Akt Phosphorylation and Stabilization of X-linked Inhibitor of Apoptosis Protein (XIAP)

Abstract

Akt negatively regulates apoptotic pathways at a premitochondrial level through phosphorylation and modulation of proteins such as Bad, Forkhead proteins, and GSK-3␤. Akt has also been shown to protect cell death at a post-mitochondrial level, although its downstream targets have not been well documented. Here, we demonstrate that Akt, including AKT1 and AKT2, interacts with and phosphorylates X-linked inhibitor of apoptosis protein (XIAP) at residue serine-87 in vitro and in vivo. Phosphorylation of XIAP by Akt protects XIAP from ubiquitination and degradation in response to cisplatin. Moreover, autoubiquitination of XIAP is also inhibited by Akt. Consistent with this, an XIAP mutant introduced into cells which mimics the Akt-phosphorylated form (i.e. XIAP-S87D) displays reduced ubiquitination and degradation as compared with wild type XIAP. The greater stability of XIAP-S87D in cells translated to increased cell survival after cisplatin treatment. Conversely, a mutant that could not be phosphorylated by Akt (XIAP-S87A) was more rapidly degraded and showed increased cisplatin-induced apoptosis. Furthermore, suppression of XIAP by either siRNA or adenovirus of antisense of XIAP induced programmed cell death and inhibited Akt-stimulated cell survival in ovarian cancer cells. These data identify XIAP as a new downstream target of Akt and a potentially important mediator of the effect of Akt on cell survival.Akt, also named protein kinase B (PKB) 1 or RAC kinase, is a family of phosphatidylinositol 3-OH-kinase-regulated serine/ threonine kinase (1-3). Three isoforms of Akt have been identified: Akt1/PKB␣, Akt2/PKB␤, and Akt3/PKB␥, all of which are activated by growth factors in a phosphatidylinositol 3-OHkinase-dependent manner (4 -6). Accumulated evidence shows that Akt and its downstream targets constitute a major cell survival pathway. Akt promotes cell survival and suppresses apoptotic death in a number of cell types induced by a variety of stimuli, including growth factor withdrawal, cell cycle discordance, and loss of cell adhesion (7). Several downstream targets containing the Akt phosphorylation consensus sequence (R-X-R-X-X-S/T) have been identified which shed light on the mechanisms by which Akt promotes cell survival and blocks apoptosis. The first anti-apoptotic Akt target identified was the pro-apoptotic protein BAD. BAD is a pro-death member of the Bcl-2 family that initiates apoptosis by binding to Bcl-x L on the outer mitochondrial membrane, causing the release of cytochrome c into the cytosol. Akt phosphorylates BAD on Ser 136 , promoting the association of BAD with 14-3-3 proteins in the cytosol and inactivating its proapoptotic function (7). The execution of cellular apoptosis also involves changes in the transcriptional program (7). Akt decreases the transcription of a subset of death genes by phosphorylation of the Forkhead family of transcription factors, which causes their nuclear exclusion and inactivation (7). Akt also phosphorylates and activates the cyclic AMP-response element-binding pr...

The Localization of Protein Kinase Cδ in Different Subcellular Sites Affects Its Proapoptotic and Antiapoptotic Functions and the Activation of Distinct Downstream Signaling Pathways

“…The survival effects of AKT are exerted by phosphorylating proteins, such as BAD, caspase-9, the forkhead transcription factors (49), or XIAP (50). Indeed, XIAP has been recently identified as a downstream target of AKT and as an important regulator of AKT survival effects.…”

Combinatorial activation of FAK and AKT by transforming growth factor-β1 confers an anoikis-resistant phenotype to myofibroblasts

“…Activation of the PKB/AKT pathway mediates growth factordependent survival of a wide variety of cell types through a number of downstream targets, including caspase-9, BAD, NF-B, fork-head family transcription factors and XIAP (X-linked inhibitor of apoptosis) [19,20]. In addition to receptor tyrosine kinase(s)-activating growth factor ligands, PI3K-AKT has also been shown to be activated by adhesion signalling via FAK [16,21,22] and integrin-linked kinase (ILK) [22].…”

Rosiglitazone sensitizes hepatocellular carcinoma cell lines to 5-fluorouracil antitumor activity through activation of the PPARγ signaling pathway

“…Chemoresistance is an active process of tumor cell survival and a consequence of multiple factors, including reduction of intercellular drug accumulation, repair of tumor cell DNA damage, activation of PI3K/Akt, Ras or MAPK signaling pathways, and dysfunction of the tumor suppression gene [9,10] . PTEN is a tumor suppressor gene that inhibits the PI3K/Akt mediated cell survival pathway [11] .…”