Ribosomal Protein S3, a New Substrate of Akt,Serves as a Signal Mediator between Neuronal Apoptosis and DNA Repair

RPS3, a conserved, eukaryotic ribosomal protein of the 40 S subunit, is required for ribosome biogenesis. Because ribosomal proteins are abundant and ubiquitous, they may have additional extraribosomal functions. Here, we show that human RPS3 is a physiological target of Akt kinase and a novel mediator of neuronal apoptosis. NGF stimulation resulted in phosphorylation of threonine 70 of RPS3 by Akt, and this phosphorylation was required for Akt binding to RPS3. RPS3 induced neuronal apoptosis, up-regulating proapoptotic proteins Dp5/Hrk and Bim by binding to E2F1 and acting synergistically with it. Akt-dependent phosphorylation of RPS3 inhibited its proapoptotic function and perturbed its interaction with E2F1. These events coincided with nuclear translocation and accumulation of RPS3, where it functions as an endonuclease. Nuclear accumulation of RPS3 results in an increase in DNA repair activity to some extent, thereby sustaining neuronal survival. Abolishment of Akt-mediated RPS3 phosphorylation through mutagenesis accelerated apoptotic cell death and severely compromised nuclear translocation of RPS3. Thus, our findings define an extraribosomal role of RPS3 as a molecular switch that accommodates apoptotic induction to DNA repair through Akt-mediated phosphorylation.     

Involvement of Akt in mitochondria-dependent apoptosis induced by a cdc25 phosphatase inhibitor naphthoquinone analog

Vitamin K-related analogs induce growth inhibition via a cell cycle arrest through cdc25A phosphatase inhibition in various cancer cell lines. We report that 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (DDN), a naphthoquinone analog, induces mitochondria-dependent apoptosis in human promyelocytic leukemia HL-60 cells. DDN induced cytochrome c release, Bax translocation, cleavage of Bid and Bad, and activation of caspase-3, -8, -9 upon the induction of apoptosis. Cleavage of Bid, the caspase-8 substrate, was inhibited by the broad caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), whereas cytochrome c release was not affected, suggesting that activation of caspase-8 and subsequent Bid cleavage occur downstream of cytochrome c release. DDN inhibited the activation of Akt detected by decreasing level of phosphorylation. Overexpression of constitutively active Akt protected cells from DDN-induced apoptosis, while dominant negative Akt moderately enhanced cell death. Furthermore, Akt prevented release of cytochrome c and cleavage of Bad in DDN-treated HL-60 cells. Taken together, DDN-induced apoptosis is associated with mitochondrial signaling which involves cytochrome c release via a mechanism inhibited by Akt.     

Nitric oxide-induced carbonylation of Bcl-2, GAPDH and ANT precedes apoptotic events in insulin-secreting RINm5F cells

Generation of high levels of nitric oxide (NO) following induction of NOS2 by interleukin-1 beta (IL-1beta) triggers beta cell apoptosis in insulin-secreting RINm5F cells. Mitochondrial and nuclear events such as downregulation of the antiapoptotic protein Bcl-2, activation of the pore responsible for the permeability transition (PT) and DNA fragmentation are involved in the process. We report in the present paper that exposure of insulin-producing RINm5F cells to NO donors and to IL-1beta leads to oxidative carbonylation of both Bcl-2 and the adenine nucleotide translocator (ANT) component of the mitochondrial PT pore. When the effect of endogenous generation of high concentrations of NO following exposure of cells to IL-1beta was studied, carbonylation of Bcl-2 preceded downregulation of the protein. Overexpression of Mn-SOD decreases substantially the extent of Bcl-2 carbonylation in SIN-1-exposed cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibition, carbonylation and translocation from cytoplasm to nucleus and DNA fragmentation were also induced by DETA/NO exposure. DETA/NO-induced carbonylation of Bcl-2 and ANT proteins takes place 6 h before apoptotic release of histone-associated DNA to cytoplasm. Time course studies also reveal a close parallel between GAPDH translocation to nucleus and carbonylation. Inhibitors of lipooxidation end products formation such as piridoxamine (PM) and aminoguanidine (AG) block NO-triggered carbonylation of Bcl-2, ANT and GAPDH, prevent NO-induced GAPDH enzyme inhibition and nuclear translocation and DNA fragmentation. Our results support the notion that the oxidative carbonylation of proteins plays a role in the control of NO-induced apoptosis.     

Akt phosphorylates MstI and prevents its proteolytic activation, blocking FOXO3 phosphorylation and nuclear translocation

Oxidative stress can induce apoptosis through activation of MstI, subsequent phosphorylation of FOXO and nuclear translocation. MstI is a common component of apoptosis initiated by various stresses. MstI kinase activation requires autophosphorylation and proteolytic degradation by caspases. The role of Akt in regulating MstI activity has not been previously examined. Here, we show that MstI is a physiological substrate of Akt. Akt phosphorylation of MstI diminishes its apoptotic cleavage by caspases and prevents its kinase activity on FOXO3. MstI directly binds to Akt, which is regulated Akt kinase activity. Akt phosphorylates MstI on the Thr(387) residue and protects MstI from apoptotic cleavage in vitro and in apoptotic cells. Interestingly, Akt phosphorylation of MstI strongly inhibits its kinase activity on FOXO3. The phosphorylation mimetic mutant MST1 T387E blocks H2O2-triggered FOXO3 nuclear translocation and apoptosis. Thus, our findings support that Akt blocks MstI-triggered FOXO3 nuclear translocation by phosphorylating MstI, promoting cell survival.     

Perifosine induces cell cycle arrest and apoptosis in human hepatocellular carcinoma cell lines by blockade of Akt phosphorylation

Hepatocellular carcinoma (HCC) is one of the most common solid cancers, representing the third cause of cancer-related death among cirrhotic patients. Treatment of advanced HCC has become a very active area of research. Perifosine, a new synthetic alkylphospholipid Akt inhibitor, has shown anti-tumor activity by inhibition of Akt phosphorylation. In this study, the effect of perifosine on the cell proliferation and apoptosis in hepatoma cells has been investigated. Cell growth inhibition was detected by MTT assay, cell cycle was analyzed by flow cytometry, AnnexinV-FITC apoptosis detection kit was used to detect cell apoptosis, and protein expression was examined by Western blotting analysis. Our present studies showed that Akt phosphorylation was inhibited by perifosine in HepG2 and Bel-7402 human hepatocellular carcinoma cells. Perifosine inhibited the growth of HepG2 cells and Bel-7402 cells in a dose-dependent manner, and arrested cell cycle progression at the G₂ phase. Apoptosis induction became more effective with increasing perifosine concentration. The caspase cascade and its downstream effectors, Poly (ADP-ribose) polymerase (PARP), were also activated simultaneously upon perifosine treatment. The proapoptotic effect of perifosine was in part depending on regulation of the phosphorylation level of ERK and JNK. Perifosine cotreatment substantially increased cytotoxic effects of cisplatin in HepG2 cells. Down-regulating the expression of Bcl-2 and up-regulating the level of Bax may be the potential mechanism for this synergistic effect. Our findings suggest that the small molecule Akt inhibitor perifosine shows substantial anti-tumor activity in human hepatoma cancer cell lines, and is a good candidate for treatment combinations with classical cytostatic compounds in hepatocellular carcinoma.     

15-Deoxy-Δ<Superscript>12,14</Superscript>-prostaglandin J<Subscript>2</Subscript> (15d-PGJ<Subscript>2</Subscript>) sensitizes human leukemic HL-60 cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis through Akt downregulation

While tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising new agent for the treatment of cancer, resistance to TRAIL remains a therapeutic challenge. Identifying agents to use in combination with TRAIL to enhance apoptosis in leukemia cells would increase the potential utility of this agent as a therapy for leukemia. Here, we show that 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂), a natural ligand for peroxisome proliferator-activated receptor γ (PPARγ), can sensitize TRAIL-resistant leukemic HL-60 cells to TRAIL-induced apoptosis. The sensitization to TRAIL-induced apoptosis by 15d-PGJ₂ was not blocked by a PPARγ inhibitor (GW9662), suggesting a PPARγ-independent mechanism. This process was accompanied by activation of caspase-8, caspase-9, and caspase-3 and was concomitant with Bid and PARP cleavage. We observed significant decreases in XIAP, Bcl-2, and c-FLIP after cotreatment with 15d-PGJ₂ and TRAIL. We also observed the inhibition of Akt expression and phosphorylation by cotreatment with 15d-PGJ₂ and TRAIL. Furthermore, inactivation of Akt by Akt inhibitor IV sensitized human leukemic HL-60 cells to TRAIL, indicating a key role for Akt inhibition in these events. Taken together, these findings indicate that 15d-PGJ₂ may augment TRAIL-induced apoptosis in human leukemia cells by down-regulating the expression and phosphorylation of Akt.     

S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) influences cytotoxicity, translocating to the nucleus during apoptosis. Here we report a signalling pathway in which nitric oxide (NO) generation that follows apoptotic stimulation elicits S-nitrosylation of GAPDH, which triggers binding to Siah1 (an E3 ubiquitin ligase), nuclear translocation and apoptosis. S-nitrosylation of GAPDH augments its binding to Siah1, whose nuclear localization signal mediates translocation of GAPDH. GAPDH stabilizes Siah1, facilitating its degradation of nuclear proteins. Activation of macrophages by endotoxin and of neurons by glutamate elicits GAPDH-Siah1 binding, nuclear translocation and apoptosis, which are prevented by NO deletion. The NO-S-nitrosylation-GAPDH-Siah1 cascade may represent an important molecular mechanism of cytotoxicity.     

α-TEA inhibits survival and enhances death pathways in cisplatin sensitive and resistant human ovarian cancer cells

RRR-α-tocopherol ether linked acetic acid analog (α-TEA), is a potential chemotherapeutic agent for ovarian cancer. Pro-death and pro-life signaling pathways were studied to understand the anti-cancer actions of α-TEA on cisplatin-sensitive (A2780S) and -resistant (A2780/cp70R) human ovarian cancer cells. Both cell lines were refractory to Fas; whereas, α-TEA sensitized them to Fas signaling. α-TEA increased levels of Fas message, protein and membrane-associated Fas. Neutralizing antibodies to Fas or Fas L partially blocked α-TEA-induced apoptosis. α-TEA induced prolonged activation of c-Jun N-terminal kinase (JNK) and its substrate c-Jun; Bax conformational change; and cleavage of Bid and caspases-8, -9 and -3. Chemical inhibitors of JNK, and caspases blocked α-TEA-induced apoptosis. α-TEA decreased phosphorylation of protein kinase B (Akt/PKB) and extracellular signal-regulated kinase (ERK1/2), as well as cellular FLICE-like inhibitory protein (c-FLIP) and Survivin protein levels. Knockdown of Akt and ERK activity using phosphoinositide- 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MKK1) inhibitors enhanced α-TEA-induced apoptosis. Over-expression of constitutively active Akt2 and MKK1 blocked α-TEA-induced apoptosis. Collectively, data show α-TEA to be a potent apoptotic inducer of both cisplatin-sensitive and -resistant human ovarian cancer cells via activating death receptor Fas signaling and suppressing anti-apoptotic AKT and ERK targets.     

Phosphorylation switches Bax from promoting to inhibiting apoptosis thereby increasing drug resistance

Akt is a pro‐survival kinase frequently activated in human cancers and is associated with more aggressive tumors that resist therapy. Here, we connect Akt pathway activation to reduced sensitivity to chemotherapy via Akt phosphorylation of Bax at residue S184, one of the pro‐apoptotic Bcl‐2 family proteins required for cells to undergo apoptosis. We show that phosphorylation by Akt converts the pro‐apoptotic protein Bax into an anti‐apoptotic protein. Mechanistically, we show that phosphorylation (i) enables Bax binding to pro‐apoptotic BH3 proteins in solution, and (ii) prevents Bax inserting into mitochondria. Together, these alterations promote resistance to apoptotic stimuli by sequestering pro‐apoptotic activator BH3 proteins. Bax phosphorylation correlates with cellular resistance to BH3 mimetics in primary ovarian cancer cells. Further, analysis of the TCGA database reveals that 98% of cancer patients with increased BAX levels also have an upregulated Akt pathway, compared to 47% of patients with unchanged or decreased BAX levels. These results suggest that in patients, increased phosphorylated anti‐apoptotic Bax promotes resistance of cancer cells to inherent and drug‐induced apoptosis.     

Downregulation of Akt2 attenuates ER stress-induced cytotoxicity through JNK-Wnt pathway in cardiomyocytes

Akt, also known as protein kinase B (PKB), is a serine/threonine kinase that promotes survival and growth in response to extracellular signals. Akt1 has been demonstrated to play vital roles in cardiovascular diseases, but the role of Akt2 in cardiomyocytes is not fully understood. This study investigated the effect of Akt2 knockdown on tunicamycin (TM)-induced cytotoxicity in cardiomyocytes and the underlying mechanisms with a focus on the JNK-Wnt pathway. TM treatment significantly increased the expression of Akt2 at both mRNA and protein levels, which was shown to be mediated by the induction of reactive oxygen species (ROS). Knockdown of Akt2 expression via siRNA transfection markedly increased cell viability, decreased lactate dehydrogenase (LDH) release and reduced cell apoptosis after TM exposure. The results of western blot showed that downregulation of Akt2 also attenuated the TM-induced activation of the unfolded protein response (UPR) factors and ER stress associated pro-apoptotic proteins. In addition, Si-Akt2 transfection partially prevented the TM-induced decrease in nuclear localization of β-catenin. By using the selective inhibitor SP-600,125 to inhibit JNK phosphorylation, we found that knockdown of Akt2-induced protection and inhibition of ER stress was mediated by reversing TM-induced decrease of Wnt through the JNK pathway. In summary, these data suggested that Akt2 play a pivotal role in regulating cardiomyocyte survival during ER stress by modulating the JNK-Wnt pathway.     

Identification of N10-substituted phenoxazines as potent and specific inhibitors of Akt signaling

A series of 30 N10-substituted phenoxazines were synthesized and screened as potential inhibitors of Akt. In cellular assays at 5 mum, 17 compounds inhibited insulin-like growth factor 1 (IGF-I)-stimulated phosphorylation of Akt (Ser-473) by at least 50% but did not inhibit IGF-I-stimulated phosphorylation of Erk-1/2 (Thr-202/Tyr-204). Substitutions at the 2-position (Cl or CF3) did not alter inhibitory activity, whereas N10-substitutions with derivatives having acetyl (20B) or morpholino (12B) side chain lost activity compared with propyl or butyl substituents (7B and 14B). Inhibition of Akt phosphorylation was associated with the inhibition of IGF-I stimulation of the mammalian target of rapamycin phosphorylation (Ser-2448 and Ser-2481), phosphorylation of p70 S6 kinase (Thr-389), and ribosomal protein S6 (Ser-235/236) in Rh1, Rh18, and Rh30 cell lines. The two most potent compounds 10-[4'-(N-diethylamino)butyl]-2-chlorophenoxazine (10B) and 10-[4'-[(beta-hydroxyethyl)piperazino]butyl]-2-chlorophenoxazine (15B) (in vitro, IC50 approximately 1-2 microM) were studied further. Inhibition of Akt phosphorylation correlated with inhibition of its kinase activity as determined in vitro after immunoprecipitation. Akt inhibitory phenoxazines did not inhibit the activity of recombinant phosphatidylinositol 3'-kinase, PDK1, or SGK1 but potently inhibited the kinase activity of recombinant Akt and Akt deltaPH, a mutant lacking the pleckstrin homology domain. Akt inhibitory phenoxazines blocked IGF-I-stimulated nuclear translocation of Akt in Rh1 cells and suppressed growth of Rh1, Rh18, and Rh30 cells (IC50 2-5 microM), whereas "inactive" derivatives were > or = 10-fold less potent inhibitors of cell growth. In contrast to rapamycin analogs, Akt inhibitory phenoxazines induced significant levels of apoptosis under serum-containing culture conditions at concentrations of agent consistent with Akt inhibition. Thus, the cellular responses to phenoxazine inhibitors of Akt appear qualitatively different from the rapamycin analogs. Modeling studies suggest inhibitory phenoxazines may bind in the ATP-binding site, although ATP competition studies were unable to distinguish between competitive and noncompetitive inhibition.     

Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3β (GSK3β), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3β. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.     

Suppression of growth of highly-metastatic human breast cancer cells by norcantharidin and its mechanisms of action

The effects of norcantharidin (NCTD) on the growth of highly-metastatic human breast cancer cells were investigated by in vitro and ex vivo assays. Our results indicated that norcantharidin inhibited the in vitro growth of human breast cancer MDA-MB-231 cell line in dose- and time-dependent manners after the cancer cells were treated with norcantharidin at the concentrations of 6, 30 and 60 μmol/L for 24, 48 and 72 h. Moreover, the sera from the NCTD-treated rabbits after intravenous injection of NCTD at 15 and 30 min significantly suppressed the growth of the cancer cells ex vivo. The analyses by Hoechst 33258 staining and flow cytometry showed that the typical apoptotic morphological changes appeared and cell cycles arrested at G2/M phase in MDA-MB-231 cells after the cells were treated for 48 h with NCTD. In addition, NCTD down-regulated the expressions of anti-apoptotic protein Bcl-2 and up-regulated the expressions of pro-apoptotic protein Bax, eventually leading to the reduction of Bcl-2/Bax ratio in MDA-MB-231 cells. Furthermore, NCTD at concentrations of 6, 30 and 60 μmol/L dose-dependently reduced the phosphorylation of Akt and NF-κB expression in the breast cancer cell line. Induction of apoptosis and cell cycle arrest as well as reduction of Bcl-2/Bax ratio by NCTD may be the important mechanisms of action of NCTD suppressing the growth of MDA-MB-231 cells, which are associated with inhibition of the Akt and NF-κB signaling. Our findings suggest that norcantharidin may have a wide therapeutic and/or adjuvant therapeutic application in the treatment of human breast cancer.     

Effects of bufalin on the proliferation of human lung cancer cells and its molecular mechanisms of action

Bufalin, a naturally occurring small-molecule compound from Traditional Chinese Medicine (TCM) Chansu showed inhibitory effects against human prostate, hepatocellular, endometrial and ovarian cancer cells, and leukemia cells. However, whether or not bufalin has inhibitory activity against the proliferation of human non–small cell lung cancer (NSCLC) cells is unclear. The aim of this study is to study the effects of bufalin on the proliferation of NSCLC and its molecular mechanisms of action. The cancer cell proliferation was measured by MTT assay. The apoptosis and cell cycle distribution were analyzed by flow cytometry. The protein expressions and phosphorylation in the cancer cells were detected by Western blot analysis. In the present study, we have demonstrated that bufalin suppressed the proliferation of human NSCLC A549 cell line in time- and dose-dependent manners. Bufalin induced the apoptosis and cell cycle arrest by affecting the protein expressions of Bcl-2/Bax, cytochrome c, caspase-3, PARP, p53, p21WAF1, cyclinD1, and COX-2 in A549 cells. In addition, bufalin reduced the protein levels of receptor expressions and/or phosphorylation of VEGFR1, VEGFR2, EGFR and/or c-Met in A549 cells. Furthermore, bufalin inhibited the protein expressions and phosphorylation of Akt, NF-κB, p44/42 MAPK (ERK1/2) and p38 MAPK in A549 cells. Our results suggest that bufalin inhibits the human lung cancer cell proliferation via VEGFR1/VEGFR2/EGFR/c-Met–Akt/p44/42/p38-NF-κB signaling pathways; bufalin may have a wide therapeutic and/or adjuvant therapeutic application in the treatment of human NSCLC.     

Retracted: Upregulated microRNA-15b alleviates ovarian cancer through inhitbition of the PI3K/Akt pathway by targeting LPAR3

Ovarian cancer characterizes as the fourth leading consequence of death associated with cancer for women. Accumulating evidence underscores the vital roles of microRNAs (miRNAs) in preventing ovarian cancer development. Besides, induction of the phosphatidylinositol-3 kinase/serine/threonine kinase (PI3K/Akt) pathway associated with the ovarian cancer cell migration and invasion. The study aims to examine the effects of miR-15b on the proliferation, apoptosis, and senescence of human ovarian cancer cells by binding to lysophosphatidic acid receptor 3 (LPAR3) with the involvement of the PI3K/Akt pathway. The positive expression of LPAR3 protein was detected by immunohistochemistry. Then the interaction between miR-15b and LPAR3 was examined. The possible role of miR-15b in ovarian cancer was explored using gain- and loss-of-function experiments. Subsequently, the functions of miR-15b on PI3K/Akt pathway, proliferation, migration, invasion, senescence and apoptosis of ovarian cancer cells were assessed. Furthermore, in vivo tumorigenicity assay in nude mice was performed. LPAR3 was overexpressed, whereas miR-15b was poorly expressed in ovarian cancer tissues. LPAR3 is a direct target of miR-15b. Restored miR-15b promoted Bax expression, apoptosis, and senescence, inhibited expression of LPAR3 and Bcl-2, the extent of PI3K and Akt phosphorylation, as well as ovarian cancer cell proliferation, migration, and invasion. Further, tumor growth was observed to be prevented by miR-15b overexpression. Collectively, our study demonstrates that miR-15b represses the proliferation and drives the senescence and apoptosis of ovarian cancer cells through the suppression of LPAR3 and the PI3K/Akt pathway, highlighting an antitumorigenic role of miR-15b.     

Conditional knock-out of integrin-linked kinase demonstrates an essential role in protein kinase B/Akt activation

Protein kinase B (PKB/Akt) plays a pivotal role in signaling pathways downstream of phosphatidylinositol 3-kinase, regulating fundamental processes such as cell survival, cell proliferation, differentiation, and metabolism. PKB/Akt activation is regulated by phosphoinositide phospholipid-mediated plasma membrane anchoring and by phosphorylation on Thr-308 and Ser-473. Whereas the Thr-308 site is phosphorylated by PDK-1, the identity of the Ser-473 kinase has remained unclear and controversial. The integrin-linked kinase (ILK) is a potential regulator of phosphorylation of PKB/Akt on Ser-473. Utilizing double-stranded RNA interference (siRNA) as well as conditional knock-out of ILK using the Cre-Lox system, we now demonstrate that ILK is essential for the regulation of PKB/Akt activity. ILK knock-out had no effect on phosphorylation of PKB/Akt on Thr-308 but resulted in almost complete inhibition of phosphorylation on Ser-473 and significant inhibition of PKB/Akt activity, accompanied by significant stimulation of apoptosis. The inhibition of PKB/Akt Ser-473 phosphorylation was rescued by kinase-active ILK but not by a kinase-deficient mutant of ILK, suggesting a role for the kinase activity of ILK in the stimulation of PKB/Akt phosphorylation. ILK knock-out also resulted in the suppression of phosphorylation of GSK-3beta on Ser-9 and cyclin D1 expression. These data establish ILK as an essential upstream regulator of PKB/Akt activation.     

CH-ILKBP regulates cell survival by facilitating the membrane translocation of protein kinase B/Akt

Cell survival depends on proper propagation of protective signals through intracellular signaling intermediates. We report here that calponin homology domain-containing integrin-linked kinase (ILK)-binding protein (CH-ILKBP), a widely expressed adaptor protein localized at plasma membrane-actin junctions, is essential for transmission of survival signals. Cells that are depleted of CH-ILKBP undergo extensive apoptosis despite the presence of cell-extracellular matrix contacts and soluble growth factors. The activating phosphorylation of protein kinase B (PKB/Akt), a key regulator of apoptosis, is impaired in the absence of CH-ILKBP. Importantly, loss of CH-ILKBP prevents the membrane translocation of PKB/Akt. Furthermore, forced membrane targeting of PKB/Akt bypasses the requirement of CH-ILKBP for the activating phosphorylation of PKB/Akt, suggesting that CH-ILKBP is required for the membrane translocation but not the subsequent phosphorylation of PKB/Akt. Finally, we show that loss of CH-ILKBP is also required for the full activation of extracellular signal-regulated kinase (ERK)1/2. However, restoration of the PKB/Akt activation is sufficient for protection of cells from apoptosis induced by the depletion of CH-ILKBP despite the persistent suppression of the ERK1/2 activation. Thus, CH-ILKBP is an important component of the prosurvival signaling pathway functioning primarily by facilitating the membrane translocation of PKB/Akt and consequently the activation of PKB/Akt in response to extracellular survival signals.