Phage-peptide display identifies the interferon-responsive, death-activated protein kinase family as a novel modifier of MDM2 and p21WAF1
Phage-peptide display identifies the interferon-responsive, death-activated protein kinase family as a novel modifier of MDM2 and p21WAF1
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DOI：
10.1016/j.jmb.2003.10.081
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发表时间：
2004-03-12
影响因子：
5.6
通讯作者：
Hupp, T
中科院分区：
文献类型：
--
作者：
Burch, LR;Scott, M;Hupp, T
噬菌体肽显示是一种多功能的工具，用于鉴定新的蛋白质-蛋白质界面。我们之前的工作重点是选择与MDM2蛋白特定亚型结合的噬菌体肽，在这项工作中，我们将假定的MDM2结合蛋白进行噬菌体肽展示，以进一步扩展假定的蛋白质相互作用图谱。与MDM2结合的一种肽与死亡活化蛋白激酶（DAPK）家族成员具有显著的同源性，而死亡活化蛋白激酶（DAPK）家族与p53途径没有直接联系。我们研究了DAPK家族的一个核成员DAPK3或ZIP激酶是否与p53通路有直接联系。ZIP激酶被克隆和纯化，该酶能够磷酸化MDM2的Ser166位点，该位点先前被Akt激酶修饰，从而证明ZIP激酶是真正的MDM2结合蛋白。然后将原生ZIP激酶片段进行噬菌体肽展示，并在p21（WAF1）中鉴定出一个ZIP激酶一致的肽基序。ZIP激酶磷酸化p21（WAF1）的Thr145位点，p21（WAF1）磷酸化位点的丙氨酸取代突变改变了其被ZIP激酶磷酸化的能力。因此，尽管ZIP激酶的一致位点被定义为含有最小的RKKx（T/S）一致基序，但由于磷酸化受体位点周围的氨基酸残基可以影响激酶的特异性活性，因此涉及ZIP激酶结合的交替接触。转染的ZIPK在体内可促进p21（WAF1）在Thr145位点的磷酸化，并可延长p21（WAF1）的半衰期，而p21（WAF1[T145A]）的半衰期不受ZIP激酶的影响。因此，噬菌体肽展示确定了一个干扰素响应蛋白激酶家族，作为p53通路MDM2和p21（WAF1）两个组分的新修饰剂，并强调了噬菌体肽展示在获得生化途径新见解方面的实用性。(C) 2003 Elsevier Ltd.版权所有。
Phage-peptide display is a versatile tool for identifying novel protein-protein interfaces. Our previous work highlighted the selection of phage-peptides that bind to specific isoforms of MDM2 protein and in this work we subjected the putative MDM2-binding proteins to phage-peptide display to expand further on putative protein interaction maps. One peptide that bound MDM2 had significant homology to members of the death-activated protein kinase (DAPK) family, an enzyme family of no known direct link to the p53 pathway. We examined whether a nuclear member of the DAPK family named DAPK3 or ZIP kinase had direct, links to the p53 pathway. ZIP kinase was cloned, purified, and the enzyme,, was able to phosphorylate MDM2 at Ser166, a site previously reported to be modified by Akt kinase, thus demonstrating that ZIP kinase is a bona fide MDM2-binding protein. Native ZIP kinase fractions were then subjected to phage-peptide display and one ZIP kinase consensus peptide motif was identified in p21(WAF1). ZIP kinase phosphorylates p21(WAF1) at Thr145 and alanine-substituted mutations in the p21(WAF1) phosphorylation site alter its ability to be phosphorylated by ZIP kinase. Thus, although ZIP kinase consensus sites were then defined as containing a minimal RKKx(T/S) consensus motif, alternate contacts in ZIP kinase binding are implicated, since amino acid residues surrounding the phospho-acceptor site can effect the specific activity of the kinase. Transfected ZIPK can promote the phosphorylation of p21(WAF1) at Thr145 in vivo and can increase the half-life of p21(WAF1), while the half-life of p21(WAF1[T145A]) is not effected by ZIP kinase. Thus, phage-peptide display identified an interferon-responsive protein kinase family as a novel modifier of two components of the p53 pathway MDM2 and p21(WAF1), and underscores the utility of phage-peptide display for gaining novel insights into biochemical pathways. (C) 2003 Elsevier Ltd. All rights reserved.