Substrate specificity analysis of protein kinase complex Dbf2-Mob I by peptide library and proteome array screening
Substrate specificity analysis of protein kinase complex Dbf2-Mob I by peptide library and proteome array screening
复制标题
DOI：
10.1186/1471-2091-6-22
复制
发表时间：
2005-10-21
期刊：
影响因子：
--
通讯作者：
Deshaies, Raymond J.
中科院分区：
文献类型：
--
作者：
Mah, Angie S.;Elia, Andrew E. H.;Deshaies, Raymond J.
背景资料：有丝分裂退出网络（MEN）是一组形成信号级联的蛋白质，其对于酿酒酵母中的细胞退出有丝分裂是必需的。MEN也被牵连在胞质分裂中发挥作用。该信号通路的两个组分是蛋白激酶Dbf 2及其激酶活性所必需的结合伴侣Mob 1。MEN中作用于Dbf 2-Mob 1上游的组分已被表征，但Dbf 2 Mob 1的生理底物尚未确定。使用肽文库选择、最佳肽变体的磷酸化和磷酸化位点阵列的筛选的组合，我们发现Dbf 2-Mob 1优先磷酸化丝氨酸而不是苏氨酸，并且需要在其底物中磷酸化丝氨酸上游三个残基的精氨酸。肽底物中对精氨酸的这种需求不能用类似电荷的赖氨酸取代。这种特异性确定为肽底物也很明显，在许多蛋白磷酸化的Dbf 2-Mob 1在蛋白质组chip analysis.Conclusion：我们已经确定了肽库的选择和磷酸化阵列筛选的蛋白激酶Dbf 2-Mob 1优先磷酸化的底物，包含RXXS基序。随后的蛋白质组微阵列筛选揭示了可以在体外被Dbf 2-Mob 1磷酸化的蛋白质。这些蛋白质富含RXXS基序，并且可能包括介导Dbf 2-Mob 1在有丝分裂退出和胞质分裂中的功能的底物。在磷酸化位点的相对低程度的序列限制表明Dbf 2通过将其底物停靠在不同于磷酸化位点的位点来实现特异性。
Background: The mitotic exit network ( MEN) is a group of proteins that form a signaling cascade that is essential for cells to exit mitosis in Saccharomyces cerevisiae. The MEN has also been implicated in playing a role in cytokinesis. Two components of this signaling pathway are the protein kinase Dbf2 and its binding partner essential for its kinase activity, Mob1. The components of MEN that act upstream of Dbf2- Mob1 have been characterized, but physiological substrates for Dbf2Mob1 have yet to be identified.Results: Using a combination of peptide library selection, phosphorylation of opitmal peptide variants, and screening of a phosphosite array, we found that Dbf2- Mob1 preferentially phosphorylated serine over threonine and required an arginine three residues upstream of the phosphorylated serine in its substrate. This requirement for arginine in peptide substrates could not be substituted with the similarly charged lysine. This specificity determined for peptide substrates was also evident in many of the proteins phosphorylated by Dbf2- Mob1 in a proteome chip analysis.Conclusion: We have determined by peptide library selection and phosphosite array screening that the protein kinase Dbf2- Mob1 preferentially phosphorylated substrates that contain an RXXS motif. A subsequent proteome microarray screen revealed proteins that can be phosphorylated by Dbf2- Mob1 in vitro. These proteins are enriched for RXXS motifs, and may include substrates that mediate the function of Dbf2- Mob1 in mitotic exit and cytokinesis. The relatively low degree of sequence restriction at the site of phosphorylation suggests that Dbf2 achieves specificity by docking its substrates at a site that is distinct from the phosphorylation site.