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The Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII) is well known for transmitting Ca2+-signals, which leads to a multitude of physiological responses. Its functionality is believed to involve CaMKII holoenzyme dynamics where trans-autophosphorylation of the crucial phosphorylation site, T286 occurs. Phosphorylation of this site does not occur when stimulated exclusively with the arrhythmia associated D130G mutant form of CaM in vitro. Here, we present evidence that the loss-of-CaMKII function correlates with premature phosphorylation of its inhibitory phosphosite T306 in CaMKIIα and T307 in CaMKIIδ as this site was up to 20-fold more phosphorylated in the presence of D130G CaM compared to wildtype CaM. Indeed, changing this phosphosite to a non-phosphorylatable alanine reversed the inhibitory effect of D130G both in vitro and in live cell experiments. In addition, several phosphosites with so far undescribed functions directing the Ca2+-sensitivity of the CaMKII sensor were also affected by the presence of the D130G mutation implicating a role of several additional autophosphosites (besides T286 and T306/T307) so far not known to regulate CaMKII Ca2+ sensitivity. Furthermore, we show that introducing a D130G mutation in the CALM2 gene of the P19CL6 pluripotent mouse embryonic carcinoma cell line using CRISPR/Cas9 decreased the spontaneous beat frequency compared to wildtype cells when differentiated into cardiomyocytes supporting an alteration of cardiomyocyte physiology caused by this point mutation. In conclusion, our observations shed for the first time light on how the D130G CaM mutation interferes with the function of CaMKII and how it affects the beating frequency of cardiomyocyte-like cells.

Calmodulin (CaM) is a ubiquitous Ca2+‐sensing protein regulating many important cellular processes. Several CaM‐associated variants have been identified in a small group of patients with cardiac arrhythmias. The mechanism remains largely unknown, even though a number of ion channels, including the ryanodine receptors and the L‐type calcium channels have been shown to be functionally affected by the presence of mutant CaM. CaM is constitutively bound to the SK channel, which underlies the calcium‐gated ISK contributing to cardiac repolarization. The CaM binding to SK channels is essential for gating, correct assembly, and membrane expression. To elucidate the effect of nine different arrhythmogenic CaM variants on SK3 channel function, HEK293 cells stably expressing SK3 were transiently co‐transfected with CaMWT or variant and whole‐cell patch‐clamp recordings were performed with a calculated free Ca2+ concentration of 400 nmol/L. MDCK cells were transiently transfected with SK3 and/or CaMWT or variant to address SK3 and CaM localization by immunocytochemistry. The LQTS‐associated variants CaMD96V, CaMD130G, and CaMF142L reduced ISK,Ca compared with CaMWT (P < 0.01, P < 0.001, and P < 0.05, respectively). The CPVT associated variant CaMN54I also reduced the ISK,Ca (P < 0.05), which was linked to an accumulation of SK3/CaMN54I channel complexes in intracellular compartments (P < 0.05). The CPVT associated variants, CaMA103V and CaMD132E only revealed a tendency toward reduced current, while the variants CaMF90L and CaMN98S, causing LQTS syndrome, did not have any impact on ISK,Ca. In conclusion, we found that the arrhythmogenic CaM variants CaMN54I, CaMD96V, CaMD130G, and CaMF142L significantly down‐regulate the SK3 channel current, but with distinct mechanism.

Calmodulin (CaM) is a Ca(2+) 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 Ca(2+)/CaM-dependent kinase II, most prominently the D129G CaM mutation, which was incapable of stimulating Thr(286) autophosphorylation. Furthermore, the CaM D129G mutation led to bradycardia in zebrafish and an arrhythmic phenotype in a subset of the analyzed zebrafish.

et al., 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., This work was financed by grants (to A. V.) from the Ministerio de Ciencia e Innovación (SAF2008-00986 and SAF2011-23494) and the European Commission (contract ITN 289033); Grant from the Danish Research Council (to M. W. B.), the Lundbeck Foundation, AP Møller Fonden, Dagmar Marshall Fonden, Willumsen Fonden, Danielsen Fonden, and Wedell Wedellsborg Fonden.

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 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.

et al., 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 Tyr(99)/Tyr(138) or trimethylated at Lys(115) survived and grew well. CaM mutated at both Ca(2+) 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., This work was supported by grants from the Danish Research Council, the Lundbeeck Foundation, AP Møller Fonden, Dagmar Marshall Fonden, Willumsen Fonden, Danielsen Fonden, and Wedell Wedellsborg Fonden, Frænkels Mindefond, Hansen Fonden (to M. W. B.). Supported by the Danish Cancer Society (R2-A183). Supported by a predoctoral fellowship from the Junta de Ampliación de Estudios, Consejo Superior de Investigaciones Científicas. Supported by the Ministerio de Ciencia e Innovación (SAF2008-00986 and SAF2011-23494), the Consejería de Educación de la Comunidad de Madrid (S2011/BMD-2349), and the European Commission (Contract ITN 289033).

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.

International audience; ALG-2 (apoptosis linked gene 2 product) is a calcium binding protein for which no clear cellular function has been established. In this study we identified Scotin as a novel ALG-2 target protein containing 6 PXY and 4 PYP repeats, earlier identified in the ALG-2 binding regions of AIP1/ALIX and TSG101, respectively. An in vitro synthesized C-terminal fragment of Scotin bound specifically to immobilized recombinant ALG-2 and tagged ALG-2 and Scotin were shown by immunoprecipitation to interact in MCF7 and U2OS cell lines. Furthermore ALG-2 bound to endogenous Scotin in extracts from mouse NIH3T3 cells. Overexpression of ALG-2 led to accumulation of Scotin in MCF7 and H1299 cells. In vitro and in vivo binding of ALG-2 to Scotin was demonstrated to be strictly calcium dependent indicating a role of this interaction in calcium signaling pathways.

A variety of stimuli can trigger intracellular calcium oscillations. Relatively little is known about the molecular mechanisms decoding these events. We show that ALG-2, a Ca 2+ -binding protein originally isolated as a protein associated with apoptosis, is directly linked to Ca 2+ signalling. We discovered that the subcellular distribution of a tagged version of ALG-2 could be directed by physiological external stimuli (including ATP, EGF, prostaglandin, histamine), which provoke intracellular Ca 2+ oscillations. Cellular stimulation led to a redistribution of ALG-2 from the cytosol to a punctate localization in an oscillatory fashion unitemporally with Ca 2+ oscillations, whereas a Ca 2+ -binding deficient mutant of ALG-2 did not redistribute. Using tagged ALG-2 as bait we identified its novel target protein Sec31A and based on the partial colocalization of endogenous ALG-2 and Sec31A we propose that ALG-2 temporarily binds to the COPII vesicles providing a link between Ca 2+ signalling and ER to Golgi trafficking.

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.