Ser67-phosphorylated inhibitor 1 is a potent protein phosphatase 1 inhibitor
Ser67-phosphorylated inhibitor 1 is a potent protein phosphatase 1 inhibitor
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DOI：
10.1073/pnas.100460897
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发表时间：
2000-05-23
影响因子：
11.1
通讯作者：
Paudel, HK
中科院分区：
文献类型：
--
作者：
Huang, KX;Paudel, HK
Inhibitor 1 (I-1) 是蛋白磷酸酶 1 (PP1)（一种主要的真核丝氨酸/苏氨酸磷酸酶）的蛋白质抑制剂，非磷酸化 I-1 没有活性，而磷酸化 I-1 是有效的 PP1 抑制剂。 I-1 在体内的 Thr(35) 和 Ser(67) 上被磷酸化。 Thr(35) 被 cAMP 依赖性蛋白激酶（A 激酶）磷酸化，Thr(35) 磷酸化 I-1 抑制 PP1。迄今为止，磷酸化 Ser(67) 的激酶尚未被鉴定，Ser(67) 磷酸化的生理作用尚不清楚。在本研究中，当使用含有 Ala 取代 Thr(35) [GST-I-1(T35A)] 的谷胱甘肽 S-转移酶 (GST) 融合 I-1 突变体作为底物时，我们在脑提取物中检测到高水平的激酶活性。脑提取物中的 GST-I-1(T35A) 激酶和神经元 cdc2 样蛋白激酶 (NCLK) 无法通过一系列连续色谱法相互分离。使用来自激酶活性柱级分的抗 NCLK 抗体对 GST-I-1(T35A) 激酶进行免疫沉淀。纯化的 NCLK 磷酸化 GST-I-1(T35A) 和 I-1（每摩尔 I-1 0.7 摩尔磷酸盐）、HPLC 磷酸肽图谱、氨基酸测序和定点诱变确定 NCLK 磷酸化 I-1 的 Ser(67)。 NCLK-磷酸化-1和I-1(T35A)抑制PP1，IC50值分别接近9.5和13.8 nM。相比之下，A激酶磷酸化I-1仅比NCLK-磷酸化I-1抑制约1.2倍。我们的数据表明，NCLK 是一种潜在的体内 I-1 激酶，Thr(35) 和 Ser(67) 磷酸化独立激活 I-1。
Inhibitor 1 (I-1) is a protein inhibitor of protein phosphatase 1 (PP1), a major eukaryotic Ser/Thr phosphatase, Nonphosphorylated I-1 is inactive, whereas phosphorylated I-1 is a potent PP1 inhibitor. I-1 is phosphorylated in vivo on Thr(35) and Ser(67). Thr(35) is phosphorylated by cAMP-dependent protein kinase (A kinase), and Thr(35)-phosphorylated I-1 inhibits PP1, Until now the kinase that phosphorylates Ser(67) had not been identified and the physiological role of Ser(67) phosphorylation was unknown. In this study we detected a high level of kinase activity in brain extract when a glutathione S-transferase (GST) fusion I-1 mutant containing an Ala substituted for Thr(35) [GST-I-1(T35A)] was used as the substrate. GST-I-1(T35A) kinase and neuronal cdc2-like protein kinase (NCLK) in the brain extract could not be separated from each other by a series of sequential chromatographies. GST-I-1(T35A) kinase immunoprecipitated with anti-NCLK antibody from kinase-active column fractions. Purified NCLK-phosphorylated GST-I-1(T35A) and I-1 (0.7 mole of phosphate per mole of I-1), HPLC phosphopeptide mapping, amino acid sequencing, and site-directed mutagenesis determined that NCLK phosphorylates Ser(67) of I-1. NCLK-phosphorylated -1 and I-1(T35A) inhibited PP1 with IC50 values approximate to 9.5 and 13.8 nM, respectively, When compared, A kinase-phosphorylated I-1 was only approximate to 1.2 times more inhibitory than NCLK-phosphorylated I-1. Our data indicate that NCLK is a potential in vivo I-1 kinase and that Thr(35) and Ser(67) phosphorylation independently activate I-1.