Role of translocation in the activation and function of protein kinase B.
We have investigated the role of subcellular localization in the regulation of protein kinase B (PKB) activation. The myristoylation/palmitylation motif from the Lck tyrosine kinase was attached to the N terminus of protein kinase B to alter its subcellular location. Myristoylated/palmitylated (m/p)-PKBalpha was associated with the plasma membrane of transfected cells, whereas the wild-type kinase was mostly cytosolic. The activity of m/p-PKBalpha was 60-fold higher compared with the unstimulated wild-type enzyme, and could not be stimulated further by growth factors or phosphatase inhibitors. In vivo 32P labeling and mutagenesis demonstrated that m/p-PKBalpha activity was due to phosphorylation on Thr308 and Ser473, that are normally induced on PKB following stimulation of the cells with insulin or insulin-like growth factor-1 (IGF-1). A dominant negative form of phosphoinositide 3-kinase (PI3-K) did not affect m/p-PKBalpha activity. The pleckstrin homology (PH) domain of m/p-PKBalpha was not required for its activation or phosphorylation on Thr308 and Ser473, suggesting that this domain may serve as a membrane-targeting module. Consistent with this view, PKBalpha was translocated to the plasma membrane within minutes after stimulation with IGF-1. This translocation required the PH domain and was sensitive to wortmannin. Our results indicate that PI3-K activity is required for translocation of PKB to the plasma membrane, where its activation occurs through phosphorylation of the same sites that are induced by insulin or IGF-1. Following activation the kinase detached from the membrane and translocated to the nucleus.
Andjelković
M
M
Friedrich Miescher-Institut, Maulbeerstrasse 66, CH-4056 Basel, Switzerland.
Alessi
D R
DR
Meier
R
R
Fernandez
A
A
Lamb
N J
NJ
Frech
M
M
Cron
P
P
Cohen
P
P
Lucocq
J M
JM
Hemmings
B A
BA
D019943:Amino Acid Substitution; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D009025:Morpholines / Q000494:pharmacology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D021382:Protein Sorting Signals / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D021382:Protein Sorting Signals; D011518:Proto-Oncogene Proteins; D013912:Threonine; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D012694:Serine; D007334:Insulin-Like Growth Factor I; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt