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

被引:177
作者
Gray, A [1 ]
Van der Kaay, J [1 ]
Downes, CP [1 ]
机构
[1] Univ Dundee, Dept Biochem, Dundee DD1 5EH, Scotland
关键词
green fluorescent protein; confocal microscopy;
D O I
10.1042/0264-6021:3440929
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We have tested the binding specificities of the pleckstrin homology (PH) domains of protein kinase B (PKB) and GRP1 (general receptor for phosphoinositides-l), 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 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)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.
引用
收藏
页码:929 / 936
页数:8
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共 29 条
  • [1] Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase B alpha
    Alessi, DR
    James, SR
    Downes, CP
    Holmes, AB
    Gaffney, PRJ
    Reese, CB
    Cohen, P
    [J]. CURRENT BIOLOGY, 1997, 7 (04) : 261 - 269
  • [2] Translocation of PDK-1 to the plasma membrane is important in allowing PDK-1 to activate protein kinase B
    Anderson, KE
    Coadwell, J
    Stephens, LR
    Hawkins, PT
    [J]. CURRENT BIOLOGY, 1998, 8 (12) : 684 - 691
  • [3] Role of translocation in the activation and function of protein kinase B
    Andjelkovic, M
    Alessi, DR
    Meier, R
    Fernandez, A
    Lamb, NJC
    Frech, M
    Cron, P
    Cohen, P
    Lucocq, JM
    Hemmings, BA
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1997, 272 (50) : 31515 - 31524
  • [4] PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2
    Balendran, A
    Casamayor, A
    Deak, M
    Paterson, A
    Gaffney, P
    Currie, R
    Downes, CP
    Alessi, DR
    [J]. CURRENT BIOLOGY, 1999, 9 (08) : 393 - 404
  • [5] A novel integrin-activated pathway forms PKB/Akt-stimulatory phosphatidylinositol 3,4-bisphosphate via phosphatidylinositol 3-phosphate in platelets
    Banfic, H
    Tang, XW
    Batty, IH
    Downes, CP
    Chen, CS
    Rittenhouse, SE
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (01) : 13 - 16
  • [6] Biphasic activation of PKBα/Akt in platelets -: Evidence for stimulation both by phosphatidylinositol 3,4-bisphosphate, produced via a novel pathway, and by phosphatidylinositol 3,4,5-trisphosphate
    Banfic, H
    Downes, CP
    Rittenhouse, SE
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (19) : 11630 - 11637
  • [7] Thrombin receptors modulate insulin-stimulated phosphatidylinositol 3,4,5-trisphosphate accumulation in 1321N1 astrocytoma cells
    Batty, IH
    Downes, CP
    [J]. BIOCHEMICAL JOURNAL, 1996, 317 : 347 - 351
  • [8] Intracellular localization of phosphatidylinositide 3-kinase and insulin receptor substrate-1 in adipocytes: Potential involvement of a membrane skeleton
    Clark, SF
    Martin, S
    Carozzi, AJ
    Hill, MM
    James, DE
    [J]. JOURNAL OF CELL BIOLOGY, 1998, 140 (05) : 1211 - 1225
  • [9] INHIBITION OF THE TRANSLOCATION OF GLUT1 AND GLUT4 IN 3T3-L1 CELLS BY THE PHOSPHATIDYLINOSITOL 3-KINASE INHIBITOR, WORTMANNIN
    CLARKE, JF
    YOUNG, PW
    YONEZAWA, K
    KASUGA, M
    HOLMAN, GD
    [J]. BIOCHEMICAL JOURNAL, 1994, 300 : 631 - 635
  • [10] Role of phosphatidylinositol 3,4,5-trisphosphate in regulating the activity and localization of 3-phosphoinositide-dependent protein kinase-1
    Currie, RA
    Walker, KS
    Gray, A
    Deak, M
    Casamayor, A
    Downes, CP
    Cohen, P
    Alessi, DR
    Lucocq, J
    [J]. BIOCHEMICAL JOURNAL, 1999, 337 : 575 - 583