through the use of recombinant protein expressed in Escherichia coli. This preparation is limited by several key differences in its properties compared with native PP1. In the present study, we have analyzed recombinant PP1 expressed in Sf9 insect cells using baculovirus. Sf9 PP1 exhibited properties identical to those of native PP1, with respect to regulation by metals, inhibitor proteins, and targeting subunits, and failure to dephosphorylate a phosphotyrosine-containing substrate or phospho-DARPP-32 (Dopamine and cAMP-regulated phosphoprotein, M(r) 32,000). Mutations at Y272 in the beta12/beta13 loop resulted in a loss of activity and reduced the sensitivity to thiophospho-DARPP-32 and inhibitor-2. Mutations of Y272 also increased the relative activity toward a phosphotyrosine-containing substrate or phospho-DARPP-32. Mutation of acidic groove residues caused no change in sensitivity to thiophospho-DARPP-32 or inhibitor-2, but one mutant (E252A:D253A:E256R) exhibited an increased K(m) for phosphorylase a. Several PP1/PP2A chimeras were prepared in which C-terminal sequences of PP2A were substituted into PP1. Replacement of residues 274-330 of PP1 with the corresponding region of PP2A resulted in a large loss of sensitivity to thiophospho-DARPP-32 and inhibitor-2, and also resulted in a loss of interaction with the targeting subunits, spinophilin and PP1 nuclear targeting subunit (PNUTS). More limited alterations in residues in beta12, beta13, and beta14 strands highlighted a key role for M290 and C291 in the interaction of PP1 with thiophospho-DARPP-32, but not inhibitor-2.
先前通过使用在大肠杆菌中表达的重组蛋白，对蛋白磷酸酶1（PP1）受蛋白抑制剂和靶向亚基的调控进行了研究。与天然PP1相比，这种制备方法受到其性质上几个关键差异的限制。在本研究中，我们分析了使用杆状病毒在Sf9昆虫细胞中表达的重组PP1。Sf9 PP1在受金属、抑制剂蛋白和靶向亚基调控方面，以及在无法使含磷酸酪氨酸的底物或磷酸化DARPP - 32（多巴胺和cAMP调节的磷蛋白，分子量32,000）去磷酸化方面，表现出与天然PP1相同的特性。β12/β13环中Y272位点的突变导致活性丧失，并降低了对硫代磷酸化DARPP - 32和抑制剂 - 2的敏感性。Y272的突变还增加了对含磷酸酪氨酸的底物或磷酸化DARPP - 32的相对活性。酸性凹槽残基的突变对硫代磷酸化DARPP - 32或抑制剂 - 2的敏感性没有影响，但一个突变体（E252A:D253A:E256R）对磷酸化酶a的米氏常数（K(m)）增加。制备了几种PP1/PP2A嵌合体，其中PP2A的C端序列被替换到PP1中。用PP2A的相应区域替换PP1的274 - 330位残基，导致对硫代磷酸化DARPP - 32和抑制剂 - 2的敏感性大幅丧失，并且还导致与靶向亚基亲嗜素和PP1核靶向亚基（PNUTS）的相互作用丧失。β12、β13和β14链中残基更有限的改变突出了M290和C291在PP1与硫代磷酸化DARPP - 32相互作用中的关键作用，但在与抑制剂 - 2的相互作用中并非如此。
