Adler Victor, Pincus Matthew R
QRNA Corp, New York, NY, USA.
Ann Clin Lab Sci. 2004 Winter;34(1):35-46.
We have previously found that the pi-isozyme of glutathione-S-transferase (GST-pi) is a strong and selective inhibitor of the phosphorylation of the transcriptional activating protein jun by its activating kinase, jun-N-terminal kinase (JNK). We further performed molecular dynamics calculations on the 3-dimensional structure of GST-pi free and bound to an inhibitor that blocks its ability to inhibit the JNK-jun activation. We thus identified 4 putative domains that may be involved in the interaction between GST-pi and the JNK-jun complex: residues 34-50, 99-121, 165-182 (with 2 overlapping sub-domains 165-175 and 169-182), and 194-201. We have synthesized each of these domains and tested them for their abilities to affect the GST-JNK-jun system, first in a cell-free system. We find that peptides corresponding to residues 99-121 and 194-201 strongly inhibit the binding of GST to the JNK-jun complex but do not inhibit JNK-induced phosphorylation of jun, while peptides corresponding to residues 34-50 and 165-182 do not inhibit GST binding but, except for the 165-175 subdomain peptide, strongly inhibit jun phosphorylation. A control peptide, X13, had no effect on either process. Peptide effects on jun phosphorylation appear to be selective for the JNK-jun system since the 34-50 peptide has no effect on other kinase systems (eg, casein kinase, MAP kinase). Three of the domain peptides, 34-50, 165-175, and 194-201 have been attached on their carboxyl-terminal ends to a penetratin sequence, enabling transmembrane transport into cells, and have been introduced into human astrocytes in which JNK was activated with anisomycin. We find that the 34-50-penetratin peptide strongly inhibits intracellular jun phosphorylation while the 194-201-penetratin peptide has no effect; the 165-175-penetratin peptide has a weak effect on this process. Thus, the effects in cells parallel those in the cell-free system. We conclude that all putative domains, identified in our prior structural studies, appear to interact with the JNK-jun complex. The 34-50 peptide may be useful in selectively blocking uncontrolled mitogenic signaling involving the JNK-jun pathway and may be a potential agent for blocking oncogenic ras-p21-induced cell transformation.
我们之前发现，谷胱甘肽-S-转移酶（GST-pi）的π同工酶是转录激活蛋白jun的激活激酶——jun氨基末端激酶（JNK）磷酸化的一种强效且选择性的抑制剂。我们进一步对游离状态以及与一种可阻断其抑制JNK-jun激活能力的抑制剂结合状态下的GST-pi三维结构进行了分子动力学计算。由此，我们确定了4个可能参与GST-pi与JNK-jun复合物相互作用的假定结构域：第34 - 50位氨基酸残基、第99 - 121位氨基酸残基、第165 - 182位氨基酸残基（有两个重叠的亚结构域，即第165 - 175位氨基酸残基和第169 - 182位氨基酸残基）以及第194 - 201位氨基酸残基。我们合成了这些结构域中的每一个，并首先在无细胞系统中测试它们影响GST-JNK-jun系统的能力。我们发现，对应于第99 - 121位氨基酸残基和第194 - 201位氨基酸残基的肽段强烈抑制GST与JNK-jun复合物的结合，但不抑制JNK诱导的jun磷酸化，而对应于第34 - 50位氨基酸残基和第165 - 182位氨基酸残基的肽段不抑制GST结合，但除了第165 - 175亚结构域肽段外，强烈抑制jun磷酸化。一个对照肽段X13对这两个过程均无影响。肽段对jun磷酸化的影响似乎对JNK-jun系统具有选择性，因为第34 - 50肽段对其他激酶系统（如酪蛋白激酶、丝裂原活化蛋白激酶）没有影响。其中三个结构域肽段，即第34 - 50位、第165 - 175位和第194 - 201位氨基酸残基的肽段，已在其羧基末端连接了一个穿膜肽序列，使其能够跨膜转运进入细胞，并已被导入用茴香霉素激活JNK的人星形胶质细胞中。我们发现，第34 - 50穿膜肽段强烈抑制细胞内jun磷酸化，而第194 - 201穿膜肽段没有影响；第165 - 175穿膜肽段对这一过程有微弱影响。因此，细胞内的效应与无细胞系统中的效应相似。我们得出结论，在我们之前的结构研究中确定的所有假定结构域似乎都与JNK-jun复合物相互作用。第34 - 50肽段可能有助于选择性阻断涉及JNK-jun途径的失控有丝分裂信号传导，并且可能是一种潜在的阻断致癌性ras-p21诱导的细胞转化的药物。
