BBA - Molecular Cell Research (v.1833, #1)

Transglutaminase 2 facilitates or ameliorates HIF signaling and ischemic cell death depending on its conformation and localization by Soner Gundemir; Gozde Colak; Julianne Feola; Richard Blouin; Gail V.W. Johnson (1-10).
Transglutaminase 2 (TG2) is a widely expressed and multifunctional protein that modulates cell death/survival processes. We have previously shown that TG2 binds to hypoxia inducible factor 1β (HIF1β) and decreases the upregulation of HIF responsive genes; however, the relationship between these observations was not investigated. In this study, we investigated whether endogenous TG2 is sufficient to suppress HIF activity and whether the interaction between TG2 and HIF1β is required for this suppression. shRNA-mediated silencing of TG2 significantly enhanced HIF activation in response to hypoxia. In addition, nuclear localization of TG2 is required for its suppressive effect on HIF activity, with TG2 being recruited to HIF responsive promoters in hypoxic conditions. These observations suggest that TG2 directly regulates hypoxic transcriptional machinery; however, its interaction with HIF1β was not required for this regulation. We also examined whether TG2's effect on cell death/survival processes in ischemia is due to its effects on HIF signaling. Our results indicate that TG2 mediated HIF suppression can be separated from TG2's effect on cell survival in hypoxic/hypoglycemic conditions. Lastly, here we show that nuclear TG2 in the closed conformation and non-nuclear TG2 in the open conformation have opposing effects on hypoxic/hypoglycemic cell death, which could explain previous controversial results. Overall, our results further clarify the role of TG2 in mediating the cellular response to ischemia and suggest that manipulating the conformation of TG2 might be of pharmacological interest as a therapeutic strategy for the treatment of ischemia-related pathologies.► Endogenous TG2 suppresses HIF activity. ► Nuclear localization of TG2 is necessary for suppression of HIF activity. ► Interaction between TG2 and HIF1β is not required for HIF activity suppression. ► TG2 is found on HIF target promoters in hypoxia. ► Conformation of TG2 is crucial in determining the fate of the cell in ischemia.
Keywords: Ischemia; Hypoxia; Transglutaminase 2; Cell death; Transcription;

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that has been implicated in a number of cellular processes. In Dictyostelium, Cdk5 localizes to the nucleus and cytoplasm, interacts with puromycin-sensitive aminopeptidase A (PsaA), and regulates endocytosis, secretion, growth, and multicellular development. Here we show that Cdk5 is a calmodulin (CaM)-binding protein (CaMBP) in Dictyostelium. Cdk5, PsaA, and CaM were all present in isolated nuclei and Cdk5 and PsaA co-immunoprecipitated with nuclear CaM. Although nuclear CaMBPs have previously been identified in Dictyostelium, the detection of CaM in purified nuclear fractions had not previously been shown. Putative CaM-binding domains (CaMBDs) were identified in Cdk5 and PsaA. Deletion of one of the two putative CaMBDs in Cdk5 (132LLINRKGELKLADFGLARAFGIP154) prevented CaM-binding indicating that this region encompasses a functional CaMBD. This deletion also increased the nuclear distribution of Cdk5 suggesting that CaM regulates the nucleocytoplasmic transport of Cdk5. A direct binding between CaM and PsaA could not be determined since deletion of the one putative CaMBD in PsaA prevented the nuclear localization of the deletion protein. Together, this study provides the first direct evidence for nuclear CaM in Dictyostelium and the first evidence in any system for Cdk5 being a CaMBP.► Cdk5 is a calmodulin-binding protein in Dictyostelium. ► Calmodulin localizes to the nucleus in Dictyostelium. ► Cdk5 and PsaA interact with CaM in Dictyostelium nuclei. ► Cdk5 contains a calmodulin-binding domain. ► Calmodulin regulates the nucleocytoplasmic transport of Cdk5.
Keywords: Cyclin-dependent kinase 5; Calmodulin; Puromycin-sensitive aminopeptidase; Calmodulin-binding protein; Dictyostelium; Nucleus;

The novel interaction between microspherule protein Msp58 and ubiquitin E3 ligase EDD regulates cell cycle progression by Mario Benavides; Lai-Fong Chow-Tsang; Jinsong Zhang; Hualin Zhong (21-32).
Microspherule protein Msp58 (or MCRS1) plays a role in numerous cellular processes including transcriptional regulation and cell proliferation. It is not well understood either how Msp58 mediates its myriad functions or how it is itself regulated. Here, by immunoprecipitation, we identify EDD (E3 identified by differential display) as a novel Msp58-interacting protein. EDD, also called UBR5, is a HECT-domain (homologous to E6-AP carboxy-terminus) containing ubiquitin ligase that plays a role in cell proliferation, differentiation and DNA damage response. Both in vitro and in vivo binding assays show that Msp58 directly interacts with EDD. Microscopy studies reveal that these two proteins co-localize in the nucleus. We have also found that depletion of EDD leads to an increase of Msp58 protein level and extends the half-life of Msp58, demonstrating that EDD negatively regulates Msp58's protein stability. Furthermore, we show that Msp58 is upregulated in multiple different cell lines upon the treatment with proteasome inhibitor MG132 and exogenously expressed Msp58 is ubiquitinated, suggesting that Msp58 is degraded by the ubiquitin–proteasome pathway. Finally, knockdown of either Msp58 or EDD in human lung fibroblast WI-38 cells affects the levels of cyclins B, D and E, as well as cell cycle progression. Together, these results suggest a role for the Msp58/EDD interaction in controlling cell cycle progression. Given that both Msp58 and EDD are often aberrantly expressed in various human cancers, our findings open a new direction to elucidate Msp58 and EDD's roles in cell proliferation and tumorigenesis.► Microspherule protein Msp58 forms a novel complex with E3 ubiquitin ligase EDD. ► Msp58 and EDD directly interact and co-localize in the nucleoplasm. ► Two regions of Msp58 are sufficient to bind EDD independently. ► EDD negatively regulates the protein level of Msp58. ► The Msp58/EDD complex regulates cyclin levels and cell cycle progression.
Keywords: Microspherule protein 1; E3 ubiquitin ligase; Cell cycle; Cyclin B; Cyclin D; Nucleoplasm;

p21-activated kinases and gastrointestinal cancer by Hong He; Graham S. Baldwin (33-39).
p21-activated kinases (PAKs) were initially identified as effector proteins downstream from GTPases of the Rho family. To date, six members of the PAK family have been discovered in mammalian cells. PAKs play important roles in growth factor signalling, cytoskeletal remodelling, gene transcription, cell proliferation and oncogenic transformation. A large body of research has demonstrated that PAKs are up-regulated in several human cancers, and that their overexpression is linked to tumour progression and resistance to therapy. Structural and biochemical studies have revealed the mechanisms involved in PAK signalling, and opened the way to the development of PAK-targeted therapies for cancer treatment. Here we summarise recent findings from biological and clinical research on the role of PAKs in gastrointestinal cancer, and discuss the current status of PAK-targeted anticancer therapies.► The p21-activated kinases (PAKs) are up-regulated in several human cancers. ► PAK overexpression is linked to tumour progression and resistance to therapy. ► PAK1 connects multiple signalling pathways important in colorectal cancer. ► PAKs are attractive targets for new therapies for gastrointestinal cancers.
Keywords: PAK; Signalling; Liver cancer; Pancreatic cancer; Gastric cancer; Colorectal cancer;

The enzyme aromatase, encoded by the CYP19A1 gene, catalyzes the production of estrogens and inhibition of aromatase has therefore become one of the key strategies in breast cancer treatment. We have studied the effects of the vitamin D analog EB1089 on aromatase gene expression and enzyme activity in breast cancer cells. We found that EB1089 was able to decrease the gene expression and enzyme activity as well as inhibit aromatase-dependent cell growth. Furthermore, a low dose of EB1089 combined with low doses of clinically used aromatase inhibitors such as anastrozole, letrozole and exemestane were able to effectively inhibit aromatase-dependent growth of breast cancer cells. The molecular mechanism for this effect of EB1089 on the aromatase gene expression was investigated and we found that it is mediated by the vitamin D receptor (VDR), vitamin D receptor interacting repressor (VDIR) and Williams syndrome transcription factor (WSTF). ChIP and Re-ChIP assays revealed that EB1089 mediates dissociation of WSTF from the CYP19A1 promoter and thereby decreases the gene expression. Regulation of aromatase via WSTF has not been reported previously. Furthermore, gene silencing of WSTF results in decreased gene expression of CYP19A1 and aromatase activity, showing that WSTF is an interesting drug target for development of new anti-cancer drugs. In summary, we report that the vitamin D analog EB1089 is able to decrease the gene expression and enzyme activity of aromatase via a novel regulatory pathway for aromatase and suggest that EB1089 may be a new treatment option for estrogen dependent breast cancer.► Vitamin D analog EB1089 decreases gene expression of CYP19A1 in breast cancer cells. ► EB1089 dissociates modulator WSTF from the CYP19A1 promoter in a VDR dependent manner. ► EB1089 reduces the aromatase activity. ► EB1089 is of interest in treatment of endocrine responsive breast cancers. ► WSTF is an interesting drug target for new treatments against breast cancer.
Keywords: Breast cancer; Aromatase; Vitamin D; Selective aromatase modulator; CYP19 regulation;

Neuronal activity and energy metabolism are tightly coupled processes. Previously, we found that nuclear respiratory factor 1 (NRF-1) transcriptionally co-regulates energy metabolism and neuronal activity by regulating all 13 subunits of the critical energy generating enzyme, cytochrome c oxidase (COX), as well as N-methyl-d-aspartate (NMDA) receptor subunits 1 and 2B, GluN1 (Grin1) and GluN2B (Grin2b). We also found that another transcription factor, nuclear respiratory factor 2 (NRF-2 or GA-binding protein) regulates all subunits of COX as well. The goal of the present study was to test our hypothesis that NRF-2 also regulates specific subunits of NMDA receptors, and that it functions with NRF-1 via one of three mechanisms: complementary, concurrent and parallel, or a combination of complementary and concurrent/parallel. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation of mouse neuroblastoma cells and rat visual cortical tissue, promoter mutations, real-time quantitative PCR, and western blot analysis, NRF-2 was found to functionally regulate Grin1 and Grin2b genes, but not any other NMDA subunit genes. Grin1 and Grin2b transcripts were up-regulated by depolarizing KCl, but silencing of NRF-2 prevented this up-regulation. On the other hand, over-expression of NRF-2 rescued the down-regulation of these subunits by the impulse blocker TTX. NRF-2 binding sites on Grin1 and Grin2b are conserved among species. Our data indicate that NRF-2 and NRF-1 operate in a concurrent and parallel manner in mediating the tight coupling between energy metabolism and neuronal activity at the molecular level.► NRF-2 functionally regulates critical Grin1 and Grin2b subunits of NMDA receptors. ► Silencing NRF-2 prevented KCl-induced up-regulation of Grin1, Grin2b, and COX. ► Over-expressing NRF-2 rescued TTX-induced down-regulation of Grin1, Grin2b, and COX. ► NRF-2 (GABP) transcriptionally coregulates energy metabolism and neuronal activity. ► NRF-2 and NRF-1 regulate NMDA receptors and COX in a concurrent and parallel manner.
Keywords: Gene regulation; NRF-2; GABP; GluN1; GluN2B; NMDA;

Poly(ADP-ribose) polymerase 1 inhibition protects against low shear stress induced inflammation by Wei-dong Qin; Shu-jian Wei; Xu-ping Wang; Juan Wang; Wen-ke Wang; Fuqiang Liu; Lei Gong; Fei Yan; Yun Zhang; Mingxiang Zhang (59-68).
Background: Atherosclerosis begins as local inflammation of vessels at sites of disturbed flow, where low shear stress (LSS) leads to mechanical irritation and plaque development and progression. Nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP-1) is associated with the inflammation response during atherosclerosis. We investigated the role and underlying mechanism of PARP-1 in LSS-induced inflammation in human umbilical vein endothelial cells (HUVECs). Methods and results: HUVECs were simulated by LSS (0.4 Pa). PARP-1 expression was inhibited by ABT888 or siRNA. The inducible nitric oxide synthase (iNOS) and intercellular adhesion molecular-1 (ICAM-1) expression was regulated by LSS in a time dependent manner. LSS could increase superoxide production and 3-nitrotyrosine formation. LSS induced DNA damage as assessed by H2A.X phosphorylation and comet assay. Compared with cells under static, LSS increased PARP-1 expression and PAR formation via MEK/ERK signaling pathway. PARP-1 inhibition increased Sirt1 activity through an increased intracellular nicotinamide adenine dinucleotide (NAD+) level. Moreover, PARP-1 inhibition attenuated LSS-induced iNOS and ICAM-1 upregulation by inhibiting nuclear factor kappa B (NF-κB) nuclear translocation and activity, with a reduced NF-κB phosphorylation. Conclusions: LSS induced oxidative damage and PARP-1 activation via MEK/ERK pathway. PARP-1 inhibition restored Sirt1 activity by increasing NAD+ level and decreased iNOS and ICAM-1 expression by inhibiting NF-κB nuclear translocation and activity as well as NF-κB phosphorylation. PARP-1 played a fundamental role in LSS induced inflammation. Inhibition of PARP-1 might be a mechanism for treatment of inflammation response during atherosclerosis.► We firstly demonstrated the critical role of PARP-1 in low shear stress induced inflammation. ► Low shear stress increased superoxide production and nitrotyrosine expression. ► Low shear stress increased PARP-1 expression and PAR formation. ► PARP-1 can be activated by LSS via ERK dependent pathway. ► PARP-1 inhibition decreased LSS-induced inflammatory cytokines upregulation by preventing NF-κB nuclear translocation.
Keywords: Atherosclerosis; Wall shear stress; Poly(ADP-ribose) polymerase 1; Sirt1; Inflammatory factor; NF-κB;

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) enhances osteoclast differentiation via the up-regulation of Notch1 protein stability by Yun-Hee Choi; Eun-Jung Ann; Ji-Hye Yoon; Jung-Soon Mo; Mi-Yeon Kim; Hee-Sae Park (69-79).
The Notch signaling pathway plays a crucial role in the regulation of cell fate decision, and is also a key regulator of cell differentiation, including bone homeostasis, in a variety of contexts. However, the role of Notch1 signaling in osteoclast differentiation is still controversial. In this study, we show that Receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation is promoted by the Notch1 intracellular domain (Notch1-IC) and Ca2 +/Calmodulin dependent protein kinase IV (CaMKIV) signaling. Notch1-IC protein level was augmented by CaMKIV through escape from ubiquitin dependent protein degradation. In addition, CaMKIV remarkably increased Notch1-IC stability, and the kinase activity of CaMKIV was essential for facilitating Notch1 signaling. CaMKIV directly interacted with Notch1-IC and phosphorylates Notch1-IC, thereby decreasing proteasomal protein degradation through F-box and WD repeat domain-containing 7 (Fbw7). We also found that Notch1-IC prevented inhibition of osteoclast differentiation by KN-93 but not the phosphorylation deficient form of Notch1-IC. These results suggest that phosphorylated Notch1-IC by CaMKIV increases Notch1-IC stability, which enhances osteoclast differentiation.► CaMKIV and Notch1 are required for RANKL-induced osteoclast differentiation. ► Notch1-IC interacts directly with CaMKIV in intact cells. ► CaMKIV up-regulates the Notch1-IC protein stability. ► CaMKIV positively regulates Notch1 signaling through an E3 ligase, Fbw7 ► CaMKIV-mediated phosphorylation of Notch1-IC inhibits the degradation of Notch1-IC.
Keywords: CaMKIV; Notch1-IC; Osteoclastogenesis; Phosphorylation; Fbw7; Ubiquitylation;

Glutathionylation of UCP2 sensitizes drug resistant leukemia cells to chemotherapeutics by Aline Pfefferle; Ryan J. Mailloux; Cyril Nii-Klu Adjeitey; Mary-Ellen Harper (80-89).
Uncoupling protein-2 (UCP2) is used by cells to control reactive oxygen species (ROS) production by mitochondria. This ability depends on the glutathionylation state of UCP2. UCP2 is often overexpressed in drug resistant cancer cells and therein controls cell ROS levels and limits drug toxicity. With our recent observation that glutathionylation deactivates proton leak through UCP2, we decided to test if diamide, a glutathionylation catalyst, can sensitize drug resistant cells to chemotherapeutic agents. Using drug sensitive HL-60 cells and the drug resistant HL-60 subline, Mx2, we show that chemical induction of glutathionylation selectively deactivates proton leak through UCP2 in Mx2 cells. Chemical glutathionylation of UCP2 disables chemoresistance in the Mx2 cells. Exposure to 200 μM diamide led to a significant increase in Mx2 cell death that was augmented when cells were exposed to either menadione or the anthracycline doxorubicin. Diamide also sensitized Mx2 cells to a number of other chemotherapeutics. Proton leak through UCP2 contributed significantly to the energetics of the Mx2 cells. Knockdown of UCP2 led to a significant decrease in both resting and state 4 (i.e., proton leak-dependent) respiration (~ 43% and 62%, respectively) in Mx2 cells. Similarly diamide inhibited proton leak-dependent respiration by ~ 64%. In contrast, diamide had very little effect on proton leak in HL-60 cells. Collectively, our observations indicate that manipulation of UCP2 glutathionylation status can serve as a therapeutic strategy for cancer treatment.► UCP2 is more deglutathionylated and active in drug resistant cancer cells. ► Induction of glutathionylation with diamide inhibits proton leak through UCP2. ► Glutathionylation of UCP2 increases sensitivity to chemotherapeutics. ► Effects of diamide on cancer cells are specific to drug resistant cells. ► Manipulation of cell UCP2 glutathionylation may serve as new method to treat cancer.
Keywords: Uncoupling protein-2; Glutathionylation; Drug resistance; Proton leak; Chemotherapy; Mitochondria;

Inhibition of ERK activation enhances the repair of double-stranded breaks via non-homologous end joining by increasing DNA-PKcs activation by Fengxiang Wei; Judy Yan; Damu Tang; Xiaozeng Lin; Lizhi He; Yanyun Xie; Lijian Tao; Shaojuan Wang (90-100).
Non-homologous end joining (NHEJ) is one of the major pathways that repairs double-stranded DNA breaks (DSBs). Activation of DNA-PK is required for NHEJ. However, the mechanism leading to DNA-PKcs activation remains incompletely understood. We provide evidence here that the MEK–ERK pathway plays a role in DNA-PKcs-mediated NHEJ. In comparison to the vehicle control (DMSO), etoposide (ETOP)-induced DSBs in MCF7 cells were more rapidly repaired in the presence of U0126, a specific MEK inhibitor, based on the reduction of γH2AX and tail moments. Additionally, U0126 increased reactivation of luciferase activity, which resulted from the repair of restriction enzyme-cleaved DSBs. Furthermore, while inhibition of ERK activation using the dominant-negative MEK1K97M accelerated the repair of DSBs, enforcing ERK activation with the constitutively active MEK1Q56P reduced DSB repair. In line with MEK activating ERK1 and ERK2 kinases, knockdown of either ERK1 or ERK2 increased DSB repair. Consistent with the activation of DNA-PKcs being required for NHEJ, we demonstrated that inhibition of ERK activation using U0126, MEK1K97M, and knockdown of ERK1 or ERK2 enhanced ETOP-induced activation of DNA-PKcs. Conversely, enforcing ERK activation by MEK1Q56P reduced ETOP-initiated DNA-PKcs activation. Taken together, we demonstrate that ERK reduces NHEJ-mediated repair of DSBs via attenuation of DNA-PKcs activation.► Enhancing ERK activation reduces repair of ETOP-induced DSBs and DNA-PK activation. ► Inhibition of ERK activation sensitizes these processes. ► ERK reduces DNA-PK-mediated NHEJ. ► ERK facilitates cells to choose whether to repair DSBs via NHEJ.
Keywords: ERK1/2 kinases; DNA damage repair; Non-homologous end joining (NHEJ); DNA damage response (DDR); Ku; DNA-PKcs;

RAGE in tissue homeostasis, repair and regeneration by Guglielmo Sorci; Francesca Riuzzi; Ileana Giambanco; Rosario Donato (101-109).
RAGE (receptor for advanced glycation end-products) is a multiligand receptor of the immunoglobulin superfamily involved in inflammation, diabetes, atherosclerosis, nephropathy, neurodegeneration, and cancer. Advanced glycation end-products, high mobility group box-1 (amphoterin), β-amyloid fibrils, certain S100 proteins, and DNA and RNA are RAGE ligands. Upon RAGE ligation, adaptor proteins (i.e., diaphanous-1, TIRAP, MyD88 and/or other as yet unidentified adaptors) associate with RAGE cytoplasmic domain resulting in signaling. However, RAGE activation may not be restricted to pathological statuses, the receptor being involved in tissue homeostasis and regeneration/repair upon acute injury, and in resolution of inflammation. RAGE effects are strongly dependent on the cell type and the context, which may condition therapeutic strategies aimed at reducing RAGE signaling.► RAGE is a multiligand receptor involved in inflammation, diabetes, atherosclerosis, neurodegeneration and cancer. ► RAGE transduces effects of AGEs, HMGB1, β-amyloid fibrils, certain S100 proteins, and DNA and RNA in several cell types. ► Adaptor proteins associate with RAGE’s cytoplasmic domain resulting in signaling. ► However, RAGE is also involved in tissue homeostasis and regeneration upon acute injury, and in resolution of inflammation.
Keywords: RAGE; Inflammation; Cancer; Tissue regeneration/repair; HMGB1; S100 protein;

Phosphorylation and nitration of tyrosine residues affect functional properties of Synaptophysin and Dynamin I, two proteins involved in exo-endocytosis of synaptic vesicles by Cinzia Mallozzi; Carmen D'Amore; Serena Camerini; Gianfranco Macchia; Marco Crescenzi; Tamara Corinna Petrucci; Anna Maria Michela Di Stasi (110-121).
Phosphorylation and nitration of protein tyrosine residues are thought to play a role in signaling pathways at the nerve terminal and to affect functional properties of proteins involved in the synaptic vesicle (SV) exo-endocytotic cycle. We previously demonstrated that the tyrosine residues in the C-terminal domain of the SV protein Synaptophysin (SYP) are targets of peroxynitrite (PN). Here, we have characterized the association between SYP and c-src tyrosine kinase demonstrating that phosphorylation of Tyr273 in the C-terminal domain of SYP is crucial in mediating SYP binding to and activation of c-src. SYP forms a complex with Dynamin I (DynI), a GTPase required for SV endocytosis, which may be regulated by tyrosine phosphorylation of SYP. We here report that, in rat brain synaptosomes treated with PN, the formation of SYP/DynI complex was impaired. Noteworthy, we found that DynI was also modified by PN. DynI tyrosine phosphorylation was down-regulated in a dose-dependent manner, while DynI tyrosine nitration increased. Using mass spectrometry analysis, we identified Tyr354 as one nitration site in DynI. In addition, we tested DynI self-assembly and GTPase activity, which are enhanced by c-src-dependent tyrosine phosphorylation of DynI, and found that both were inhibited by PN. Our results suggest that the site-specific tyrosine residue modifications may modulate the association properties of SV proteins and serve as a regulator of DynI function via control of self-assembly, thus influencing the physiology of the exo-endocytotic cycle.► Peroxynitrite affects functions of synaptic proteins involved in exo-endocytosis. ► Phosphorylated Tyr273 in C-terminal tail of Synaptophysin binds to and activates c-src. ► Formation of Synaptophysin/Dynamin I complex is impaired by peroxynitrite. ► Peroxynitrite inhibits Dynamin I GTPase activity and self-assembly. ► We identified in Dynamin I one nitration site at Tyr354 using MS/MS analysis.
Keywords: Peroxynitrite; Phosphotyrosine; Nitrotyrosine; Src tyrosine kinase; Redox signaling; Synaptic vesicle;

Protein tyrosine kinase regulation by ubiquitination: Critical roles of Cbl-family ubiquitin ligases by Bhopal Mohapatra; Gulzar Ahmad; Scott Nadeau; Neha Zutshi; Wei An; Sarah Scheffe; Lin Dong; Dan Feng; Benjamin Goetz; Priyanka Arya; Tameka A. Bailey; Nicholas Palermo; Gloria E.O. Borgstahl; Amarnath Natarajan; Srikumar M. Raja; Mayumi Naramura; Vimla Band; Hamid Band (122-139).
Protein tyrosine kinases (PTKs) coordinate a broad spectrum of cellular responses to extracellular stimuli and cell–cell interactions during development, tissue homeostasis, and responses to environmental challenges. Thus, an understanding of the regulatory mechanisms that ensure physiological PTK function and potential aberrations of these regulatory processes during diseases such as cancer are of broad interest in biology and medicine. Aside from the expected role of phospho-tyrosine phosphatases, recent studies have revealed a critical role of covalent modification of activated PTKs with ubiquitin as a critical mechanism of their negative regulation. Members of the Cbl protein family (Cbl, Cbl-b and Cbl-c in mammals) have emerged as dominant “activated PTK-selective” ubiquitin ligases. Structural, biochemical and cell biological studies have established that Cbl protein-dependent ubiquitination targets activated PTKs for degradation either by facilitating their endocytic sorting into lysosomes or by promoting their proteasomal degradation. This mechanism also targets PTK signaling intermediates that become associated with Cbl proteins in a PTK activation-dependent manner. Cellular and animal studies have established that the relatively broadly expressed mammalian Cbl family members Cbl and Cbl-b play key physiological roles, including their critical functions to prevent the transition of normal immune responses into autoimmune disease and as tumor suppressors; the latter function has received validation from human studies linking mutations in Cbl to human leukemia. These newer insights together with embryonic lethality seen in mice with a combined deletion of Cbl and Cbl-b genes suggest an unappreciated role of the Cbl family proteins, and by implication the ubiquitin-dependent control of activated PTKs, in stem/progenitor cell maintenance. Future studies of existing and emerging animal models and their various cell lineages should help test the broader implications of the evolutionarily-conserved Cbl family protein-mediated, ubiquitin-dependent, negative regulation of activated PTKs in physiology and disease.► Cbl-family ubiquitin ligases function as negative regulators of PTK signaling ► Recruitment and activation mechanisms ensure Cbl selectivity for activated PTKs ► Cbl-dependent ubiquitination regulates PTK degradation in lysosome and proteasome ► Genetic studies demonstrate essential roles of Cbl proteins in physiological pathways ► Mutational inactivation of Cbl is causally linked to human leukemia
Keywords: Cbl; E3 ubiquitin ligase; Tyrosine kinase binding domain; Ubiquitination; Signal transduction; Animal model;

Ascorbic acid rescues cardiomyocyte development in Fgfr1 −/− murine embryonic stem cells by Elisabetta Crescini; Laura Gualandi; Daniela Uberti; Chiara Prandelli; Marco Presta; Patrizia Dell'Era (140-147).
Fibroblast growth factor receptor 1 (Fgfr1) gene knockout impairs cardiomyocyte differentiation in murine embryonic stem cells (mESC). Here, various chemical compounds able to enhance cardiomyocyte differentiation in mESC [including dimethylsulfoxide, ascorbic acid (vitC), free radicals and reactive oxygen species] were tested for their ability to rescue the cardiomyogenic potential of Fgfr1 −/− mESC. Among them, only the reduced form of vitC, l-ascorbic acid, was able to recover beating cell differentiation in Fgfr1 −/− mESC. The appearance of contracting cells was paralleled by the expression of early and late cardiac gene markers, thus suggesting their identity as cardiomyocytes. In the attempt to elucidate the mechanism of action of vitC on Fgfr1 −/− mESC, we analyzed several parameters related to the intracellular redox state, such as reactive oxygen species content, Nox4 expression, and superoxide dismutase activity. The results did not show any relationship between the antioxidant capacity of vitC and cardiomyocyte differentiation in Fgfr1 −/− mESC. No correlation was found also for the ability of vitC to modulate the expression of pluripotency genes. Then, we tested the hypothesis that vitC was acting as a prolyl hydroxylase cofactor by maintaining iron in a reduced state. We first analyze hypoxia inducible factor (HIF)-1α mRNA and protein levels that were found to be slightly upregulated in Fgfr1 −/− cells. We treated mESC with Fe2 + or the HIF inhibitor CAY10585 during the first phases of the differentiation process and, similar to vitC, the two compounds were able to rescue cardiomyocyte formation in Fgfr1 −/− mESC, thus implicating HIF-1α modulation in Fgfr1-dependent cardiomyogenesis.► Treatment with vitamin (vit) C rescues cardiomyocyte differentiation in Fgfr1 −/− mESC. ► The phenotype rescue of vitC is not due to its antioxidant effect. ► The phenotype rescue of vitC is not due to modulation of pluripotency genes. ► FeCl2 rescues cardiomyocyte differentiation in Fgfr1 −/− mESC. ► The HIF-1α inhibitor CAY10585 rescues cardiomyocyte differentiation in Fgfr1 −/− mESC.
Keywords: FGFR1; Cardiomyocyte differentiation; Embryonic stem cell; Ascorbic acid;

Mitochondrial dynamics and physiology by Heidi McBride; Luca Scorrano (148-149).

Recent advances into the understanding of mitochondrial fission by Kirstin Elgass; Julian Pakay; Michael T. Ryan; Catherine S. Palmer (150-161).
Mitochondria exist as a highly dynamic tubular network, and their morphology is governed by the delicate balance between frequent fusion and fission events, as well as by interactions with the cytoskeleton. Alterations in mitochondrial morphology are associated with changes in metabolism, cell development and cell death, whilst several human pathologies have been associated with perturbations in the cellular machinery that coordinate these processes. Mitochondrial fission also contributes to ensuring the proper distribution of mitochondria in response to the energetic requirements of the cell. The master mediator of fission is Dynamin related protein 1 (Drp1), which polymerises and constricts mitochondria to facilitate organelle division. The activity of Drp1 at the mitochondrial outer membrane is regulated through post-translational modifications and interactions with mitochondrial receptor and accessory proteins. This review will concentrate on recent advances made in delineating the mechanism of mitochondrial fission, and will highlight the importance of mitochondrial fission in health and disease. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.► Mitochondrial morphology is governed by the balanced fusion and fission events. ► Dynamin related protein 1 (Drp1) is the master mediator of mitochondrial fission. ► Drp1 is regulated by receptor-like proteins and post-translational modifications. ► Mitochondrial fission is observed in numerous diseases and during cell death.
Keywords: Mitochondrion; Fission; Dynamin related protein 1; Mff; MiD49; MiD51;

Mechanistic perspective of mitochondrial fusion: Tubulation vs. fragmentation by Mafalda Escobar-Henriques; Fabian Anton (162-175).
Mitochondrial fusion is a fundamental process driven by dynamin related GTPase proteins (DRPs), in contrast to the general SNARE-dependence of most cellular fusion events. The DRPs Mfn1/Mfn2/Fzo1 and OPA1/Mgm1 are the key effectors for fusion of the mitochondrial outer and inner membranes, respectively. In order to promote fusion, these two DRPs require post-translational modifications and proteolysis. OPA1/Mgm1 undergoes partial proteolytic processing, which results in a combination between short and long isoforms. In turn, ubiquitylation of mitofusins, after oligomerization and GTP hydrolysis, promotes and positively regulates mitochondrial fusion. In contrast, under conditions of mitochondrial dysfunction, negative regulation by proteolysis on these DRPs results in mitochondrial fragmentation. This occurs by complete processing of OPA1 and via ubiquitylation and degradation of mitofusins. Mitochondrial fragmentation contributes to the elimination of damaged mitochondria by mitophagy, and may play a protective role against Parkinson's disease. Moreover, a link of Mfn2 to Alzheimer's disease is emerging and mutations in Mfn2 or OPA1 cause Charcot–Marie–Tooth type 2A neuropathy or autosomal-dominant optic atrophy. Here, we summarize our current understanding on the molecular mechanisms promoting or inhibiting fusion of mitochondrial membranes, which is essential for cellular survival and disease control. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.► Mitochondrial fusion key mediators are conserved DRPs called mitofusins and OPA1/Mgm1. ► Mammalian cell-free mitochondrial fusion assays can now be used in disease models. ► Recent data allow to propose new mechanisms for both OM and IM fusion. ► Tubulation and fragmentation: opposite processes requiring similar proteolytic events.
Keywords: Mitochondria; Dynamics; Fusion; DRP; Mitofusin/Mfn1/Mfn2/Fzo1; OPA1/Mgm1;

The dynamin GTPase OPA1: More than mitochondria? by Pascale Belenguer; Luca Pellegrini (176-183).
The studies addressing the molecular mechanisms governing mitochondrial fusion and fission have brought to light a small group of dynamin-like GTPases (Guanosine-Triphosphate hydrolase) as central regulators of mitochondrial morphology and cristae remodeling, apoptosis, calcium signaling, and metabolism. One of them is the mammalian OPA1 (Optic atrophy 1) protein, which resides inside the mitochondrion anchored to the inner membrane and, in a cleaved form, is associated to oligomeric complexes, in the intermembrane space of the organelle. Here, we review the studies that have made OPA1 emerge as the best understood regulator of mitochondrial inner membrane fusion and cristae remodeling. Further, we re-examine the findings behind the recent claim that OPA1 mediates adrenergic control of lipolysis by functioning as a cytosolic A-kinase anchoring protein (AKAP), on the hemimembrane that envelops the lipid droplet. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.► OPA1 is the best characterized mitochondrial fusion protein. ► Its structure and function are evolutionarily conserved. ► A complex proteolytic cascade governs its role in mitochondria dynamics, apoptosis, and quality control. ► The recently proposed localization of OPA1 on the lipid droplets, and function as an AKAP, remains controversial.
Keywords: OPA1; Mitochondria; Membrane dynamics; Apoptosis; mtDNA; Dominant optic atrophy;

The meaning of mitochondrial movement to a neuron's life by Jonathan R. Lovas; Xinnan Wang (184-194).
Cells precisely regulate mitochondrial movement in order to balance energy needs and avoid cell death. Neurons are particularly susceptible to disturbance of mitochondrial motility and distribution due to their highly extended structures and specialized function. Regulation of mitochondrial motility plays a vital role in neuronal health and death. Here we review the current understanding of regulatory mechanisms that govern neuronal mitochondrial transport and probe their implication in health and disease. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.► Transport machineries that move mitochondria along microtubules and actin cytoskeleton ► Cellular signaling pathways that regulate mitochondrial movement in neurons ► Implication of the regulation of mitochondrial motility in health and disease and future directions
Keywords: Mitochondrial movement; Neuron; Neurodegeneration; Ca++; Parkinson's; Mitophagy;

Proteolytic control of mitochondrial function and morphogenesis by Ruchika Anand; Thomas Langer; Michael James Baker (195-204).
Mitochondrial proteostasis depends on a hierarchical system of tightly controlled quality surveillance mechanisms. Proteases within mitochondria take center stage in this network. They eliminate misfolded and damaged proteins and ensure the biogenesis and morphogenesis of mitochondria by processing or degrading short-lived regulatory proteins. Mitochondrial gene expression, the mitochondrial phospholipid metabolism and the fusion of mitochondrial membranes are under proteolytic control. Furthermore, in response to stress and mitochondrial dysfunction, proteolysis inhibits fusion and facilitates mitophagy and apoptosis. Defining these versatile activities of mitochondrial proteases will be pivotal for understanding the pathogenesis of various neurodegenerative disorders associated with defective mitochondria-associated proteolysis. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.► A hierarchical system of quality control mechanisms maintains mitochondrial integrity. ► Mitochondrial proteases act as quality control and regulatory enzymes. ► Mitochondrial dynamics and biogenesis is under proteolytic control. ► Mitophagy is regulated by mitochondrial proteases and the UPS.
Keywords: Mitochondria; Quality control; Mitophagy; Mitochondrial dynamics; AAA protease; Lon protease;

Mitochondrial morphology in mitophagy and macroautophagy by Ligia C. Gomes; Luca Scorrano (205-212).
Mitochondria are critical organelles in energy conversion, metabolism and amplification of signalling. They are however also major sources of reactive oxygen species and when dysfunctional they consume cytosolic ATP. Maintenance of a cohort of healthy mitochondria is therefore crucial for the overall cell fitness. Superfluous or damaged organelles are mainly degraded by mitophagy, a selective process of autophagy. In response to the triggers of mitophagy, mitochondria fragment: this morphological change accompanies the exposure of “eat-me” signals, resulting in the engulfment of the organelle by the autophagosomes. Conversely, during macroautophagy mitochondria fuse to be spared from degradation and to sustain ATP production in times of limited nutrient availability. Thus, mitochondrial shape defines different types of autophagy, highlighting the interplay between morphology of the organelle and complex cellular responses. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.► Mitochondria are target of specific and unselective autophagy. ► Different signals target mitochondria to autophagy in physiological and pathological conditions. ► Morphology of the organelle differs during mitophagy and autophagy, contributing to classify the two processes.
Keywords: Autophagy; Mitophagy; Mitochondrion; Mitochondrial fusion; Mitochondrial fission;

More than a billion years ago, bacterial precursors of mitochondria became endosymbionts in what we call eukaryotic cells today. The true significance of the word “endosymbiont” has only become clear to cell biologists with the discovery that the endoplasmic reticulum (ER) superorganelle dedicates a special domain for the metabolic interaction with mitochondria. This domain, identified in all eukaryotic cell systems from yeast to man and called the mitochondria-associated membrane (MAM), has a distinct proteome, specific tethers on the cytosolic face and regulatory proteins in the ER lumen of the ER. The MAM has distinct biochemical properties and appears as ER tubules closely apposed to mitochondria on electron micrographs. The functions of the MAM range from lipid metabolism and calcium signaling to inflammasome formation. Consistent with these functions, the MAM is enriched in lipid metabolism enzymes and calcium handling proteins. During cellular stress situations, like an altered cellular redox state, the MAM alters its set of regulatory proteins and thus alters MAM functions. Notably, this set prominently comprises ER chaperones and oxidoreductases that connect protein synthesis and folding inside the ER to mitochondrial metabolism. Moreover, ER membranes associated with mitochondria also accommodate parts of the machinery that determines mitochondrial membrane dynamics and connect mitochondria to the cytoskeleton. Together, these exciting findings demonstrate that the physiological interactions between the ER and mitochondria are so bilateral that we are tempted to compare their relationship to the one of a married couple: distinct, but inseparable and certainly dependent on each other. In this paradigm, the MAM stands for the intracellular location where the two organelles tie the knot. Resembling “real life”, the happy marriage between the two organelles prevents the onset of diseases that are characterized by disrupted metabolism and decreased lifespan, including neurodegeneration and cancer. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.► This review describes the discovery of the mitochondria-associated membrane (MAM). ► The MAM accommodates lipid metabolism, but also ER–mitochondria calcium signaling. ► Oxidoreductases (e.g. Ero1α) and chaperones (e.g. calnexin) regulate MAM signaling. ► Targeting to the MAM depends on redox, phosphorylation or palmitoylation. ► MAM tethers play important roles in neurodegenerative diseases.
Keywords: Endoplasmic reticulum (ER); Mitochondria; Mitochondria-associated membrane (MAM); Lipid metabolism; Calcium signaling;

Mitochondrial-mediated antiviral immunity by Takumi Koshiba (225-232).
Mitochondria, cellular powerhouses of eukaryotes, are known to act as central hubs for multiple signal transductions. Recent research reveals that mitochondria are involved in cellular innate antiviral immunity in vertebrates, particularly mammals. Mitochondrial-mediated antiviral immunity depends on the activation of the retinoic acid-inducible gene I (RIG-I)-like receptors signal transduction pathway and on the participation of a mitochondrial outer membrane adaptor protein, called the “mitochondrial antiviral signaling (MAVS)”. In this review, we discuss unexpected discoveries that are revealing how the organelles contribute to the innate immune response against RNA viruses. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.► Innate immunity is an essential and ubiquitous system that defends organisms from infectious pathogens. ► Mitochondria play a fundamental role in antiviral immunity in mammals. ► A mitochondrial outer membrane protein, MAVS, plays an important role in this process.
Keywords: Antiviral innate immunity; MAVS; Mitochondrial dynamics; RLR pathway; Signal transduction;

Mitochondrial dynamics in heart disease by Gerald W. Dorn (233-241).
Mitochondrial fission and fusion have been observed, and their importance revealed, in almost every tissue and cell type except adult cardiac myocytes. As each human heart is uniquely dependent upon mitochondria to generate massive amounts of ATP that fuel its approximately 38 million contractions per year, it seems odd that cardiac myocytes are the sole exception to the general rule that mitochondrial dynamism is important to function. Here, I briefly review the mechanisms for mitochondrial fusion and fission and examine current data that dispel the previous notion that mitochondrial fusion is dispensable in the heart. Rare and generally overlooked examples of cardiomyopathies linked either to naturally-occurring mutations or to experimentally-induced mutagenesis of mitochondrial fusion/fission genes are described. New findings from genetically targeted Drosophila and mouse models wherein mitochondrial fusion deficiency has specifically been induced in cardiac myocytes are discussed. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.► Mitochondrial fusion/fission has not been observed in normal adult cardiomyocytes (84). ► Genetic inhibition of mitochondrial fusion causes fragmentation and heart failure (84). ► Thus, mitochondrial fusion/fission is essential to normal cardiac health (75). ► Mitochondrial fission is associated with programmed cardiomyocyte death (74). ► Inhibiting mitochondrial fission may protect against cardiac injury (71).
Keywords: Mitochondrial fusion; Mitochondrial fission; Cardiomyopathy; Drosophila; Genetic mouse model; Human mutation;