title: The Interplay between Calmodulin and Membrane Interactions with the Pleckstrin Homology Domain of Akt.
abstract: The Akt protein, a serine/threonine kinase, plays important roles in cell survival, apoptosis, and oncogenes. Akt is translocated to the plasma membrane for activation. Akt-membrane binding is mediated by direct interactions between its pleckstrin homology domain (PHD) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). It has been shown that Akt activation in breast cancer cells is modulated by calmodulin (CaM). However, the molecular mechanism of the interplay between CaM and membrane binding is not established. Here, we employed nuclear magnetic resonance (NMR) and biochemical and biophysical techniques to characterize how PI(3,4,5)P3, CaM, and membrane mimetics (nanodisc) bind to Akt(PHD). We show that PI(3,4,5)P3binding to Akt(PHD) displaces the C-terminal lobe of CaM but not the weakly binding N-terminal lobe. However, binding of a PI(3,4,5)P3-embedded membrane nanodisc to Akt(PHD) with a 103-fold tighter affinity than PI(3,4,5)P3is able to completely displace CaM. We also show that Akt(PHD) binds to both layers of the nanodisc, indicating proper incorporation of PI(3,4,5)P3on the nanodisc surface. No detectable binding has been observed between Akt(PHD) and PI(3,4,5)P3-free nanodiscs, demonstrating that PI(3,4,5)P3is required for membrane binding, CaM displacement, and Akt activation. Using pancreatic cancer cells, we demonstrate that inhibition of Akt-CaM binding attenuated Akt activation. Our findings support a model by which CaM binds to Akt to facilitate its translocation to the membrane. Elucidation of the molecular details of the interplay between membrane and CaM binding to Akt may help in the development of potential targets to control the pathophysiological processes of cell survival.
authors.affiliation: From the Departments of Microbiology and.
authors.forename: Constance
authors.initials: C
authors.lastname: Agamasu
authors.affiliation: From the Departments of Microbiology and.
authors.forename: Ruba H
authors.initials: RH
authors.lastname: Ghanam
authors.affiliation: Pathology, University of Alabama at Birmingham and.
authors.forename: Fei
authors.initials: F
authors.lastname: Xu
authors.affiliation: Pathology, University of Alabama at Birmingham and.|the Research Department, Birmingham Veterans Affairs Medical Center, Birmingham, Alabama 35294.
authors.forename: Yong
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Pathology, University of Alabama at Birmingham and.|the Research Department, Birmingham Veterans Affairs Medical Center, Birmingham, Alabama 35294.
authors.forename: Yabing
authors.initials: Y
authors.lastname: Chen
authors.affiliation: From the Departments of Microbiology and saad@uab.edu.
authors.forename: Jamil S
authors.identifier: http://orcid.org/0000-0001-9503-6484
authors.initials: JS
authors.lastname: Saad
mesh_terms: D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D000070539:Pleckstrin Homology Domains; D011485:Protein Binding; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D018129:Phosphatidylinositol Phosphates; C060974:phosphatidylinositol 3,4,5-triphosphate; D051057:Proto-Oncogene Proteins c-akt
keywords: breast cancer; calmodulin (CaM); isothermal titration calorimetry (ITC); membrane nanodisc; nuclear magnetic resonance (NMR); pancreatic cancer; phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3); plasma membrane; pleckstrin homology domain (PHD)