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

Matthias Frech, Mirjana Andjelkovic, Evan Ingley, K. Kishta Reddy, J R Falck, Brian A. Hemmings

Research output: Contribution to journalArticle

345 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)8474-8481
Number of pages8
JournalJournal of Biological Chemistry
Volume272
Issue number13
DOIs
StatePublished - Mar 28 1997

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Proto-Oncogene Proteins c-akt
Inositol Phosphates
Phosphatidylinositols
Phosphotransferases
Fluorescence
Sf9 Cells
Pleckstrin Homology Domains
platelet protein P47
Baculoviridae
COS Cells
Mutant Proteins
Anchors
Tryptophan
Modulators
Quenching
Assays
Protein Isoforms
Derivatives
Membranes

ASJC Scopus subject areas

  • Biochemistry

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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, Matthias; Andjelkovic, Mirjana; Ingley, Evan; Reddy, K. Kishta; Falck, J R; Hemmings, Brian A.

In: Journal of Biological Chemistry, Vol. 272, No. 13, 28.03.1997, p. 8474-8481.

Research output: Contribution to journalArticle

Frech, Matthias ; Andjelkovic, Mirjana ; Ingley, Evan ; Reddy, K. Kishta ; Falck, J R ; Hemmings, Brian A. / High affinity binding of inositol phosphates and phosphoinositides to the pleckstrin homology domain of RAC/protein kinase B and their influence on kinase activity. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 13. pp. 8474-8481.
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AU - Hemmings, Brian A.

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