K252a suppresses neuronal cells apoptosis through inhibiting the translocation of Bax to mitochondria induced by the MLK3/JNK signaling after transient global brain ischemia in rat hippocampal CA1 subregion

It is demonstrated that the c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. Our previous studies have suggested that K252a can obviously inhibit JNK activation induced by ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. Here, we further discussed the potential mechanism of ischemic brain injury induced by the activation of JNK after 15?min of transient global cerebral ischemia. As a result, through inhibiting phosphorylation of Bcl-2 (a cytosolic target of JNK) and 14-3-3 protein (a cytoplasmic anchor of Bax) induced by the activation of JNK, K252a decreased the release of Bax from Bcl-2/Bax and 14-3-3/Bax dimers, further attenuating the translocation of Bax from cytosol to mitochondria and the release of cytochrome c induced by ischemia/reperfusion, which related to mitochondria-mediated apoptosis. Importantly, pre-infusion of K2525a 20?min before ischemia showed neuroprotective effect against neuronal cells apoptosis. These findings imply that K252a induced neuroprotection against ischemia/reperfusion in rat hippocampal CA1 subregion via inhibiting the mitochondrial apoptosis pathway induced by JNK activation.     

Mitochondria and Neuronal Death/Survival Signaling Pathways in Cerebral Ischemia

Apoptotic cell death pathways have been implicated in acute brain injuries, including cerebral ischemia, brain trauma, and spinal cord injury, and in chronic neurodegenerative diseases. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and suggest the involvement of mitochondria and the cell survival/death signaling pathways in cell death/survival cascades. Recent studies have implicated mitochondria-dependent apoptosis involving pro- and anti-apoptotic protein binding, the release of cytochrome c and second mitochondria-derived activator of caspase, the activation of downstream caspases-9 and –3, and DNA fragmentation. Reactive oxygen species are known to be significantly generated in the mitochondrial electron transport chain in the dysfunctional mitochondria during reperfusion after ischemia, and are also implicated in the survival signaling pathway that involves phosphatidylinositol-3-kinase (PI3-K), Akt, and downstream signaling molecules, like Bad, 14-3-3, and the proline-rich Akt substrate (PRAS), and their bindings. Further studies of these survival pathways may provide novel therapeutic strategies for clinical stroke.Special issue dedicated to Lawrence F. Eng.     

Hexokinase-mitochondria interaction mediated by Akt is required to inhibit apoptosis in the presence or absence of Bax and Bak

The serine/threonine kinase Akt inhibits mitochondrial cytochrome c release and apoptosis induced by a variety of proapoptotic stimuli. The antiapoptotic activity of Akt is coupled, at least in part, to its effects on cellular metabolism. Here, we provide genetic evidence that Akt is required to maintain hexokinase association with mitochondria. Targeted disruption of this association impairs the ability of growth factors and Akt to inhibit cytochrome c release and apoptosis. Targeted disruption of mitochondria-hexokinase (HK) interaction or exposure to proapoptotic stimuli that promote rapid dissociation of hexokinase from mitochondria potently induce cytochrome c release and apoptosis, even in the absence of Bax and Bak. These effects are inhibited by activated Akt, but not by Bcl-2, implying that changes in outer mitochondrial membrane (OMM) permeability leading to apoptosis can occur in the absence of Bax and Bak and that Akt inhibits these changes through maintenance of hexokinase association with mitochondria.     

Opposing effects of Bad phosphorylation at two distinct sites by Akt1 and JNK1/2 on ischemic brain injury

Increasing evidence suggests that the Bcl-2 family proteins play pivotal roles in regulation of the mitochondria cell-death pathway on transient cerebral ischemia. Bad, a BH3-only proapoptotic Bcl-2 family protein, has been shown to be phosphorylated extensively on serine by kinds of kinases. However, the exact mechanisms of the upstream kinases in regulation of Bad signaling pathway remain unknown. Here, we reported that Bad could be phosphorylated not only by Akt1 but also by JNK1/2 after transient global ischemia in rat hippocampal CA1 region. Our data demonstrated that Akt1 mediated the phosphorylation of Bad at serine 136, which increased the interaction of serine 136-phosphorylated Bad with 14-3-3 proteins and prevented the dimerization of Bad with Bcl-Xl, inhibited the release of cytochrome c to the cytosol and the death effector caspase-3 activation, leading to the survival of neuron. In contrast, JNK1/2 induced the phosphorylation of Bad at a novel site of serine 128 after brain ischemia/reperfusion, which inhibited the interaction of PI3K/Akt-induced serine 136-phosphorylated Bad with 14-3-3 proteins, thereby promoted the apoptotic effect of Bad. In addition, activated Akt1 inhibited the activation of Bad(S128) through downregulating JNK1/2 activation, thus inhibiting JNK-mediated Bad apoptosis pathway. Furthermore, the fate of cell to survive or to die was determined by a balance between prosurvival and proapoptotic signals. Taken together, our studies reveal that Bad phosphorylation at two distinct sites induced by Akt1 and JNK1/2 have opposing effects on ischemic brain injury, and present the possibility of Bad as a potential therapeutic target for stroke treatment.     

14,15-EET Suppresses Neuronal Apoptosis in Ischemia–Reperfusion Through the Mitochondrial Pathway

Neuronal apoptosis mediated by the mitochondrial apoptosis pathway is an important pathological process in cerebral ischemia–reperfusion injury. 14,15-EET, an intermediate metabolite of arachidonic acid, can promote cell survival during ischemia/reperfusion. However, whether the mitochondrial apoptotic pathway is involved this survival mechanism is not fully understood. In this study, we observed that infarct size in ischemia–reperfusion injury was reduced in sEH gene knockout mice. In addition, Caspase 3 activation, cytochrome C release and AIF nuclear translocation were also inhibited. In this study, 14,15-EET pretreatment reduced neuronal apoptosis in the oxygen–glucose deprivation and re-oxygenation group in vitro. The mitochondrial apoptosis pathway was also inhibited, as evidenced by AIF translocation from the mitochondria to nucleus and the reduction in the expressions of cleaved-caspase 3 and cytochrome C in the cytoplasm. 14,15-EET could reduce neuronal apoptosis through upregulation of the ratio of Bcl-2 (anti-apoptotic protein) to Bax (apoptosis protein) and inhibition of Bax aggregation onto mitochondria. PI3K/AKT pathway is also probably involved in the reduction of neuronal apoptosis by EET. Our study suggests that 14,15-EET could suppress neuronal apoptosis and reduce infarct volume through the mitochondrial apoptotic pathway. Furthermore, the PI3K/AKT pathway also appears to be involved in the neuroprotection against ischemia–reperfusion by 14,15-EET.     

大鼠脑缺血诱导的细胞色素c的释放和Bcl-2表达的上调

利用全脑缺血模型,采用免疫印迹和免疫沉淀方法,探讨N-甲基-D-天冬氨酸受体和L-型电压门控钙通道拮抗剂对细胞色素c从线粒体中的释放和Bcl-2的表达变化影响。缺血／复灌后24h,线粒体中细胞色素c明显降低而胞浆中细胞色素c的成分相应增加。Bcl-2的表达呈时间依赖性,其表达在缺血／复灌后6h达到最大。在所有样品中,线粒体呼吸链蛋白细胞色素氧化酶没有变化,表明线粒体的制备方法是可靠的。线粒体中Bcl-2的表达减少和细胞色素c的释放可以被NMDA受体拮抗剂氯胺酮和L-型电压门控钙通道拮抗剂尼氟地平抑制。结果表明,N-甲基-D-天冬氨酸受体和L-型电压门控钙通道可能介导了脑缺血后细胞色素c从线粒体中的释放和Bcl-2的上调表达。缺血诱导的细胞色素c释放具有损伤作用而Bcl-2的上调表达则对脑缺血具有一定的保护作用。     

NXY-059, a nitrone with free radical trapping properties inhibits release of cytochrome c after focal cerebral ischemia.

Recent studies have demonstrated that disodium 2,4-disulfophenyl-N-tert-butylnitrone (NXY-059), a novel nitrone with free radical trapping properties, has a considerable neuroprotective effect against cerebral ischemic injury. The mechanisms of its action have not been fully defined. In order to evaluate whether NXY-059 exerts its protective effects by inhibiting the release of cytochrome c, a key initiator of programmed cell death pathway, we have studied the effects of NXY-059 on reducing infarct volume and on inhibiting cytochrome c release from the mitochondria after transient focal cerebral ischemia. Wistar rats were subjected to 2 hr of middle cerebral artery occlusion and perfusion-fixed after 4, 6, 12, and 24 hr of reperfusion. NXY-059 (30 mg/kg) was i.v. injected 1 hr after reperfusion and followed immediately by 30 mg/kg/hr continuous i.v. infusion for the entire reperfusion period. The results showed that NXY-059 reduced infarct volume from 37.2% to 12.5% (p<0.0001). Immunocytochemistry demonstrated that the release of cytochrome c increased at 6 hr, peaked at 12 and 24 hr of reperfusion. NXY-059 treatment prevented ischemia-induced cytochrome c release. NXY-059 may reduce ischemic brain damage through suppressing the cell death pathway that is initiated by cytochrome c release.     

The phosphatidylinositol 3-kinase (PI3K)-Akt pathway suppresses Bax translocation to mitochondria

Bax, a proapoptotic member of the Bcl-2 family, localizes largely in the cytoplasm but redistributes to mitochondria in response to apoptotic stimuli, where it induces cytochrome c release. In this study, we show that the phosphatidylinositol 3-OH kinase (PI3K)-Akt pathway plays an important role in the regulation of Bax subcellular localization. We found that LY294002, a PI3K inhibitor, blocked the effects of serum to prevent Bax translocation to mitochondria and that expression of an active form of PI3K suppressed staurosporine-induced Bax translocation, suggesting that PI3K activity is essential for retaining Bax in the cytoplasm. In contrast, both U0126, a MEK inhibitor, and active MEK had little effect on Bax localization. In respect to downstream effectors of PI3K, we found that expression of active Akt, but not serum and glucocorticoid-induced protein kinase (SGK), suppressed staurosporine-induced translocation of Bax, whereas dominant negative Akt moderately promoted Bax translocation. Expression of Akt did not alter the levels of Bax, Bcl-2, Bcl-X(L), or phosphorylated JNK under the conditions used, suggesting that there were alternative mechanisms for Akt in the suppression of Bax translocation. Collectively, these results suggest that the PI3K-Akt pathway inhibits Bax translocation from cytoplasm to mitochondria and have revealed a novel mechanism by which the PI3K-Akt pathway promotes survival.     

Apoptosis repressor with caspase recruitment domain protects against cell death by interfering with Bax activation

Myocardial ischemia/reperfusion (I/R) is associated with an extensive loss of myocardial cells. The apoptosis repressor with caspase recruitment domain (ARC) is a protein that is highly expressed in heart and skeletal muscle and has been demonstrated to protect the heart against I/R injury (Gustafsson, A. B., Sayen, M. R., Williams, S. D., Crow, M. T., and Gottlieb, R. A. (2002) Circulation 106, 735-739). In this study, we have shown that transduction of TAT-ARCL31F, a mutant of ARC in the caspase recruitment domain, did not reduce creatine kinase release and infarct size after I/R. TAT-ARCL31F also failed to protect against hydrogen peroxide-mediated cell death in H9c2 cells, suggesting that the caspase recruitment domain is important in mediating ARC's protective effects. In addition, we report that ARC co-immunoprecipitated with the pro-apoptotic protein Bax, which causes cytochrome c release when activated. TAT-ARC, but not TAT-ARCL31F, prevented Bax activation and cytochrome c release in hydrogen peroxide-treated H9c2 cells. TAT-ARC was also effective in blocking cytochrome c release after ischemia and reperfusion, whereas TAT-ARCL31F had no effect on cytochrome c release. In addition, recombinant ARC protein abrogated Bax-induced cytochrome c release from isolated mitochondria. This suggests that ARC can protect against cell death by interfering with activation of the mitochondrial death pathway through the interaction with Bax, preventing mitochondrial dysfunction and release of pro-apoptotic factors.     

Initiation of mitochondrial-mediated apoptosis during cardiac reperfusion

Reperfusion of myocardial tissue can result in programmed cell death. Nevertheless, relatively little information exists concerning pathways initiated in vivo that ultimately commit cardiac cells to apoptosis during ischemia/reperfusion. The goal of the present study was to determine whether mitochondrial-mediated mechanisms of apoptosis are initiated during in vivo cardiac ischemia/reperfusion. We provide evidence that the content of cytochrome c in the cytosol increases exclusively during reperfusion. Over the same time interval Bax, a pro-apoptotic protein implicated in release of cytochrome c from mitochondria, was found to disappear from cytosolic extracts. This was associated with the appearance of tightly associated Bax in the mitochondrial fraction. Cytochrome c from reperfused cytosolic extracts is present as a high molecular weight oligomer consistent with formation of the apoptosome. In addition, pro-caspase-9 was found to disappear exclusively during reperfusion. Therefore, the results of the current study indicate that the mitochondrial-mediated pathway of apoptosis is initiated as a result of in vivo cardiac ischemia/reperfusion.     

Prolyl hydroxylase inhibitors delay neuronal cell death caused by trophic factor deprivation

Nerve growth factor (NGF) serves a critical survival-promoting function for developing sympathetic neurons. Following removal of NGF, sympathetic neurons undergo apoptosis characterized by the activation of c-Jun N-terminal kinases (JNKs), up-regulation of BH3-only proteins including BcL-2-interacting mediator of cell death (BIM)_EL, release of cytochrome c from mitochondria, and activation of caspases. Here we show that two small-molecule prolyl hydroxylase inhibitors frequently used to activate hypoxia-inducible factor (HIF) – ethyl 3,4-dihydroxybenzoic acid (DHB) and dimethyloxalylglycine (DMOG) – can inhibit apoptosis caused by trophic factor deprivation. Both DHB and DMOG blocked the release of cytochrome c from mitochondria after NGF withdrawal, whereas only DHB blocked c-Jun up-regulation and phosphorylation. DHB, but not DMOG, also attenuated the induction of BIM_EL in NGF-deprived neurons, suggesting a possible mechanism whereby DHB could inhibit cytochrome c release. DMOG, on the other hand, was substantially more effective at stabilizing HIF-2α and inducing expression of the HIF target gene hexokinase 2 than was DHB. Thus, while HIF prolyl hydroxylase inhibitors can delay cell death in NGF-deprived neurons, they do so through distinct mechanisms that, at least in the case of DHB, are partly independent of HIF stabilization.     

Postnatal brain development and neural cell differentiation modulate mitochondrial Bax and BH3 peptide-induced cytochrome c release

Bax mediates cytochrome c release and apoptosis during neurodevelopment. Brain mitochondria that were isolated from 8-day, 17-day, and adult rats displayed decreasing levels of mitochondrial Bax. The amount of cytochrome c released from brain mitochondria by a peptide containing the BH3 cell death domain decreased with increasing age. However, approximately 60% of cytochrome c in adult brain mitochondria could be released by the BH3 peptide in the presence of exogenous human recombinant Bax. Mitochondrial Bax was downregulated in PC12S neural cells differentiated with nerve growth factor, and mitochondria isolated from these cells demonstrated decreased sensitivity to BH3-peptide-induced cytochrome c release. These results demonstrate that immature brain mitochondria and mitochondria from undifferentiated neural cells are particularly sensitive to cytochrome c release mediated by endogenous Bax and a BH3 death domain peptide. Postnatal developmental changes in mitochondrial Bax levels may contribute to the increased susceptibility of neurons to pathological apoptosis in immature animals.     

cAMP inhibits bile acid-induced apoptosis by blocking caspase activation and cytochrome c release

We have previously shown that cAMP protects against bile acid-induced apoptosis in cultured rat hepatocytes in a phosphoinositide 3-kinase (PI3K)-dependent manner. In the present studies, we investigated the mechanisms involved in this anti-apoptotic effect. Hepatocyte apoptosis induced by glycodeoxycholate (GCDC) was associated with mitochondrial depolarization, activation of caspases, the release of cytochrome c from the mitochondria, and translocation of BAX from the cytosol to the mitochondria. cAMP inhibited GCDC-induced apoptosis, caspase 3 and caspase 9 activation, and cytochrome c release in a PI3K-dependent manner. cAMP activated PI3K in p85 immunoprecipitates and resulted in PI3K-dependent activation of the survival kinase Akt. Chemical inhibition of Akt phosphorylation with SB-203580 partially blocked the protective effect of cAMP. cAMP resulted in wortmannin-independent phosphorylation of BAD and was associated with translocation of BAD from the mitochondria to the cytosol. These results suggest that GCDC-induced apoptosis in cultured rat hepatocytes proceeds through a caspase-dependent intracellular stress pathway and that the survival effect of cAMP is mediated in part by PI3K-dependent Akt activation at the level of the mitochondria.     

Akt/Protein Kinase B Inhibits Cell Death by Preventing the Release of Cytochrome c from Mitochondria

Growth factors signaling through the phosphoinositide 3-kinase/Akt pathway promote cell survival. The mechanism by which the serine/threonine kinase Akt prevents cell death remains unclear. We have previously shown that Akt inhibits the activity of DEVD-targeted caspases without changing the steady-state levels of Bcl-2 and Bcl-x(L). Here we show that Akt inhibits apoptosis and the processing of procaspases to their active forms by delaying mitochondrial changes in a caspase-independent manner. Akt activation is sufficient to inhibit the release of cytochrome c from mitochondria and the alterations in the inner mitochondrial membrane potential. However, Akt cannot inhibit apoptosis induced by microinjection of cytochrome c. We also demonstrated that Akt inhibits apoptosis and cytochrome c release induced by several proapoptotic Bcl-2 family members. Taken together, our results show that Akt promotes cell survival by intervening in the apoptosis cascade before cytochrome c release and caspase activation via a mechanism that is distinct from Bad phosphorylation.     

Akt regulates cell survival and apoptosis at a postmitochondrial level

Phosphoinositide 3 kinase/Akt pathway plays an essential role in neuronal survival. However, the cellular mechanisms by which Akt suppresses cell death and protects neurons from apoptosis remain unclear. We previously showed that transient expression of constitutively active Akt inhibits ceramide-induced death of hybrid motor neuron 1 cells. Here we show that stable expression of either constitutively active Akt or Bcl-2 inhibits apoptosis, but only Bcl-2 prevents the release of cytochrome c from mitochondria, suggesting that Akt regulates apoptosis at a postmitochondrial level. Consistent with this, overexpressing active Akt rescues cells from apoptosis without altering expression levels of endogenous Bcl-2, Bcl-x, or Bax. Akt inhibits apoptosis induced by microinjection of cytochrome c and lysates from cells expressing active Akt inhibit cytochrome c induced caspase activation in a cell-free assay while lysates from Bcl-2-expressing cells have no effect. Addition of cytochrome c and dATP to lysates from cells expressing active Akt do not activate caspase-9 or -3 and immunoprecipitated Akt added to control lysates blocks cytochrome c-induced activation of the caspase cascade. Taken together, these data suggest that Akt inhibits activation of caspase-9 and -3 by posttranslational modification of a cytosolic factor downstream of cytochrome c and before activation of caspase-9.     

PI3K inhibitors changed the p53-induced response of Saos-2 cells from growth arrest to apoptosis

p53 is activated by stress leading to oncogenic alteration, which induces either cell cycle arrest or apoptosis, although the mechanism involved in this decision has not been fully clarified as yet. This work was undertaken to change the cellular response by inducing apoptosis with PI3K inhibitors to Saos-2 cells that had been growth-arrested in both G1 and G2/M by the wild-type activity of temperature-sensitive (ts) p53. We found that the PI3K/Akt inhibitors LY294002 and wortmannin, but not the MEK inhibitor U0126, were capable of inducing apoptosis in growth-arrested Saos-2 cells, as assessed by an increase in the sub-G1 population, pyknotic nuclei, and DNA ladder formation. We detected the cleavage of caspases 9 and 3, and PARP after LY294002 addition, accompanied by a loss of cytochrome c from the mitochondria, and observed Bax translocation to the mitochondria and down-regulation of phospho-Akt, suggesting that blocking of survival signals triggered the apoptotic signal through the mitochondrial apoptotic pathway. It is thus suggested that the PI3K/Akt pathway played an important role in determining cell fate between growth arrest and apoptosis.     

PKC and Raf-1 inhibition-related apoptotic signalling in N2a cells

In this study, a neuroblastoma N2a cell line was applied to investigate mechanisms of apoptosis induced either by selective inhibition of protein kinase C (PKC) by low amounts of staurosporine (STS(10) ) or by inhibition PI3-K after wortmannin (WM) treatment. We present evidence that, in the absence of serum in the medium, decreased phosphorylation of Raf-1 and BAD112, as well as Akt and BAD136, proteins and their translocation to mitochondria coincided with STS10 - or WM-induced apoptosis, respectively. Concomitantly, release of cytochrome c into the cytosol indicated a BCL-2-dependent mode of cell death after both treatments. Furthermore, in typical 'gain of function' experiments, cells with overexpression of permanently active Raf-1 or Akt transgenes displayed a significantly higher and independent resistance to either STS10 or WM. Thus, our results indicate that PKC/Raf-1/BAD112, as well as PI3-K/Akt/BAD136 signalling pathways, are both necessary for N2a cell survival and thus are unable to functionally substitute for each other as long as the cells do not receive additional signal(s) derived from serum. However, in the presence of serum, undefined trophic signal(s) can stimulate cross-talk between these two pathways at a level upstream from Raf-1 and Akt phosphorylation. In this case, only simultaneous inhibition of PKC and PI3-K is able to induce apoptosis.     

Delayed ischemic postconditioning protects hippocampal CA1 neurons by preserving mitochondrial integrity via Akt/GSK3β signaling

Delayed ischemic postconditioning (Post C), which involves a brief ischemia followed by reperfusion 2 days after 8-10 min global cerebral ischemia (GCI), has been shown to exert a remarkable protection of the vulnerable hippocampal CA1 region of the brain and attenuation of behavioral deficits, although the mechanisms remain poorly understood. The purpose of the current study was to explore the effect of Post C upon mitochondrial integrity, cytochrome c release and Bax translocation as a potential key mechanism for Post C protection of the critical hippocampal CA1 region neurons. The results of the study revealed that ischemic Post C (3 min) administered 2 days after 8-min GCI exerted a robust preservation from GCI injury, as evidenced by the increase of NeuN-positive and the decrease of TUNEL-positive cells, as well as morphological features of mitochondrial integrity in the hippocampal CA1 region. We also found that Post C significantly blocked inner mitochondrial membrane potential depolarization, as shown by JC-1 staining, and attenuates cytochrome c release and Bax translocation induced by GCI. Pre-treatment of the PI3K inhibitor LY294002, 20 min prior to Post C, significantly attenuated Post C-induced elevation of p-Akt and p-GSK3β, as well as prevented Post C enhancement of mitochondrial integrity and Post C neuroprotection. The results suggest that phosphorylation of Akt and subsequent inactivation of GSK3β signaling is critical in mediating Post C beneficial effects upon mitochondrial integrity, function and neuroprotection following GCI injury.     

Hsp27 inhibits Bax activation and apoptosis via a phosphatidylinositol 3-kinase-dependent mechanism

Hsp27 inhibits mitochondrial injury and apoptosis in both normal and cancer cells by an unknown mechanism. To test the hypothesis that Hsp27 decreases apoptosis by inhibiting Bax, Hsp27 expression was manipulated in renal epithelial cells before transient metabolic stress, an insult that activates Bax, induces mitochondrial injury, and causes apoptosis. Compared with control, enhanced Hsp27 expression inhibited conformational Bax activation, oligomerization, and translocation to mitochondria, reduced the leakage of both cytochrome c and apoptosis-inducing factor, and significantly improved cell survival by >50% after stress. In contrast, Hsp27 down-regulation using RNA-mediated interference promoted Bax activation, increased Bax translocation, and reduced cell survival after stress. Immunoprecipitation did not detect Hsp27-Bax interaction before, during, or after stress, suggesting that Hsp27 indirectly inhibits Bax. During stress, Hsp27 expression prevented the inactivation of Akt, a pro-survival kinase, and increased the interaction between Akt and Bax, an Akt substrate. In contrast, Hsp27 RNA-mediated interference promoted Akt inactivation during stress. Hsp27 up- or down-regulation markedly altered the activity of phosphatidylinositol 3-kinase (PI3-kinase), a major regulator of Akt. Furthermore, distinct PI3-kinase inhibitors completely abrogated the protective effect of Hsp27 expression on Akt activation, Bax inactivation, and cell survival. These data show that Hsp27 antagonizes Bax-mediated mitochondrial injury and apoptosis by promoting Akt activation via a PI3-kinase-dependent mechanism.