Interacting domains of P14-3-3 and actin involved in protein–protein interactions of living cells
Interacting domains of P14-3-3 and actin involved in protein–protein interactions of living cells
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DOI：
10.1007/s00203-011-0707-8
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发表时间：
2011-04
影响因子：
2.8
通讯作者：
Daliang Luo;Yi Yang;Jing Guo;Jianhua Zhang;Zhongzhen Guo;Shide Liu;Sheng-li Tian
中科院分区：
文献类型：
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作者：
Daliang Luo;Yi Yang;Jing Guo;Jianhua Zhang;Zhongzhen Guo;Shide Liu;Sheng-li Tian
14-3-3蛋白是存在于所有真核细胞中的保守调节蛋白，其通过靶蛋白相互作用控制许多细胞活性。为了研究多头绒泡菌P14-3-3与肌动蛋白在活细胞中的相互作用，利用PCR和DNA重组技术构建了多种P14-3-3和肌动蛋白的构建体。利用酵母双杂交技术和FRET技术研究了P14-3-3与肌动蛋白的相互作用。双杂交实验表明，P14-3-3 N端76-108个氨基酸和C端207-216个氨基酸在介导与肌动蛋白的相互作用中发挥重要作用，肌动蛋白N端1-54个氨基酸和C端326-376个氨基酸在与mPa的相互作用中也至关重要，a在Ser 62处具有突变（Ser 62 → Gly 62）的P14-3-3。潜在磷酸化位点的突变并不影响P14-3-3和肌动蛋白之间的相互作用。FRET结果表明，P14-3-3与肌动蛋白共定位，FRET效率为22.2%，距离为7.4 nm，P14-3-3 N端76-108和C端207-216个氨基酸在介导这种相互作用中是重要的，没有N-末端1-54或C-末端326-376氨基酸的截短的肌动蛋白肽与P14-3-3相互作用，与从酵母双杂交测定获得的结果一致。根据获得的数据，我们确定了关键的肌动蛋白和P14-3-3接触区域。
14-3-3 proteins are conserved regulatory proteins present in all eukaryotic cells that control numerous cellular activities via targeted protein interactions. To elucidate the interaction between P14-3-3 fromPhysarum polycephalumand actin in living cells, PCR and DNA recombination were used to generate various P14-3-3 and actin constructs. Yeast two-hybrid assay and FRET were employed to characterize the interaction between P14-3-3 and actin. The two-hybrid assay indicated that P14-3-3 N-terminal 76–108 amino acids and the C-terminal 207–216 amino acids played an important role in mediating interactions with actin, and the actin N-terminal 1–54 amino acids and the C-terminal 326–376 amino acids are also crucial in the interactions with the mPa, a P14-3-3 with mutations at Ser62 (Ser62 → Gly62). Mutations to potential phosphorylation sites did not affect interactions between P14-3-3 and actin. FRET results demonstrated that P14-3-3 co-localized with actin with a FRET efficiency of 22.2% and a distance of 7.4 nm and that P14-3-3 N-terminal 76–108 and C-terminal 207–216 amino acids were important in mediating this interaction, the truncated actin peptides without either the N-terminal 1–54 or C-terminal 326–376 amino acids interacted with P14-3-3, consistent with the results obtained from the yeast two-hybrid assay. Based on data obtained, we identified critical actin and P14-3-3 contact regions.