Identification of functional domains in AKT responsible for distinct roles of AKT isoforms in pressure-stimulated cancer cell adhesion.
Cell adhesion is a critical step in cancer metastasis, activated by extracellular forces such as pressure and shear. Reducing AKT1, but not AKT2, ablates the increase in cancer cell adhesion associated with 15 mm Hg increased extracellular pressure. To identify the determinants of this AKT isoform specificity, we exchanged the pleckstrin homology (PH) domains and/or hinge regions of AKT1 and AKT2. Wild type isoforms or these chimeras were overexpressed in Caco-2 cells in the absence or presence of isoform-specific siRNA to suppress endogenous AKT1. Pressure-induced AKT translocation and phosphorylation to the membrane were compared, along with the stimulation of cell adhesion by pressure. Pressure stimulated translocation of AKT1, but not AKT2 to the plasma membrane. Among our chimeras, only the chimeric AKT2 (chimera2), in which both the AKT2 PH domain and hinge region had been replaced by those of AKT1, translocated to the membrane in response to pressure. Similarly, only chimera2 rescued the function of AKT1 in mediating pressure-stimulated adhesion after endogenous AKT1 had been reduced. Pressure also promoted phosphorylation of AKT1 but not AKT2, and expression of a nonphosphorylatable double point mutant prevented pressure-stimulated adhesion. Among the chimeras, pressure promoted only chimera2 phosphorylation. These results identify the AKT1 PH domain and hinge region as functional domains which jointly permit AKT1 translocation and phosphorylation in response to extracellular pressure and distinguish determine the specificity of AKT1 in mediating the effects of extracellular pressure on cancer cell adhesion. These may be useful targets for interventions to inhibit metastasis.
Wang
Shouye
S
Surgical Service, John D. Dingell VA Medical Center, Wayne State University, Detroit, MI 48201, USA.
Basson
Marc D
MD
D018938:Caco-2 Cells; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002678:Chimera / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D009369:Neoplasms / Q000201:enzymology*; D010716:Phosphatidylinositols / Q000494:pharmacology; D010766:Phosphorylation; D011312:Pressure; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D034741:RNA, Small Interfering / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism