Isolation of the active form of RAC-protein kinase (PKB/Akt) from transfected COS-7 cells treated with heat shock stress and effects of phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate on its enzyme activity.
RAC-protein kinase (PKB/Akt) has been shown to be activated by growth factor stimulation as a downstream target of phosphatidylinositol 3-kinase and also by heat shock through a pathway independent of phosphatidylinositol 3-kinase. RAC-protein kinase was purified by antibody affinity chromatography from COS-7 cells transfected with the epitope-tagged expression plasmid. The protein kinase activity of RAC-protein kinase purified from heat-treated cells was 9-fold higher than the enzyme isolated from untreated control cells. Phosphatidylinositol 3,4,5-trisphosphate did not enhance the activity of RAC-protein kinase purified from either heat-treated cells or control cells, whereas phosphatidylinositol 4,5-bisphosphate suppressed the enzyme isolated from heat-treated cells. These results indicate that RAC-protein kinase may interact with phosphoinositides, however, it could not be activated by simple association with the product of phosphatidylinositol 3-kinase reaction.
Department of Biology, Faculty of Science, Kobe University, Japan.
H
H
Matsuzaki
H
H
Konishi
M
M
Tanaka
Y
Y
Ono
T
T
Takenawa
Y
Y
Watanabe
S
S
Ozaki
S
S
Kuroda
U
U
Kikkawa
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D004789:Enzyme Activation; D006358:Hot Temperature; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology*; D018129:Phosphatidylinositol Phosphates / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D011994:Recombinant Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt