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

Verline Justilien, Lee Jameison, Channing J. Der, Kent L. Rossman, Alan P Fields

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

The Rho GTPase guanine nucleotide exchange factor Ect2 is genetically and biochemically linked to the PKCι oncogene in non-small cell lung cancer (NSCLC). Ect2 is overexpressed and mislocalized to the cytoplasm of NSCLC cells where it binds the oncogenic PKCι-Par6 complex, leading to activation of the Rac1 small GTPase. Here, we identify a previously uncharacterized phosphorylation site on Ect2, threonine 328, that serves to regulate the oncogenic activity of Ect2 in NSCLC cells. PKCι directly phosphorylates Ect2 at Thr-328 in vitro, and RNAi-mediated knockdown of either PKCι or Par6 leads to a decrease in phospho-Thr-328 Ect2, indicating that PKCι regulates Thr-328 Ect2 phosphorylation in NSCLC cells. Both wild-type Ect2 and a phosphomimetic T328D Ect2 mutant bind the PKCι-Par6 complex, activate Rac1, and restore transformed growth and invasion when expressed in NSCLC cells made deficient in endogenous Ect2 by RNAi-mediated knockdown. In contrast, a phosphorylation-deficient T328A Ect2 mutant fails to bind the PKCι-Par6 complex, activate Rac1, or restore transformation. Our data support a model in which PKCι-mediated phosphorylation regulates Ect2 binding to the oncogenic PKCι-Par6 complex thereby activating Rac1 activity and driving transformed growth and invasion.

Original languageEnglish (US)
Pages (from-to)8149-8157
Number of pages9
JournalJournal of Biological Chemistry
Volume286
Issue number10
DOIs
StatePublished - Mar 11 2011

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Phosphorylation
Non-Small Cell Lung Carcinoma
Protein Kinase C
RNA Interference
Guanine Nucleotide Exchange Factors
rho GTP-Binding Proteins
Rho Guanine Nucleotide Exchange Factors
Monomeric GTP-Binding Proteins
Threonine
Growth
Oncogenes
Chemical activation
Cells
Cytoplasm

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Oncogenic activity of Ect2 is regulated through protein kinase Cι-mediated phosphorylation. / Justilien, Verline; Jameison, Lee; Der, Channing J.; Rossman, Kent L.; Fields, Alan P.

In: Journal of Biological Chemistry, Vol. 286, No. 10, 11.03.2011, p. 8149-8157.

Research output: Contribution to journalArticle

Justilien, Verline ; Jameison, Lee ; Der, Channing J. ; Rossman, Kent L. ; Fields, Alan P. / Oncogenic activity of Ect2 is regulated through protein kinase Cι-mediated phosphorylation. In: Journal of Biological Chemistry. 2011 ; Vol. 286, No. 10. pp. 8149-8157.
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abstract = "The Rho GTPase guanine nucleotide exchange factor Ect2 is genetically and biochemically linked to the PKCι oncogene in non-small cell lung cancer (NSCLC). Ect2 is overexpressed and mislocalized to the cytoplasm of NSCLC cells where it binds the oncogenic PKCι-Par6 complex, leading to activation of the Rac1 small GTPase. Here, we identify a previously uncharacterized phosphorylation site on Ect2, threonine 328, that serves to regulate the oncogenic activity of Ect2 in NSCLC cells. PKCι directly phosphorylates Ect2 at Thr-328 in vitro, and RNAi-mediated knockdown of either PKCι or Par6 leads to a decrease in phospho-Thr-328 Ect2, indicating that PKCι regulates Thr-328 Ect2 phosphorylation in NSCLC cells. Both wild-type Ect2 and a phosphomimetic T328D Ect2 mutant bind the PKCι-Par6 complex, activate Rac1, and restore transformed growth and invasion when expressed in NSCLC cells made deficient in endogenous Ect2 by RNAi-mediated knockdown. In contrast, a phosphorylation-deficient T328A Ect2 mutant fails to bind the PKCι-Par6 complex, activate Rac1, or restore transformation. Our data support a model in which PKCι-mediated phosphorylation regulates Ect2 binding to the oncogenic PKCι-Par6 complex thereby activating Rac1 activity and driving transformed growth and invasion.",
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