Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Rua Giuseppe Máximo Scolfaro 10.000, C.P.6192, 13084-971 Campinas, São Paulo Brazil ; Programa de Pós-graduação em Biologia Funcional e Molecular, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, São Paulo Brazil.
Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Rua Giuseppe Máximo Scolfaro 10.000, C.P.6192, 13084-971 Campinas, São Paulo Brazil.
Proteome Sci. 2015 Feb 26;13:11. doi: 10.1186/s12953-015-0065-6. eCollection 2015.
Neks are serine-threonine kinases that are similar to NIMA, a protein found in Aspergillus nidulans which is essential for cell division. In humans there are eleven Neks which are involved in different biological functions besides the cell cycle control. Nek4 is one of the largest members of the Nek family and has been related to the primary cilia formation and in DNA damage response. However, its substrates and interaction partners are still unknown. In an attempt to better understand the role of Nek4, we performed an interactomics study to find new biological processes in which Nek4 is involved. We also described a novel Nek4 isoform which lacks a region of 46 amino acids derived from an insertion of an Alu sequence and showed the interactomics profile of these two Nek4 proteins.
Isoform 1 and isoform 2 of Nek4 were expressed in human cells and after an immunoprecipitation followed by mass spectrometry, 474 interacting proteins were identified for isoform 1 and 149 for isoform 2 of Nek4. About 68% of isoform 2 potential interactors (102 proteins) are common between the two Nek4 isoforms. Our results reinforce Nek4 involvement in the DNA damage response, cilia maintenance and microtubule stabilization, and raise the possibility of new functional contexts, including apoptosis signaling, stress response, translation, protein quality control and, most intriguingly, RNA splicing. We show for the first time an unexpected difference between both Nek4 isoforms in RNA splicing control. Among the interacting partners, we found important proteins such as ANT3, Whirlin, PCNA, 14-3-3ε, SRSF1, SRSF2, SRPK1 and hNRNPs proteins.
This study provides new insights into Nek4 functions, identifying new interaction partners and further suggests an interesting difference between isoform 1 and isoform 2 of this kinase. Nek4 isoform 1 may have similar roles compared to other Neks and these roles are not all preserved in isoform 2. Besides, in some processes, both isoforms showed opposite effects, indicating a possible fine controlled regulation.
Neks 是丝氨酸苏氨酸激酶，与 Aspergillus nidulans 中的 NIMA 蛋白相似，后者对细胞分裂至关重要。在人类中，有 11 种 Nek 参与细胞周期控制以外的不同生物学功能。Nek4 是 Nek 家族中最大的成员之一，与初级纤毛形成和 DNA 损伤反应有关。然而，其底物和相互作用伙伴仍不清楚。为了更好地理解 Nek4 的作用，我们进行了相互作用组学研究，以发现 Nek4 参与的新生物学过程。我们还描述了一种新的 Nek4 同工型，它缺乏一个由 Alu 序列插入衍生的 46 个氨基酸的区域，并展示了这两种 Nek4 蛋白的相互作用组学特征。
Nek4 的同工型 1 和同工型 2 在人细胞中表达，经过免疫沉淀和质谱分析，鉴定出同工型 1 的 474 个相互作用蛋白和同工型 2 的 149 个相互作用蛋白。同工型 2 的潜在相互作用蛋白（102 个蛋白）中有约 68%是两种 Nek4 同工型共有的。我们的结果进一步证实了 Nek4 参与 DNA 损伤反应、纤毛维持和微管稳定，并提出了新的功能背景的可能性，包括细胞凋亡信号转导、应激反应、翻译、蛋白质质量控制，最有趣的是 RNA 剪接。我们首次显示了两种 Nek4 同工型在 RNA 剪接控制方面的意外差异。在相互作用的伴侣中，我们发现了重要的蛋白质，如 ANT3、Whirlin、PCNA、14-3-3ε、SRSF1、SRSF2、SRPK1 和 hNRNPs 蛋白。
这项研究为 Nek4 的功能提供了新的见解，确定了新的相互作用伙伴，并进一步表明该激酶的同工型 1 和同工型 2 之间存在有趣的差异。与其他 Nek 相比，Nek4 同工型 1 可能具有相似的作用，但这些作用并非在同工型 2 中全部保留。此外，在某些过程中，两种同工型表现出相反的效果，表明可能存在精细的调控。
