Mdm2 and MdmX RING Domains Play Distinct Roles in the Regulation of p53 Responses: A Comparative Study of Mdm2 and MdmX RING Domains in U2OS Cells

Dysfunction of the tumor suppressor p53 occurs in most human cancers. Mdm2 and MdmX are homologous proteins from the Mdm (Murine Double Minute) protein family, which play a critical role in p53 inactivation and degradation. The two proteins interact with one another via the intrinsic RING (Really Interesting New Gene) domains to achieve the negative regulation of p53. The downregulation of p53 is accomplished by Mdm2-mediated p53 ubiquitination and proteasomal degradation through the ubiquitin proteolytic system and by Mdm2 and MdmX mediated inhibition of p53 transactivation. To investigate the role of the RING domain of Mdm2 and MdmX, an analysis of the distinct functionalities of individual RING domains of the Mdm proteins on p53 regulation was conducted in human osteosarcoma (U2OS) cell line. Mdm2 RING domain was observed mainly localized in the cell nucleus, contrasting the localization of MdmX RING domain in the cytoplasm. Mdm2 RING was found to possess an endogenous E3 ligase activity, whereas MdmX RING did not. Both Mdm2 and MdmX RING domains were able to dimerize with endogenous full-length Mdm2 and MdmX protein and affect their cellular function. The results showed that overexpression of the Mdm2 or MdmX RING domains interfered with the endogenous full-length Mdm2 and MdmX activity and resulted in p53 stabilization and p53 target gene activation. However, both Mdm RING domains showed oncogenic activity in a colony formation assay, suggesting that the Mdm RING domains possess p53-independent oncogenic properties. This study highlights the distinct structural and functional traits of the RING domain of Mdm2 and MdmX and characterized their role in cellular responses through interfering with p53 dependent signaling pathway.

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Calcineurin homologous proteins regulate the membrane localization and activity of sodium/proton exchangers in C. elegans

AJP Cell Physiology ◽

10.1152/ajpcell.00291.2015 ◽

2016 ◽

Vol 310 (3) ◽

pp. C233-C242 ◽

Cited By ~ 4

Author(s):

Erik Allman ◽

Qian Wang ◽

Rachel L. Walker ◽

Molly Austen ◽

Maureen A. Peters ◽

...

Keyword(s):

Genetic Model ◽

Expression Patterns ◽

Critical Role ◽

Model Organism ◽

Loss Of Function ◽

Homologous Proteins ◽

Relative Significance ◽

C Elegans ◽

Fluorescent Tags

Calcineurin B homologous proteins (CHP) are N-myristoylated, EF-hand Ca2+-binding proteins that bind to and regulate Na+/H+ exchangers, which occurs through a variety of mechanisms whose relative significance is incompletely understood. Like mammals, Caenorhabditis elegans has three CHP paralogs, but unlike mammals, worms can survive CHP loss-of-function. However, mutants for the CHP ortholog PBO-1 are unfit, and PBO-1 has been shown to be required for proton signaling by the basolateral Na+/H+ exchanger NHX-7 and for proton-coupled intestinal nutrient uptake by the apical Na+/H+ exchanger NHX-2. Here, we have used this genetic model organism to interrogate PBO-1's mechanism of action. Using fluorescent tags to monitor Na+/H+ exchanger trafficking and localization, we found that loss of either PBO-1 binding or activity caused NHX-7 to accumulate in late endosomes/lysosomes. In contrast, NHX-2 was stabilized at the apical membrane by a nonfunctional PBO-1 protein and was only internalized following its complete loss. Additionally, two pbo-1 paralogs were identified, and their expression patterns were analyzed. One of these contributed to the function of the excretory cell, which acts like a kidney in worms, establishing an alternative model for testing the role of this protein in membrane transporter trafficking and regulation. These results lead us to conclude that the role of CHP in Na+/H+ exchanger regulation differs between apical and basolateral transporters. This further emphasizes the importance of proper targeting of Na+/H+ exchangers and the critical role of CHP family proteins in this process.

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Critical Role of Hypoxia Sensor - HIF-1α in VEGF Gene Activation. Implications for Angiogenesis and Tissue Injury Healing

Current Medicinal Chemistry ◽

10.2174/092986712803413944 ◽

2012 ◽

Vol 19 (1) ◽

pp. 90-97 ◽

Cited By ~ 154

Author(s):

A. Ahluwalia ◽

A. S. Tarnawski

Keyword(s):

Tissue Injury ◽

Critical Role ◽

Gene Activation ◽

Vegf Gene

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Evidence for a critical role for the cytoplasmic region of the interleukin 2 (IL-2) receptor gamma chain in IL-2, IL-4, and IL-7 signalling

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5433-5440.1994 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5433-5440

Author(s):

A Kawahara ◽

Y Minami ◽

T Taniguchi

Keyword(s):

Cellular Proliferation ◽

Critical Role ◽

Gene Activation ◽

Interleukin 2 ◽

Selective Inhibition ◽

Cell Interior ◽

Gamma Chain ◽

Cytoplasmic Region ◽

Hematopoietic Cell Line

The high-affinity interleukin 2 receptor (IL-2R) consists of at least three distinct subunits: the IL-2R alpha chain (IL-2R alpha), beta chain (IL-2R beta), and gamma chain (IL-2R gamma). It has been shown that the cytoplasmic region of IL-2R beta, but not of IL-2R alpha, is essential for IL-2 signalling to the cell interior. In the present study, we examined the functional role of the IL-2R gamma cytoplasmic region in the IL-3-dependent mouse hematopoietic cell line BAF-B03, which expresses the endogenous IL-2R alpha and IL-2R gamma, or its subline F7, which additionally expresses human IL-2R beta cDNA. We show that overexpression of a mutant IL-2R gamma, lacking all but 7 amino acids of its cytoplasmic region, results in the selective inhibition of IL-2-induced c-fos gene activation and cellular proliferation in F7 cells. When two chimeric receptor molecules in which the cytoplasmic regions of IL-2R beta and IL-2R gamma had been swapped with each other (IL-2R beta/gamma and IL-2R gamma/beta) were coexpressed in BAF-B03, the cells responded to IL-2. These results indicate the critical importance of the IL-2-induced functional cooperation of the two cytoplasmic regions. Finally, we provide evidence that the IL-2R gamma cytoplasmic region is also critical for the IL-4 and IL-7-induced growth signal transduction in BAF-B03.

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Evidence for a critical role for the cytoplasmic region of the interleukin 2 (IL-2) receptor gamma chain in IL-2, IL-4, and IL-7 signalling.

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5433 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5433-5440 ◽

Cited By ~ 46

Author(s):

A Kawahara ◽

Y Minami ◽

T Taniguchi

Keyword(s):

Cellular Proliferation ◽

Critical Role ◽

Gene Activation ◽

Interleukin 2 ◽

Selective Inhibition ◽

Cell Interior ◽

Gamma Chain ◽

Cytoplasmic Region ◽

Hematopoietic Cell Line

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Coordinate regulation of 11 beta-HSD and Ke 6 genes in cpk mouse: implications for steroid metabolic defect in PKD

AJP Renal Physiology ◽

10.1152/ajprenal.1994.267.5.f791 ◽

1994 ◽

Vol 267 (5) ◽

pp. F791-F797

Author(s):

N. Aziz ◽

M. M. Maxwell ◽

B. M. Brenner

Keyword(s):

Critical Role ◽

Renal Cysts ◽

Polycystic Kidney ◽

Coordinate Regulation ◽

Histocompatibility Complex ◽

Dehydrogenase Enzyme ◽

Significant Homology ◽

New Gene ◽

Cpk Mouse

Polycystic kidney disease (PKD) is characterized by multiple renal cysts, which ultimately result in renal failure. We have reported the identification of a new gene, Ke 6, within the major histocompatibility complex, which is downregulated in two different mouse models of heritable PKD (N. Aziz, M. Maxwell, B. St.-Jacques, and B.M. Brenner. Mol. Cell. Biol. 13: 1847-1853, 1993). The Ke 6 gene gives rise to two transcripts, Ke 6a and Ke 6b. Ke 6a protein has significant homology to several mammalian and bacterial steroid dehydrogenases. The homology of Ke 6a protein to specific functional domains of the human and rat 11 beta-hydroxysteroid dehydrogenase enzyme (11 beta-HSD), which inactivates glucocorticoids, is substantial. We report here that the Ke 6 gene and the 11 beta-HSD gene are regulated in the same aberrant pattern in the cpk mouse. The strong evidence for a critical role of steroids in cystogenesis leads us to propose that a possible elevation of intrahepatic and intrarenal steroid levels occurs in the cpk mouse as a result of repression of steroid metabolic enzymes, which ultimately leads to development of cysts.

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Necdin, a p53 target gene, regulates the quiescence and response to genotoxic stress of hematopoietic stem/progenitor cells

Blood ◽

10.1182/blood-2011-11-393983 ◽

2012 ◽

Vol 120 (8) ◽

pp. 1601-1612 ◽

Cited By ~ 34

Author(s):

Takashi Asai ◽

Yan Liu ◽

Silvana Di Giandomenico ◽

Narae Bae ◽

Delphine Ndiaye-Lobry ◽

...

Keyword(s):

Cell Cycle ◽

Steady State ◽

Progenitor Cells ◽

Target Gene ◽

Critical Role ◽

Genotoxic Stress ◽

Dependent Manner ◽

Hematopoietic Stem ◽

P53 Target Gene

Abstract We recently defined a critical role for p53 in regulating the quiescence of adult hematopoietic stem cells (HSCs) and identified necdin as a candidate p53 target gene. Necdin is a growth-suppressing protein and the gene encoding it is one of several that are deleted in patients with Prader-Willi syndrome. To define the intrinsic role of necdin in adult hematopoiesis, in the present study, we transplanted necdin-null fetal liver cells into lethally irradiated recipients. We show that necdin-null adult HSCs are less quiescent and more proliferative than normal HSCs, demonstrating the similar role of necdin and p53 in promoting HSC quiescence during steady-state conditions. However, wild-type recipients repopulated with necdin-null hematopoietic stem/progenitor cells show enhanced sensitivity to irradiation and chemotherapy, with increased p53-dependent apoptosis, myelosuppression, and mortality. Necdin controls the HSC response to genotoxic stress via both cell-cycle–dependent and cell-cycle–independent mechanisms, with the latter occurring in a Gas2L3-dependent manner. We conclude that necdin functions as a molecular switch in adult hematopoiesis, acting in a p53-like manner to promote HSC quiescence in the steady state, but suppressing p53-dependent apoptosis in response to genotoxic stress.

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Critical Role of Ser-520 Phosphorylation for Membrane Recruitment and Activation of the ZAP-70 Tyrosine Kinase in T Cells

Molecular and Cellular Biology ◽

10.1128/mcb.23.21.7667-7677.2003 ◽

2003 ◽

Vol 23 (21) ◽

pp. 7667-7677 ◽

Cited By ~ 4

Author(s):

Yaoming Yang ◽

Patricia Villain ◽

Tomas Mustelin ◽

Clément Couture

Keyword(s):

T Cells ◽

Plasma Membrane ◽

Tyrosine Kinases ◽

Critical Role ◽

Gene Activation ◽

Interleukin 2 ◽

Antigen Receptor ◽

Membrane Recruitment ◽

Receptor Ligation

ABSTRACT Regulation of protein tyrosine kinases (PTKs) by tyrosine phosphorylation is well recognized; in fact, nearly all PTKs require phosphorylation of tyrosine residues in their “activation loop” for catalytic activity. In contrast, the phosphorylation of PTKs on serine and threonine residues has not been studied nearly as much. We report that the ZAP-70 PTK contains predominately phosphoserine in normal T lymphocytes as well as in Jurkat T leukemia cells. We have identified one site of phosphorylation as Ser-520 and find this site to be important for the recruitment and activation of ZAP-70 in T cells. Mutant ZAP-70-S520A had reduced ability to autophosphorylate and to mediate antigen receptor-induced interleukin 2 gene activation and was not enriched at the plasma membrane. These defects were rescued by addition of a myristylation signal to the N terminus of ZAP-70-S520A to force its plasma membrane and lipid raft localization. We conclude that phosphorylation of ZAP-70 at Ser-520 plays an important role in the correct localization of ZAP-70 and in priming ZAP-70 for its acute recruitment and activation upon antigen receptor ligation.

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ParB dynamics and the critical role of the CTD in DNA condensation unveiled by combined force-fluorescence measurements

10.1101/477836 ◽

2018 ◽

Author(s):

Julene Madariaga-Marcos ◽

Cesar L. Pastrana ◽

Gemma L. M. Fisher ◽

Mark S. Dillingham ◽

Fernando Moreno-Herrero

Keyword(s):

Dna Binding ◽

Protein Binding ◽

Network Formation ◽

Critical Role ◽

Magnetic Tweezers ◽

Dna Condensation ◽

Full Length ◽

Tirf Microscopy ◽

Fluorescence Measurements

AbstractBacillus subtilis ParB forms multimeric networks involving non-specific DNA binding leading to DNA condensation. In our previous work (Fisher et al., 2017), we found that an excess of the free C-terminal domain (CTD) of ParB impeded DNA condensation or promoted decondensation of pre-assembled networks. However, interpretation of the molecular basis for this phenomenon was complicated by our inability to uncouple protein binding from DNA condensation. Here, we have combined lateral magnetic tweezers with TIRF microscopy to simultaneously control the restrictive force against condensation and to visualize ParB protein binding by fluorescence. At non-permissive forces for condensation, ParB binds non-specifically and highly dynamically to DNA. Our new approach concluded that the free CTD blocks the formation of ParB networks by heterodimerization with full length DNA-bound ParB. This strongly supports a model in which the CTD acts as a key bridging interface between distal DNA binding loci within ParB networks.Significance StatementUsing combined Magnetic Tweezers and TIRF microscopy we show that the CTD of ParB blocks ParB network formation by heterodimerization with the full-length protein, which remains bound to the DNA.

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Functional Dissection of the C Terminus of CBFβ-SMMHC Indicates a Critical Role of the Multimerization Domain during Hematopoiesis and Leukemogenesis

Blood ◽

10.1182/blood.v124.21.2218.2218 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2218-2218

Author(s):

Ling Zhao ◽

Lemlem Alemu ◽

Jun Cheng ◽

Tao Zhen ◽

Alan D. Friedman ◽

...

Keyword(s):

Cell Population ◽

Transcriptional Repression ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Critical Role ◽

Full Length ◽

C Terminus ◽

Definitive Hematopoiesis

Abstract The recurrent chromosome 16 inversion (inv(16)) in acute myeloid leukemia (AML) subtype M4Eo results in a fusion between CBFB and MYH11 genes, which encodes a chimeric protein CBFβ-SMMHC (core binding factor β - smooth muscle myosin heavy chain). We previously generated mouse CBFB-MYH11 knock-in models that mimic the human inv(16) AML and demonstrated that the CBFβ-SMMHC fusion protein blocks RUNX1 and CBFβ function during definitive hematopoiesis and plays a driving role in leukemogenesis. Our recent studies indicated that the C-terminus of CBFβ-SMMHC, which contains domains for multimerization and transcriptional repression, is important for leukemogenesis by CBFβ-SMMHC (Kamikubo et al, Blood 121:638, 2013). In this study we generated a new CBFB-MYH11 knock-in mouse model to determine the role of the multimerization domain of CBFβ-SMMHC during hematopoiesis and leukemogenesis. Previous studies have dissected the assembly competence domain (ACD) of the CBFβ-SMMHC C-terminus to identify the critical amino acid residuals for multimerization (Zhang et al., Oncogene 25:7289, 2006). Among them, mutations in helices D and E are the ones that affect multimerization the most. Importantly, the helices D and E mutations do not interfere with the repression function of CBFβ-SMMHC. Therefore, we generated knock-in mice expressing CBFβ-SMMHC with mutated helices D &E in the ACD of the C-terminus (mDE) to determine the role of multimerization for the in vivo function of CBFβ-SMMHC. The embryonic hematopoietic phenotype in the mDE knockin embryos is very similar to what we have observed in the knockin embryos expressing C-terminally-deleted CBFβ-SMMHC (Kamikubo et al, Blood 121:638, 2013), i.e., heterozygous embryos (Cbfb+/mDE) were viable and showed no defects in fetal liver definitive hematopoiesis, while homozygous embryos (CbfbmDE/mDE) showed hemorrhage in the central nervous system and died around E12.5, as seen in the full length CBFβ-SMMHC heterozygous knockin mice and the Cbfb-/- and Runx1-/- mice. Analysis of peripheral blood from adult Cbfb+/mDE mice showed decreased B cell population and increased T cell population, while the myeloid compartment was unchanged. Preliminary findings suggest that leukemogenesis is at least delayed in the Cbfb+/mDE mice as compared to mice expressing full-length CBFβ-SMMHC. Therefore the multimerization function of CBFβ-SMMHC is critical for its ability to induce defects in embryonic hematopoiesis and for leukemogenesis. Disclosures No relevant conflicts of interest to declare.

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A Role of Poly (ADP-Ribose) Polymerase in NF- B Transcriptional Activation

Biological Chemistry ◽

10.1515/bc.1999.118 ◽

1999 ◽

Vol 380 (7-8) ◽

pp. 953-959 ◽

Cited By ~ 203

Author(s):

P. O. Hassa ◽

M. O. Hottiger

Keyword(s):

Transcriptional Activation ◽

Target Genes ◽

Inflammatory Responses ◽

Critical Role ◽

Gene Activation ◽

Nuclear Complex ◽

Activation Cascade ◽

Ltr Promoter

AbstractThe transcription factor NF-κB plays a critical role in immune and inflammatory responses. Here we show that poly (ADP ribose) polymerase (PARP) is required for specific NF-κB transcriptional activationin vivo. The activation of the HIV-LTR promoter and an NF-κBdependent artificial promoter was drastically reduced in PARP (_/_) cells, independently of the signaling pathway through which NF-bB was induced. Furthermore NF-κB-dependent gene activation was restoredin vivoby the expression of PARP in PARP (_/_) cells. Finally, we show that both NF-κB and PARP formed a stable immunoprecipitable nuclear complex. This interaction did not need DNA binding. Our results suggest that PARP is an important cofactor in the activation cascade of NF-κB-dependent target genes.

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