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]

 
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]

 
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]

  
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]

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]

 
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]

 
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]

 
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]

 
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]

  
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]

 
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]

  
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]

 
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]

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]

 
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]

 
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]

 
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]

 
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]

 
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]

 
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]

 
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]

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]

  
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]

  
Transcriptional switch by activating transcription factor 2-derived peptide sensitizes melanoma cells to apoptosis and inhibits their tumorigenicity.

Bhoumik A, Jones N, Ronai Z.

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

The notorious resistance of melanoma cells to drug treatment can be overcome by expression of a 50-aa peptide derived from activating transcription factor 2 (ATF2(50-100)). Here we demonstrate that ATF2(50-100) induced apoptosis by sequestering ATF2 to the cytoplasm, thereby inhibiting its transcriptional activities. Furthermore, ATF2(50-100) binds to c-Jun N-terminal kinase (JNK) and increases its activity. Mutation within ATF2(50-100) that impairs association with JNK and the inhibition of JNK or c-Jun expression by RNA interference (RNAi) reduces the degree of ATF2(50-100)-induced apoptosis. In contrast, TAM67, a dominant negative of the Jun family of transcription factors, or JunD RNAi attenuates sensitization of melanoma cells expressing ATF2(50-100) to apoptosis after treatment with anisomycin, which is used as a model drug. Mutations within the JNK binding region of ATF2(50-100) or expression of TAM67 or JunD RNAi attenuates inhibition of melanoma's tumorigenicity by ATF2(50-100). We conclude that inhibition of ATF2 in concert with increased JNK/Jun and JunD activities is central for the sensitization of melanoma cells to apoptosis and inhibition of their tumorigenicity.

PMID: 15010535 [PubMed - indexed for MEDLINE]

 
Structural mechanism of the bromodomain of the coactivator CBP in p53 transcriptional activation.

Mujtaba S, He Y, Zeng L, Yan S, Plotnikova O, Sachchidanand, Sanchez R, Zeleznik-Le NJ, Ronai Z, Zhou MM.

Department of Physiology and Biophysics, New York University, 1425 Madison Avenue, New York, NY 10029, USA.

Lysine acetylation of the tumor suppressor protein p53 in response to a wide variety of cellular stress signals is required for its activation as a transcription factor that regulates cell cycle arrest, senescence, or apoptosis. Here, we report that the conserved bromo-domain of the transcriptional coactivator CBP (CREB binding protein) binds specifically to p53 at the C-terminal acetylated lysine 382. This bromodomain/acetyl-lysine binding is responsible for p53 acetylation-dependent coactivator recruitment after DNA damage, a step essential for p53-induced transcriptional activation of the cyclin-dependent kinase inhibitor p21 in G1 cell cycle arrest. We further present the three-dimensional nuclear magnetic resonance structure of the CBP bromodomain in complex with a lysine 382-acetylated p53 peptide. Using structural and biochemical analyses, we define the molecular determinants for the specificity of this molecular recognition.

PMID: 14759370 [PubMed - indexed for MEDLINE]

  
Ubiquitination and translocation of TRAF2 is required for activation of JNK but not of p38 or NF-kappaB.

Habelhah H, Takahashi S, Cho SG, Kadoya T, Watanabe T, Ronai Z.

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

TRAF2 is a RING finger protein that regulates the cellular response to stress and cytokines by controlling JNK, p38 and NF-kappaB signaling cascades. Here, we demonstrate that TRAF2 ubiquitination is required for TNFalpha-induced activation of JNK but not of p38 or NF-kappaB. Intact RING and zinc finger domains are required for TNFalpha-induced TRAF2 ubiquitination, which is also dependent on Ubc13. TRAF2 ubiquitination coincides with its translocation to the insoluble cellular fraction, resulting in selective activation of JNK. Inhibition of Ubc13 expression by RNAi resulted in inhibition of TNFalpha-induced TRAF2 translocation and impaired activation of JNK but not of IKK or p38. TRAF2 aggregates in the cytoplasm, as seen in Hodgkin-Reed-Sternberg lymphoma cells, resulting in constitutive NF-kappaB activity but failure to activate JNK. These findings demonstrate that the TRAF2 RING is required for Ubc13-dependent ubiquitination, resulting in translocation of TRAF2 to an insoluble fraction and activation of JNK, but not of p38 or NF-kappaB. Altogether, our findings highlight a novel mechanism of TRAF2-dependent activation of diverse signaling cascades that is impaired in Hodgkin-Reed-Sternberg cells.

PMID: 14713952 [PubMed - indexed for MEDLINE]

 
Subcellular localization of activating transcription factor 2 in melanoma specimens predicts patient survival.

Berger AJ, Kluger HM, Li N, Kielhorn E, Halaban R, Ronai Z, Rimm DL.

Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.

The transcription factor activating transcription factor 2 (ATF2) has been shown to be associated with melanocytic oncogenesis and melanoma tumor proliferation in preclinical models. The clinical significance of ATF2 expression is unknown. To determine the prognostic value of ATF2 in melanoma, we evaluated the pattern and level of ATF2 expression in a large cohort of melanoma specimens. Immunohistochemical staining was performed on a tissue microarray representing 544 patients with a mean follow-up time of 60 months. Expression was evaluated semiquantitatively and correlated with overall survival and other clinicopathological data. Strong cytoplasmic ATF2 expression was associated with primary specimens rather than metastases (P < 0.0001) and with better survival (P = 0.0003). Strong nuclear ATF2 expression was associated with metastatic specimens (P < 0.0001) and with poor survival (P = 0.0008). Patients who had both weak cytoplasmic and strong nuclear ATF2 staining had the worst outcome, both among the full cohort of patients (P < 0.0001) and among the patients with localized disease (n = 269; P < 0.0001). On multivariate analysis of the primary cutaneous specimens, weak cytoplasmic staining and strong nuclear staining was an independent predictor of poor outcome, as was Clark level. Nuclear ATF2 is likely to be transcriptionally active, whereas cytoplasmic ATF2 probably represents an inactive form. These findings support other preclinical findings in which transcriptionally active ATF2 is involved in tumor progression-proliferation in melanoma. Moreover, our findings suggest that ATF2 might be a useful prognostic marker in early-stage melanoma.

PMID: 14678960 [PubMed - indexed for MEDLINE]

 
c-Jun-NH2 kinase (JNK) contributes to the regulation of c-Myc protein stability.

Alarcon-Vargas D, Ronai Z.

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

In accord with the central role c-Myc plays in control of cell growth and death, the stability of this protein is tightly regulated. Although the NH2-terminal domain of c-Myc has been implicated in the regulation of its stability, c-Myc-S, which lacks this domain, is equally unstable, pointing to the role of additional domains in the regulation of c-Myc stability. Our former studies revealed that amino acids (aa) 127-189 of c-Myc are responsible for stress-induced stability of the c-Myc protein. This region of c-Myc shares homology with the delta domain of c-Jun, which is required for JNK association and subsequent targeting of c-Jun for ubiquitination under non-stressed growth conditions. Here we demonstrate that JNK associates with, and mediates, c-Myc ubiquitination and degradation. Addition of JNK increased the degree of c-Myc ubiquitination in in vitro ubiquitination reactions. Increased c-Myc stability following MEKK1/JNK stimuli is abolished upon mutation within the delta-like domain of c-Myc (aa 166-181), as well as deletion of aa 127-189. Significantly, inhibition of JNK expression via small interfering RNA increased c-Myc protein expression. Similarly, squelching JNK association with c-Myc by overexpression of a peptide corresponding to aa 127-189 of c-Myc increased endogenous c-Myc stability and elevated the fraction of cells within the G2/M phase of the cell cycle. In all, these findings point to the contribution of JNK to the regulation of c-Myc protein stability under normal growth conditions.

PMID: 14625288 [PubMed - indexed for MEDLINE]

  
RNF5, a RING finger protein that regulates cell motility by targeting paxillin ubiquitination and altered localization.

Didier C, Broday L, Bhoumik A, Israeli S, Takahashi S, Nakayama K, Thomas SM, Turner CE, Henderson S, Sabe H, Ronai Z.

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

RNF5 is a RING finger protein found to be important in the growth and development of Caenorhabditis elegans. The search for RNF5-associated proteins via a yeast two-hybrid screen identified a LIM-containing protein in C. elegans which shows homology with human paxillin. Here we demonstrate that the human homologue of RNF5 associates with the amino-terminal domain of paxillin, resulting in its ubiquitination. RNF5 requires intact RING and C-terminal domains to mediate paxillin ubiquitination. Whereas RNF5 mediates efficient ubiquitination of paxillin in vivo, protein extracts were required for in vitro ubiquitination, suggesting that additional modifications and/or an associated E3 ligase assist RNF5 targeting of paxillin ubiquitination. Mutant Ubc13 efficiently inhibits RNF5 ubiquitination, suggesting that RNF5 generates polychain ubiquitin of the K63 topology. Expression of RNF5 increases the cytoplasmic distribution of paxillin while decreasing its localization within focal adhesions, where it is primarily seen under normal growth. Concomitantly, RNF5 expression results in inhibition of cell motility. Via targeting of paxillin ubiquitination, which alters its localization, RNF5 emerges as a novel regulator of cell motility.

PMID: 12861019 [PubMed - indexed for MEDLINE]

 
ERK and PI3K negatively regulate STAT-transcriptional activities in human melanoma cells: implications towards sensitization to apoptosis.

Krasilnikov M, Ivanov VN, Dong J, Ronai Z.

Institute of Carcinogenesis, NN Blochin Cancer Research Center, Moscow, Russia.

Signal transducers and activators of transcription (STAT) proteins nuclear translocation and transcriptional activity are regulated by diverse protein kinases in response to extracellular stimuli by cytokines, growth factors and stress. Using two melanoma-derived cell lines that exhibit marked differences in basal activities of MAPKs and PI3K-AKT, we studied changes both in STAT activities and in their sensitization to apoptosis. Activating mutations of B-RAF (T1796A) and impaired expression of PTEN are detected in LU1205, but not in FEMX melanoma cells, and are reflected in high basal levels of expression and activities of MAPKs and PI3K-AKT. Treatment with either PD98059 (PD) or LY294002 (LY), the pharmacological inhibitors of MEK-ERK and PI3K, respectively, markedly increased GAS-Luc activity in LU1205, but not in FEMX cells. Tyrosine phosphorylation of STAT3/5 and of JAK2 also increased upon treatment of LU1205 cells with either PD or LY, suggesting that constitutive active MAPK and PI3K signals inhibit tyrosine phosphorylation of JAK/STATs. Treatment of FEMX and LU1205 with PD sensitized the cells to apoptosis, albeit by TNFalpha and TRAIL death cascades, respectively, indicating that additional yet distinct targets are affected by each signaling pathway. Indeed, the combination of LY and PD treatment synergistically increased the apoptosis of LU1205 and FEMX cells. Overall, whereas PI3K and MAPK downregulate JAK-STAT signaling, additional targets are affected by these kinases and sensitizes melanoma to apoptosis via distinct death cascades.

PMID: 12821943 [PubMed - indexed for MEDLINE]

 
Death receptors and melanoma resistance to apoptosis.

Ivanov VN, Bhoumik A, Ronai Z.

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

Impaired ability to undergo programmed cell death in response to a wide range of external stimuli acquires melanomas a selective advantage for progression and metastasis as well as their notorious resistance to therapy. Better understanding of mechanisms that govern apoptosis has enabled identification of diverse routes by which melanomas manage to escape stimuli of apoptosis. Changes at genomic, transcriptional and post-translational levels of G-proteins and protein kinases (Ras, B-Raf) and their transcription factor effectors (c-Jun, ATF2, Stat3 and NF-kappaB) affects TNF, Fas and TRAIL receptors, which play important roles in acquiring melanoma's resistance to apoptosis. Here, we summarize our current understanding of changes that alters the regulation of death receptors during melanoma development.

PMID: 12789291 [PubMed - indexed for MEDLINE]

  
FAP-1 association with Fas (Apo-1) inhibits Fas expression on the cell surface.

Ivanov VN, Lopez Bergami P, Maulit G, Sato TA, Sassoon D, Ronai Z.

Ruttenberg Cancer Center, Department of Molecular and Developmental Biology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029-6574, USA.

As revealed by intracellular pools of nonactive Fas (Apo-1), export of Fas to the cell surface is often impaired in human tumors, thereby inactivating Fas ligand-mediated apoptosis. Here, we demonstrate that association with Fas-associated phosphatase 1 (FAP-1) attenuates Fas export to the cell surface. Forced expression of FAP-1 reduces cell surface Fas levels and increases the intracellular pool of Fas within the cytoskeleton network. Conversely, expression of dominant-negative forms of FAP-1, or inhibition of FAP-1 expression by short interfering RNA, efficiently up-regulates surface expression of Fas. Inhibition of Fas surface expression by FAP-1 depends on its association with the C terminus of Fas. Mutation within amino acid 275 results in decreased association with FAP-1 and greater export of Fas to the cell surface in melanomas, normal fibroblasts, or Fas null cells. Identifying the role of FAP-1 in binding to, and consequently inhibition of, Fas export to the cell surface provides novel insight into the mechanism underlying the regulation of Fas trafficking, which is commonly impaired in advanced tumors with FAP-1 overexpression.

PMID: 12724420 [PubMed - indexed for MEDLINE]

 
SUMO in cancer--wrestlers wanted.

Alarcon-Vargas D, Ronai Z.

The Ruttenberg Cancer Center; Mount Sinai School of Medicine; New York, New York 10029, USA.

SUMO (small ubiquitin-related modifier) represents a class of ubiquitin-like proteins that is conjugated, like ubiquitin, by a set of enzymes to cellular regulatory proteins, including oncogenes and tumor suppressor genes, that play key roles in the control of cell growth, differentiation and apoptosis. SUMO conjugation affects substrates' subcellular localization and stability as well as transcriptional activities. Given the substrates involved, protein SUMOylation would be expected to be important in the course of tumorigenesis and, accordingly, altered in human cancer. Although evidence to support this notion is still scarce, this review summarizes the current knowledge of protein sumoylation and highlights the challenges to be addressed in the context of human cancer.

PMID: 12432270 [PubMed - indexed for MEDLINE]

 
Stress-induced decrease in TRAF2 stability is mediated by Siah2.

Habelhah H, Frew IJ, Laine A, Janes PW, Relaix F, Sassoon D, Bowtell DD, Ronai Z.

Ruttenberg Cancer Center and Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, NY 10029, USA.

TRAF2 serves as a central regulator of the cellular response to stress and cytokines through the regulation of key stress-signaling cascades. Here we demonstrate that wild-type, but not RING mutant, Siah2 targets TRAF2 for ubiquitylation and degradation in vitro. Siah2 mediates equally efficient ubiquitylation of RING mutant TRAF2. In vivo, Siah2 primarily targets TRAF2 for degradation under stress conditions. Tumor necrosis factor-alpha (TNF-alpha) and actinomycin D treatment results in accelerated TRAF2 degradation in wild-type mouse embryo fibroblasts (MEFs), as compared with Siah2(-/-) cells. Similarly, TRAF2 half-life is prolonged in Siah2(-/-) compared with wild-type MEFs subjected to stress stimuli. Siah2 efficiently decreases TNF-alpha-dependent induction of JNK activity and transcriptional activation of NF-kappaB. Apoptosis induced by TNF-alpha and actinomycin D treatment is increased upon expression of Siah2, or attenuated upon expression of TRAF2 or RING mutant Siah2. Identifying Siah2 as a regulator of TRAF2 stability reveals its role in the regulation of TRAF2 signaling following exposure to stress.

PMID: 12411493 [PubMed - indexed for MEDLINE]

  
An ATF2-derived peptide sensitizes melanomas to apoptosis and inhibits their growth and metastasis.

Bhoumik A, Huang TG, Ivanov V, Gangi L, Qiao RF, Woo SL, Chen SH, Ronai Z.

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

Melanomas are among the aggressive tumor types because of their notorious resistance to treatment and their high capacity to metastasize. ATF2 is among transcription factors implicated in the progression of melanoma and its resistance to treatment. Here we demonstrate that the expression of a peptide spanning amino acids 50-100 of ATF2 (ATF2(50-100)) reduces ATF2 transcriptional activities while increasing the expression and activity of c-Jun. Altering the balance of Jun/ATF2 transcriptional activities sensitized melanoma cells to apoptosis, an effect that could be attenuated by inhibiting c-Jun. Inhibition of ATF2 via RNA interference likewise increased c-Jun expression and primed melanoma cells to undergo apoptosis. Growth and metastasis of SW1 and B16F10 mouse melanomas were inhibited by ATF2(50-100) to varying degrees up to a complete regression, depending on the mode (inducible, constitutive, or adenoviral delivery) of its expression.

PMID: 12208865 [PubMed - indexed for MEDLINE]

 
Regulation of c-myc stability by selective stress conditions and by MEKK1 requires aa 127-189 of c-myc.

Alarcon-Vargas D, Tansey WP, Ronai Z.

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

C-myc availability is central for its ability to serve as a regulator of cell growth and death. Here we study the regulation of c-myc protein stability and identify domains of c-myc that are important for its stabilization in response to stress kinases activated following selective stress conditions. UV-irradiation elicited an increase in c-myc protein levels, which could be attenuated by inhibitors of stress kinases but also by actinomycin D-inhibitor of transcription. Inhibition of protein synthesis results in a noticeable decrease in c-myc levels, further pointing to the short half-life of the protein. However, in combination with tumor necrosis factor-alpha (TNF-alpha), cycloheximide efficiently increases steady-state levels of c-myc, suggesting that selective stress conditions are required to increase c-myc protein stability. Expression of MEKK1, an upstream regulator of protein kinases that has been implicated in mediating the response to diverse stress conditions, also results in an efficient increase in the half-life of c-myc protein. To map c-myc domains that are responsive to stress kinases, we monitored changes in the level of c-myc deletion mutants following MEKK1 expression. Of the seven c-myc deletion mutants analysed, the domain spanning amino acids 127-189 was found to be required for MEKK1-dependent increase in c-myc stability. In all, the present study identifies a novel domain that is important for the regulation of c-myc stability by stress kinases in response to selective stress conditions.

PMID: 12080469 [PubMed - indexed for MEDLINE]

 
p53-Mdm2--the affair that never ends.

Alarcon-Vargas D, Ronai Z.

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

The p53-Mdm2 paradigm represents the best-studied relationship between a tumor suppressor gene which functions as a transcription factor and an oncogene, which functions primarily as an E3 protein ligase. The intimate relationship between these two partners has expanded to include almost every cellular biological system - from development, to growth control and programmed cell death. The affair between Mdm2 and p53 is closely controlled by a complex array of post-translational modifications, which in turn dictates the stability and activity of p53 and Mdm2. Functional diversity depends on the association with a large subset of partner proteins, which dictates the type of activity and corresponding selectivity. Here we summarize the current understanding of post-translational modifications and their effect on conformation-based functional relationship between Mdm2 and p53, as it pertains to their diverse cellular biological functions.

PMID: 11960904 [PubMed - indexed for MEDLINE]

 
Regulation of Fas expression by STAT3 and c-Jun is mediated by phosphatidylinositol 3-kinase-AKT signaling.

Ivanov VN, Krasilnikov M, Ronai Z.

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

Cooperation between STAT3 and c-Jun results in suppression of Fas Receptor (FasR) transcription, which is often seen in advanced human tumors. To identify requirements for STAT3-Jun cooperation, we elucidated the role of protein kinases that affect both transcription factors. The phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway was found capable of down-regulating both STAT3- and c-Jun-dependent transcription, resulting in derepression of FasR transcription. Conversely, inhibition of PI3K-AKT signaling via the specific pharmacological inhibitor LY294002 up-regulated AP1/Jun- and STAT-dependent transcriptional activities, resulting in suppression of the FasR promoter activities and decreased FasR surface expression. PI3K-AKT's ability to affect FasR transcription was not observed in c-jun null fibroblasts, suggesting that c-Jun is required for PI3K/AKT-mediated regulation of FasR transcription. Interestingly, the dominant negative form of Rac1 (RacN17) was also efficient in relieving FasR expression, suggesting that the increase in FasR expression following AKT stimuli could be mediated via AKT ability to elicit suppression of Rac1, which in turn decreases JNK activities and c-Jun phosphorylation. Overall, our findings demonstrate that through its negative effects on both c-Jun and STAT3, the PI3K-AKT pathway disrupts cooperation between c-Jun and STAT3, which is required for silencing the FasR promoter, resulting in increased expression of surface FasR and concomitant sensitization to FasL-mediated programmed cell death.

PMID: 11733515 [PubMed - indexed for MEDLINE]

 
Gene mutation as a target for early detection in cancer diagnosis.

Minamoto T, Ronai Z.

Cancer Research Institute, Kanazawa University, Kanazawa, Japan.

The increasing number of genetic aberrations implicated in the development of human cancer has prompted a search to detect them at the earliest possible stage of their formation. Of the many such genetic changes identified thus far, relatively few meet the standard for markers in early diagnosis and prognosis, namely that the genetic modifications occur during the early onset phase of cancer development. Parallel to the increasing number of such genes is the growing availability of technologies using more powerful and cost-efficient methods that enable mass screening for genetic alterations. The purpose of this review is to summarize the currently available genes that can serve as markers for early detection of cancers and methods that allow their detection.

PMID: 11738944 [PubMed - indexed for MEDLINE]

  
TIP49b, a regulator of activating transcription factor 2 response to stress and DNA damage.

Cho SG, Bhoumik A, Broday L, Ivanov V, Rosenstein B, Ronai Z.

The Ruttenberg Cancer Center, Mount Sinai School of Medicine, 1 Gustave Levy Place, New York, NY 10029, USA.

Activating transcription factor 2 (ATF2/CRE-BP1) is implicated in transcriptional control of stress-responsive genes. A yeast two-hybrid screen identified TBP-interacting protein 49b (TIP49b), a component of the INO80 chromatin-remodeling complex, as a novel ATF2-interacting protein. TIP49b's association with ATF2 is phosphorylation dependent and requires amino acids 150 to 248 of ATF2 (ATF2(150-248)), which are implicated in intramolecular inhibition of ATF2 transcriptional activities. Forced expression of TIP49b efficiently attenuated ATF2 transcriptional activities under normal growth conditions as well as after UV treatment, ionizing irradiation, or activation of p38 kinase, all of which induced ATF2 phosphorylation and increased TIP49b-ATF2 association. Constitutive expression of ATF2(150-248) peptide outcompeted TIP49b interaction with ATF2 and alleviated the suppression of ATF2 transcriptional activities. Expression of ATF2(150-248) in fibroblasts or melanoma but not in ATF2-null cells caused a profound G(2)M arrest and increased degree of apoptosis following irradiation. The interaction between ATF2 and TIP49b constitutes a novel mechanism that serves to limit ATF2 transcriptional activities and highlights the central role of ATF2 in the control of the cell cycle and apoptosis in response to stress and DNA damage.

PMID: 11713276 [PubMed - indexed for MEDLINE]

To check out earlier publications from the RONAI lab – please visit Pub Med