Guesdon F, Knight C G, Rawlinson L M, Saklatvala J
Department of Cell Adhesion and Signaling, Strangeways Research Laboratory, Cambridge, CB1 4RN, United Kingdom.
J Biol Chem. 1997 Nov 28;272(48):30017-24. doi: 10.1074/jbc.272.48.30017.
Tumor necrosis factor (TNF) and interleukin 1 (IL1) activate a protein kinase, TIP kinase, which phosphorylates beta casein in vitro. We have now identified its main phosphorylation site on beta casein, Ser124 (Km approximately 28 mu M), and a minor phosphorylation site, Ser142 (Km approximately 0.7 mM). The sequence motif that determined the phosphorylation of Ser124 by the kinase was studied with synthetic peptides bearing deletions or substitutions of the neighboring residues. This allowed synthesis of improved substrates (Km approximately 6 mu M) and showed that efficient phosphorylation of Ser124 was favored by the presence of large hydrophobic residues at positions +1, +9, +11, and +13 (counted relative to the position of the phosphoacceptor amino acid) and of a cysteine at position -2. Peptides in which Ser124 was replaced by tyrosine were also phosphorylated by TIP kinase, showing it to have dual specificity. It is unable to phosphorylate the MAP kinases in vitro and is therefore not directly involved in their activation. Its biochemical characteristics indicate that TIP kinase is a novel dual specificity kinase, perhaps related to the mixed lineage kinases. It copurified with a phosphoprotein of about 95 kDa, which could correspond either to the autophosphorylated kinase or to an associated substrate.
肿瘤坏死因子（TNF）和白细胞介素1（IL1）可激活一种蛋白激酶，即TIP激酶，该激酶可在体外使β-酪蛋白磷酸化。我们现已确定其在β-酪蛋白上的主要磷酸化位点为Ser124（米氏常数约为28μM），次要磷酸化位点为Ser142（米氏常数约为0.7mM）。利用带有邻近残基缺失或取代的合成肽研究了决定该激酶对Ser124进行磷酸化的序列基序。这使得能够合成改良的底物（米氏常数约为6μM），并表明在+1、+9、+11和+13位（相对于磷酸受体氨基酸的位置计数）存在大的疏水残基以及在-2位存在半胱氨酸有利于Ser124的高效磷酸化。Ser124被酪氨酸取代的肽也可被TIP激酶磷酸化，表明其具有双重特异性。它在体外无法使丝裂原活化蛋白激酶磷酸化，因此不直接参与其激活过程。其生化特性表明TIP激酶是一种新型的双重特异性激酶，可能与混合谱系激酶有关。它与一种约95kDa的磷蛋白共纯化，该磷蛋白可能对应于自身磷酸化的激酶或相关底物。
