Abp1p is an actin-binding protein that plays a central role in the organization of Saccharomyces cerevisiae actin cytoskeleton. By a combination of two-hybrid and phage-display approaches, we have identified six new ligands of the Abp1-SH3 domain. None of these SH3-mediated novel interactions was detected in recent all genome high throughput protein interaction projects. Here we show that the SH3-mediated association of Abp1p with the Ser/Thr kinases Prk1p and Ark1p is essential for their localization to actin cortical patches. The Abp1-SH3 domain has a rather unusual binding specificity, because its target peptides contain the tetrapentapeptide ؉XXXPXXPX؉PXXL with positive charges flanking the polyproline core on both sides. Here we present the structure of the Abp1-SH3 domain solved at 1.3-Å resolution. The peptide-binding pockets in the SH3 domain are flanked by two acidic residues that are uncommon at those positions in the SH3 domain family. We have shown by site-directed mutagenesis that one of these negatively charged side chains may be the key determinant for the preference for non-classical ligands.The actin cytoskeleton plays a key role in many essential cellular processes, such as motility, endocytosis, secretion, and membrane recycling (1-3). As a consequence, its organization and dynamic rearrangements need to be tightly controlled spatially and temporally. A thorough understanding of the interaction network connecting all the actin-associated proteins, the scaffolds and the anchoring proteins, is likely to help to clarify the mechanisms underlying its coordinated regulation (4).Most of the components of the yeast cell cytoskeleton have homologues in mammals where they often play similar roles (5-7). Abp1p 1 (actin-binding protein) is a Saccharomyces cerevisiae protein homologue to the mouse mAbp1p-SH3P7, which is a Src kinase target involved in polarized cell growth and motility (8, 9). The yeast Abp1 protein is 592 amino acids long and includes an actin depolymerizing factor-homology domain at the N terminus and a SH3 domain at the C terminus of the protein (10). Abp1p is found concentrated in the actin patches that are enriched at the sites of polarized cell surface growth in the bud of budding yeast and in the mating projection of mating yeast. The overexpression of Abp1p disturbs the actin cytoskeleton and leads to an aberrant budding pattern and cortical actin assembly (11). Deletion of the ABP1 gene, on the other hand, does not cause any apparent cytoskeletal defect (12). Abp1p, however, is found to be essential when any of the genes encoding for Sac6p (the actin filament-bundling protein, fimbrin), Sla1p, Sla2p, or Prk1p is deleted (13, 51). These proteins, which functionally interact with Abp1p, are also localized in the cortical actin cytoskeleton (11, 13). The observed negative synergism in yeast cells carrying combinations of deletions in ABP1-SH3 and in the SLA1, SLA2, and SAC6 genes suggests that these gene products perform redundant essential function(s) that require the presence of a...

A novel pathway of fenretinide-induced apoptosis is mediated by acidic sphingomyelinase, glucosylceramide synthase, and GD3 synthase, which may represent targets for future drug development. GD3 may be a key signaling intermediate leading to apoptosis via the activation of 12-lipoxygenase.

Cells may use multiple pathways to commit suicide. In certain contexts, dying cells generate large amounts of autophagic vacuoles and clear large proportions of their cytoplasm, before they finally die, as exemplified by the treatment of human mammary carcinoma cells with the anti-estrogen tamoxifen (TAM, <= 1 mu M). Protein analysis during autophagic cell death revealed distinct proteins of the nuclear fraction including CST-pi and some proteasomal subunit constituents to be affected during autophagic cell death. Depending on the functional status of caspase-3, MCF-7 cells may switch between autophagic and apoptotic features of cell death [Fazi, B., Bursch, W., Fimia, G.M., Nardacci R., Piacentini, M., Di Sano, F., Piredda. L., 2008. Fenretinide induces autophagic cell death in caspase-defective breast cancer cells. Autophagy 4(4), 435-441]. Furthermore, the self-destruction of MCF-7 cells was found to be completed by phagocytosis of cell residues [Petrovski, G., Zahuczky, G., Katona, K., Vereb, G., Martinet. W., Nemes, Z., Bursch, W., Fesus, L., 2007. Clearance of dying autophagic cells of different origin by professional and non-professional phagocytes. Cell Death Diff. 14 (6),1117-1128]. Autophagy also constitutes a cell's strategy of defense upon cell damage by eliminating damaged bulk proteins/organelles. This biological condition may be exemplified by the treatment of MCF-7 cells with a necrogenic TAM-dose (10 mu M), resulting in the lysis of almost all cells within 24 h. However, a transient (1 h) challenge of MCF-7 cells with the same dose allowed the recovery of cells involving autophagy. Enrichment of chaperones in the insoluble cytoplasmic protein fraction indicated the formation of aggresomes, a potential trigger for autophagy. In a further experimental model HL60 cells were treated with TAM, causing dose-dependent distinct responses: 1-5 mu M TAM, autophagy predominant; 7-9 mu M, apoptosis predominant; 15 mu M, necrosis. These phenomena might be attributed to the degree of cell damage caused by tamoxifen, either by generating ROS, increasing membrane fluidity or forming DNA-adducts. Finally, autophagy constitutes a cell's major adaptive (survival) strategy in response to metabolic challenges such as glucose or amino acid deprivation, or starvation in general. Notably, the role of autophagy appears not to be restricted to nutrient recycling in order to maintain energy supply of cells and to adapt cell(organ) size to given physiological needs. For instance, using a newly established hepatoma cell line HCC-1.2, amino acid and glucose deprivation revealed a pro-apoptotic activity, additive to TGF-beta 1.The proapoptotic action of glucose deprivation was antagonized by 2-deoxyglucose, possibly by stabilizing the mitochondrial membrane involving the action of hexokinase II. These observations suggest that signaling cascades steering autophagy appear to provide links to those regulating cell number. Taken together, our data exemplify that a given cell may flexibly respond to type and degree of (micro)envir...

Some chemotherapeutic agents can elicit apoptotic cancer cell death, thereby activating an anticancer immune response that influences therapeutic outcome. We previously reported that anthracyclins are particularly efficient in inducing immunogenic cell death, correlating with the pre-apoptotic exposure of calreticulin (CRT) on the plasma membrane surface of anthracyclintreated tumor cells. Here, we investigated the role of cellular Ca 2 þ homeostasis on CRT exposure. A neuroblastoma cell line (SH-SY5Y) failed to expose CRT in response to anthracyclin treatment. This defect in CRT exposure could be overcome by the overexpression of Reticulon-1C, a manipulation that led to a decrease in the Ca 2 þ concentration within the endoplasmic reticulum lumen. The combination of Reticulon-1C expression and anthracyclin treatment yielded more pronounced endoplasmic reticulum Ca 2 þ depletion than either of the two manipulations alone. Chelation of intracellular (and endoplasmic reticulum) Ca 2 þ , targeted expression of the ligand-binding domain of the IP 3 receptor and inhibition of the sarco-endoplasmic reticulum Ca 2 þ -ATPase pump reduced endoplasmic reticulum Ca 2 þ load and promoted pre-apoptotic CRT exposure on the cell surface, in SH-SY5Y and HeLa cells. These results provide evidence that endoplasmic reticulum Ca 2 þ levels control the exposure of CRT. In contrast to prior belief, apoptotic cell death can be immunogenic and hence elicits an active immune response against dying tumor cells. 1 This is therapeutically relevant because immune defects that compromise the response against apoptotic cells reduce the efficacy of anticancer chemotherapy, both in suitable animal models and in patients. 2 We found that anthracyclins and g-irradiation are particularly efficient in inducing immunogenic cell death, at least in mouse models. [3][4][5] The immunogenicity of cellular demise correlates with the exposure of calreticulin (CRT) on the plasma membrane (PM) surface of stressed tumor cells, a phenomenon that manifests before the cells acquire signs of apoptosis such as phosphatidylserine exposure. Inhibition of CRT exposure curtails the capacity of anthracyclin-treated or irradiated tumor cells to vaccinate against cancer and reduces the therapeutic efficacy of anthracyclins on tumors established in immunocompetent hosts. 3-5 Provision of recombinant CRT or drug-mediated enforcement of CRT exposure rendered per se nonimmunogenic chemotherapies (for instance with etoposide and mitomycin C) immunogenic and boosted their therapeutic efficacy. 5 These results suggest that CRT exposure is both necessary and sufficient to render conventional chemotherapies immunogenic.CRT is an abundant Ca 2 þ -binding chaperone that is mostly present in the endoplasmic reticulum (ER) lumen, although it can also be found in other subcellular localizations. 6,7 When present on the surface of damaged cells, it can serve as an 'eat-me' signal and hence facilitate the recognition and later engulfment of the dying cells by macrophages 8 or by dendrit...

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