Guesdon F, Waller R J, Saklatvala J
Cytokine Laboratory, The Babraham Institute, Cambridge, United Kingdom.
Cytokine. 1997 Jul;9(7):471-9. doi: 10.1006/cyto.1996.0190.
Interleukin 1 (IL-1) and tumour necrosis factor (TNF) activate a novel protein kinase, TIP kinase, which phosphorylates beta-casein in vitro. We have identified and purified to homogeneity a tryptic fragment of beta-casein, called T1, which was phosphorylated by TIP kinase with kinetics similar to those of the intact protein (K[m] = 27 +/- 6 microM). Phosphopeptide maps of in vitro phosphorylated T1 and beta-casein were identical, confirming that T1 contained the main phosphorylation site of the protein. T1 corresponded to residues 114 to 169 of beta-casein. It was phosphorylated by constitutively active protein kinases to a much lesser extent than beta-casein and thus constituted a specific substrate of the cytokine-activated enzyme. This made possible the detection of TIP kinase in extracts of IL-1-stimulated HeLa and KB cells, which had been hampered by high background phosphorylation when beta-casein was used as substrate. Our results show that the use of fragment T1 allows detection of low levels of TIP kinase in crude samples. They also suggest that its activation, which had previously been observed only in connective tissue cells, may be a general response of many cell types to IL-1 or TNF.
白细胞介素1（IL-1）和肿瘤坏死因子（TNF）可激活一种新型蛋白激酶——TIP激酶，该激酶可在体外使β-酪蛋白磷酸化。我们已鉴定并纯化出一种β-酪蛋白的胰蛋白酶片段，称为T1，它被TIP激酶磷酸化的动力学与完整蛋白相似（米氏常数K[m]=27±6微摩尔）。体外磷酸化的T1和β-酪蛋白的磷酸肽图谱相同，证实T1包含该蛋白的主要磷酸化位点。T1对应于β-酪蛋白的114至169位氨基酸残基。它被组成型活性蛋白激酶磷酸化的程度远低于β-酪蛋白，因此是细胞因子激活酶的特异性底物。这使得在IL-1刺激的HeLa细胞和KB细胞提取物中检测TIP激酶成为可能，而此前当使用β-酪蛋白作为底物时，由于高背景磷酸化而受到阻碍。我们的结果表明，使用片段T1能够检测粗样品中低水平的TIP激酶。它们还表明，其激活作用此前仅在结缔组织细胞中观察到，可能是许多细胞类型对IL-1或TNF的普遍反应。
