Guesdon F, Freshney N, Waller R J, Rawlinson L, Saklatvala J
Cytokine Biochemistry Department, Strangeways Research Laboratory, Worts' Causeway, Cambridge, United Kingdom.
J Biol Chem. 1993 Feb 25;268(6):4236-43.
We have partially purified and characterized two protein kinases that were strongly activated by interleukin-1 (IL-1) or tumor necrosis factor (TNF) in MRC-5 fibroblasts. The kinases were separated by anion exchange chromatography of cytosolic fractions. They phosphorylated in vitro the small heat shock protein (hsp27) or beta-casein and were stimulated 3- and 4.5-fold, respectively, in cells that had been exposed to IL-1 or TNF for 10 min. They were distinct from the mitogen-activated protein kinases, whose activation by IL-1 or TNF has been reported recently. The hsp27 kinase phosphorylated its substrate on serine residues. Its molecular mass was estimated to be 45-kDa by gel filtration. It is probably involved in the increase in hsp27 phosphorylation seen in intact cells. The beta-casein kinase behaved as a 65-kDa protein. It phosphorylated its substrate on serine and threonine residues and had little activity on alpha-casein. The hsp27 and beta-casein kinases were not activated after stimulation of the cells with phorbol myristate acetate (PMA). In contrast, the MAP kinases were activated to a similar extent (2-3-fold) by the cytokines and by PMA. The hsp27- and beta-casein kinases probably correspond to novel enzymes whose mechanisms of activation may be independent of protein kinase C or MAP kinases.
我们已经部分纯化并鉴定了两种蛋白激酶，它们在MRC-5成纤维细胞中被白细胞介素-1（IL-1）或肿瘤坏死因子（TNF）强烈激活。这些激酶通过胞质组分的阴离子交换色谱法分离。它们在体外使小热休克蛋白（hsp27）或β-酪蛋白磷酸化，并且在暴露于IL-1或TNF 10分钟的细胞中分别被刺激3倍和4.5倍。它们与丝裂原活化蛋白激酶不同，最近有报道称IL-1或TNF可激活丝裂原活化蛋白激酶。hsp27激酶在丝氨酸残基上使其底物磷酸化。通过凝胶过滤估计其分子量为45 kDa。它可能参与了完整细胞中hsp27磷酸化的增加。β-酪蛋白激酶表现为一种65 kDa的蛋白质。它在丝氨酸和苏氨酸残基上使其底物磷酸化，而对α-酪蛋白几乎没有活性。在用佛波酯肉豆蔻酸酯（PMA）刺激细胞后，hsp27和β-酪蛋白激酶未被激活。相反，丝裂原活化蛋白激酶被细胞因子和PMA激活到相似的程度（2至3倍）。hsp27和β-酪蛋白激酶可能对应于新的酶，其激活机制可能独立于蛋白激酶C或丝裂原活化蛋白激酶。
