title: Oncogenic activity of Ect2 is regulated through protein kinase C iota-mediated phosphorylation.
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.
authors.affiliation: From the Department of Cancer Biology, Mayo Clinic College of Medicine, Jacksonville, Florida 32224 and.
authors.forename: Verline
authors.initials: V
authors.lastname: Justilien
authors.affiliation: From the Department of Cancer Biology, Mayo Clinic College of Medicine, Jacksonville, Florida 32224 and.
authors.forename: Lee
authors.initials: L
authors.lastname: Jameison
authors.affiliation: the Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599.
authors.forename: Channing J
authors.initials: CJ
authors.lastname: Der
authors.affiliation: the Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599.
authors.forename: Kent L
authors.initials: KL
authors.lastname: Rossman
authors.affiliation: From the Department of Cancer Biology, Mayo Clinic College of Medicine, Jacksonville, Florida 32224 and. Electronic address: fields.alan@mayo.edu.
authors.forename: Alan P
authors.initials: AP
authors.lastname: Fields
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D055785:Gene Knockdown Techniques; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D020125:Mutation, Missense; D009361:Neoplasm Invasiveness; D010766:Phosphorylation / Q000235:genetics; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism; D000099293:Protein Kinase C-lambda
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D007527:Isoenzymes; D011493:Protein Kinase C; D011518:Proto-Oncogene Proteins; D020830:rac1 GTP-Binding Protein; D000099293:Protein Kinase C-lambda; C489850:ECT2 protein, human; C499657:PARD6A protein, human; C107953:RAC1 protein, human