p38 map kinase substrate specificity differs greatly for protein and peptide substrates
p38 map kinase substrate specificity differs greatly for protein and peptide substrates
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DOI：
10.1006/abbi.2000.2005
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发表时间：
2000-10-15
影响因子：
3.9
通讯作者：
LoGrasso, P
中科院分区：
文献类型：
--
作者：
Hawkins, J;Zheng, S;LoGrasso, P
为了了解产生 p38 底物特异性的分子特征，我们比较了各种 p38 底物的特异性常数 kcat/Km 以及与蛋白质与肽底物结合相关的自由能。我们的结果表明，与肽对应物相比，蛋白质是 p38 的底物好 1320 倍。此外，序列元件的比较表明，在肽底物而不是蛋白质底物的情况下，序列 PXT/SP 更适合催化。最后，在肽的背景下，如蛋白激酶 A (PKA) 的肽抑制剂所见，由于 p-11 位点的结合亲和力增加，2 在含有 p-11 残基的 p38 肽底物中不存在。这些结果是在 p38 的有序连续动力学机制的背景下得出的，表明 p38 底物的选择可能会对测量到的 ATP 和抑制剂的结合产生影响。 p38 是丝裂原激活蛋白 (MAP) 激酶家族的成员，它会响应脂多糖 (LPS)、TNF- 和 IL-1 等刺激而被激活 (1–3)。 TNF-和 IL-1 是促炎细胞因子，在慢性炎症中起主要作用 (4)，阻断其功能的药物已在临床上成功治疗类风湿性关节炎和克罗恩病 (5-9)。此外，选择性 p38 抑制剂可降低血浆 TNF 水平，并且对关节炎动物模型有效 (10)。 MAP 激酶激酶 (MKK) (3, 11–13) 激活 p38 的许多方面、其动力学机制 (14) 以及与 ATP 竞争结合的化合物对 p38 的抑制作用均已得到描述 (14–18)。然而，除了 p38 下游底物 (3, 18-20) 的描述外，还没有对引起 p38 底物特异性的分子特征进行表征。虽然已经描述了关于细胞外调节激酶（ERK 1 和 2）MAP 激酶家族成员的肽底物特异性的报告 (21)，但尚未研究对其他 MAP 激酶家族成员（例如 p38 和 c-jun N 末端激酶 (JNK)）的蛋白质和肽底物的特异性决定因素的理解。使用与已知 p38 底物中天然磷酸化位点相对应的合成肽，我们比较了 kcat/Km 值，以确定哪些底物最适合 p38 以及哪些氨基酸残基负责特异性。此外，较大底物（例如蛋白质）中由 X 表示的一个或多个基团的结合能可以与较小底物（例如肽）中的结合能进行比较，并由 (22) 给出
To understand the molecular features that give rise to p38 substrate specificity, we compared the specificity constants, kcat/Km, and the free energy associated with protein versus peptide substrate binding for a variety of p38 substrates. Our results showed that proteins were up to 1320-fold better substrates for p38 compared to their peptide counterparts. Moreover, comparison of sequence elements revealed that in the context of a peptide substrate, but not a protein substrate, the sequence PXT/SP was preferred for catalysis. Finally, in the context of a peptide, the increased binding affinity ascribed to the p-11 position as seen with peptide inhibitors of protein kinase A (PKA), 2 was not present in p38 peptide substrates containing a p-11 residue. These results taken in the context of the ordered sequential kinetic mechanism for p38 suggest that the choice of substrate for p38 will likely have effects on the measured binding of ATP and inhibitor. p38 is a member of the mitogen-activated protein (MAP) kinase family that is activated in response to stimuli such as lipopolysaccharide (LPS), TNF-, and IL-1 (1–3). TNF-and IL-1 are proinflammatory cytokines which play a predominant role in chronic inflammation (4), and agents that block their function have been clinically successful in the treatment of rheumatoid arthritis and Crohn’s disease (5–9). Moreover, selective p38 inhibitors lower plasma TNF-levels and are efficacious in animal models of arthritis (10). Many aspects of p38 activation by MAP kinase kinases (MKKs)(3, 11–13), its kinetic mechanism (14), and its inhibition by compounds that compete with ATP for binding have been described (14–18). However, with the exception of the description of downstream substrates for p38 (3, 18–20), there has been no characterization of the molecular features that give rise to p38 substrate specificity. While reports on the peptide substrate specificity of extracellular-regulated kinase (ERK 1 and 2) MAP kinase family members have been described (21), understanding of the specificity determinants for protein and peptide substrates for other MAP kinase family members such as p38 and c-jun N-terminal kinase (JNK) have not been investigated. Using synthetic peptides that correspond to natural phosphorylation sites in known p38 substrates, we compared kcat/Km values to determine what substrates were best for p38 and what amino acid residues were responsible for the specificity. Furthermore, the binding energy of a group or groups represented by X in a larger substrate such as a protein can be compared to that in a smaller substrate such as a peptide and is given by (22)