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High affinity binding of inositol phosphates and phosphoinositides to the pleckstrin homology domain of RAC/protein kinase B and their influence on kinase activity

Frech, M., Andjelkovic, M., Ingley, E., Reddy, K.K., Falck, J.R. and Hemmings, B.A. (1997) 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, 272 (13). pp. 8474-8481.

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Link to Published Version: https://doi.org/10.1074/jbc.272.13.8474
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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.

Publication Type: Journal Article
Publisher: American Society for Biochemistry and Molecular Biology Inc.
URI: http://researchrepository.murdoch.edu.au/id/eprint/39588
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