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    : PLoS ONE. 2008 Feb 13;3(2):e1609.Click here to read Click here to read Links
     

    The ER-Bound RING Finger Protein 5 (RNF5/RMA1) Causes Degenerative Myopathy in Transgenic Mice and Is Deregulated in Inclusion Body Myositis.

    Signal Transduction, The Burnham Institute for Medical Research, La Jolla, California, United States of America.

    Growing evidence supports the importance of ubiquitin ligases in the pathogenesis of muscular disorders, although underlying mechanisms remain largely elusive. Here we show that the expression of RNF5 (aka RMA1), an ER-anchored RING finger E3 ligase implicated in muscle organization and in recognition and processing of malfolded proteins, is elevated and mislocalized to cytoplasmic aggregates in biopsies from patients suffering from sporadic-Inclusion Body Myositis (sIBM). Consistent with these findings, an animal model for hereditary IBM (hIBM), but not their control littermates, revealed deregulated expression of RNF5. Further studies for the role of RNF5 in the pathogenesis of s-IBM and more generally in muscle physiology were performed using RNF5 transgenic and KO animals. Transgenic mice carrying inducible expression of RNF5, under control of beta-actin or muscle specific promoter, exhibit an early onset of muscle wasting, muscle degeneration and extensive fiber regeneration. Prolonged expression of RNF5 in the muscle also results in the formation of fibers containing congophilic material, blue-rimmed vacuoles and inclusion bodies. These phenotypes were associated with altered expression and activity of ER chaperones, characteristic of myodegenerative diseases such as s-IBM. Conversely, muscle regeneration and induction of ER stress markers were delayed in RNF5 KO mice subjected to cardiotoxin treatment. While supporting a role for RNF5 Tg mice as model for s-IBM, our study also establishes the importance of RNF5 in muscle physiology and its deregulation in ER stress associated muscular disorders.

    PMID: 18270596 [PubMed - in process]


     
    Proc Natl Acad Sci U S A. 2008 Feb 5;105(5):1674-9.  .Click here to read
     

    Suppressor role of activating transcription factor 2 (ATF2) in skin cancer.

    Burnham Institute for Medical Research, La Jolla, CA 92037, USA.

    Activating transcription factor 2 (ATF2) regulates transcription in response to stress and growth factor stimuli. Here, we use a mouse model in which ATF2 was selectively deleted in keratinocytes. Crossing the conditionally expressed ATF2 mutant with K14-Cre mice (K14.ATF2(f/f)) resulted in selective expression of mutant ATF2 within the basal layer of the epidermis. When subjected to a two-stage skin carcinogenesis protocol [7,12-dimethylbenz[a]anthracene/phorbol 12-tetradecanoate 13-acetate (DMBA/TPA)], K14.ATF2(f/f) mice showed significant increases in both the incidence and prevalence of papilloma development compared with the WT ATF2 mice. Consistent with these findings, keratinocytes of K14.ATF2(f/f) mice exhibit greater anchorage-independent growth compared with ATF2 WT keratinocytes. Papillomas of K14.ATF2(f/f) mice exhibit reduced expression of presenilin1, which is associated with enhanced beta-catenin and cyclin D1, and reduced Notch1 expression. Significantly, a reduction of nuclear ATF2 and increased beta-catenin expression were seen in samples of squamous and basal cell carcinoma, as opposed to normal skin. Our data reveal that loss of ATF2 transcriptional activity serves to promote skin tumor formation, thereby indicating a suppressor activity of ATF2 in skin tumor formation.


     
    : Clin Cancer Res. 2007 Nov 15;13(22 Pt 1):6769-78.Click here to read Links
     

    Preclinical studies of celastrol and acetyl isogambogic acid in melanoma.

    Signal Transduction Program, Cancer Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.

    PURPOSE: Sensitize melanomas to apoptosis and inhibit their growth and metastatic potential by compounds that mimic the activities of activating transcription factor 2 (ATF2)-driven peptides. EXPERIMENTAL DESIGN: Small-molecule chemical library consisting of 3,280 compounds was screened to identify compounds that elicit properties identified for ATF2 peptide, including (a) sensitization of melanoma cells to apoptosis, (b) inhibition of ATF2 transcriptional activity, (c) activation of c-Jun NH(2)-terminal kinase (JNK) and c-Jun transcriptional activity, and (d) inhibition of melanoma growth and metastasis in mouse models. RESULTS: Two compounds, celastrol (CSL) and acetyl isogambogic acid, could, within a low micromolar range, efficiently elicit cell death in melanoma cells. Both compounds efficiently inhibit ATF2 transcriptional activities, activate JNK, and increase c-Jun transcriptional activities. Similar to the ATF2 peptide, both compounds require JNK activity for their ability to inhibit melanoma cell viability. Derivatives of CSL were identified as potent inducers of cell death in mouse and human melanomas. CSL and a derivative (CA19) could also efficiently inhibit growth of human and mouse melanoma tumors and reduce the number of lung metastases in syngeneic and xenograft mouse models. CONCLUSIONS: These studies show for the first time the effect of CSL and acetyl isogambogic acid on melanoma. These compounds elicit activities that resemble the well-characterized ATF2 peptide and may therefore offer new approaches for the treatment of this tumor type.

    PMID: 18006779 [PubMed - indexed for MEDLINE]


     
    Cancer Res. 2007 Sep 1;67(17):8172-9.Click here to read
     

    Increased expression of the E3 ubiquitin ligase RNF5 is associated with decreased survival in breast cancer.

    Signal Transduction Program, The Burnham Institute for Medical Research, La Jolla, CA 92037, USA.

    The selective ubiquitination of proteins by ubiquitin E3 ligases plays an important regulatory role in control of cell differentiation, growth, and transformation and their dysregulation is often associated with pathologic outcomes, including tumorigenesis. RNF5 is an E3 ubiquitin ligase that has been implicated in motility and endoplasmic reticulum stress response. Here, we show that RNF5 expression is up-regulated in breast cancer tumors and related cell lines. Elevated expression of RNF5 was seen in breast cancer cell lines that became more sensitive to cytochalasin D- and paclitaxel-induced apoptosis following its knockdown with specific short interfering RNA. Inhibition of RNF5 expression markedly decreased cell proliferation and caused a reorganization of the actin cytoskeleton in response to stress in MCF-7 but not in p53 mutant breast cancer cells, suggesting a p53-dependent function. Significantly, high levels of RNF5 were associated with decreased survival in human breast cancer specimens. Similarly, RNF5 levels were higher in metastatic melanoma specimens and in melanoma, leukemia, ovarian, and renal tumor-derived cell lines, suggesting that increased RNF5 expression may be a common event during tumor progression. These results indicate that RNF5 is a novel regulator of breast cancer progression through its effect on actin cytoskeletal alterations, which also affect sensitivity of breast cancer cells to cytoskeletal targeting antineoplastic agents.

    PMID: 17804730 [PubMed - indexed for MEDLINE]

     

    Cancer Cell 2007 May 11(5):447-60
     

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            Rewired ERK-JNK signaling pathways in melanoma.
     

    Lopez-Bergami P, Huang C, Goydos JS, Yip D, Bar-Eli M, Herlyn M, Smalley KS, Mahale A, Eroshkin A, Aaronson S, Ronai Z
     

    Constitutive activation of MEK-ERK signaling is often found in melanomas. Here, we identify a mechanism that links ERK with JNK signaling in human melanoma. Constitutively active ERK increases c-Jun transcription and stability, which are mediated by CREB and GSK3, respectively. Subsequently, c-Jun increases transcription of target genes, including RACK1, an adaptor protein that enables PKC to phosphorylate and enhance JNK activity, enforcing a feed-forward mechanism of the JNK-Jun pathway. Activated c-Jun is also responsible for elevated cyclin D1 expression, which is frequently overexpressed in human melanoma. Our data reveal that, in human melanoma, the rewired ERK signaling pathway upregulates JNK and activates the c-Jun oncogene and its downstream targets, including RACK1 and cyclin D1.

    PMID: 17482134 [PubMed - indexed for MEDLINE]

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    : Proc Natl Acad Sci U S A. 2007 Apr 10;104(15):6371-6. Epub 2007 Apr 2.

     

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    Ubiquitin-conjugating enzyme Ubc13 is a critical component of TNF receptor-associated factor (TRAF)-mediated inflammatory responses.

    Fukushima T, Matsuzawa S, Kress CL, Bruey JM, Krajewska M, Lefebvre S, Zapata JM, Ronai Z, Reed JC.

    Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037.

    Ubc13 is a ubiquitin-conjugating enzyme responsible for noncanonical ubiquitination of TNF receptor-associated factor (TRAF)-family adapter proteins involved in Toll-like receptor and TNF-family cytokine receptor signaling, which are regulators of innate immunity. Gene ablation was used to study the function of Ubc13 in mice. Whereas homozygous ubc13 gene disruption resulted in embryonic lethality, heterozygous ubc13(+/-) mice appeared normal, without alterations in immune cell populations. Haploinsufficient ubc13(+/-) mice were resistant to lipopolysaccharide-induced lethality, and demonstrated reduced in vivo ubiquitination of TRAF6. Macrophages and splenocytes isolated from ubc13(+/-) mice exhibited reduced lipopolysaccharide-inducible cytokine secretion and impaired activation of TRAF-dependent signal transduction pathways (NF-kappaB, JNK, and p38 MAPK). These findings document a critical role for Ubc13 in inflammatory responses and suggest that agents reducing Ubc13 activity could have therapeutic utility.

    PMID: 17404240 [PubMed - in process]


    : Cell. 2006 Nov 17;127(4):675-7.

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    Comment on:

    ·         Cell. 2006 Nov 17;127(4):775-88.


    A new function for p53 ubiquitination.

    Hirano Y, Ronai Z.

    Burnham Institute for Medical Research, La Jolla, CA 92130, USA.

    The amount of p53 protein in a cell is normally limited by ubiquitin-dependent degradation. In this issue of Cell, Le Cam et al. (2006) reveal that p53 ubiquitination contributes to transcriptional activation rather than protein stability. These results may provide insight into how p53 can modulate diverse cellular processes such as growth arrest and apoptosis.

    PMID: 17110328 [PubMed - indexed for MEDLINE]



    : J Biol Chem. 2006 Nov 17;281(46):35316-26. Epub 2006 Sep 25.

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    Regulation of the ring finger E3 ligase Siah2 by p38 MAPK.

    Khurana A, Nakayama K, Williams S, Davis RJ, Mustelin T, Ronai Z.

    Signal Transduction Program, Burnham Institute for Medical Research, La Jolla, California 92037, USA.

    The RING finger ubiquitin ligase Siah2 controls the stability of various substrates involved in stress and hypoxia responses, including the PHD3, which controls the stability of HIF-1alpha. In the present study we determined the role of Siah2 phosphorylation in the regulation of its activity toward PHD3. We show that Siah2 is subject to phosphorylation by p38 MAPK, which increases Siah2-mediated degradation of PHD3. Consistent with these findings, MKK3/MKK6 double-deficient cells, which cannot activate p38 kinases, exhibit impaired Siah2-dependent degradation of PHD3. Phosphopeptide mapping identified T24 and S29 as the primary phospho-acceptor sites. Phospho-mutant forms of Siah2 (S29A or T24A/S29A) exhibit impaired degradation of PHD3, particularly after hypoxia. Conversely, a phospho-mimic form of Siah2 (T24E/S29D) exhibits stronger degradation of PHD3, compared with wild type Siah2. Whereas phospho-mutant Siah2 exhibits weaker association with PHD3, phospho-mimic Siah2 associates as well as wild type and is localized within the perinuclear region, suggesting that phosphorylation of Siah2 affects its subcellular localization and, consequently, the degree of its association with PHD3. In all, our findings reveal the phosphorylation of Siah2 by p38 and the implications of such phosphorylation for Siah2 activity toward PHD3.

    PMID: 17003045 [PubMed - indexed for MEDLINE]


    : Mol Cell Biol. 2006 Dec;26(23):8901-13. Epub 2006 Sep 25.

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    Regulation of p53 localization and activity by Ubc13.

    Laine A, Topisirovic I, Zhai D, Reed JC, Borden KL, Ronai Z.

    Signal Transduction Program, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.

    The abundance and activity of p53 are regulated largely by ubiquitin ligases. Here we demonstrate a previously undisclosed regulation of p53 localization and activity by Ubc13, an E2 ubiquitin-conjugating enzyme. While increasing p53 stability, Ubc13 decreases p53 transcriptional activity and increases its localization to the cytoplasm, changes that require its ubiquitin-conjugating activity. Ubc13 elicits K63-dependent ubiquitination of p53, which attenuates Hdm2-induced polyubiquitination of p53. Ubc13 association with p53 requires an intact C-terminal domain of p53 and is markedly stronger with a p53 mutant that cannot tetramerize. Expression of Ubc13 in vivo increases the pool of monomeric p53, indicating that Ubc13 affects tetramerization of p53. Significantly, wild-type but not mutant Ubc13 is associated with polysomes and enriches p53 within this fraction. In response to DNA damage, Ubc13 is no longer capable of facilitating p53 monomerization, in part due to a decrease in its own levels which is p53 dependent. Our findings point to a newly discerned mechanism important in the regulation of p53 organization, localization, and activity by Ubc13.

    PMID: 17000756 [PubMed - indexed for MEDLINE]


    : Nat Cell Biol. 2006 Oct;8(10):1084-94. Epub 2006 Sep 10.

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    Meiotic regulation of the CDK activator RINGO/Speedy by ubiquitin-proteasome-mediated processing and degradation.

    Gutierrez GJ, Vogtlin A, Castro A, Ferby I, Salvagiotto G, Ronai Z, Lorca T, Nebreda AR.

    EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany. gustavo@burnham.org

    Xenopus RINGO/Speedy (XRINGO) is a potent inducer of oocyte meiotic maturation that can directly activate Cdk1 and Cdk2. Here, we show that endogenous XRINGO protein accumulates transiently during meiosis I entry and then is downregulated. This tight regulation of XRINGO expression is the consequence of two interconnected mechanisms: processing and degradation. XRINGO processing involves recognition of at least three distinct phosphorylated recognition motifs by the SCF(betaTrCP) ubiquitin ligase, followed by proteasome-mediated limited degradation, resulting in an amino-terminal XRINGO fragment. XRINGO processing is directly stimulated by several kinases, including protein kinase A and glycogen synthase kinase-3beta, and may contribute to the maintenance of G2 arrest. On the other hand, XRINGO degradation after meiosis I is mediated by the ubiquitin ligase Siah-2, which probably requires phosphorylation of XRINGO on Ser 243 and may be important for the omission of S phase at the meiosis-I-meiosis-II transition in Xenopus oocytes.

    PMID: 16964245 [PubMed - indexed for MEDLINE]


    : Biochem J. 2007 Jan 1;401(1):217-26.

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    Hypoxia-induced assembly of prolyl hydroxylase PHD3 into complexes: implications for its activity and susceptibility for degradation by the E3 ligase Siah2.

    Nakayama K, Gazdoiu S, Abraham R, Pan ZQ, Ronai Z.

    Signal Transduction Program, Burnham Institute for Medical Research, La Jolla, CA 92037, USA.

    PHD1-3 (prolyl hydroxylases 1-3) catalyse the hydroxylation of HIF (hypoxia-inducible factor)-alpha subunit that triggers the substrate ubiquitination and subsequent degradation. The RING (really interesting new gene) finger E3 ligase Siah2 preferentially targets PHD3 for degradation. Here, we identify the requirements for such selective targeting. Firstly, PHD3 lacks an N-terminal extension found in PHD1 and PHD2; deletion of this domain from PHD1 and PHD2 renders them susceptible to degradation by Siah2. Secondly, PHD3 can homo- and hetero-multimerize with other PHDs. Consequently, PHD3 is found in high-molecular-mass fractions that were enriched in hypoxia. Interestingly, within the lower-molecular-mass complex, PHD3 exhibits higher specific activity towards hydroxylation of HIF-1alpha and co-localizes with Siah2, suggesting that Siah2 limits the availability of the more active form of PHD3. These findings provide new insight into the mechanism underlying the regulation of PHD3 availability and activity in hypoxia by the E3 ligase Siah2.

    PMID: 16958618 [PubMed - indexed for MEDLINE]


    : Oncogene. 2007 Mar 1;26(10):1477-83. Epub 2006 Aug 21.

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    Regulation of p53 localization and transcription by the HECT domain E3 ligase WWP1.

    Laine A, Ronai Z.

    Signal Transduction Program, The Burnham Institute for Medical Research, La Jolla, CA 92037, USA.

    As a key cellular regulatory protein p53 is subject to tight regulation by several E3 ligases. Here, we demonstrate the role of HECT domain E3 ligase, WWP1, in regulating p53 localization and activity. WWP1 associates with p53 and induces p53 ubiquitylation. Unlike other E3 ligases, WWP1 increases p53 stability; inhibition of WWP1 expression or expression of a ligase-mutant form results in decreased p53 expression. WWP1-mediated stabilization of p53 is associated with increased accumulation of p53 in cytoplasm with a concomitant decrease in its transcriptional activities. WWP1 effects are independent of Mdm2 as they are seen in cells lacking Mdm2 expression. Whereas WWP1 limits p53 activity, p53 reduces expression of WWP1, pointing to a possible feedback loop mechanism. Taken together, these findings identify the first instance of a ubiquitin ligase that causes stabilization of p53 while inactivating its transcriptional activities.

    PMID: 16924229 [PubMed - indexed for MEDLINE]


    : Nat Cell Biol. 2006 Aug;8(8):790-1.

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    Comment on:

    ·         Nat Cell Biol. 2006 Aug;8(8):855-62.


    Balancing Mdm2 - a Daxx-HAUSP matter.

    Ronai Z.

    PMID: 16880812 [PubMed - indexed for MEDLINE]


    : Trends Biochem Sci. 2006 Jun;31(6):324-32. Epub 2006 May 2.

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    Ubiquitin and SUMO systems in the regulation of mitotic checkpoints.

    Gutierrez GJ, Ronai Z.

    Signal Transduction Program, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. gustavo@burnham.org

    Proteolysis mediated by the ubiquitin-proteasome system is a crucial regulatory mechanism in signal transduction cascades of temporal cellular processes such as cell division. Two principal subtypes of modular ubiquitin ligase, the anaphase-promoting complex or cyclosome (APC/C) and the Skp1/Cullin-1/F-box protein complex, have emerged as essential regulators of key events in the cell cycle. The importance of these ligases is best illustrated by their roles in the checkpoint and repair pathways or in response to multiple stresses, where they affect activation of the M-phase-promoting factor or proper formation and/or maintenance of the mitotic spindle. Recent studies have considerably improved our understanding of the function of the concerted action of the phosphorylation and ubiquitin or SUMO systems in the regulation of the stability and activity of key components of the mitotic checkpoint.

    PMID: 16647857 [PubMed - indexed for MEDLINE]


    : Mol Cell Biol. 2006 Jan;26(2):413-24.

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    RACK1 recruits STAT3 specifically to insulin and insulin-like growth factor 1 receptors for activation, which is important for regulating anchorage-independent growth.

    Zhang W, Zong CS, Hermanto U, Lopez-Bergami P, Ronai Z, Wang LH.

    Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029-6574, USA.

    Current understanding of the activation of STATs is through binding between the SH2 domain of STATs and phosphotyrosine of tyrosine kinases. Here we demonstrate a novel role of RACK1 as an adaptor for insulin and insulin-like growth factor 1 receptor (IGF-1R)-mediated STAT3 activation specifically. Intracellular association of RACK1 via its N-terminal WD domains 1 to 4 (WD1-4) with insulin receptor (IR)/IGF-1R is augmented upon respective ligand stimulation, whereas association with STAT3 is constitutive. Purified RACK1 or RACK1 WD1-4 associates directly with purified IR, IGF-1R, and STAT3 in vitro. Insulin induces multiprotein complex formation of RACK1, IR, and STAT3. Overexpression or downregulation of RACK1 greatly enhances or decreases, respectively, IR/IGF-1R-mediated activation of STAT3 and its target gene expression. Site-specific mutants of IR and IGF-1R impaired in RACK1 binding are ineffective in mediating recruitment and activation of STAT3 as well as in insulin- or IGF-1-induced protection of cells from anoikis. RACK1-mediated STAT3 activation is important for insulin and IGF-1-induced anchorage-independent growth in certain ovarian cancer cells. We conclude that RACK1 mediates recruitment of STAT3 to IR and IGF-1R specifically for activation, suggesting a general paradigm for the need of an adaptor in mediating activation of STATs by receptor protein tyrosine kinases.

    PMID: 16382134 [PubMed - indexed for MEDLINE]


    : J Biol Chem. 2006 Jan 20;281(3):1840-52. Epub 2005 Nov 23.

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    Opposite roles of FAP-1 and dynamin in the regulation of Fas (CD95) translocation to the cell surface and susceptibility to Fas ligand-mediated apoptosis.

    Ivanov VN, Ronai Z, Hei TK.

    Center for Radiological Research, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA. vni3@columbia.edu

    Human melanoma is the most aggressive form of skin cancer and is extremely resistant to radiation and chemotherapy. One of the critical parameters of this resistance is down-regulation of Fas (CD95) cell-surface expression. Using TIG3 normal human fibroblasts and human melanoma cell lines, we investigated transcriptional regulation of FAP-1, a regulator of Fas translocation in the cell. Protein-tyrosine phosphatase FAP-1 (PTPN13, PTP-BAS) interacts with human Fas protein and prevents its export from the cytoplasm to the cell surface. In contrast, dynamin-2 facilitates Fas protein translocation from the Golgi apparatus via the trans-Golgi network to the cell surface. Suppression of dynamin functions by dominant negative dynamin K44A blocks Fas export, whereas the down-regulation of FAP-1 expression by specific RNA interference restores Fas export (a phenomenon that could still be down-regulated in the presence of dominant-negative dynamin). Based on the FAP-1- and dynamin-dependent regulation of Fas translocation, we have created human melanoma lines with different levels of surface expression of Fas. Treatment of these melanoma lines with soluble Fas ligand resulted in programmed cell death that was proportional to the pre-existing levels of surface Fas. Taking into consideration the well known observations that FAP-1 expression is often up-regulated in metastatic tumors, we have established a causal connection between high basal NF-kappaB transcription factor activity (which is a hallmark of many types of metastatic tumors) and NF-kappaB-dependent transcriptional regulation of FAP-1 gene expression that finally restricts Fas protein trafficking, thereby, facilitating the survival of cancer cells.

    PMID: 16306044 [PubMed - indexed for MEDLINE]


    : Mol Cell Biol. 2005 Oct;25(19):8619-30.

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    JAMP, a Jun N-terminal kinase 1 (JNK1)-associated membrane protein, regulates duration of JNK activity.

    Kadoya T, Khurana A, Tcherpakov M, Bromberg KD, Didier C, Broday L, Asahara T, Bhoumik A, Ronai Z.

    Signal Transduction Program, The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA.

    We report the identification and characterization of JAMP (JNK1 [Jun N-terminal kinase 1]-associated membrane protein), a predicted seven-transmembrane protein that is localized primarily within the plasma membrane and associates with JNK1 through its C-terminal domain. JAMP association with JNK1 outcompetes JNK1 association with mitogen-activated protein kinase phosphatase 5, resulting in increased and prolonged JNK1 activity following stress. Elevated expression of JAMP following UV or tunicamycin treatment results in sustained JNK activity and a higher level of JNK-dependent apoptosis. Inhibition of JAMP expression by RNA interference reduces the degree and duration of JNK activation and concomitantly the level of stress-induced apoptosis. Through its regulation of JNK1 activity, JAMP emerges as a membrane-anchored regulator of the duration of JNK1 activity in response to diverse stress stimuli.

    PMID: 16166642 [PubMed - indexed for MEDLINE]


    : Mol Cell. 2005 Aug 5;19(3):309-20.

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    Erratum in:

    ·         Mol Cell. 2005 Aug 19;19(4):578-9.


    RACK1 mediates activation of JNK by protein kinase C [corrected]

    Lopez-Bergami P, Habelhah H, Bhoumik A, Zhang W, Wang LH, Ronai Z.

    Department of Oncological Sciences, Mount Sinai School of Medicine, New York, New York 10029, USA.

    Activation of the Jun-N-terminal kinase (JNK) signaling cascade by phorbol esters (TPA) or protein kinase C (PKC) is well documented, although the underlying mechanism is not known. Here, we demonstrate that the receptor for activated C kinase 1 (RACK1) serves as an adaptor for PKC-mediated JNK activation. Phosphorylation of JNK by PKC occurs on Ser129 and requires the presence of RACK1. Ser129 phosphorylation augments JNK phosphorylation by MKK4 and/or MKK7 and is required for JNK activation by TPA, TNFalpha, UV irradiation, and PKC, but not by anisomycin or MEKK1. Inhibition of RACK1 expression by siRNA attenuates JNK activation, sensitizes melanoma cells to UV-induced apoptosis, and reduces their tumorigenicity in nude mice. In finding the role of RACK1 in activation of JNK by PKC, our study also highlights the nature of crosstalk between these two signal-transduction pathways.

    PMID: 16061178 [PubMed - indexed for MEDLINE]


    : Mol Cell. 2005 May 27;18(5):577-87.

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    ATM-dependent phosphorylation of ATF2 is required for the DNA damage response.

    Bhoumik A, Takahashi S, Breitweiser W, Shiloh Y, Jones N, Ronai Z.

    Signal Transduction Program, The Burnham Institute, La Jolla, California 92037, USA.

    Activating transcription factor 2 (ATF2) is regulated by JNK/p38 in response to stress. Here, we demonstrate that the protein kinase ATM phosphorylates ATF2 on serines 490 and 498 following ionizing radiation (IR). Phosphoantibodies to ATF2(490/8) reveal dose- and time-dependent phosphorylation of ATF2 by ATM that results in its rapid colocalization with gamma-H2AX and MRN components into IR-induced foci (IRIF). Inhibition of ATF2 expression decreased recruitment of Mre11 to IRIF, abrogated S phase checkpoint, reduced activation of ATM, Chk1, and Chk2, and impaired radioresistance. ATF2 requires neither JNK/p38 nor its DNA binding domain for recruitment to IRIF and the S phase checkpoint. Our findings identify a role for ATF2 in the DNA damage response that is uncoupled from its transcriptional activity.

    PMID: 15916964 [PubMed - indexed for MEDLINE]


    : Sci STKE. 2005 Apr 26;2005(281):re5.

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    Ubiquitin chains in the ladder of MAPK signaling.

    Laine A, Ronai Z.

    Signal Transduction Program, The Burnham Institute, La Jolla, CA 92037, USA.

    With a better understanding of the cellular stress response, it has become evident that catalytic modules consisting of kinases that mediate the activation of downstream effector components are subject to multiple layers of regulation. Such regulatory mechanisms are not limited to those involving scaffold proteins or protein phosphatases, and they appear to include a growing number of modifications by ubiquitin and ubiquitin-like proteins. The role of ubiquitin in the regulation of mitogen-activated protein kinase (MAPK) emerges as a paradigm for understanding the role of ubiquitination in regulating other signal transduction pathways. Ubiquitination influences signal diversification and limits the duration of the signal through its role in the assembly of protein kinase complexes, subcellular localization, and the actual degradation of the kinase or its substrate. This review summarizes our current understanding of the roles of ubiquitin in regulating MAPK signaling.

    PMID: 15855411 [PubMed - indexed for MEDLINE]


    : Oncogene. 2005 Apr 7;24(15):2574-9.

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    Erratum in:

    ·         Oncogene. 2005 Jun 9;24(25):4164.


    Phosphorylation of MdmX by CDK2/Cdc2(p34) is required for nuclear export of Mdm2.

    Elias B, Laine A, Ronai Z.

    Department of Oncological Sciences, Cancer Center, Mount Sinai School of Medicine, One Gustive L. Levy Place, Box 1130, New York, NY 10029, USA.

    Mdm2 and MdmX function as cellular regulators of the p53 tumor suppressor protein. Intriguingly, the activities of these proteins are interdependent; MdmX stabilizes Mdm2, enabling its activities towards p53, but it also requires Mdm2 for its nuclear localization. Here we demonstrate that via its phosphorylation by CDK2/Cdc2p34, MdmX regulates nuclear export of Mdm2. Cdc2p34 phosphorylates MdmX on Ser 96 in vitro. Mutation within this site (MdmX(S96A)) impairs, whereas phosphomimic substitution (MdmX(S96D)) increases the cytoplasmic localization of MdmX, suggesting that CDK2/Cdc2p34 phosphorylation is required for export of MdmX from the nucleus. Consequently, cells that express MdmX(S96A) retain Mdm2 in their nuclei, suggesting that export of Mdm2 to the cytoplasm is MdmX-dependent. Similarly, treatment of cells with the pharmacological inhibitor of CDK2/Cdc2p34 or with a dominant-negative Cdc2 results in nuclear localization of MdmX and Mdm2 and decreases the level of Mdm2 expression. Since Cdc2p34 is active in nonstressed conditions, our finding provides a novel insight into the signaling cascade involved in the regulation of MdmX localization and for regulation of Mdm2 localization and stability.

    PMID: 15735705 [PubMed - indexed for MEDLINE]


    : Cancer Res. 2004 Nov 15;64(22):8222-30.

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    Inhibition of melanoma growth and metastasis by ATF2-derived peptides.

    Bhoumik A, Gangi L, Ronai Z.

    Department of Oncological Sciences, Mount Sinai School of Medicine, New York, New York 10029, USA.

    The resistance of melanoma to apoptosis, as well as its growth and metastasis capabilities, can be overcome by expression of a peptide derived from amino acid (aa) 51 to 100 of ATF2. Here we show that expression of ATF2((51-100)) in human melanoma cells reduced their growth in nude mice, which was additionally inhibited upon treatment with protein kinase inhibitors UCN-01 or SB203580. Injection of a fusion protein consisting of HIV-TAT and aa 51 to 100 of ATF2 into SW1 melanomas efficiently inhibits their growth and their metastasis up to complete regression. Additionally, expression of a 10aa peptide that corresponds to aa 51 to 60 of ATF2 sensitizes melanoma cells to spontaneous apoptosis, which coincides with activation of caspase 9 and poly(ADP-ribose) polymerase cleavage, and inhibit their growth in vivo. The 10aa peptide increases the association of c-Jun NH(2)-terminal kinase with c-Jun but not with ATF2, resulting in concomitant increase in TRE-mediated transcription. Our study points to mechanisms underlying the activities of the ATF2 peptide while highlighting its possible use in drug design.

    PMID: 15548688 [PubMed - indexed for MEDLINE]


    : J Biol Chem. 2004 Dec 17;279(51):53782-8. Epub 2004 Oct 5.

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    Regulation of 2-oxoglutarate (alpha-ketoglutarate) dehydrogenase stability by the RING finger ubiquitin ligase Siah.

    Habelhah H, Laine A, Erdjument-Bromage H, Tempst P, Gershwin ME, Bowtell DD, Ronai Z.

    Department of Oncological Sciences, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029, USA.

    The 2-oxoglutarate dehydrogenase complex (OGHDC) (also known as the alpha-ketoglutarate dehydrogenase complex) is a rate-limiting enzyme in the mitochondrial Krebs cycle. Here we report that the RING finger ubiquitin-protein isopeptide ligase Siah2 binds to and targets OGDHC-E2 for ubiquitination-dependent degradation. OGDHC-E2 expression and activity are elevated in Siah2(-/-) cells compared with Siah2(+)(/)(+) cells. Deletion of the mitochondrial targeting sequence of OGDHC-E2 results in its cytoplasmic localization and rapid proteasome-dependent degradation in Siah2(+)(/)(+) but not in Siah2(-/-) cells. Significantly, because of its overexpression or disruption of the mitochondrial membrane potential, the release of OGDHC-E2 from mitochondria to the cytoplasm also results in its concomitant degradation. The role of the Siah family of ligases in the regulation of OGDHC-E2 stability is expected to take place under pathological conditions in which the levels of OGDHC-E2 are altered.

    PMID: 15466852 [PubMed - indexed for MEDLINE]


    : Genes Dev. 2004 Oct 1;18(19):2380-91.

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    The small ubiquitin-like modifier (SUMO) is required for gonadal and uterine-vulval morphogenesis in Caenorhabditis elegans.

    Broday L, Kolotuev I, Didier C, Bhoumik A, Gupta BP, Sternberg PW, Podbilewicz B, Ronai Z.

    Department of Oncological Sciences, Mount Sinai School of Medicine, New York, New York 10029, USA. limor.broday@mssm.edu

    The small ubiquitin-like modifier (SUMO) modification alters the subcellular distribution and function of its substrates. Here we show the major role of SUMO during the development of the Caenorhabditis elegans reproductive system. smo-1 deletion mutants develop into sterile adults with abnormal somatic gonad, germ line, and vulva. SMO-1::GFP reporter is highly expressed in the somatic reproductive system. smo-1 animals lack a vulval-uterine connection as a result of impaired ventral uterine pi-cell differentiation and anchor cell fusion. Mutations in the LIN-11 LIM domain transcription factor lead to a uterine phenotype that resembles the smo-1 phenotype. LIN-11 is sumoylated, and its sumoylation is required for its activity during uterine morphogenesis. Expression of a SUMO-modified LIN-11 in the smo-1 background partially rescued pi-cell differentiation and retained LIN-11 in nuclear bodies. Thus, our results identify the reproductive system as the major SUMO target during postembryonic development and highlight LIN-11 as a physiological substrate whose sumoylation is associated with the formation of a functional vulval-uterine connection. Copyright 2004 Cold Spring Harbor Laboratory Press

    PMID: 15466489 [PubMed - indexed for MEDLINE]

    : Mol Cell. 2004 Sep 24;15(6):843-4.

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    Comment on:

    ·         Mol Cell. 2004 Sep 10;15(5):713-25.


    JNKing Revealed.

    Ronai Z.

    Department of Oncological Sciences, Mount Sinai School of Medicine, New York, NY 10029, USA.

    In a recent issue of Molecular Cell, Sabapathy et al. (2004) demonstrate that JNK2 destabilizes c-Jun under nonstimulating conditions, whereas after stress, activated JNK1 phosphorylates c-Jun, resulting in its activation and stabilization, thereby highlighting functional differences between JNK1 and JNK2. Copyright 2004 Cell Press

    PMID: 15383272 [PubMed - indexed for MEDLINE]


    : J Cell Biol. 2004 Jun 21;165(6):857-67.

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    The LIM domain protein UNC-95 is required for the assembly of muscle attachment structures and is regulated by the RING finger protein RNF-5 in C. elegans.

    Broday L, Kolotuev I, Didier C, Bhoumik A, Podbilewicz B, Ronai Z.

    Ruttenberg Cancer Center, Mount Sinai School of Medicine, Box 1130, New York, NY 10029, USA.

    Here, we describe a new muscle LIM domain protein, UNC-95, and identify it as a novel target for the RING finger protein RNF-5 in the Caenorhabditis elegans body wall muscle. unc-95(su33) animals have disorganized muscle actin and myosin-containing filaments as a result of a failure to assemble normal muscle adhesion structures. UNC-95 is active downstream of PAT-3/beta-integrin in the assembly pathways of the muscle dense body and M-line attachments, and upstream of DEB-1/vinculin in the dense body assembly pathway. The translational UNC-95::GFP fusion construct is expressed in dense bodies, M-lines, and muscle-muscle cell boundaries as well as in muscle cell bodies. UNC-95 is partially colocalized with RNF-5 in muscle dense bodies and its expression and localization are regulated by RNF-5. rnf-5(RNAi) or a RING domain deleted mutant, rnf-5(tm794), exhibit structural defects of the muscle attachment sites. Together, our data demonstrate that UNC-95 constitutes an essential component of muscle adhesion sites that is regulated by RNF-5.

    PMID: 15210732 [PubMed - indexed for MEDLINE]


    : Cell. 2004 Jun 25;117(7):941-52.

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    Comment in:

    ·         Cell. 2004 Jun 25;117(7):851-3.


    Siah2 regulates stability of prolyl-hydroxylases, controls HIF1alpha abundance, and modulates physiological responses to hypoxia.

    Nakayama K, Frew IJ, Hagensen M, Skals M, Habelhah H, Bhoumik A, Kadoya T, Erdjument-Bromage H, Tempst P, Frappell PB, Bowtell DD, Ronai Z.

    Department of Oncological Sciences, Mount Sinai School of Medicine, New York, New York 10029, USA.

    Hypoxia-inducible factor-1alpha (HIF1alpha) is a central regulator of the cellular response to hypoxia. Prolyl-hydroxylation of HIF1alpha by PHD enzymes is prerequisite for HIF1alpha degradation. Here, we demonstrate that the abundance of PHD1 and PHD3 are regulated via their targeting for proteasome-dependent degradation by the E3 ubiquitin ligases Siah1a/2, under hypoxia conditions. Siah2 null fibroblasts exhibit prolonged PHD3 half-life, resulting in lower levels of HIF1alpha expression during hypoxia. Significantly, hypoxia-induced HIF1alpha expression was completely inhibited in Siah1a/2 null cells, yet could be rescued upon inhibition of PHD3 by RNAi. Siah2 targeting of PHD3 for degradation increases upon exposure to even mild hypoxic conditions, which coincides with increased Siah2 transcription. Siah2 null mice subjected to hypoxia displayed an impaired hyperpneic respiratory response and reduced levels of hemoglobin. Thus, the control of PHD1/3 by Siah1a/2 constitutes another level of complexity in the regulation of HIF1alpha during hypoxia.

    PMID: 15210114 [PubMed - indexed for MEDLINE]


    : Proc Natl Acad Sci U S A. 2004 Apr 6;101(14):4990-5. Epub 2004 Mar 29.

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    Melanoma cells transfected to express CD83 induce antitumor immunity that can be increased by also engaging CD137.

    Yang S, Yang Y, Raycraft J, Zhang H, Kanan S, Guo Y, Ronai Z, Hellstrom I, Hellstrom KE.

    Pacific Northwest Research Institute, Seattle, WA 98122, USA.

    Interactions between CD83 and its ligand(s) can up-regulate immune responses. M2-CD83 cells, derived by transfecting the M2 clone of mouse melanoma K1735 cells to express mouse CD83, were rejected by syngeneic mice, unless they were injected with a CD83Ig fusion protein. Rejection was mediated by CD4+ and CD8+ T cells plus natural killer cells, whereas rejection of M2-1D8 cells, which express anti-CD137 single-chain variable region fragments (scFv), occurs in the absence of CD8+ T cells. Furthermore, the tumor specificity of the immunity induced by the two cell lines differed. Immunization with live or mitomycin C-treated M2-CD83 cells prevented outgrowth of transplanted M2-WT cells and had therapeutic efficacy against established M2-WT tumors. A highly metastatic clone of K1735 cells, SW1-C, and its subline SW1-P2, which expresses an activating transcription factor 2-driven peptide, were then studied because they have particularly low immunogenicity. Neither SW1-C nor SW1-P2 cells became rejectable after expression of CD83 or anti-CD137 scFv. However, outgrowth of cells from either line was delayed in mice immunized against M2-CD83 or M2-1D8 cells, and immunization with a mixture of mitomycin C-treated cells from M2-CD83 plus M2-1D8 prevented tumor formation by SW1-P2 cells in five of five and by SW1-C cells in three of five mice. We conclude that M2 cells expressing CD83 can induce a tumor-destructive immune response also against SW1 cells and that this response can be made more effective by combining them with M2 cells expressing anti-CD137 scFv. A similar approach may be therapeutically beneficial against certain human cancers.

    PMID: 15051893 [PubMed - indexed for MEDLINE]


    : Proc Natl Acad Sci U S A. 2004 Mar 23;101(12):4222-7. Epub 2004 Mar 9.

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