Journal article
High affinity binding of inositol phosphates and phosphoinositides to the pleckstrin homology domain of RAC/protein kinase B and their influence on kinase activity
Journal of Biological Chemistry, Vol.272(13), pp.8474-8481
1997
Abstract
The influence of inositol phosphates and phosphoinositides on the α isoform of the RAC-protein kinase B (RAC/PKB) was studied using purified wild type and mutant kinase preparations and a recombinant pleckstrin homology (PH) domain. Binding of inositol phosphates and phosphoinositides to the PH domain was measured as the quenching of intrinsic tryptophan fluorescence. Inositol phosphates and D3-phosphorylated phosphoinositides bound with affinities of 1-10 μM and 0.5 μM, respectively. Similar values were obtained using RAC/PKB expressed and purified from baculovirus-infected Sf9 cells in the fluorescence assay. The influence of synthetic dioctanoyl derivatives of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate on the activity of RAC/PKB purified from transfected COS- 1 cells was studied. Phosphatidylinositol 3,4,5-trisphosphate was found to inhibit the RAC/PKB kinase activity with half-maximal inhibition at 2.5 μM. In contrast, phosphatidylinositol 3,4-bisphosphate stimulated kinase activity (half-maximal stimulation at 2.5 μM). A mutant RAC/PKB protein lacking the PH domain was not affected by D3-phosphorylated phosphoinositides. These results demonstrate that the PH domain of RAC/PKB binds inositol phosphates and phosphoinositides with high affinity, and suggest that the products of the phosphatidylinositide 3-kinase can act as both a membrane anchor and modulator of RAC/PKB activity. The data also provide further evidence for a link between phosphatidylinositide 3-kinase and RAC/PKB regulation.
Details
- Title
- High affinity binding of inositol phosphates and phosphoinositides to the pleckstrin homology domain of RAC/protein kinase B and their influence on kinase activity
- Authors/Creators
- M. Frech (Author/Creator) - Friedrich Miescher InstituteM. Andjelkovic (Author/Creator) - Friedrich Miescher InstituteE. Ingley (Author/Creator) - Friedrich Miescher InstituteK.K. Reddy (Author/Creator) - The University of Texas Southwestern Medical CenterJ.R. Falck (Author/Creator) - The University of Texas Southwestern Medical CenterB.A. Hemmings (Author/Creator) - Friedrich Miescher Institute
- Publication Details
- Journal of Biological Chemistry, Vol.272(13), pp.8474-8481
- Publisher
- American Society for Biochemistry and Molecular Biology Inc.
- Identifiers
- 991005540073207891
- Murdoch Affiliation
- Murdoch University
- Language
- English
- Resource Type
- Journal article
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