Huang K X, Paudel H K
Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, and Department of Neurology and Neurosurgery, McGill University, 3755 Cote Ste-Catherine Road, Montreal, QC, H3T 1E2.
Proc Natl Acad Sci U S A. 2000 May 23;97(11):5824-9. doi: 10.1073/pnas.100460897.
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 I-1 and I-1(T35A) inhibited PP1 with IC(50) values approximately 9.5 and 13. 8 nM, respectively. When compared, A kinase-phosphorylated I-1 was only approximately 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.
抑制剂1（I-1）是蛋白磷酸酶1（PP1）的一种蛋白抑制剂，PP1是一种主要的真核丝氨酸/苏氨酸磷酸酶。未磷酸化的I-1无活性，而磷酸化的I-1是一种有效的PP1抑制剂。I-1在体内苏氨酸（Thr）35和丝氨酸（Ser）67位点被磷酸化。Thr35由环磷酸腺苷（cAMP）依赖性蛋白激酶（A激酶）磷酸化，且Thr35磷酸化的I-1可抑制PP1。到目前为止，磷酸化Ser67的激酶尚未被鉴定出来，且Ser67磷酸化的生理作用也不清楚。在本研究中，当使用含丙氨酸替代Thr35的谷胱甘肽S-转移酶（GST）融合I-1突变体[GST-I-1（T35A）]作为底物时，我们在脑提取物中检测到了高水平的激酶活性。脑提取物中的GST-I-1（T35A）激酶和神经元细胞周期蛋白依赖性激酶2样蛋白激酶（NCLK）不能通过一系列连续色谱法彼此分离。GST-I-1（T35A）激酶与抗NCLK抗体从激酶活性柱级分中共同免疫沉淀。纯化的NCLK使GST-I-1（T35A）和I-1磷酸化（每摩尔I-1磷酸化0.7摩尔磷酸盐）。高效液相色谱（HPLC）磷酸肽图谱分析、氨基酸测序和定点诱变确定NCLK使I-1的Ser67磷酸化。NCLK磷酸化的I-1和I-1（T35A）抑制PP1的半数抑制浓度（IC50）值分别约为9.5和13.8 nM。相比较而言，A激酶磷酸化的I-1的抑制作用仅比NCLK磷酸化的I-1约高1.2倍。我们的数据表明NCLK是一种潜在的体内I-1激酶，且Thr35和Ser67的磷酸化独立激活I-1。
