Characterization of the CLASP2 Protein Interaction Network Identifies SOGA1 as a Microtubule-Associated Protein
Characterization of the CLASP2 Protein Interaction Network Identifies SOGA1 as a Microtubule-Associated Protein
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DOI：
10.1074/mcp.ra117.000011
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发表时间：
2017-10-01
影响因子：
7
通讯作者：
Langlais, Paul R.
中科院分区：
文献类型：
--
作者：
Kruse, Rikke;Krantz, James;Langlais, Paul R.
CLASP2 是一种微管相关蛋白，会经历胰岛素刺激的磷酸化，并与重组肌动蛋白和 GLUT4 在质膜上共定位。为了深入了解 CLASP2 在此系统中的作用，我们开发并成功执行了简化的相互作用组方法，并在 3T3-L1 脂肪细胞中构建了 CLASP2 蛋白网络。使用两种不同的市售 CLASP2 抗体和一种表位标记的过表达 CLASP2 抗体，我们进行了多重亲和纯化与质谱 (AP-MS) 实验结合无标记定量蛋白质组学，并使用生物信息学工具相互作用组显着性分析 (SAINT) 分析数据。我们发现 CLASP2 共免疫沉淀 (co-IP) 新型蛋白 SOGA1、微管相关蛋白激酶 MARK2 和微管/肌动蛋白调节蛋白 G2L1。 GTP酶激活蛋白AGAP1和AGAP3也在CLASP2相互作用组中富集，尽管随后的AGAP3和CLIP2相互作用组分析表明AGAP3优先于CLIP2。后续的 MARK2 相互作用组分析证实了 CLASP2 的相互共免疫沉淀，并揭示 MARK2 可以共免疫沉淀 SOGA1、糖原合酶和糖原蛋白。对 SOGA1 相互作用组的研究证实 SOGA1 可以相互共免疫 CLASP2 和 MARK2 以及糖原合酶和糖原蛋白。 SOGA1 被证实与 CLASP2 和微管蛋白共定位，这表明 SOGA1 是一种新的微管相关蛋白。这些结果介绍了这些提出的新型蛋白质网络的代谢功能及其与微管的关系，作为细胞骨架相关蛋白质生物学的新领域。
CLASP2 is a microtubule-associated protein that undergoes insulin-stimulated phosphorylation and co-localization with reorganized actin and GLUT4 at the plasma membrane. To gain insight to the role of CLASP2 in this system, we developed and successfully executed a streamlined interactome approach and built a CLASP2 protein network in 3T3-L1 adipocytes. Using two different commercially available antibodies for CLASP2 and an antibody for epitope-tagged, overexpressed CLASP2, we performed multiple affinity purification coupled with mass spectrometry (AP-MS) experiments in combination with label-free quantitative proteomics and analyzed the data with the bioinformatics tool Significance Analysis of Interactome (SAINT). We discovered that CLASP2 coimmunoprecipitates (co-IPs) the novel protein SOGA1, the microtubule- associated protein kinase MARK2, and the microtubule/actin-regulating protein G2L1. The GTPaseactivating proteins AGAP1 and AGAP3 were also enriched in the CLASP2 interactome, although subsequent AGAP3 and CLIP2 interactome analysis suggests a preference of AGAP3 for CLIP2. Follow-up MARK2 interactome analysis confirmed reciprocal co-IP of CLASP2 and revealed MARK2 can co-IP SOGA1, glycogen synthase, and glycogenin. Investigating the SOGA1 interactome confirmed SOGA1 can reciprocal co-IP both CLASP2 and MARK2 as well as glycogen synthase and glycogenin. SOGA1 was confirmed to colocalize with CLASP2 and with tubulin, which identifies SOGA1 as a new microtubule-associated protein. These results introduce the metabolic function of these proposed novel protein networks and their relationship with microtubules as new fields of cytoskeletonassociated protein biology.