INVOLVEMENT OF THE PLECKSTRIN HOMOLOGY DOMAIN IN THE INSULIN-STIMULATED ACTIVATION OF PROTEIN-KINASE-B

Involvement of the pleckstrin homology (PR) domain in the insulin-stim ulated activation of protein kinase B (PKB) was investigated in human embryonic kidney 293 cells. Different PKB constructs that contain muta tions or deletions in the PH domain were transfected into cells, and t he results on the basal and insulin-induced kinase activities were ana lyzed. Deletion of the entire PH domain (Delta PH-PKB) did not impair the kinase activity; in contrast, the basal activity was elevated with respect to wild-type PKB. In addition, Delta PH-PKB was responsive to insulin, and as for wild-type PKB, this was dependent on phosphoinosi tide 3-kinase. By contrast, a point mutation within the PH domain that impairs phospholipid binding (R25C) resulted in a construct that was not responsive to insulin. However, this defect was overcome by mutati ons that mimic the phosphorylation state of the active kinase. The inc rease in the basal activity of Delta PH-PKB was shown to be due to an elevation in the level of phosphorylation of this construct. In additi on, the subcellular localization of Delta PH-PKB, as determined by bot h immunofluorescence and fractionation, was predominately cytosolic, a nd Delta PH-PKB was present in the plasma membrane at much lower level s compared with wild-type PKB. These data show that phosphorylation is the major factor regulating the activity of PKB and that either remov al of the PH domain or binding of phospholipids is required to permit this phosphorylation. In addition, membrane localization does not appe ar to be required for the activation process, but instead, binding of PKB to membrane phospholipids permits a conformational change in the m olecule that allows for phosphorylation.