Li Ying-Hua, Wang Yin-Yin, Zhong Shan, Rong Zhi-Li, Ren Yong-Ming, Li Zhi-Yong, Zhang Shu-Ping, Chang Zhi-Jie, Liu Li
Institute of Biomedicine and School of Medicine, Tsinghua University, Beijing, 100084, China.
Mol Cells. 2009 Jan 31;27(1):39-45. doi: 10.1007/s10059-009-0003-5. Epub 2009 Feb 5.
Ligand-dependent or independent oligomerization of receptor protein tyrosine kinase (RPTK) is often an essential step for receptor activation and intracellular signaling. The novel oncogene with kinase-domain (NOK) is a unique RPTK that almost completely lacks an ectodomain, expresses intracellularly and activates constitutively. However, it is unknown whether NOK can form oligomer or what function oligomerization would have. In this study, two NOK deletion mutants were generated by either removing the ectodomain (NOKDeltaECD) or including the endodomain (NOK-ICD). Co-immunoprecipitation demonstrated that the transmembrane (TM) domain of NOK was essential for its intermolecular interaction. The results further showed that NOK aggregated more closely as lower order oligomers (the dimer- and trimer-sized) than either deletion mutant did since NOK could be cross-linked by both Sulfo-EGS and formaldehyde, whereas either deletion mutant was only sensitive to Sulfo-EGS. Removing the NOK TM domain (NOK-ICD) not only markedly promoted higher order oligomerization, but also altered the subcellular localization of NOK and dramatically elevated the NOK-mediated constitutive activation of extracellular signal-regulated kinase (ERK). Moreover, NOK-ICD but not NOK or NOKDeltaECD was co-localized with the upstream signaling molecule RAS on cell membrane. Thus, TM-mediated intermolecular contacting may be mainly responsible for the constitutive activation of NOK and contribute to the autoinhibitory effect on RAS/MAPK signaling.
受体蛋白酪氨酸激酶（RPTK）的配体依赖性或非依赖性寡聚化通常是受体激活和细胞内信号传导的关键步骤。新型激酶结构域癌基因（NOK）是一种独特的RPTK，几乎完全缺乏胞外结构域，在细胞内表达并组成性激活。然而，尚不清楚NOK是否能形成寡聚体，以及寡聚化会有什么功能。在本研究中，通过去除胞外结构域（NOKDeltaECD）或包含胞内结构域（NOK-ICD）产生了两种NOK缺失突变体。免疫共沉淀表明NOK的跨膜（TM）结构域对其分子间相互作用至关重要。结果进一步表明，与任何一种缺失突变体相比，NOK以更低阶的寡聚体（二聚体和三聚体大小）更紧密地聚集，因为NOK可被磺基-EGS和甲醛交联，而任何一种缺失突变体仅对磺基-EGS敏感。去除NOK的TM结构域（NOK-ICD）不仅显著促进了高阶寡聚化，还改变了NOK的亚细胞定位，并显著提高了NOK介导的细胞外信号调节激酶（ERK）的组成性激活。此外，NOK-ICD而非NOK或NOKDeltaECD与上游信号分子RAS在细胞膜上共定位。因此，TM介导的分子间接触可能是NOK组成性激活的主要原因，并有助于对RAS/MAPK信号传导的自抑制作用。
