title: Structural and Biophysical Characterization of the Interactions between Calmodulin and the Pleckstrin Homology Domain of Akt.
abstract: The translocation of Akt, a serine/threonine kinase, to the plasma membrane is a critical step in the Akt activation pathway. It is established that membrane binding of Akt is mediated by direct interactions between its pleckstrin homology domain (PHD) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). There is now evidence that Akt activation in many breast cancer cells is also modulated by the calcium-binding protein, calmodulin (CaM). Upon EGF stimulation of breast cancer cells, CaM co-localizes with Akt at the plasma membrane to enhance activation. However, the molecular details of Akt(PHD) interaction with CaM are not known. In this study, we employed NMR, biochemical, and biophysical techniques to characterize CaM binding to Akt(PHD). Our data show that CaM forms a tight complex with the PHD of Akt (dissociation constant = 100 nm). The interaction between CaM and Akt(PHD) is enthalpically driven, and the affinity is greatly dependent on salt concentration, indicating that electrostatic interactions are important for binding. The CaM-binding interface in Akt(PHD) was mapped to two loops adjacent to the PI(3,4,5)P3 binding site, which represents a rare CaM-binding motif and suggests a synergistic relationship between CaM and PI(3,4,5)P3 upon Akt activation. Elucidation of the mechanism by which Akt interacts with CaM will help in understanding the activation mechanism, which may provide insights for new potential targets to control the pathophysiological processes of cell survival.
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: Constance
authors.initials: C
authors.lastname: Agamasu
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: Ruba H
authors.initials: RH
authors.lastname: Ghanam
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294. Electronic address: saad@uab.edu.
authors.forename: Jamil S
authors.initials: JS
authors.lastname: Saad
mesh_terms: D055592:Biophysical Phenomena; D001943:Breast Neoplasms / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010716:Phosphatidylinositols / Q000378:metabolism; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D013816:Thermodynamics
chemical_list: D002147:Calmodulin; D010716:Phosphatidylinositols; D011994:Recombinant Proteins; C118303:phosphoinositide-3,4,5-triphosphate; C494918:AKT1 protein, human; D051057:Proto-Oncogene Proteins c-akt
keywords: Akt; breast cancer; calmodulin (CaM); isothermal titration calorimetry (ITC); nuclear magnetic resonance (NMR); phosphatidylinositol (3,4,5)-trisphosphate; plasma membrane; pleckstrin homology domain