Lim Jeewon, Hwang Yo-Sep, Kim Jong-Tae, Yoon Hyang-Ran, Park Hyo-Min, Han Jahyeong, Kwon Taeho, Lee Kyung-Ho, Cho Hee-Jun, Lee Hee-Gu
Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea.
Department of Biomolecular Science, University of Science and Technology (UST), Daejeon 34113, Republic of Korea.
Cells. 2024 Dec 16;13(24):2072. doi: 10.3390/cells13242072.
Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1) plays a critical role in regulating the activity of Rho guanosine triphosphatases (GTPases). Phosphorylation of RhoGDI1 dynamically modulates the activation of Rho GTPases, influencing cell proliferation and migration. This study explored the involvement of Never In Mitosis A (NIMA)-related serine/threonine protein kinase 2 (NEK2) in phosphorylating RhoGDI1 and its implications in cancer cell behavior associated with tumor progression. We employed GST pull-down assays and immunoprecipitation to investigate the interaction between NEK2 and RhoGDI1. Truncation fragments identified the region of RhoGDI1 responsible for binding with NEK2. Phosphorylation assays determined the site of NEK2-mediated phosphorylation on RhoGDI1. Functional assays were conducted using overexpression of the RhoGDI1 substitution mutant to assess their impact on cancer cell behavior. NEK2 directly bound to RhoGDI1 and phosphorylated it at Ser174. This phosphorylation event facilitated cancer cell proliferation and motility by activating RhoA and Rac1. The RhoGDI1 aa 112-134 region was critical for the binding to NEK2. Disruption of the NEK2-RhoGDI1 interaction through overexpression of a RhoGDI1 truncated fragment (aa 112-134) led to diminished RhoGDI1 phosphorylation and RhoA/Rac1 activation induced by NEK2, resulting in reduced cancer cell proliferation and migration. Moreover, in vivo studies showed reduced tumor growth and lung metastasis when the NEK2-RhoGDI1 interaction was disrupted. This study indicates that NEK2 promotes the metastatic behaviors of cancer cells by activating RhoA and Rac1 by phosphorylating RhoGDI1.
Rho鸟嘌呤核苷酸解离抑制剂1（RhoGDI1）在调节Rho鸟苷三磷酸酶（GTPases）的活性中起关键作用。RhoGDI1的磷酸化动态调节Rho GTPases的激活，影响细胞增殖和迁移。本研究探讨了与有丝分裂无关A（NIMA）相关的丝氨酸/苏氨酸蛋白激酶2（NEK2）在RhoGDI1磷酸化中的作用及其在与肿瘤进展相关的癌细胞行为中的意义。我们采用谷胱甘肽S-转移酶（GST）下拉试验和免疫沉淀法研究NEK2与RhoGDI1之间的相互作用。截短片段确定了RhoGDI1与NEK2结合的区域。磷酸化试验确定了NEK2介导的RhoGDI1磷酸化位点。使用RhoGDI1替代突变体的过表达进行功能试验，以评估它们对癌细胞行为的影响。NEK2直接与RhoGDI1结合并在Ser174位点使其磷酸化。这一磷酸化事件通过激活RhoA和Rac1促进癌细胞增殖和运动。RhoGDI1的112-134氨基酸区域对于与NEK2的结合至关重要。通过过表达RhoGDI1截短片段（112-134氨基酸）破坏NEK2-RhoGDI1相互作用，导致NEK2诱导的RhoGDI1磷酸化和RhoA/Rac1激活减少，从而导致癌细胞增殖和迁移减少。此外，体内研究表明，破坏NEK2-RhoGDI1相互作用时，肿瘤生长和肺转移减少。本研究表明，NEK2通过磷酸化RhoGDI1激活RhoA和Rac1，促进癌细胞的转移行为。
