BBA - Molecular Cell Research (v.1793, #8)

Apoptin, a tumor-selective killer by Marek Los; Soumya Panigrahi; Iran Rashedi; Sanat Mandal; Joerg Stetefeld; Frank Essmann; Klaus Schulze-Osthoff (1335-1342).
Apoptin, a small protein from chicken anemia virus, has attracted great attention, because it specifically kills tumor cells while leaving normal cells unharmed. The subcellular localization of apoptin appears to be crucial for this tumor-selective activity. In normal cells, apoptin resides in the cytoplasm, whereas in cancerous cells it translocates into the nucleus. The nuclear translocation of apoptin is largely controlled by its phosphorylation. In tumor cells, apoptin causes the nuclear accumulation of survival kinases including Akt and is phosphorylated by CDK2. Thereby, apoptin redirects survival signals into cell death responses. Apoptin also binds as a multimeric complex to DNA and interacts with several nuclear targets, such as the anaphase-promoting complex, resulting in a G2/M phase arrest. The proapoptotic signal of apoptin is then transduced from the nucleus to cytoplasm by Nur77, which triggers a p53-independent mitochondrial death pathway. In this review, we summarize recent discoveries of apoptin's mechanism of action that might provide intriguing insights for the development of novel tumor-selective anticancer drugs.
Keywords: Apoptin; Apoptosis; CDK2; Nur77; Tumor;

HSPB7 is a SC35 speckle resident small heat shock protein by Michel J. Vos; Bart Kanon; Harm H. Kampinga (1343-1353).
Background: The HSPB family is one of the more diverse families within the group of HSP families. Some members have chaperone-like activities and/or play a role in cytoskeletal stabilization. Some members also show a dynamic, stress-induced translocation to SC35 splicing speckles. If and how these features are interrelated and if they are shared by all members are yet unknown. Methods: Tissue expression data and interaction and co-regulated gene expression data of the human HSPB members was analyzed using bioinformatics. Using a gene expression library, sub-cellular distribution of the diverse members was analyzed by confocal microscopy. Chaperone activity was measured using a cellular luciferase refolding assay. Results: Online databases did not accurately predict the sub-cellular distribution of all the HSPB members. A novel and non-predicted finding was that HSPB7 constitutively localized to SC35 splicing speckles, driven by its N-terminus. Unlike HSPB1 and HSPB5, that chaperoned heat unfolded substrates and kept them folding competent, HSPB7 did not support refolding. Conclusion: Our data suggest a non-chaperone-like role of HSPB7 at SC35 speckles. General significance: The functional divergence between HSPB members seems larger than previously expected and also includes non-canonical members lacking classical chaperone-like functions.
Keywords: HSPB7; CVHSP; HSPB; Small heat shock protein; SC35;

Cyclooxygenase-2 functionally inactivates p53 through a physical interaction with p53 by Eun Mi Choi; So Ra Kim; Eun Jeong Lee; Jeong A. Han (1354-1365).
Cyclooxygenase-2 (COX-2), an endoplasmic reticulum-resident protein, has been known to promote tumorigenesis, but the exact mechanisms involved have not been identified. We have previously reported that COX-2 physically interacts with the tumor suppressor p53 and regulates its function. However, it remains to be elucidated how COX-2 can interact with p53 residing in different compartments and whether their interaction is involved in the regulation of p53 function. We here demonstrated that upon genotoxic stress, COX-2 and p53 accumulate in the nucleus, where they physically interact with one another. We also showed that an amino-terminal region (amino acids 1–126) of COX-2 interacts with the DNA-binding domain of p53. The p53-interacting region was critical for COX-2-mediated inhibition of p53 DNA-binding and transcriptional activity as well as p53- and genotoxic stress-induced apoptosis. In addition, an active site mutant of COX-2 (S516Q) as well as wild-type COX-2 potently inhibited p53 transcriptional activity and genotoxic stress-induced apoptosis. These results suggest that COX-2 principally inhibits p53 function through a catalytic activity-independent mechanism and that COX-2 inhibits p53 function through a physical interaction with p53 in the nucleus. These findings provide novel insight into the action mechanisms of COX-2 and strongly suggest that the functional inactivation of p53 by COX-2 can be one of the mechanisms by which COX-2 promotes tumorigenesis.
Keywords: Apoptosis; Cyclooxygenase-2; Interaction; Nucleus; p53; Transactivation;

HIV-1 gp120 primes lymphocytes for opioid-induced, β-arrestin 2-dependent apoptosis by Jonathan Moorman; Yi Zhang; Bindong Liu; Gene LeSage; Yangchao Chen; Charles Stuart; Deborah Prayther; Deling Yin (1366-1371).
The mechanisms by which opioids affect progression of human immunodeficiency virus type 1 (HIV-1) infection are not well-defined. HIV-1 gp120 is important in the apoptotic death of uninfected, bystander T cells. In this study, we show that co-treatment of human peripheral blood mononuclear cells (PBMC) with HIV-1 gp120/morphine synergistically induces apoptosis in PBMC. Co-treatment of murine splenocytes from μ opiate receptor knockout mice with gp120/morphine resulted in decreased apoptosis when compared to splenocytes from wild type mice. Co-treatment of human PBMC or murine splenocytes with gp120/morphine led to decreased expression of β-arrestin 2, a protein required for opioid-mediated signaling. The role of β-arrestin 2 was confirmed in Jurkat lymphocytes, in which 1) over-expression of β-arrestin 2 inhibited gp120/morphine-induced apoptosis and 2) RNA interference of β-arrestin 2 expression enhanced gp120/morphine-induced apoptosis. These data suggest a novel mechanism by which HIV-1 gp120 and opioids induce lymphocyte cell death.
Keywords: HIV; Opioid; β-arrestin; gp120; Drug use; Lymphocyte; Apoptosis;

Insulin induced phosphorylation of prohibitin at tyrosine114 recruits Shp1 by Sudharsana R. Ande; Yuanyuan Gu; B.L.G. Nyomba; Suresh Mishra (1372-1378).
Prohibitin (PHB or PHB1) is an evolutionarily conserved ubiquitously expressed multifunctional protein and is present in various cellular compartments. Phosphorylation of PHB has been suggested as one of the potential mechanisms in the regulation of its various functions however exact sites of phosphorylation remain to be determined. To better understand the functional relevance of phosphorylation of PHB, we have explored the potential sites of phosphorylation using combination of approaches including phosphoamino specific immunoblotting, proteolysis, two-dimensional gel electrophoresis, phosphoamino acid analysis and site-directed mutagenesis techniques and report that tyrosine 114 (Tyr114) in PHB is phosphorylated in response to insulin stimulation. In addition, using active insulin receptor (IR) and synthetic biotinylated PHB peptide (PHB107–121) we have shown that IR also phosphorylates Tyr114 in an in vitro kinase assay. Phosphorylation of PHB at Tyr114 was confirmed by immunoblotting using anti-phosphoTyr114 specific antibody. Furthermore, we demonstrate that SH2 domain containing tyrosine phosphatase-1 (Shp1) co-immunoprecipitate with PHB antiserum after insulin induced phosphorylation of PHB. Biotinylated-PHB107–121 peptide phosphorylated at Tyr114 was also able to pull down Shp1 in pull down assays. Non-phosphorylated PHB107–121 peptide, corresponding PHB2121–135 peptide and Tyr114Phe mutant-PHB fail to pull down Shp1. In summary, we have identified Tyr114 in PHB as an important site of phosphorylation and phosphorylation at this residue creates a binding site for Shp1 both in vivo and in vitro.
Keywords: Prohibitin; Insulin; Tyrosine phosphorylation; Phosphotyrosine binding; Protein–protein interaction;

A dual mechanism of cytoprotection afforded by M-LDH in embryonic heart H9C2 cells by Sofija Jovanović; Qingyou Du; Andriy Sukhodub; Aleksandar Jovanović (1379-1386).
Muscle form of lactate dehydrogenase (M-LDH), a minor LDH form in cardiomyocytes, physically interacts with ATP-sensitive K+ (KATP) channel-forming subunits. Here, we have shown that expression of 193gly-M-LDH, an inactive mutant of M-LDH, inhibit regulation of the KATP channels activity by LDH substrates in embryonic rat heart H9C2 cells. In cells expressing 193gly-M-LDH chemical hypoxia has failed to activate KATP channels. The similar results were obtained in H9C2 cells expressing Kir6.2AFA, a mutant form of Kir6.2 with largely decreased K+ conductance. Kir6.2AFA has slightly, but significantly, reduced cellular survival under chemical hypoxia while the deleterious effect of 193gly-M-LDH was significantly more pronounced. The levels of total and subsarcolemmal ATP in H9C2 cells were not affected by Kir6.2AFA, but the expression of 193gly-M-LDH led to lower levels of subsarcolemmal ATP during chemical hypoxia. We conclude that M-LDH regulates both the channel activity and the levels of subsarcolemmal ATP and that both mechanism contribute to the M-LDH-mediated cytoprotection.
Keywords: Lactate dehydrogenase; KATP channel; H9C2 cell; Hypoxia;

Ursodeoxycholic acid (UDCA) has been shown to prevent colon tumorigenesis in animal models and in humans. In vitro work indicates that this bile acid can suppress cell growth and mitogenic signaling suggesting that UDCA may be an anti-proliferative agent. However, the mechanism by which UDCA functions is unclear. Previously we showed that bile acids may alter cellular signaling by acting at the plasma membrane. Here we utilized EGFR as a model membrane receptor and examined the effects that UDCA has on its functioning. We found that UDCA promoted an interaction between EGFR and caveolin-1 and this interaction enhanced UDCA-mediated suppression of MAP kinase activity and cell growth. Importantly, UDCA treatment led to recruitment of the ubiquitin ligase, c-Cbl, to the membrane, ubiquitination of EGFR, and increased receptor degradation. Moreover, suppression of c-Cbl activity abrogated UDCA's growth suppression activities suggesting that receptor ubiquitination plays an important role in UDCA's biological activities. Taken together these results suggest that UDCA may act to suppress cell growth by inhibiting the mitogenic activity of receptor tyrosine kinases such as EGFR through increased receptor degradation.
Keywords: Bile acid; Colon cancer; Chemoprevention; c-Cbl; Endocytosis;