Role of translocation in the activation and function of protein kinase B

TY - JOUR

T1 - Role of translocation in the activation and function of protein kinase B

AU - Andjelković, Mirjana

AU - Alessi, Dario R.

AU - Meier, Roger

AU - Fernandez, Anne

AU - Lamb, Ned J C

AU - Frech, Matthias

AU - Cron, Peter

AU - Cohen, Philip

AU - Lucocq, John M.

AU - Hemmings, Brian A.

PY - 1997/12/12

Y1 - 1997/12/12

N2 - 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)-PKBα was associated with the plasma membrane of transfected cells, whereas the wild-type kinase was mostly cytosolic. The activity of m/p-PKBα 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-PKBα activity was due to phosphorylation on Thr308 and Ser478, 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/pPKBα activity. The pleckstrin homology (PH) domain of m/p-PKBα 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, PKBα 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.

AB - 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)-PKBα was associated with the plasma membrane of transfected cells, whereas the wild-type kinase was mostly cytosolic. The activity of m/p-PKBα 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-PKBα activity was due to phosphorylation on Thr308 and Ser478, 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/pPKBα activity. The pleckstrin homology (PH) domain of m/p-PKBα 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, PKBα 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.

UR - https://www.scopus.com/pages/publications/15644381754

U2 - 10.1074/jbc.272.50.31515

DO - 10.1074/jbc.272.50.31515

M3 - Article

C2 - 9395488

AN - SCOPUS:15644381754

SN - 0021-9258

VL - 272

SP - 31515

EP - 31524

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 50

ER -