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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. 1999 Dec 15; 344 Pt 3:929-36.BJ

Abstract

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)P(3) 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)P(3) and PtdIns(3,4)P(2) 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 H(2)O(2)). Concomitantly, only PH(PKB)GFP, and not PH(GRP1)GFP, was localized at plasma membranes after 30 min of treatment with H(2)O(2). The fusion proteins appear accurately to report the spatial and temporal distribution of PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) 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)P(3) and PtdIns(3,4)P(2).

Authors+Show Affiliations

Department of Biochemistry, University of Dundee, Dundee DD1 5EH, Scotland, U.K. agray@bad.dundee.ac.ukNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

10585883

Citation

Gray, A, et al. "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." The Biochemical Journal, vol. 344 Pt 3, 1999, pp. 929-36.
Gray A, Van Der Kaay J, Downes CP. 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. 1999;344 Pt 3:929-36.
Gray, A., Van Der Kaay, J., & Downes, C. P. (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. The Biochemical Journal, 344 Pt 3, 929-36.
Gray A, Van Der Kaay J, Downes CP. 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. 1999 Dec 15;344 Pt 3:929-36. PubMed PMID: 10585883.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Gray,A, AU - Van Der Kaay,J, AU - Downes,C P, PY - 1999/12/10/pubmed PY - 2000/3/25/medline PY - 1999/12/10/entrez SP - 929 EP - 36 JF - The Biochemical journal JO - Biochem. J. VL - 344 Pt 3 N2 - 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)P(3) 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)P(3) and PtdIns(3,4)P(2) 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 H(2)O(2)). Concomitantly, only PH(PKB)GFP, and not PH(GRP1)GFP, was localized at plasma membranes after 30 min of treatment with H(2)O(2). The fusion proteins appear accurately to report the spatial and temporal distribution of PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) 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)P(3) and PtdIns(3,4)P(2). SN - 0264-6021 UR - https://www.unboundmedicine.com/medline/citation/10585883/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_34_bisphosphate_and/or_phosphatidylinositol_345_trisphosphate_in_vivo_ L2 - https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/10585883/ DB - PRIME DP - Unbound Medicine ER -