High affinity binding of inositol phosphates and phosphoinositides to the pleckstrin homology domain of RAC/protein kinase B and their influence on kinase activity.
The influence of inositol phosphates and phosphoinositides on the alpha 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 microM and 0.5 microM, 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 microM. In contrast, phosphatidylinositol 3, 4-bisphosphate stimulated kinase activity (half-maximal stimulation at 2.5 microM). 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.
Frech
M
M
Friedrich Miescher Institute, P. O. Box 2543, CH-4002 Basel, Switzerland.
Andjelkovic
M
M
Ingley
E
E
Reddy
K K
KK
Falck
J R
JR
Hemmings
B A
BA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism*; D019556:COS Cells; D034281:Dynamins; D020558:GTP Phosphohydrolases / Q000737:chemistry; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism*; D008969:Molecular Sequence Data; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D010750:Phosphoproteins; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D016415:Sequence Alignment; D013049:Spectrin / Q000737:chemistry; D013054:Spectrophotometry, Atomic
D001798:Blood Proteins; D007295:Inositol Phosphates; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010750:Phosphoproteins; C060974:phosphatidylinositol 3,4,5-triphosphate; C039077:platelet protein P47; D013049:Spectrin; C051854:inositol 3,4-bisphosphate; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020558:GTP Phosphohydrolases; D034281:Dynamins