title: Physical association of PDK1 with AKT1 is sufficient for pathway activation independent of membrane localization and phosphatidylinositol 3 kinase.
abstract: Frequent activation of the AKT serine-threonine kinase in cancer confers resistance to therapy. AKT is activated by a multi-step process involving phosphatidylinositide (PtdIns) phosphate-mediated recruitment of AKT and its upstream kinases, including 3-Phosphoinositide-dependent kinase 1 (PDK1), to the inner surface of the cell membrane. PDK1 in the appropriate context phosphorylates AKT at threonine 308 (T308) to activate AKT. Whether PtdIns(3,4,5)Ps (PtdInsP3) binding and AKT membrane translocation mediate functions other than formation of a functional PDK1::AKT complex have not been fully elucidated. We fused complementary fragments of intensely fluorescent protein (IFP) to AKT1 and PDK1 to induce a stable complex to study the prerequisites of AKT1 phosphorylation and function. In the stabilized PDK1-IFPC::IFPN-AKT1 complex, AKT1 T308 phosphorylation was independent of PtdIns, as demonstrated by treatment with Phosphatidylinositol 3 Kinase (PI3K) inhibitors. Further when interaction with PtdIns and the cell membrane was prevented by creating PH-domain mutants of AKT1 (R25A) and PDK1 (R474A), AKT1 phosphorylation on T308 was maintained in the PDK1-IFPC::IFPN-AKT1 complex. The PDK1-IFPC::IFPN-AKT1 complex was sufficient for phosphorylation of known AKT substrates, and conferred resistance to inhibitors of PI3K (LY294002, PI103, GDC0941 and TGX286) but not inhibitors of the downstream TORC1 complex (rapamycin). Thus the locus of action of targeted therapeutics can be elucidated by the constitutively active AKT1 complex. Our data indicate that PtdIns and membrane localization are not required for AKT phosphorylation and activation, but rather serve to induce a functional physical interaction between PDK1 and AKT. The PDK1-IFPC::IFPN-AKT1 complex provides a cell-based platform to examine specificity of drugs targeting PI3K pathway components.
authors.affiliation: Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America. zding@mdanderson.org
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Ding
authors.forename: Jiyong
authors.initials: J
authors.lastname: Liang
authors.forename: Jin
authors.initials: J
authors.lastname: Li
authors.forename: Yiling
authors.initials: Y
authors.lastname: Lu
authors.forename: Vathsala
authors.initials: V
authors.lastname: Ariyaratna
authors.forename: Zhimin
authors.initials: Z
authors.lastname: Lu
authors.forename: Michael A
authors.initials: MA
authors.lastname: Davies
authors.forename: John K
authors.initials: JK
authors.lastname: Westwick
authors.forename: Gordon B
authors.initials: GB
authors.lastname: Mills
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D005456:Fluorescent Dyes / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D034741:RNA, Small Interfering / Q000378:metabolism; D013912:Threonine / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D034741:RNA, Small Interfering; D013912:Threonine; C000631429:PDK1 protein, human