RhoG regulates anoikis through a phosphatidylinositol 3-kinase-dependent mechanism

In normal epithelial cells, cell-matrix interaction is required for cell survival and proliferation, whereas disruption of this interaction causes epithelial cells to undergo apoptosis called anoikis. Here we show that the small GTPase RhoG plays an important role in the regulation of anoikis. HeLa cells are capable of anchorage-independent cell growth and acquire resistance to anoikis. We found that RNA interference-mediated knockdown of RhoG promoted anoikis in HeLa cells. Previous studies have shown that RhoG activates Rac1 and induces several cellular functions including promotion of cell migration through its effector ELMO and the ELMO-binding protein Dock180 that function as a Rac-specific guanine nucleotide exchange factor. However, RhoG-induced suppression of anoikis was independent of the ELMO- and Dock180-mediated activation of Rac1. On the other hand, the regulation of anoikis by RhoG required phosphatidylinositol 3-kinase (PI3K) activity, and constitutively active RhoG bound to the PI3K regulatory subunit p85alpha and induced the PI3K-dependent phosphorylation of Akt. Taken together, these results suggest that RhoG protects cells from apoptosis caused by the loss of anchorage through a PI3K-dependent mechanism, independent of its activation of Rac1.     

CUB domain-containing protein 1 is a novel regulator of anoikis resistance in lung adenocarcinoma

Malignant tumor cells frequently achieve resistance to anoikis, a form of apoptosis induced by detachment from the basement membrane, which results in the anchorage-independent growth of these cells. Although the involvement of Src family kinases (SFKs) in this alteration has been reported, little is known about the signaling pathways involved in the regulation of anoikis under the control of SFKs. In this study, we identified a membrane protein, CUB-domain-containing protein 1 (CDCP1), as an SFK-binding phosphoprotein associated with the anchorage independence of human lung adenocarcinoma. Using RNA interference suppression and overexpression of CDCP1 mutants in lung cancer cells, we found that tyrosine-phosphorylated CDCP1 is required to overcome anoikis in lung cancer cells. An apoptosis-related molecule, protein kinase Cdelta, was found to be phosphorylated by the CDCP1-SFK complex and was essential for anoikis resistance downstream of CDCP1. Loss of CDCP1 also inhibited the metastatic potential of the A549 cells in vivo. Our findings indicate that CDCP1 is a novel target for treating cancer-specific disorders, such as metastasis, by regulating anoikis in lung adenocarcinoma.     

Disruption of epithelial cell-matrix interactions induces apoptosis

Cell-matrix interactions have major effects upon phenotypic features such as gene regulation, cytoskeletal structure, differentiation, and aspects of cell growth control. Programmed cell death (apoptosis) is crucial for maintaining appropriate cell number and tissue organization. It was therefore of interest to determine whether cell- matrix interactions affect apoptosis. The present report demonstrates that apoptosis was induced by disruption of the interactions between normal epithelial cells and extracellular matrix. We have termed this phenomenon "anoikis." Overexpression of bcl-2 protected cells against anoikis. Cellular sensitivity to anoikis was apparently regulated: (a) anoikis did not occur in normal fibroblasts; (b) it was abrogated in epithelial cells by transformation with v-Ha-ras, v-src, or treatment with phorbol ester; (c) sensitivity to anoikis was conferred upon HT1080 cells or v-Ha-ras-transformed MDCK cells by reverse- transformation with adenovirus E1a; (d) anoikis in MDCK cells was alleviated by the motility factor, scatter factor. The results suggest that the circumvention of anoikis accompanies the acquisition of anchorage independence or cell motility.     

Mechanotransduction and anoikis: Death and the homeless cell

Developed organs display strict spatial organization of differentiated cells which is required for proper organ function. One important device that prevents tissue disorganization is the death of cells that lose anchorage to their native matrix, a signal that indicates potential loss of proper tissue context. Termed anoikis (Greek for Homelessness), this form of cell death is a specialized form of apoptosis. Interestingly, at certain stages of development and tissue repair, cells are required to migrate in an unanchored state, suggesting that anoikis must be strictly regulated at some level. Likewise, cellular transformation is often accompanied by an inappropriate loss of anoikis and subsequent acquisition of a metastatic phenotype. Despite its importance, the molecular pathways involved in the regulation of anoikis and the proximal signals reporting loss of anchorage are poorly understood. Recent studies suggest that attachment may be reported by a mechanosensory testing of the cell's physical environment.     

Mitotic modulation of translation elongation factor 1 leads to hindered tRNA delivery to ribosomes

Translation elongation in eukaryotes is mediated by the concerted actions of elongation factor 1A (eEF1A), which delivers aminoacylated tRNA to the ribosome; elongation factor 1B (eEF1B) complex, which catalyzes the exchange of GDP to GTP on eEF1A; and eEF2, which facilitates ribosomal translocation. Here we present evidence in support of a novel mode of translation regulation by hindered tRNA delivery during mitosis. A conserved consensus phosphorylation site for the mitotic cyclin-dependent kinase 1 on the catalytic delta subunit of eEF1B (termed eEF1D) is required for its posttranslational modification during mitosis, resulting in lower affinity to its substrate eEF1A. This modification is correlated with reduced availability of eEF1A·tRNA complexes, as well as reduced delivery of tRNA to and association of eEF1A with elongating ribosomes. This mode of regulation by hindered tRNA delivery, although first discovered in mitosis, may represent a more globally applicable mechanism employed under other physiological conditions that involve down-regulation of protein synthesis at the elongation level.     

Cell cycle control by anchorage signaling

Virtually all the cells constituting solid organs in adult animals require anchorage to the extracellular matrix for their proliferation and survival. When deprived of anchorage, those cells arrest in G(1) phase of the cell cycle and die of apoptosis known as anoikis. However, if malignantly transformed, cells no longer require such an anchorage to proliferate and survive, and it is generally thought that the acquirement of this ability underlies the tumorigenic and metastatic capability of malignant cells. Therefore, for the past two decades, great efforts have been devoted to uncovering the nature of the anchorage signal and the mechanism by which this signal controls the G(1)-S transition in the cell cycle with little progress. However, several critical findings were recently made on anchorage signaling and the control of the cell cycle and cell death by this signaling. This review focuses on the newly emerging understanding and perspective of this highly important cell cycle and cell death regulation.     

Evidence that eukaryotic translation elongation factor 1A (eEF1A) binds the Gcn2 protein C terminus and inhibits Gcn2 activity

The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. The ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His(6)-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. The purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2Δ cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His(6)-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2α, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis.     

Direct association between caspase 3 and alpha5beta1 integrin and its role during anoikis of rat fibroblasts

We have demonstrated a direct association between alpha5beta1 integrin and caspase 3, both pro- and mature enzyme, in various sub-cellular compartments of rat fibroblasts undergoing anoikis. Integrin associated caspase 3 showed high activity in the plasma membranes, whereas in the cytosol and microsomal fraction it showed little or no activity. Our results suggest a possible role for recycled un-ligated alpha5beta1 integrin molecules between cytosol and plasma membrane, in regulation of caspase-3 activity and induction of cell death in adhesion-deprived cells.     

Raf kinases mediate the phosphorylation of eukaryotic translation elongation factor 1A and regulate its stability in eukaryotic cells

We identified eukaryotic translation elongation factor 1A (eEF1A) Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and of apoptosis of human cancer cells. Mass spectrometry identified in vitro S21 and T88 as phosphorylation sites mediated by B-Raf but not C-Raf on eEF1A1 whereas S21 was phosphorylated on eEF1A2 by both B- and C-Raf. Interestingly, S21 belongs to the first eEF1A GTP/GDP-binding consensus sequence. Phosphorylation of S21 was strongly enhanced when both eEF1A isoforms were preincubated prior the assay with C-Raf, suggesting that the eEF1A isoforms can heterodimerize thus increasing the accessibility of S21 to the phosphate. Overexpression of eEF1A1 in COS 7 cells confirmed the phosphorylation of T88 also in vivo. Compared with wt, in COS 7 cells overexpressed phosphodeficient (A) and phospho-mimicking (D) mutants of eEF1A1 (S21A/D and T88A/D) and of eEF1A2 (S21A/D), resulted less stable and more rapidly proteasome degraded. Transfection of S21 A/D eEF1A mutants in H1355 cells increased apoptosis in comparison with the wt isoforms. It indicates that the blockage of S21 interferes with or even supports C-Raf induced apoptosis rather than cell survival. Raf-mediated regulation of this site could be a crucial mechanism involved in the functional switching of eEF1A between its role in protein biosynthesis and its participation in other cellular processes.     

Eukaryotic elongation factor 1A interacts with sphingosine kinase and directly enhances its catalytic activity

Sphingosine 1-phosphate (S1P) has many important roles in mammalian cells, including contributing to the control of cell survival and proliferation. S1P is generated by sphingosine kinases (SKs), of which two mammalian isoforms have been identified (SK1 and SK2). To gain a better understanding of SK regulation, we have used a yeast two-hybrid screen to identify SK1-interacting proteins and established elongation factor 1A (eEF1A) as one such protein that associates with both SK1 and SK2. We show the direct interaction of eEF1A with the SKs in vitro, whereas the physiological relevance of this association was demonstrated by co-immunoprecipitation of the endogenous proteins from cell lysates. Although the canonical role of eEF1A resides in protein synthesis, it has also been implicated in other roles, including regulating the activity of some signaling enzymes. Thus, we examined the potential role of eEF1A in regulation of the SKs and show that eEF1A is able to directly increase the activity of SK1 and SK2 approximately 3-fold in vitro. Substrate kinetics demonstrated that eEF1A increased the catalytic rate of both SKs, while having no observable effect on substrate affinities of these enzymes for either ATP or sphingosine. Overexpression of eEF1A in quiescent Chinese hamster ovary cells increased cellular SK activity, whereas a small interfering RNA-mediated decrease in eEF1A levels in MCF7 cells substantially reduced cellular SK activity and S1P levels, supporting the in vivo physiological relevance of this interaction. Thus, this study has established a novel mechanism of regulation of both SK1 and SK2 that is mediated by their interaction with eEF1A.     

Curcumin sensitizes non-small cell lung cancer cell anoikis through reactive oxygen species-mediated Bcl-2 downregulation

Anoikis, an apoptosis triggered by loss of cell anchorage, has been shown to be a principal mechanism of inhibition of tumor metastasis. Recently, anti-apoptotic Bcl-2 and Cav-1 proteins have been demonstrated to be highly associated with tumor metastasis and apoptosis resistance. Curcumin, a major active component of turmeric, Curcuma longa, has been shown to inhibit neoplastic evolution and tumor progression; however, the underlying mechanisms are unclear. In this study, we investigated the effect of curcumin on cell anoikis as a possible mechanism of anti-tumorigenic action of curcumin, and evaluated the potential role of Bcl-2 and Cav-1 in this process. Our results showed that ectopic expression of either Bcl-2 or Cav-1 induced anoikis resistance of lung carcinoma H460 cells. Curcumin downregulated Bcl-2 protein during anoikis and sensitized the cells to detachment-induced apoptosis, whereas it had no significant effect on Cav-1 protein expression. Bcl-2 down-regulation as well as anoikis enhancement by curcumin were inhibited by superoxide anion scavenger, Mn(III)tetrakis(4-benzoic acid) porphyrin chloride, but were unaffected by other ROS scavengers including catalase and deferoxamine, suggesting that superoxide anion is a key player in the downregulation of Bcl-2 by curcumin. Furthermore, we provided evidence that curcumin decreased Bcl-2 level through ubiquitin-proteasomal degradation which sensitized cells to detachment-induced apoptosis. These findings indicate a novel pathway for curcumin regulation of Bcl-2 and provide a key mechanism of anoikis regulation that may be exploited for metastatic cancer treatment.     

The eukaryotic translation elongation factor 1A regulation of actin stress fibers is important for infectious RSV production

Cellular protein eukaryotic translation elongation factor 1A (eEF1A) is an actin binding protein that plays a role in the formation of filamentous actin (F-actin) bundles. F-Actin regulates multiple stages of respiratory syncytial virus (RSV) replication including assembly and budding. Our previous study demonstrated that eEF1A knock-down significantly reduced RSV replication. Here we investigated if the eEF1A function in actin bundle formation was important for RSV replication and release. To investigate this, eEF1A function was impaired in HEp-2 cells by either knock-down of eEF1A with siRNA, or treatment with an eEF1A inhibitor, didemnin B (Did B). Cell staining and confocal microscopy analysis showed that both eEF1A knock-down and treatment with Did B resulted in disruption of cellular stress fiber formation and elevated accumulation of F-actin near the plasma membrane. When treated cells were then infected with RSV, there was also reduced formation of virus-induced cellular filopodia. Did B treatment, similarly to eEF1A knock-down, reduced the release of infectious RSV, but unlike eEF1A knock-down, did not significantly affect RSV genome replication. The lower infectious virus production in Did B treated cells also reduced RSV-induced cell death. In conclusion, the cellular factor eEF1A plays an important role in the regulation of F-actin stress fiber formation required for RSV assembly and release.     

A novel post-translational modification of yeast elongation factor 1A. Methylesterification at the C terminus

Protein methylation reactions can play important roles in cell physiology. After labeling intact Saccharomyces cerevisiae cells with S-adenosyl-l-[methyl-(3)H]methionine, we identified a major methylated 49-kDa polypeptide containing [(3)H]methyl groups in two distinct types of linkages. Peptide sequence analysis of the purified methylated protein revealed that it is eukaryotic elongation factor 1A (eEF1A, formerly EF-1alpha), the protein that forms a complex with GTP and aminoacyl-tRNAs for binding to the ribosomal A site during protein translation. Previous studies have shown that eEF1A is methylated on several internal lysine residues to give mono-, di-, and tri-N-epsilon-methyl-lysine derivatives. We confirm this finding but also detect methylation that is released as volatile methyl groups after base hydrolysis, characteristic of ester linkages. In cycloheximide-treated cells, methyl esterified eEF1A was detected largely in the ribosome and polysome fractions; little or no methylated protein was found in the soluble fraction. Because the base-labile, volatile [methyl-(3)H]radioactivity of eEF1A could be released by trypsin treatment but not by carboxypeptidase Y or chymotrypsin treatment, we suggest that the methyl ester is present on the alpha-carboxyl group of its C-terminal lysine residue. From the results of pulse-chase experiments using radiolabeled intact yeast cells, we find that the N-methylated lysine residues of eEF1A are stable over 4 h, whereas the eEF1A carboxyl methyl ester has a half-life of less than 10 min. The rapid turnover of the methyl ester suggests that the methylation/demethylation of eEF1A at the C-terminal carboxyl group may represent a novel mode of regulation of the activity of this protein in yeast.     

Translation elongation factor eEF1A binds to a novel myosin binding protein-C-like protein

Eukaryotic translation elongation factor 1A (eEF1A) is a guanine-nucleotide binding protein, which transports aminoacylated tRNA to the ribosomal A site during protein synthesis. In a yeast two-hybrid screening of a human skeletal muscle cDNA library, a novel eEF1A binding protein, immunoglobulin-like and fibronectin type III domain containing 1 (IGFN1), was discovered, and its interaction with eEF1A was confirmed in vitro. IGFN1 is specifically expressed in skeletal muscle and presents immunoglobulin I and fibronectin III sets of domains characteristic of sarcomeric proteins. IGFN1 shows sequence and structural homology to myosin binding protein-C fast and slow-type skeletal muscle isoforms. IGFN1 is substantially upregulated during muscle denervation. We propose a model in which this increased expression of IGFN1 serves to down-regulate protein synthesis via interaction with eEF1A during denervation.     

A genome wide shRNA screen identifies α/β hydrolase domain containing 4 (ABHD4) as a novel regulator of anoikis resistance

Acquisition of resistance to anchorage dependant cell death, a process termed anoikis, is a requirement for cancer cell metastasis. However, the molecular determinants of anoikis resistance and sensitivity are poorly understood. To better understand resistance to anoikis we conducted a genome wide lentiviral shRNA screen to identify genes whose knockdown render anoikis-sensitive RWPE-1 prostate cells resistant to anoikis. RWPE-1 cells were infected with a pooled lentiviral shRNA library with 54,021 shRNA targeting 11,255 genes. After infection, an anoikis-resistant cell population was selected and shRNA sequences were amplified and sequenced. Thirty-four shRNA sequences reproducibly protected RWPE-1 cells from anoikis after culture under suspension conditions including the top validated hit, α/β hydrolase domain containing 4 (ABHD4). In validation studies, ABHD4 knockdown inhibited anoikis in RWPE-1 cells as well as anoikis sensitive NP69 nasopharyngeal and OVCAR3 ovarian cancer cells, while over-expression of the gene increased sensitivity. Induction of anoikis after ABHD4 knockdown was associated with cleavage of PARP and activation of caspases-3, but was independent in changes of FLIP, FAK and Src expression. Interestingly, induction of anoikis after ABHD4 knockdown was independent of the known role of ABHD4 in the anandamide synthesis pathway and the generation of glycerophospho-N-acyl ethanolamines. Thus, ABHD4 is a novel genetic regulator of anoikis sensitivity.     

BAG-1 p29 protein prevents drug-induced cell death in the presence of EGF and enhances resistance to anoikis in SKOV3 human ovarian cancer cells

BAG-1 is a multi-functional protein that exists in three major isoforms, BAG-1 p50, p46, and p36. A fourth isoform of 29 kDa also exists but its function remains mostly unknown. To further understand the role of this smaller isoform in ovarian cancer cells, the SKOV3 cell line was transfected with a doxycycline-inducible human BAG-1 p29 isoform or control plasmid. Ovexpression of BAG-1 p29 promotes protection from apoptosis in the presence of EGF as shown by decreased cell death measured by XTT assay and caspase-3 activity. Unexpectedly, however, BAG-1 p29 does not associate with the EGF receptor. When BAG-1 p29 transfectants were incubated in hydrogel-coated plates, BAG-1 p29-expressing SKOV3 cells were significantly more resistant to anoikis as compared to controls, and this correlated with decreased activation of caspase-3. The results of this study implicate BAG-1 p29 in the regulation of both the EGF signaling cascade and the apoptotic cascade induced by loss of anchorage.     

Overexpression of Eukaryotic Translation Elongation Factor 3 Impairs Gcn2 Protein Activation

In eukaryotes, phosphorylation of translation initiation factor 2α (eIF2α) by the kinase Gcn2 (general control nonderepressible 2) is a key response to amino acid starvation. Sensing starvation requires that Gcn2 directly contacts its effector protein Gcn1, and both must contact the ribosome. We have proposed that Gcn2 is activated by uncharged tRNA bound to the ribosomal decoding (A) site, in a manner facilitated by ribosome-bound Gcn1. Protein synthesis requires cyclical association of eukaryotic elongation factors (eEFs) with the ribosome. Gcn1 and Gcn2 are large proteins, raising the question of whether translation and monitoring amino acid availability can occur on the same ribosome. Part of the ribosome-binding domain in Gcn1 has homology to one of the ribosome-binding domains in eEF3, suggesting that these proteins utilize overlapping binding sites on the ribosome and consequently cannot function simultaneously on the same ribosome. Supporting this idea, we found that eEF3 overexpression in Saccharomyces cerevisiae diminished growth on amino acid starvation medium (Gcn⁻ phenotype) and decreased eIF2α phosphorylation, and that the growth defect associated with constitutively active Gcn2 was diminished by eEF3 overexpression. Overexpression of the eEF3 HEAT domain, or C terminus, was sufficient to confer a Gcn⁻ phenotype, and both fragments have ribosome affinity. eEF3 overexpression did not significantly affect Gcn1-ribosome association, but it exacerbated the Gcn⁻ phenotype of Gcn1-M7A that has reduced ribosome affinity. Together, this suggests that eEF3 blocks Gcn1 regulatory function on the ribosome. We propose that the Gcn1-Gcn2 complex only functions on ribosomes with A-site-bound uncharged tRNA, because eEF3 does not occupy these stalled complexes.