title: Acquired substrate preference for GAB1 protein bestows transforming activity to ERBB2 kinase lung cancer mutants.
abstract: Activating mutations in the αC-β4 loop of the ERBB2 kinase domain, such as ERBB2(YVMA) and ERBB2(G776VC), have been identified in human lung cancers and found to drive tumor formation. Here we observe that the docking protein GAB1 is hyper-phosphorylated in carcinomas from transgenic mice and in cell lines expressing these ERBB2 cancer mutants. Using dominant negative GAB1 mutants lacking canonical tyrosine residues for SHP2 and PI3K interactions or lentiviral shRNA that targets GAB1, we demonstrate that GAB1 phosphorylation is required for ERBB2 mutant-induced cell signaling, cell transformation, and tumorigenesis. An enzyme kinetic analysis comparing ERBB2(YVMA) to wild type using physiologically relevant peptide substrates reveals that ERBB2(YVMA) kinase adopts a striking preference for GAB1 phosphorylation sites as evidenced by ∼150-fold increases in the specificity constants (kcat/Km) for several GAB1 peptides, and this change in substrate selectivity was predominantly attributed to the peptide binding affinities as reflected by the apparent Km values. Furthermore, we demonstrate that ERBB2(YVMA) phosphorylates GAB1 protein ∼70-fold faster than wild type ERBB2 in vitro. Notably, the mutation does not significantly alter the Km for ATP or sensitivity to lapatinib, suggesting that, unlike EGFR lung cancer mutants, the ATP binding cleft of the kinase is not significantly changed. Taken together, our results indicate that the acquired substrate preference for GAB1 is critical for the ERBB2 mutant-induced oncogenesis.
authors.affiliation: Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Bethesda, Maryland 20892. Electronic address: ying-xin.fan@fda.hhs.gov.
authors.forename: Ying-Xin
authors.initials: YX
authors.lastname: Fan
authors.affiliation: Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Bethesda, Maryland 20892.
authors.forename: Lily
authors.initials: L
authors.lastname: Wong
authors.affiliation: Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Bethesda, Maryland 20892.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Marino
authors.affiliation: Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Bethesda, Maryland 20892.
authors.forename: Wu
authors.initials: W
authors.lastname: Ou
authors.affiliation: Division of Monoclonal Antibodies, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892.
authors.forename: Yi
authors.initials: Y
authors.lastname: Shen
authors.affiliation: Division of Monoclonal Antibodies, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892.
authors.forename: Wen Jin
authors.initials: WJ
authors.lastname: Wu
authors.affiliation: Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Kwok-Kin
authors.initials: KK
authors.lastname: Wong
authors.affiliation: Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Bethesda, Maryland 20892.
authors.forename: Jakob
authors.initials: J
authors.lastname: Reiser
authors.affiliation: Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Bethesda, Maryland 20892. Electronic address: gibbes.johnson@fda.hhs.gov.
authors.forename: Gibbes R
authors.initials: GR
authors.lastname: Johnson
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D005260:Female; D006801:Humans; D000077341:Lapatinib; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D008822:Mice, Transgenic; D020125:Mutation, Missense; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000235:genetics; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000235:genetics / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013379:Substrate Specificity / Q000187:drug effects / Q000235:genetics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000255:Adenosine Triphosphate; D000970:Antineoplastic Agents; D000077341:Lapatinib; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D011799:Quinazolines; D018719:Erb-b2 Receptor Tyrosine Kinases; C098587:GAB1 protein, human; C490897:Gab1 protein, mouse; C508053:ERBB2 protein, human; C524056:Erbb2 protein, mouse; C516725:Ptpn11 protein, mouse
keywords: Adaptor Proteins; ERBB2/HER2; Enzyme Mechanisms; Epidermal Growth Factor Receptor (EGFR); GAB1; Lung Cancer; Oncogenic Mutation; Tyrosine-protein Kinase (Tyrosine Kinase)