Inhibition of PKB/Akt1 by C2-Ceramide Involves Activation of Ceramide-Activated Protein Phosphatase in PC12 Cells Marta Salinas, Raquel Lo´pez-Valdaliso, Daniel Martı ´n, Alberto Alvarez, and Antonio Cuadrado 1 Departamento de Bioquı ´mica, Facultad de Medicina, Universidad Auto´noma de Madrid, Arzobispo Morcillo, 4 Madrid, 28029 Spain Accumulation of ceramide has been reported in stress- and receptor-induced apoptosis in the nervous system. However, its role in apoptosis signaling remains elusive. We describe here the inhibition of the NGF-activated phosphoinositide 3-kinase (PI3K)-PKB/Akt1 survival path- way by the cell permeable analog C2-ceramide. C2- ceramide did not inhibit ERK, PI3K, or PDK1 activities and did not alter the translocation of PDK1 and Akt1 to the plasma membrane, but blocked nuclear translocation of Akt1. Down-regulation of the Akt pathway was due to enhanced dephosphorylation of Akt1 at residues T308 and S473. Moreover, Akt1 was dephosphorylated in vitro by a cation-independent phosphatase involving ceramide- activated protein phosphatase (CAPP). Membrane-an- chored Akt1 was more resistant to dephosphorylation/ inactivation by C2-ceramide than wild-type Akt1. Consistently, N-myristylated-Akt1 conferred resistance to the apoptosis induced by C2-ceramide in PC12 cells. These results provide a novel mechanism for induction of apoptosis by ceramide in nerve-derived cells. INTRODUCTION Apoptotic cell death is a feature of nervous system development and neurodegenerative diseases and is regulated partly by neurotrophins (D’Mello, 1998) such as nerve-derived growth factor (NGF) (Kaplan and Miller, 1997). NGF binding to its receptor tyrosine kinase, TrkA, triggers several intracellular signaling cascades including activation of phosphoinositide 3-ki- nase (PI3K). Neuron survival has been reported to be dependent upon PI3K activation (Kennedy et al., 1997; Yao and Cooper, 1995). Activation of PI3K results in the generation of phosphatidylinositol-3,4,5-trisphos- phate (PtdIns(3,4,5)P 3 ) and phosphatidylinositol 3,4- bisphosphate, which is produced from PtdIns(3,4,5)P 3 by a polyphosphate5-phosphatase activity (Woscholski and Parker, 1997). These 38D-phosphoinositides provide docking sites for several PH domain-containing kinases including the PKB/Akt family of Ser/Thr protein ki- nases (Andjelkovic et al., 1998). Akt1 kinase, also termed PKBa, phosphorylates substrates in the motif RXRXX(S/T). These substrates have been correlated to metabolic and survival responses (Coffer et al., 1998; Downward, 1998). At the plasma membrane, Akt1 is phosphorylated by two 38phosphoinositide-dependent kinases, termed PDK1 and PDK2. PDK1 phosphorylates Akt1 at T308 within the activation P-loop (Alessi et al., 1997; Stephens et al., 1998). PDK2 has not been character- ized molecularly, but phosphorylates S473 at the car- boxyl terminus. Both phosphorylation events are neces- sary for full activation of the kinase since mutation of any of these critical residues to alanine drastically reduces Akt1 activity (Alessi et al., 1996; Andjelkovic et al., 1996). After its phosphorylation at both sites, Akt detaches from the membrane and phosphorylates its targets within the cell cytoplasm and nucleus (Meier et al., 1997). Negative regulation of Akt1 activity is prob- ably performed by dephosphorylation. Andjelkovic et al. (1996) showed that cells treated with the phosphatase inhibitors okadaic acid or vanadate exhibited enhanced Akt1 activity in the presence of serum. Moreover, in vitro treatment of Akt1 with partially purified PP2A phospha- tase reduced its intrinsic kinase activity. Meier et al. (1998) and Chen et al. (1999) have also described the inactivation of Akt1 under hyperosmotic stress condi- tions by PP2A-mediated dephosphorylation. 1 To whom correspondence and reprint requests should be ad- dressed. Fax: 3491 858 4587. E-mail: antonio.cuadrado@uam.es. MCN Molecular and Cellular Neuroscience 15, 156–169 (2000) doi:10.1006/mcne.1999.0813, available online at http://www.idealibrary.com on 156 1044-7431/00 $35.00 Copyright r 2000 by Academic Press All rights of reproduction in any form reserved.