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Overexpression of the Akt‐pleckstrin homology domain induces branched neurites in hippocampal neurons and NG108‐15 cells
Author(s) -
Leemhuis Jost,
Fischer Catharina,
Meyer Dieter K.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a950-d
Subject(s) - pleckstrin homology domain , phosphatidylinositol , neurite , wortmannin , microbiology and biotechnology , protein kinase b , green fluorescent protein , chemistry , pi3k/akt/mtor pathway , hippocampal formation , kinase , signal transduction , biology , biochemistry , gene , neuroscience , in vitro
Activation of class I phosphatidylinositol 3‐kinases (PI3‐Ks) induces the production of membrane‐bound phosphatidylinositol 3,4,5‐triphosphate (PtdIns(3,4,5)P 3 ). 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)P 3 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)P 3 ‐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)P 3 . This data shows that interactions of PtdIns(3,4,5)P 3 with PH‐domains is not the only mechanism by which PH‐domains interfere with signaling mechanism.