Phosphorylation of Plant Translation Initiation Factors by CK2 Enhances the in Vitro Interaction of Multifactor Complex Components
Phosphorylation of Plant Translation Initiation Factors by CK2 Enhances the in Vitro Interaction of Multifactor Complex Components
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DOI：
10.1074/jbc.m109.007658
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发表时间：
2009-07-31
影响因子：
4.8
通讯作者：
Browning, Karen S.
中科院分区：
文献类型：
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作者：
Dennis, Michael D.;Person, Maria D.;Browning, Karen S.
CK 2磷酸化多种底物，包括翻译起始因子。利用质谱技术鉴定了CK 2磷酸化的残基，这些残基可能在植物翻译起始过程中调节起始因子的活性。在小麦和拟南芥eIF 2 α、eIF 2 β、eIF 5和小麦eIF 3c中鉴定了CK 2体外磷酸化位点。发现天然小麦eIF 5和eIF 2 α具有对应于一些体外CK 2磷酸化位点的磷酸化位点。本研究中鉴定的大量CK 2位点位于与酵母多因子复合物（eIF 1-eIF 3-eIF 5-eIF 2-GTP-Met-tRNA（i）（Met））有关的保守结合结构域中。这是首次证明植物起始因子能够在体外形成多因子复合物的研究。此外，这些复合物内的因子的相互作用在体外和天然提取物中通过CK 2磷酸化一种或多种起始因子而增强。通过定点突变eIF 5以去除CK 2磷酸化位点来评估eIF 5的CK 2磷酸化的重要性。在pulldown试验中，去除eIF 5的CK 2磷酸化位点可抑制CK 2介导的eIF 5与eIF 1和eIF 3c相互作用的增加，并降低eIF 5介导的体外翻译起始刺激。这些结果表明CK 2磷酸化在植物翻译起始中的功能作用。
CK2 phosphorylates a wide variety of substrates, including translation initiation factors. A mass spectrometric approach was used to identify residues phosphorylated by CK2, which may regulate the activity of initiation factors during the translation initiation process in plants. CK2 in vitro phosphorylation sites were identified in wheat and Arabidopsis thaliana eIF2 alpha, eIF2 beta, eIF5, and wheat eIF3c. Native wheat eIF5 and eIF2 alpha were found to have phosphorylation sites that corresponded to some of the in vitro CK2 phosphorylation sites. A large number of the CK2 sites identified in this study are in conserved binding domains that have been implicated in the yeast multifactor complex ( eIF1-eIF3-eIF5-eIF2-GTP-Met-tRNA(i)(Met)). This is the first study to demonstrate that plant initiation factors are capable of forming a multifactor complex in vitro. In addition, the interaction of factors within these complexes was enhanced both in vitro and in native extracts by phosphorylation of one or more initiation factors by CK2. The importance of CK2 phosphorylation of eIF5 was evaluated by site-directed mutagenesis of eIF5 to remove CK2 phosphorylation sites. Removal of CK2 phosphorylation sites from eIF5 inhibits the CK2-mediated increase in eIF5 interaction with eIF1 and eIF3c in pulldown assays and reduces the eIF5-mediated stimulation of translation initiation in vitro. These results suggest a functional role for CK2 phosphorylation in the initiation of plant translation.