University of Connecticut Health Center, Farmington, CT 06030, USA.
Biochemistry. 2011 Jun 14;50(23):5279-91. doi: 10.1021/bi200089z. Epub 2011 May 16.
The ubiquitously expressed and highly promiscuous protein phosphatase 1 (PP1) regulates many cellular processes. Targeting PP1 to specific locations within the cell allows for the regulation of PP1 by conferring substrate specificity. In the present study, we identified AKAP79 as a novel PP1 regulatory subunit. Immunoprecipitaiton of the AKAP from rat brain extract found that the PP1 catalytic subunit copurified with the anchoring protein. This is a direct interaction, demonstrated by pulldown experiments using purified proteins. Interestingly, the addition of AKAP79 to purified PP1 catalytic subunit decreased phosphatase activity with an IC(50) of 811 ± 0.56 nM of the anchoring protein. Analysis of AKAP79 identified a PP1 binding site that conformed to a consensus PP1 binding motif (FxxR/KxR/K) in the first 44 amino acids of the anchoring protein. This was confirmed when a peptide mimicking this region of AKAP79 was able to bind PP1 by both pulldown assay and surface plasmon resonance. However, PP1 was still able to bind to AKAP79 upon deletion of this region, suggesting additional sites of contact between the anchoring protein and the phosphatase. Importantly, this consensus PP1 binding motif was found not to be responsible for PP1 inhibition, but rather enhanced phosphatase activity, as deletion of this domain resulted in an increased inhibition of PP1 activity. Instead, a second interaction domain localized to residues 150-250 of AKAP79 was required for the inhibition of PP1. However, the inhibitory actions of AKAP79 on PP1 are substrate dependent, as the anchoring protein did not inhibit PP1 dephosphorylation of phospho-PSD-95, a substrate found in AKAP79 complexes in the brain. These combined observations suggest that AKAP79 acts as a PP1 regulatory subunit that can direct PP1 activity toward specific targets in the AKAP79 complex.
普遍表达且高度混杂的蛋白磷酸酶 1（PP1）调节许多细胞过程。将 PP1 靶向细胞内的特定位置可以通过赋予底物特异性来调节 PP1。在本研究中，我们鉴定了 AKAP79 为一种新型的 PP1 调节亚基。从大鼠脑提取物中免疫沉淀 AKAP 发现，PP1 催化亚基与锚定蛋白共沉淀。这是一种直接相互作用，通过使用纯化蛋白进行下拉实验证明。有趣的是，将 AKAP79 添加到纯化的 PP1 催化亚基中降低了磷酸酶活性，其锚定蛋白的 IC50 为 811 ± 0.56 nM。对 AKAP79 的分析确定了一个 PP1 结合位点，该位点符合锚定蛋白前 44 个氨基酸中 PP1 结合基序（FxxR/KxR/K）。当模拟 AKAP79 该区域的肽能够通过下拉测定和表面等离子体共振来结合 PP1 时，证实了这一点。然而，即使删除该区域，PP1 仍然能够与 AKAP79 结合，这表明锚定蛋白和磷酸酶之间存在其他接触位点。重要的是，该共识 PP1 结合基序不是负责 PP1 抑制的原因，而是增强了磷酸酶活性，因为删除该结构域导致 PP1 活性的抑制增加。相反，需要 AKAP79 中残基 150-250 处的第二个相互作用结构域来抑制 PP1。然而，AKAP79 对 PP1 的抑制作用是底物依赖性的，因为锚定蛋白不会抑制 PP1 去磷酸化 PSD-95，而 PSD-95 是脑中 AKAP79 复合物中的一种底物。这些综合观察结果表明，AKAP79 作为一种 PP1 调节亚基，可将 PP1 活性导向 AKAP79 复合物中的特定靶标。
