Jonas M. la Cour

Up-regulation of ALG-2 in hepatomas and lung cancer tissue.

ALG-2 was isolated in a screen for proteins involved in programmed cell death and is the first Ca(2+)-binding protein found to be directly involved in apoptosis. We have generated polyclonal antibodies that are suitable for detecting ALG-2 using different immunological methods. Three commercial antibodies against ALG-2 did neither detect mouse recombinant ALG-2 nor endogenous ALG-2 in Jurkat cell lysates, whereas our own affinity-purified antibody recognized recombinant as well as endogenous ALG-2. The specificity of the antibody was shown by preabsorbtion experiments and on ALG-2-deficient cells using Western blot analysis and immunohistochemistry. Western blot analysis of 15 different adult mouse tissues demonstrated that ALG-2 is ubiquitously expressed. We found that ALG-2 was more than threefold overexpressed in rat liver hepatoma compared to normal rat liver using Western blot analysis, a result confirmed by immunohistochemical analysis. Staining of four different lung cancer tissue microarrays including specimens of 263 patients showed that ALG-2 is mainly localized to epithelial cells and significantly up-regulated in small-cell lung cancers and in non-small-cell lung cancers. Our results lead to the conclusion that ALG-2 beside its known proapoptotic functions may be a player in survival pathways.

The apoptosis linked gene ALG-2 is dysregulated in tumors of various origin and contributes to cancer cell viability

The apoptosis linked gene‐2 (ALG‐2), discovered as a proapoptotic calcium binding protein, has recently been found upregulated in lung cancer tissue indicating that this protein may play a role in the pathology of cancer cells and/or may be a tumor marker. Using immunohistochemistry on tissue microarrays we analysed the expression of ALG‐2 in 7371 tumor tissue samples of various origin as well as in 749 normal tissue samples. Most notably, ALG‐2 was upregulated in mesenchymal tumors. No correlation was found between ALG‐2 staining intensity and survival of patients with lung, breast or colon cancer. siRNA mediated ALG‐2 downregulation led to a significant reduction in viability of HeLa cells indicating that ALG‐2 may contribute to tumor development and expansion.

ALG-2 knockdown in HeLa cells results in G2/M cell cycle phase accumulation and cell death.

ALG-2 (apoptosis-linked gene-2 encoded protein) has been shown to be upregulated in a variety of human tumors questioning its previously assumed pro-apoptotic function. The aim of the present study was to obtain insights into the role of ALG-2 in human cancer cells. We show that ALG-2 downregulation induces accumulation of HeLa cells in the G2/M cell cycle phase and increases the amount of early apoptotic and dead cells. Caspase inhibition by the pan-caspase inhibitor zVAD-fmk attenuated the increase in the amount of dead cells following ALG-2 downregulation. Thus, our results indicate that ALG-2 has an anti-apoptotic function in HeLa cells by facilitating the passage through checkpoints in the G2/M cell cycle phase.

ALG-2 attenuates COPII budding in vitro and stabilizes the Sec23/Sec31A complex.

Coated vesicles mediate the traffic of secretory and membrane cargo proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. The coat protein complex (COPII) involved in vesicle budding is constituted by a GTPase, Sar1, the inner coat components of Sec23/Sec24 and the components of the outer coat Sec13/Sec31A. The Ca2+-binding protein ALG-2 was recently identified as a Sec31A binding partner and a possible link to Ca2+ regulation of COPII vesicle budding. Here we show that ALG-2/Ca2+ is capable of attenuating vesicle budding in vitro through interaction with an ALG-2 binding domain in the proline rich region of Sec31A. Binding of ALG-2 to Sec31A and inhibition of COPII vesicle budding is furthermore dependent on an intact Ca2+-binding site at EF-hand 1 of ALG-2. ALG-2 increased recruitment of COPII proteins Sec23/24 and Sec13/31A to artificial liposomes and was capable of mediating binding of Sec13/31A to Sec23. These results introduce a regulatory role for ALG-2/Ca2+ in COPII tethering and vesicle budding.

Regulation of the Ligand-dependent Activation of the Epidermal Growth Factor Receptor by Calmodulin

Background: The EGFR binds calmodulin (CaM). Results: CaM antagonists, CaM down-regulation in conditional CaM-KO cells, chelation of Ca2+, and mutagenesis of the CaM-binding domain inhibit EGFR activation. Conclusion: The Ca2+/CaM complex is a positive regulator of the EGFR. Significance: This is the first work with a multi-approach strategy demonstrating that CaM directly regulates the EGFR in living cells. Calmodulin (CaM) is the major component of calcium signaling pathways mediating the action of various effectors. Transient increases in the intracellular calcium level triggered by a variety of stimuli lead to the formation of Ca2+/CaM complexes, which interact with and activate target proteins. In the present study the role of Ca2+/CaM in the regulation of the ligand-dependent activation of the epidermal growth factor receptor (EGFR) has been examined in living cells. We show that addition of different cell permeable CaM antagonists to cultured cells or loading cells with a Ca2+ chelator inhibited ligand-dependent EGFR auto(trans)phosphorylation. This occurred also in the presence of inhibitors of protein kinase C, CaM-dependent protein kinase II and calcineurin, which are known Ca2+- and/or Ca2+/CaM-dependent EGFR regulators, pointing to a direct effect of Ca2+/CaM on the receptor. Furthermore, we demonstrate that down-regulation of CaM in conditional CaM knock out cells stably transfected with the human EGFR decreased its ligand-dependent phosphorylation. Substitution of six basic amino acid residues within the CaM-binding domain (CaM-BD) of the EGFR by alanine resulted in a decreased phosphorylation of the receptor and of its downstream substrate phospholipase Cγ1. These results support the hypothesis that Ca2+/CaM regulates the EGFR activity by directly interacting with the CaM-BD of the receptor located at its cytosolic juxtamembrane region.

The PEF family proteins sorcin and grancalcin interact in vivo and in vitro

The penta‐EF hand (PEF) family of calcium binding proteins includes grancalcin, peflin, sorcin, calpain large and small subunits as well as ALG‐2. Systematic testing of the heterodimerization abilities of the PEF proteins using the yeast two‐hybrid and glutathione S‐transferase pull‐down assays revealed the new finding that grancalcin interacts strongly with sorcin. In addition, sorcin and grancalcin can be co‐immunoprecipitated from lysates of human umbilical vein endothelial cells. Our results indicate that heterodimerization, in addition to differential interactions with target proteins, might be a way to regulate and fine tune processes mediated by calcium binding proteins of the penta‐EF hand type.

Significance of Calcium Binding, Tyrosine Phosphorylation, and Lysine Trimethylation for the Essential Function of Calmodulin in Vertebrate Cells Analyzed in a Novel Gene Replacement System

Background: Calmodulin is a Ca2+ binding protein and a major regulator of multiple signaling pathways. Results: Inactivation of the Ca2+ binding sites in the N- and C-terminal lobe of CaM affects cell viability differentially. Conclusion: Ca2+ binding to CaM is required for vertebrate cell survival. Significance: A novel vertebrate knock-out/knock-in system for studying the function of CaM is described. Calmodulin (CaM) was shown to be essential for survival of lower eukaryotes by gene deletion experiments. So far, no CaM gene deletion was reported in higher eukaryotes. In vertebrates, CaM is expressed from several genes, which encode an identical protein, making it difficult to generate a model system to study the effect of CaM gene deletion. Here, we present a novel genetic system based on the chicken DT40 cell line, in which the two functional CaM genes were deleted and one allele replaced with a CaM transgene that can be artificially regulated. We show that CaM is essential for survival of vertebrate cells as they die in the absence of CaM expression. Reversal of CaM repression or ectopic expression of HA-tagged CaM rescued the cells. Cells exclusively expressing HA-CaM with impaired individual calcium binding domains as well as HA-CaM lacking the ability to be phosphorylated at residues Tyr99/Tyr138 or trimethylated at Lys115 survived and grew well. CaM mutated at both Ca2+ binding sites 3 and 4 as well as at both sites 1 and 2, but to a lesser degree, showed decreased ability to support cell growth. Cells expressing CaM with all calcium binding sites impaired died with kinetics similar to that of cells expressing no CaM. This system offers a unique opportunity to analyze CaM structure-function relationships in vivo without the use of pharmacological inhibitors and to analyze the function of wild type and mutated CaM in modulating the activity of different target systems without interference of endogenous CaM.

The Arrhythmogenic Calmodulin Mutation D129G Dysregulates Cell Growth, Calmodulin-dependent Kinase II Activity, and Cardiac Function in Zebrafish

Calmodulin (CaM) is a Ca2+ binding protein modulating multiple targets, several of which are associated with cardiac pathophysiology. Recently, CaM mutations were linked to heart arrhythmia. CaM is crucial for cell growth and viability, yet the effect of the arrhythmogenic CaM mutations on cell viability, as well as heart rhythm, remains unknown, and only a few targets with relevance for heart physiology have been analyzed for their response to mutant CaM. We show that the arrhythmia-associated CaM mutants support growth and viability of DT40 cells in the absence of WT CaM except for the long QT syndrome mutant CaM D129G. Of the six CaM mutants tested (N53I, F89L, D95V, N97S, D129G, and F141L), three showed a decreased activation of Ca2+/CaM-dependent kinase II, most prominently the D129G CaM mutation, which was incapable of stimulating Thr286 autophosphorylation. Furthermore, the CaM D129G mutation led to bradycardia in zebrafish and an arrhythmic phenotype in a subset of the analyzed zebrafish.

ALG-2 participates in recovery of cells after plasma membrane damage by electroporation and digitonin treatment

The calcium binding protein ALG-2 is upregulated in several types of cancerous tissues and cancer cell death may be a consequence of ALG-2 downregulation. Novel research suggests that ALG-2 is involved in membrane repair mechanisms, in line with several published studies linking ALG-2 to processes of membrane remodeling and transport, which may contribute to the fitness of cells or protect them from damage. To investigate the involvement of ALG-2 in cell recovery after membrane damage we disrupted the PDCD6 gene encoding the ALG-2 protein in DT-40 cells and exposed them to electroporation. ALG-2 knock-out cells were more sensitive to electroporation as compared to wild type cells. This phenotype could be reversed by reestablishing ALG-2 expression confirming that ALG-2 plays an important role in cell recovery after plasma membrane damage. We found that overexpression of wild type ALG-2 but not a mutated form unable to bind Ca2+ partially protected HeLa cells from digitonin-induced cell death. Further, we were able to inhibit the cell protective function of ALG-2 after digitonin treatment by adding a peptide with the ALG-2 binding sequence of ALIX, which has been proposed to serve as the ALG-2 downstream target in a number of processes including cell membrane repair. Our results suggest that ALG-2 may serve as a novel therapeutic target in combination with membrane damaging interventions.