NEK2 Phosphorylates RhoGDI1 to Promote Cell Proliferation, Migration and Invasion Through the Activation of RhoA and Rac1 in Colon Cancer Cells

Cells. 2024 Dec 16;13(24):2072. doi: 10.3390/cells13242072.

Abstract

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.

Keywords: NEK2; Rho GTPase; RhoGDI1; cancer; migration; proliferation.

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Cell Movement*
  • Cell Proliferation*
  • Colonic Neoplasms* / genetics
  • Colonic Neoplasms* / metabolism
  • Colonic Neoplasms* / pathology
  • Humans
  • Mice
  • Mice, Nude
  • NIMA-Related Kinases* / genetics
  • NIMA-Related Kinases* / metabolism
  • Neoplasm Invasiveness
  • Phosphorylation
  • Protein Binding
  • rac1 GTP-Binding Protein* / metabolism
  • rho Guanine Nucleotide Dissociation Inhibitor alpha* / genetics
  • rho Guanine Nucleotide Dissociation Inhibitor alpha* / metabolism
  • rhoA GTP-Binding Protein* / metabolism

Substances

  • NIMA-Related Kinases
  • NEK2 protein, human
  • rhoA GTP-Binding Protein
  • rac1 GTP-Binding Protein
  • rho Guanine Nucleotide Dissociation Inhibitor alpha
  • RHOA protein, human
  • RAC1 protein, human