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Oncogenic activity of Ect2 is regulated through protein kinase C iota-mediated phosphorylation

Verline Justilien1, Lee Jameison1, Channing J Der2

  • 1From the Department of Cancer Biology, Mayo Clinic College of Medicine, Jacksonville, Florida 32224 and.

The Journal of Biological Chemistry
|December 30, 2010
PubMed

Insights

Phosphorylation of Ect2 at threonine 328 by PKCι regulates its oncogenic activity in non-small cell lung cancer (NSCLC). This phosphorylation controls Ect2 binding to the PKCι-Par6 complex, activating Rac1 and promoting tumor growth.

Area of Science:

  • Oncology
  • Molecular Biology
  • Cell Signaling

Background:

  • Ect2, a Rho GTPase guanine nucleotide exchange factor, is linked to the PKCι oncogene in non-small cell lung cancer (NSCLC).
  • Ect2 overexpression and cytoplasmic mislocalization in NSCLC cells facilitate binding with the oncogenic PKCι-Par6 complex, activating Rac1.
  • Rac1 small GTPase activation is a key pathway in cancer cell proliferation and invasion.

Purpose of the Study:

  • To identify and characterize a novel phosphorylation site on Ect2 that regulates its oncogenic function in NSCLC.
  • To elucidate the role of protein kinase C iota (PKCι)-mediated phosphorylation of Ect2 in NSCLC pathogenesis.
  • To investigate the mechanism by which Ect2 phosphorylation influences its interaction with the PKCι-Par6 complex and Rac1 activation.

Main Methods:

  • In vitro kinase assays to determine direct phosphorylation of Ect2 by PKCι.
  • RNA interference (RNAi) to knockdown PKCι and Par6, assessing the impact on Ect2 phosphorylation.
  • Expression of wild-type, phosphomimetic (T328D), and phosphorylation-deficient (T328A) Ect2 mutants in RNAi-treated NSCLC cells.
  • Assessment of Ect2-PKCι-Par6 complex formation, Rac1 activation, and cellular transformation/invasion assays.

Main Results:

  • A previously uncharacterized phosphorylation site, threonine 328 (Thr-328), was identified on Ect2.
  • PKCι was confirmed to directly phosphorylate Ect2 at Thr-328 in vitro.
  • PKCι-mediated phosphorylation of Ect2 at Thr-328 is crucial for its binding to the PKCι-Par6 complex and subsequent activation of Rac1.
  • The T328A mutant failed to restore transformed growth and invasion, while wild-type and T328D mutants rescued these phenotypes in Ect2-deficient cells.

Conclusions:

  • PKCι-mediated phosphorylation of Ect2 at Thr-328 is a critical regulatory mechanism for its oncogenic activity in NSCLC.
  • Phosphorylation at Thr-328 facilitates Ect2's interaction with the PKCι-Par6 complex, leading to Rac1 activation.
  • Targeting this phosphorylation event could offer a therapeutic strategy for NSCLC by disrupting Ect2-driven oncogenesis.

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