Overexpression of the Akt‐pleckstrin homology domain induces branched neurites in hippocampal neurons and NG108‐15 cells

Abstract

Activation of class I phosphatidylinositol 3-kinases (PI3-Ks) induces the production of membrane-bound phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P3). By binding to the phospholipids via a pleckstrin homology (PH) domain certain proteins are translocated to the membrane, where they are activated. The accumulation of PtdIns(3,4,5)P3 at the cell membrane can be indirectly visualized with use of a fusion protein consisting of the green fluorescent protein (GFP) tagged to the PH domain of Akt (PHAKT-GFP). Overexpressed PHAKT-GFP is also quite potent in antagonizing the PtdIns(3,4,5)P3-mediated activation of the Akt protein kinase. Class I PI3-Ks expressed in neurons, are involved in neurite formation. In cultured embryonic hippocampal neurons, the PHAKT-GFP induced neurites with multiple branches. In neural NG108-15 cells, overexpression of PHAKT-GFP induced numerous branched extensions. The effect on neurite branch formation was only partially mimicked by treatment of the neurons with the PI3-K inhibitor wortmannin together with the Rhokinase (ROCK) inhibitor Y-27632. Biochemical analysis provided evidence that overexpression of PHAKT indeed inhibited PI3-Ks. ROCK was inhibited and Rac and Cdc42 were activated independently of the effects of PHAKT-GFP on PtdIns(3,4,5)P3. This data shows that interactions of PtdIns(3,4,5)P3 with PH-domains is not the only mechanism by which PH-domains interfere with signaling mechanism.