We have tested the binding specificities of the pleckstrin homology (PH) domains of protein kinase B (PKB) and GRP1 (general receptor for phosphoinositides-1), expressed as green fluorescent protein (GFP) fusion proteins [PH(PKB)GFP and PH(GRP1)GFP respectively] in HEK 293 cells and Swiss 3T3 cells, using confocal microscopy. Stimulation of HEK 293 cells with insulin caused a small, but sustained, increase in PtdIns(3,4,5)P3 levels, detected using a radioligand displacement assay, which was mirrored by the translocation of PH(PKB)GFP and PH(GRP1)GFP from the cytosol to the plasma membrane of live, transfected cells. Similar results were obtained using Swiss 3T3 cells stimulated with platelet-derived growth factor (PDGF) and expressing either PH(PKB)GFP or PH(GRP1)GFP. Biochemical analyses confirmed the accumulation of both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in response to PDGF, but only the latter was present at increased levels in Swiss 3T3 cells 30 min after an oxidative stress (1 mM H2O2). Concomitantly, only PH(PKB)GFP, and not PH(GRP1)GFP, was localized at plasma membranes after 30 min of treatment with H2O2. The fusion proteins appear accurately to report the spatial and temporal distribution of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in intact cells. These results establish the lipid selectivity of these PH domains in vivo, and further emphasize the overlapping, but distinct, second messenger roles of PtdIns(3,4,5)P3 and PtdIns(3,4)P2.
Skip Nav Destination
Article navigation
December 1999
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
Research Article|
December 08 1999
The pleckstrin homology domains of protein kinase B and GRP1 (general receptor for phosphoinositides-1) are sensitive and selective probes for the cellular detection of phosphatidylinositol 3,4-bisphosphate and/or phosphatidylinositol 3,4,5-trisphosphate in vivo
Alexander GRAY;
Alexander GRAY
1Department of Biochemistry, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
Search for other works by this author on:
Jeroen V AN DER KAAY;
Jeroen V AN DER KAAY
1Department of Biochemistry, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
Search for other works by this author on:
C. Peter DOWNES
C. Peter DOWNES
1Department of Biochemistry, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
June 21 1999
Revision Received:
September 07 1999
Accepted:
September 23 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 344 (3): 929–936.
Article history
Received:
June 21 1999
Revision Received:
September 07 1999
Accepted:
September 23 1999
Citation
Alexander GRAY, Jeroen V AN DER KAAY, C. Peter DOWNES; The pleckstrin homology domains of protein kinase B and GRP1 (general receptor for phosphoinositides-1) are sensitive and selective probes for the cellular detection of phosphatidylinositol 3,4-bisphosphate and/or phosphatidylinositol 3,4,5-trisphosphate in vivo. Biochem J 15 December 1999; 344 (3): 929–936. doi: https://doi.org/10.1042/bj3440929
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.