We have identified a novel mitogen‐ and stress‐activated protein kinase (MSK1) that contains two protein kinase domains in a single polypeptide. MSK1 is activated in vitro by MAPK2/ERK2 or SAPK2/p38. Endogenous MSK1 is activated in 293 cells by either growth factor/phorbol ester stimulation, or by exposure to UV radiation, and oxidative and chemical stress. The activation of MSK1 by growth factors/phorbol esters is prevented by PD 98059, which suppresses activation of the MAPK cascade, while the activation of MSK1 by stress stimuli is prevented by SB 203580, a specific inhibitor of SAPK2/p38. In HeLa, PC12 and SK‐N‐MC cells, PD 98059 and SB 203580 are both required to suppress the activation of MSK1 by TNF, NGF and FGF, respectively, because these agonists activate both the MAPK/ERK and SAPK2/p38 cascades. MSK1 is localized in the nucleus of unstimulated or stimulated cells, and phosphorylates CREB at Ser133 with a K m value far lower than PKA, MAPKAP‐K1(p90Rsk) and MAPKAP‐K2. The effects of SB 203580, PD 98059 and Ro 318220 on agonist‐induced activation of CREB and ATF1 in four cell‐lines mirror the effects of these inhibitors on MSK1 activation, and exclude a role for MAPKAP‐K1 and MAPKAP‐K2/3 in this process. These findings, together with other observations, suggest that MSK1 may mediate the growth‐factor and stress‐induced activation of CREB.

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 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-PKBα 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.