Catalog Home Page

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.ORCID: 0000-0002-8112-9134, 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.

[img]
Preview
PDF - Published Version
Download (637kB) | Preview
Link to Published Version: https://doi.org/10.1074/jbc.272.13.8474
*Subscription may be required

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.

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

Downloads

Downloads per month over past year