MCB

Regulation of {beta}-catenin by a Novel Nongenomic Action of Thyroid Hormone {beta} Receptor.

Publication Date: 2008 May 12 PMID: 18474620
Authors: Guigon, C. J. - Zhao, L. - Lu, C. - Willingham, M. C. - Cheng, S. Y.
Journal: Mol Cell Biol

We previously created a knockin mutant mouse harboring a dominantly negative mutant thyroid hormone receptor beta (TRbeta(PV/PV) mouse) that spontaneously develops follicular thyroid carcinoma similar to human thyroid cancer. We found that beta-catenin, which plays a critical role in oncogenesis, was highly elevated in thyroid tumors of TRbeta(PV/PV) mice. We sought to understand the molecular basis underlying aberrant accumulation of beta-catenin by mutations of TRbeta in vivo. Cell-based studies showed that thyroid hormone (T3) induced the degradation of beta-catenin in cells expressing TRbeta via proteasomal pathways. In contrast, no T3-induced degradation occurred in cells expressing PV. In vitro binding studies and cell-based analyses revealed that beta-catenin physically associated with the unliganded TRbeta or PV. However, in the presence of T3, beta-catenin was dissociated from TRbeta/beta-catenin complexes, but not from PV/beta-catenin complexes. beta-catenin signaling was repressed by T3 in TRbeta-expressing cells through decreasing beta-catenin-mediated transcription activity and target gene expression, whereas sustained beta-catenin signaling was observed in PV-expressing cells. The stabilization of beta-catenin, via association with a mutated TRbeta, represents a novel activating mechanism of the oncogenic beta-catenin that could contribute to thyroid carcinogenesis in TRbeta(PV/PV) mice.

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Dynamics of RASSF1A/MOAP-1 Association with Death Receptors.

Publication Date: 2008 May 12 PMID: 18474619
Authors: Foley, C. J. - Freedman, H. - Choo, S. L. - Onyskiw, C. - Fu, N. Y. - Yu, V. C. - Tuszynski, J. - Pratt, J. C. - Baksh, S.
Journal: Mol Cell Biol

RASSF1A is a tumor suppressor protein involved in death receptor-dependent apoptosis utilizing the Bax-interacting protein, MOAP-1 (previously referred to as MAP-1). However, the dynamics of death receptor recruitment of RASSF1A and MOAP-1 are still not understood. We have now detailed recruitment to death receptors (TNF-R1 and TRAIL-R1/DR4), and identified domains of RASSF1A and MOAP-1 that are required for death receptor interaction. Upon TNFalpha stimulation, the C-terminal region of MOAP-1 associated with the death domain of TNF-R1; subsequently, RASSF1A was recruited to MOAP-1/TNF-R1 complexes. Prior to recruitment to TNF-R1/MOAP-1 complexes, RASSF1A homodimerization was lost. RASSF1A associated with the TNF-R1/MOAP-1 or TRAIL-R1/MOAP-1 complex via its N-terminal cysteine-rich (C1) domain containing a potential zinc finger binding motif. Importantly, TNF-R1 association domains on both MOAP-1 and RASSF1A were essential for death receptor-dependent apoptosis. The association of RASSF1A and MOAP-1 with death receptors involves an ordered recruitment to receptor complexes to promote cell death and inhibit tumor formation.

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MicroRNA-126 Regulates HOXA9 by Binding to the Homeobox.

Publication Date: 2008 May 12 PMID: 18474618
Authors: Shen, W. F. - Hu, Y. L. - Uttarwar, L. - Passegue, E. - Largman, C.
Journal: Mol Cell Biol

The PicTar program predicted that miR-126, miR-145, and let-7s target highly conserved sites within the Hoxa9 homeobox. There is increased nucleotide constraint in the three microRNA seed sites among Hoxa9 genes beyond that required to maintain protein identity, suggesting additional functional conservation. In preliminary experiments, forced expression of these microRNAs in Hoxa9-immortalized bone marrow cells down-regulated HOXA9 protein and caused loss of biological activity. The microRNAs were shown to target their predicted sites within the homeobox. miR-126 and Hoxa9 mRNA are co-expressed in hematopoietic stem cells, and down regulated in parallel during progenitor cell differentiation, but miR-145 is barely detectable in hematopoietic cells and let-7s are highly expressed in bone marrow progenitors, suggesting that miR-126 may function in normal hematopoietic cells to modulate HOXA9 protein. In support of this hypothesis, expression of miR-126 alone in MLL-ENL-immortalized bone marrow cells decreased endogenous HOXA9 protein while, inhibition of endogenous miR-126 increased expression of HOXA9 in F9 cells.

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COMBINED VHLH AND PTEN MUTATION CAUSES GENITAL TRACT CYSTADENOMA AND SQUAMOUS METAPLASIA.

Publication Date: 2008 May 12 PMID: 18474617
Authors: Frew, I. J. - Minola, A. - Georgiev, S. - Hitz, M. - Moch, H. - Richard, S. - Vortmeyer, A. O. - Krek, W.
Journal: Mol Cell Biol

Patients with von Hippel-Lindau (VHL) disease develop tumours in a range of tissues but existing mouse models of Vhlh mutation have failed to reproduce these lesions. Epididymal cystadenomas arise frequently in VHL patients but VHL mutation alone is believed to be insufficient for tumour formation, implying a requirement for cooperating mutations in epididymal pathogenesis. Here we show that epididymal cystadenomas from VHL patients frequently also lack expression of the PTEN tumour suppressor and display activation of PI3K pathway signalling. Strikingly, while conditional inactivation of either Vhlh or Pten in epithelia of the mouse genital tract fails to produce a tumour phenotype, their combined deletion causes benign genital tract tumours with regions of squamous metaplasia and cystadenoma. The latter are histologically identical to lesions found in VHL patients. Importantly, these lesions are characterised by expansion of basal stem cells, high levels of expression and activity of HIF1alpha and HIF2alpha and dysregulation of PI3K signalling. Our studies suggest a model for cooperative tumour suppression in which inactivation of PTEN facilitates epididymal cystadenoma genesis initiated by loss of VHL.

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PR-Set7 establishes a repressive trans-tail histone code that regulates differentiation.

Publication Date: 2008 May 12 PMID: 18474616
Authors: Sims, J. K. - Rice, J. C.
Journal: Mol Cell Biol

Post-translational modifications of the DNA-associated histone proteins play fundamental roles in eukaryotic transcriptional regulation. We previously discovered a novel trans-tail histone code involving monomethylated histone H4 lysine 20 (H4K20) and H3 lysine 9 (H3K9), however, the mechanisms that establish this code and its function in transcription were unknown. In this report we demonstrate that H3K9 monomethylation is dependent upon the PR-Set7 H4K20 monomethyltransferase, but independent of its catalytic function, indicating that PR-Set7 recruits an H3K9 monomethyltransferase to establish the trans-tail histone code. We determined that this histone code is involved in a transcriptional regulatory pathway in vivo whereby monomethylated H4K20 binds the L3MBTL1 repressor protein to repress specific genes including RUNX1, a critical regulator of hematopoietic differentiation. The selective loss of monomethylated H4K20 at the RUNX1 promoter resulted in the displacement of L3MBTL1 and concomitant increase in RUNX1 transcription. Importantly, the lack of monomethylated H4K20 in the human K562 multipotent cell line was specifically associated with spontaneous megakaryocytic differentiation, in part, by activating RUNX1. Our findings demonstrate that this newly described repression pathway is required for regulating proper megakaryopoeisis and suggests that it is likely to function similarly in other multipotent cell types to regulate specific differentiation pathways.

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Visual Analysis of the Yeast 5S rRNA Gene Transcriptome: Regulation and Role of La Protein.

Publication Date: 2008 May 12 PMID: 18474615
Authors: French, S. L. - Osheim, Y. N. - Schneider, D. A. - Sikes, M. L. - Fernandez, C. F. - Copela, L. A. - Misra, V. A. - Nomura, M. - Wolin, S. L. - Beyer, A. L.
Journal: Mol Cell Biol

5S rRNA genes from Saccharomyces cerevisiae were examined by Miller chromatin spreading, representing the first quantitative analysis of RNA polymerase III genes in situ by electron microscopy. These very short genes, approximately 132 nt, were engaged by one to three RNA polymerases. Analysis in different growth conditions and in strains with a 4-fold range in gene copy number revealed regulation at two levels: number of active genes and polymerase loading per gene. Repressive growth conditions (rapamycin or post-exponential growth) led first to fewer active genes, followed by lower polymerase loading per active gene. Pol III elongation rate was estimated in the range of 60-75 nt/sec with a reinitiation interval of approximately 1.2 sec. The yeast La protein, Lhp1, was associated with 5S genes. Its absence had no discernible effect on amount or size of 5S RNA produced, yet resulted in more polymerases per gene on average, consistent with a non-rate limiting role for Lhp1 in a process such as polymerase release/recycling upon transcription termination.

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Receptor-Type Protein Tyrosine Phosphatase {zeta} -Pleiotrophin Signaling Controls Endocytic Trafficking of DNER that Regulates Neuritogenesis.

Publication Date: 2008 May 12 PMID: 18474614
Authors: Fukazawa, N. - Yokoyama, S. - Eiraku, M. - Kengaku, M. - Maeda, N.
Journal: Mol Cell Biol

PTPzeta is a receptor-type protein tyrosine phosphatase that uses pleiotrophin as a ligand. Pleiotrophin inactivates the phosphatase activity of PTPzeta, resulting in the increase of tyrosine phosphorylation levels of its substrates. We studied the functional interaction between PTPzeta and DNER, a Notch-related transmembrane protein highly expressed in cerebellar Purkinje cells. PTPzeta and DNER displayed patchy co-localization in the dendrites of Purkinje cells, and immunoprecipitation experiments indicated that these proteins formed complexes. Several tyrosine residues in and adjacent to the tyrosine-based and the second C-terminal sorting motifs of DNER were phosphorylated and were dephosphorylated by PTPzeta, and phosphorylation of these tyrosine residues resulted in the accumulation of DNER on the plasma membrane. DNER mutants lacking sorting motifs accumulated on the plasma membrane of Purkinje cells and Neuro-2A cells, and induced their process extension. While normal DNER was actively endocytosed and inhibited the retinoic acid-induced neurite outgrowth of Neuro-2A cells, pleiotrophin stimulation increased tyrosine phosphorylation level of DNER and suppressed the endocytosis of this protein, which led to the reversal of this inhibition, thus allowing neurite extension. These observations suggest that pleiotrophin-PTPzeta signaling controls subcellular localization of DNER, and thereby regulates neuritogenesis.

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APLF (C2orf13) is a Novel Component of Poly (ADP-ribose) Signalling in Mammalian Cells.

Publication Date: 2008 May 12 PMID: 18474613
Authors: Rulten, S. - Cortes-Ledesma, F. - Guo, L. - Caldecott, K. W.
Journal: Mol Cell Biol

APLF is a novel protein of unknown function that accumulates at sites of chromosomal DNA strand breakage via forkhead-associated (FHA) domain-mediated interactions with XRCC1 and XRCC4. APLF can also accumulate at sites of chromosomal DNA strand breaks independently of the FHA domain, via an unidentified mechanism that requires a highly conserved C-terminal tandem zinc finger domain. Here, we show that the zinc finger domain binds tightly to poly (ADP-ribose), a polymeric post-translational modification synthesized transiently at sites of chromosomal damage to accelerate DNA strand break repair reactions. Protein poly ADP-ribosylation is tightly regulated and defects in either its synthesis or degradation slow global rates of chromosomal single-strand break repair. Interestingly, APLF negatively affects poly ADP-ribosylation in vitro, and this activity is dependant on its capacity to bind the polymer. In addition, transient over-expression in human A549 cells of full length APLF or a C-terminal fragment encoding the tandem zinc finger domain greatly suppresses the appearance of poly (ADP-ribose), in a zinc finger-dependent manner. We conclude that APLF can accumulate at sites of chromosomal damage via zinc finger-mediated binding to poly (ADP-ribose) and is a novel component of poly (ADP-ribose) signalling in mammalian cells.

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Rhythmic E-box binding by CLK-CYC controls daily cycles in per and tim transcription and chromatin modifications.

Publication Date: 2008 May 12 PMID: 18474612
Authors: Taylor, P. - Hardin, P. E.
Journal: Mol Cell Biol

The Drosophila circadian oscillator is comprised of interlocked per/tim and Clk transcriptional feedback loops. In the per/tim loop, CLK-CYC dependent transcriptional activation is rhythmically repressed by PER or PER-TIM to control circadian gene expression that peaks around dusk. Here we show that rhythmic transcription of per and tim involves time-of-day specific binding of CLK-CYC and associated cycles in chromatin modifications. Activation of per and tim transcription occurs in concert with CLK-CYC binding to upstream and/or intronic E-boxes, acetylation of histone H3-K9, and trimethylation of histone H3-K4. These events are associated with RNA Pol II binding to the tim promoter and transcriptional elongation by RNA Pol II that is constitutively bound to the per promoter. Repression of per and tim transcription is associated with PER dependent reversal of these events. Rhythms in H3-K9 acetylation and H3-K4 trimethylation are also associated with CLOCK-BMAL1 dependent transcription in mammals, indicating that the mechanism that controls rhythmic transcription is a conserved feature of the circadian clock even though feedback repression is mediated by different proteins.

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The MODY1 gene HNF4{alpha} and a feedback loop control COUP-TFII expression in pancreatic beta cells.

Publication Date: 2008 May 12 PMID: 18474611
Authors: Perilhou, A. - Tourrel-Cuzin, C. - Zhang, P. - Kharroubi, I. - Wang, H. - Fauveau, V. - Scott, D. K. - Wollheim, C. B. - Vasseur-Cognet, M.
Journal: Mol Cell Biol

Pancreatic islet beta cell differentiation and function are dependent upon a group of transcription factors that maintain the expression of key genes and suppress others. Knockout mice with heterozygous deletion of the Chicken Ovalbumin Upstream Promoter- Transcription Factor II (COUP-TFII) gene or complete disruption of the Hepatocyte Nuclear Factor 4 alpha (HNF4alpha) gene in pancreatic beta cells have similar insulin secretion defects leading us to hypothesize that there is transcriptional cross-talk between these two nuclear receptors. Here we show specific HNF4alpha activation of a reporter plasmid containing the COUP-TFII promoter region in transfected pancreatic beta cells. A stable association of the endogenous HNF4alpha with a region of the COUP-TFII gene promoter that contains a direct repeat 1 (DR-1) binding site was revealed by chromatin immunoprecipitation. Mutation experiments showed that this DR-1 site is essential for HNF4alpha transactivation of COUP-TFII. Dominant negative suppression of HNF4alpha function decreased endogenous COUP-TFII expression and specific inactivation of COUP-TFII by short interfering (si)RNA caused HNF4alpha mRNA levels to decrease in 832/13 INS-1 cells. This positive regulation of HNF4alpha by COUP-TFII was confirmed by adenoviral overexpression of human (h)COUP-TFII which increased HNF4alpha mRNA in 832/13 INS-1 cells and in mouse pancreatic islets. Finally, hCOUP-TFII overexpression showed that there is direct COUP-TFII autorepression as COUP-TFII occupies the proximal DR-1 binding site of its own gene in vivo. Therefore COUP-TFII could contribute to the control of insulin secretion through the complex HNF4alpha/maturity-onset diabetes of the young 1 (MODY1) transcription factor network operating in beta cells.

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Elongation factor eEF1A2 cooperates with phosphatidylinositol-4 kinaseIII {beta} to stimulate filopodia production through increased PI(4,5)P2 generation.

Publication Date: 2008 May 12 PMID: 18474610
Authors: Jeganathan, S. - Morrow, A. - Amiri, A. - Lee, J. M.
Journal: Mol Cell Biol

eEF1A2 (eukaryotic elongation factor 1 alpha 2) is a transforming gene highly expressed in human tumors of the ovary, lung and breast (2, 24, 25, 40). eEF1A2 also stimulates actin remodeling and its expression is sufficient to induce the formation of filopodia, long cellular processes composed of bundles of parallel actin filaments (1). Here, we find that eEF1A2 stimulates filopodia formation by increasing the cellular abundance of cytosolic and plasma membrane-bound phosphatidylinositol-4,5 bisphosphate (PI(4,5)P2). We have previously reported that the eEF1A2 protein binds and activates phosphatidylinositol-4 kinase III beta (PI4KIIIbeta) (21) and we find that eEF1A2-dependent PI(4,5)P2 production and filopodia generation requires PI4KIIIbeta. Furthermore, PI4KIIIbeta is itself capable of activating both PI(4,5)P2 production and filopodia creation. We propose a model for filopodia extrusion where eEF1A2 activates PI4KIIIbeta and activated PI4KIIIbeta stimulates PI(4,5)P2 and filopodia production by increasing PI4P abundance. Our work suggests an important role for both eEF1A2 and PI4KIIIbeta in the control of PI(4,5)P2 signaling and actin remodeling.

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Chaperone Control of the Activity and Specificity of the Histone H3 Acetyltransferase Rtt109.

Publication Date: 2008 May 5 PMID: 18458063
Authors: Fillingham, J. - Recht, J. - Silva, A. C. - Suter, B. - Emili, A. - Stagljar, I. - Krogan, N. J. - Allis, C. D. - Keogh, M. C. - Greenblatt, J. F.
Journal: Mol Cell Biol

Acetylation of S.cerevisiae histone H3 on K56 by the histone acetyltransferase (HAT) Rtt109 is important for repairing replication-associated lesions. Rtt109 purifies from yeast in complex with the histone chaperone Vps75, which stabilizes the HAT in vivo. A whole genome screen to identify genes whose deletions have synthetic genetic interactions with rtt109Delta suggests Rtt109 has additional functions to DNA repair. We show that, in addition to its known H3K56 acetylation activity, Rtt109 is also a H3-K9 HAT, and that Rtt109 and Gcn5 are the only H3-K9 HATs in vivo. Rtt109's H3-K9 acetylation activity in vitro is enhanced strongly by Vps75. Another histone chaperone, Asf1, as well as Vps75 are both required for H3-K9ac in vivo by Rtt109, whereas H3-K56ac in vivo requires only Asf1. Asf1 also physically interacts with the nuclear Hat1/Hat2/Hif1 complex that acetylates H4-K5 and H4-K12. We suggest Asf1 is capable of assembling into chromatin H3-H4 dimers di-acetylated on both H4-K5,12 and H3-K9,56.

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Zinc-induced formation of a co-activator complex containing the zinc-sensing transcription factor MTF-1, p300/CBP and Sp1.

Publication Date: 2008 May 5 PMID: 18458062
Authors: Li, Y. - Kimura, T. - Huyck, R. W. - Laity, J. H. - Andrews, G. K.
Journal: Mol Cell Biol

Herein, the mechanisms of transactivation of gene expression by mouse metal-response element binding transcription factor-1 (MTF-1) were investigated. Evidence obtained from co-immunoprecipitation assays revealed that exposure of the cells to zinc resulted in the rapid formation of a multiprotein complex containing MTF-1, the histone acetyltransferase p300/CBP, and the transcription factor Sp1. Down-regulation of endogenous p300 expression by siRNA transfection significantly decreased zinc-dependent metallothionein-I (MT-I) gene transcription without altering induction of zinc transporter-1 (ZnT1). MTF-1 independently facilitated the recruitment of Sp1 and p300 to the protein complex in response to zinc. Mutagenesis demonstrated that the acidic domain, one of three transactivation domains of MTF-1, is required for recruitment of p300 but not Sp1, as well as for zinc-dependent activation of MT-I gene transcription. Furthermore, mutation of leucine residues (L -> A) within a nuclear exclusion signal in the MTF-1 acidic domain impaired recruitment of p300 and zinc-dependent activation of the MT-I gene. NMR structural characterization of an isolated protein fragment corresponding to the MTF-1 acidic region demonstrated that it is largely unstructured in the presence and absence of excess stoichiometric amounts of zinc. This suggests that the mechanism of MTF-1 recruiting p300 to this complex involves extrinsic zinc-dependent steps. These studies reveal a novel zinc-responsive mechanism requiring an acidic region of MTF-1 that functions as a nuclear exclusion signal as well as participating in formation of a co-activator complex essential for transactivation MT-I gene expression.

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Electrostatic interactions positively regulate K-Ras nanocluster formation and function.

Publication Date: 2008 May 5 PMID: 18458061
Authors: Plowman, S. J. - Ariotti, N. - Goodall, A. - Parton, R. G. - Hancock, J. F.
Journal: Mol Cell Biol

The organization of Ras proteins into plasma membrane nanoclusters is essential for high fidelity signal transmission, but whether the nanoscale enviroments of different Ras nanoclusters regulate effector interactions is unknown. We show using high-resolution spatial mapping that Raf-1 is recruited to and retained in K-Ras.GTP nanoclusters. In contrast, Raf-1 recruited to the plasma membrane by H-Ras is not retained in H-Ras.GTP nanoclusters. Similarly, upon EGF receptor activation, Raf-1 is preferentially recruited to K-Ras.GTP and not H-Ras.GTP nanoclusters. The formation of K-Ras.GTP nanoclusters is inhibited by phosphorylation of S181 in the C-terminal polybasic domain or enhanced by blocking S181 phosphorylation, with a concomitant reduction or increase in Raf-1 plasma membrane recruitment, respectively. Phosphorylation of S181 does not however regulate in vivo interactions with the nanocluster scaffold galectin-3 (Gal3), indicating separate roles for the polybasic domain and Gal3 in driving K-Ras nanocluster formation. Together these data illustrate that Ras nanocluster composition regulates effector recruitment and highlight the importance of lipid/protein nanoscale environments to the activation of signaling cascades.

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GTPase-mediated regulation of the Unfolded Protein Response in C. elegans is dependent on the AAA+ ATPase CDC-48.

Publication Date: 2008 May 5 PMID: 18458060
Authors: Caruso, M. E. - Jenna, S. - Bouchecareilh, M. - Baillie, D. - Boismenu, D. - Halawani, D. - Latterich, M. - Chevet, E.
Journal: Mol Cell Biol

When Endoplasmic Reticulum (ER) homeostasis is perturbed, an adaptive mechanism is triggered and named the Unfolded Protein Response (UPR). Thus far, three known UPR signaling branches (IRE-1, PERK, ATF6) mediate the re-establishment of ER functions, but can also lead to apoptosis if ER stress is not alleviated. However, the understanding of the molecular mechanisms integrating the UPR to other ER functions such as membrane traffic or endomembrane signaling, remains incomplete. We consequently sought to identify new regulators of UPR-dependent transcriptional mechanisms and focused on a family of proteins known to mediate, amongst other, ER-related functions: the small GTP-binding proteins of the RAS super-family. To this end, we used transgenic UPR-reporter C. elegans strains as a model to specifically silence small GTPases expression. We show that the Rho subfamily member CRP-1 is an essential component of UPR-induced transcriptional events through its physical and genetic interactions with the AAA(+) ATPase CDC-48. In addition, we describe a novel signaling module involving CRP-1 and CDC-48 which may directly link the UPR to DNA remodeling and transcription control.

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Cancer Cell-Derived Clusterin Modulates the PI3K-Akt Pathway through Attenuation of IGF-1 during Serum Deprivation.

Publication Date: 2008 May 5 PMID: 18458059
Authors: Jo, H. - Jia, Y. - Subramanian, K. K. - Hattori, H. - Luo, H. R.
Journal: Mol Cell Biol

Cancer cells in their respective microenvironment must endure various growth constraint stresses. Under these conditions, the cancer cell-derived factors are thought to modulate the signaling pathways between cell growth and dormancy. Here we describe a cancer cell-derived regulatory system that modulates the PI3K-Akt pathway under serum deprivation stress. Through biochemical purification, we reveal that cancer cell-secreted IGF-1 and Clusterin, an extracellular stress protein, constitute this regulatory system. We show that secreted Clusterin associates with IGF-1 and inhibits its binding to IGF-1 receptor, hence negatively regulating the PI3K-Akt pathway during serum deprivation. This inhibitory function of Clusterin appears to be preferential to IGF-1 as it fails to exert any effects on EGF signaling. We further demonstrate that constitutive activation of oncogenic signaling downstream of IGF-1 confers the insensitivity to inhibitory effects of Clusterin. Thus, the interplays between cancer cell-derived Clusterin and IGF-1 may dictate the outcome of cell growth and dormancy during tumorigenic progression.

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Nuclear factor 45 (NF45) is a regulatory subunit of complexes with NF90/110 involved in mitotic control.

Publication Date: 2008 May 5 PMID: 18458058
Authors: Guan, D. - Altan-Bonnet, N. - Parrott, A. M. - Arrigo, C. J. - Li, Q. - Khaleduzzaman, M. - Li, H. - Lee, C. G. - Pe'ery, T. - Mathews, M. B.
Journal: Mol Cell Biol

Nuclear factor 90 (NF90) and its C-terminally extended isoform, NF110, have been isolated as DNA- and RNA-binding proteins together with the less-studied protein NF45. These complexes have been implicated in gene regulation but little is known about their cellular roles and whether they are redundant or functionally distinct. We show that heterodimeric core complexes, NF90.NF45 and NF110.NF45, exist within larger complexes that are more labile and contain multiple NF90/110 isoforms and additional proteins. Depletion of the NF45 subunit by RNA interference is accompanied by a dramatic decrease in the levels of NF90 and NF110. Reciprocally, depletion of NF90 but not of NF110 greatly reduces the level of NF45. Co-regulation of NF90 and NF45 is a post-transcriptional phenomenon, resulting from protein destabilization in the absence of partners. Depletion of NF90.NF45 complexes retards cell growth by inhibition of DNA synthesis. Giant multinucleated cells containing nuclei attached by constrictions accumulate when either NF45 or NF90, but not NF110, is depleted. This study identified NF45 as an unstable regulatory subunit of NF90.NF45 complexes and uncovered their critical role in normal cell division. Furthermore, the study revealed that NF90 is functionally distinct from NF110 and is more important for cell growth.

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The Assembly Pathway of the Mitochondrial Carrier Translocase Involves four Preprotein Translocases.

Publication Date: 2008 May 5 PMID: 18458057
Authors: Wagner, K. - Gebert, N. - Guiard, B. - Brandner, K. - Truscott, K. N. - Wiedemann, N. - Pfanner, N. - Rehling, P.
Journal: Mol Cell Biol

The mitochondrial inner membrane contains preprotein translocases that mediate insertion of hydrophobic proteins. Little is known about how the individual components of these inner membrane preprotein translocases combine to form multi-subunit complexes. We have analyzed the assembly pathway of the three membrane-integral subunits Tim18, Tim22 and Tim54 of the twin-pore carrier translocase. Tim54 displayed the most complex pathway involving four preprotein translocases. The precursor is translocated across the intermembrane space in a supercomplex of outer and inner membrane translocases. The TIM10 complex, which translocates the precursor of Tim22 through the intermembrane space, functions in a new post-translocational manner in case of Tim54, it is required for the integration of Tim54 into the carrier translocase. Tim18, the function of which has been unknown so far, stimulates integration of Tim54 into the carrier translocase. We show that the carrier translocase is built via a modular process and that each subunit follows a different assembly route. Membrane insertion and assembly into the oligomeric complex are uncoupled for each precursor protein. We propose that the mitochondrial assembly machinery has adapted to the needs of each membrane-integral subunit and that the uncoupling of translocation and oligomerization is an important principle to ensure continuous import and assembly of protein complexes in a highly active membrane.

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Site-specific release of nascent chains from ribosomes at a sense codon.

Publication Date: 2008 May 5 PMID: 18458056
Authors: Doronina, V. A. - Wu, C. - de Felipe, P. - Sachs, M. S. - Ryan, M. D. - Brown, J. D.
Journal: Mol Cell Biol

"2A" oligopeptides are autonomous elements containing a -D(V/I)ExNPGP- motif at their C-terminus. Protein synthesis from an open reading frame containing an internal 2A coding sequence yields two separate polypeptides, corresponding to sequences up to and including 2A and those downstream. We show that the 2A reaction occurs in the ribosomal peptidyl transferase centre. Ribosomes pause at the end of the 2A coding sequence, over the glycine and proline codons, and the nascent chain up to and including this glycine is released. Translation terminating release factors eRF1 and eRF3 play key roles in the reaction. On depletion of eRF1 a greater proportion of ribosomes extend through the 2A coding sequence yielding the full length protein. In contrast, impaired eRF3 GTPase activity leads to many ribosomes failing to translate beyond 2A. Further, high-level expression of a 2A-peptide containing protein inhibits growth of cells compromised for RF activity and leads to errors in stop codon recognition. We propose that the nascent 2A peptide interacts with ribosomes to drive a highly unusual and specific "termination" reaction, despite the presence of a proline codon in the A site. After this the majority of ribosomes continue translation, generating the separate downstream product.

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Extracellular signals regulate rapid coactivator recruitment at AP-1 sites by altered phosphorylation of both CREB binding protein (CBP) and c-jun proteins.

Publication Date: 2008 Apr 28 PMID: 18443043
Authors: Tsai, L. N. - Ku, T. K. - Salib, N. K. - Crowe, D. L.
Journal: Mol Cell Biol

Retinoic acid (RA) inhibits matrix metalloproteinase 9 (MMP-9) expression due to AP-1 inhibition resulting from RARs competing for limiting amounts of coactivator proteins. However given the rapid kinetics of MMP-9 transcription, it seems unlikely that these interactions can be explained passively. Our previous studies indicated that coactivator and transcription factor phosphorylation may allow for rapid regulation of MMP-9 expression. In the present study we tested this hypothesis directly. CBP and PCAF were displaced from transcription factor binding sites on the MMP-9 promoter within minutes of RA treatment. The RAR interaction domains of CBP and PCAF were not required for this displacement. RA and EGF had opposing effects on phosphorylation of CBP by ERK1 which correlated with altered CBP occupancy of AP-1 sites and differential MMP-9 promoter activation. We identified a novel phosphorylation site in the CBP carboxyl terminus that mediated association with AP-1 sites in the MMP-9 promoter. Inhibition of c-jun phosphorylation displaced PCAF from AP-1 sites and reduced promoter activity. Phosphorylation deficient c-jun was less able to recruit PCAF to AP-1 sites. We also demonstrated novel interactions between coactivators and AP-1 proteins. We propose that extracellular signal mediated coactivator exchange at AP-1 sites is mediated via protein kinase pathways.

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c-Jun controls histone modifications, NF-{kappa}B recruitment and RNA polymerase II function to activate the ccl2 gene.

Publication Date: 2008 Apr 28 PMID: 18443042
Authors: Wolter, S. - Doerrie, A. - Weber, A. - Schneider, H. - Hoffmann, E. - von der Ohe, J. - Bakiri, L. - Wagner, E. F. - Resch, K. - Kracht, M.
Journal: Mol Cell Biol

IL-1-induced mRNA expression of ccl2 (also called MCP-1), a prototypic highly regulated inflammatory gene, is severely suppressed in cells lacking c-Jun or JNK1/JNK2 genes and is only partially restored in cells expressing a c-Jun(SS63/73AA) mutant. By chromatin immunoprecipitation we identified three c-Jun-binding sites located in the far 5' region, close to the transcriptional start site and in the far 3' region of murine and human ccl2 genes. Mutational analysis revealed that the latter two contribute to ccl2 transcription in response to IL-1, or to ectopically expressed c-Jun-ATF-2 dimers. Further experiments comparing wild type and c-Jun-deficient cells revealed that c-Jun regulates ser10 phosphorylation of histone H3, acetylation of histones H3 and H4 and recruitment of HDAC3, NF-kappaB subunits and RNA polymerase II across the ccl2 locus. c-Jun also co-immunoprecipitated with p65 NF-kappaB and HDAC3. Based on DNA microarray analysis, c-Jun was required for full expression of 133 out of 162 IL-1-induced genes. For inflammatory genes, these data support an activator function of c-Jun that is executed by multiple mechanisms, including phosphorylation-dependent interaction with p65 NF-kappaB and HDAC3 at the level of chromatin.

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Regulation of multiple core spliceosomal proteins by alternative splicing-coupled nonsense-mediated mRNA decay.

Publication Date: 2008 Apr 28 PMID: 18443041
Authors: Saltzman, A. L. - Kim, Y. K. - Pan, Q. - Fagnani, M. M. - Maquat, L. E. - Blencowe, B. J.
Journal: Mol Cell Biol

Alternative splicing (AS) can regulate gene expression by introducing premature termination codons (PTCs) into spliced mRNA that subsequently elicit transcript degradation by the nonsense-mediated mRNA decay (NMD) pathway. However, the range of cellular functions controlled by this process and the factors required are poorly understood. Using quantitative AS microarray profiling, we find that there are significant overlaps among the sets of PTC-introducing AS events affected by individual knockdown of the three core human NMD factors, Up-Frameshift 1 (UPF1), UPF2 and UPF3X/B. However, the levels of some PTC-containing splice variants are less or not detectably affected by the knockdown of UPF2 and/or UPF3X, compared with the knockdown of UPF1. The intron sequences flanking the affected alternative exons are often highly conserved, suggesting important regulatory roles for these AS events. The corresponding genes represent diverse cellular functions and, surprisingly, many encode core spliceosomal proteins and assembly factors. We further show that conserved, PTC-introducing AS events are enriched in genes encoding core spliceosomal proteins. Where tested, altering the expression levels of these core spliceosomal components affects the regulation of PTC-containing splice variants from the corresponding genes. Together, our results show that AS-coupled NMD can have different UPF factor requirements and is likely to regulate many general components of the spliceosome. The results further implicate general spliceosomal components in AS regulation.

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Cmc1p is a Conserved Mitochondrial Twin Cx9C Protein Involved in Cytochrome c Oxidase Biogenesis.

Publication Date: 2008 Apr 28 PMID: 18443040
Authors: Horn, D. - Al-Ali, H. - Barrientos, A.
Journal: Mol Cell Biol

Copper is an essential cofactor of two mitochondrial enzymes: cytochrome c oxidase (COX) and Cu-Zn superoxide dismutase (Sod1p). Copper incorporation into these enzymes is facilitated by metallochaperone proteins which probably use copper from a mitochondrial matrix localized pool. Here we describe a novel conserved mitochondrial metallochaperone-like protein, Cmc1p, whose function affects both COX and Sod1p. In Saccharomyces cerevisiae, Cmc1p localizes to the mitochondrial inner membrane facing the intermembrane space. Cmc1p is essential for full expression of COX and respiration, contains a twin Cx9C domain conserved in other COX assembly copper chaperons and has the ability to bind copper(I). Additionally, mutant cmc1 cells display increased mitochondrial Sod1p activity while CMC1 overexpression results in decreased Sod1p activity. Our results suggest that Cmc1p could play a direct or indirect role in copper trafficking and distribution to COX and Sod1p.

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Ydj1 protects nascent protein kinases from degradation and controls the rate of their maturation.

Publication Date: 2008 Apr 28 PMID: 18443039
Authors: Mandal, A. K. - Nillegoda, N. - Chen, J. A. - Caplan, A. J.
Journal: Mol Cell Biol

Ydj1 is an S. cerevisiae Hsp40 molecular chaperone that functions with Hsp70 to promote polypeptide folding. We identified Ydj1 as being important for maintaining steady state levels of protein kinases after screening several chaperones and co-chaperones in gene deletion mutant strains. Pulse-chase analyses revealed that a portion of Tpk2 kinase was degraded shortly after synthesis in a ydj1Delta mutant, while the remainder was capable of maturing but with reduced kinetics compared to the wild type. Cdc28 maturation was also delayed in the ydj1Delta mutant strain. Ydj1 protects nascent kinases in different contexts, such as when Hsp90 is inhibited with geldanamycin or when CDC37 is mutated. The protective function of Ydj1 is due partly to its intrinsic chaperone function, but this is minor compared with the protective effect resulting from its interaction with Hsp70. SIS1, a type II Hsp40 was unable to suppress defects in kinase accumulation in the ydj1Delta mutant, suggesting some specificity in Ydj1 chaperone action. However, analysis of chimeric proteins that contained the chaperone modules of Ydj1 or Sis1 indicated that Ydj1 promotes kinase accumulation independently of its client-binding specificity. Our results suggest that Ydj1 can both protect nascent chains against degradation and accelerate the rate of kinase maturation.

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A T>G TRANSVERSION AT NT -567 UPSTREAM OF HBG2 IN A GATA-1 BINDING MOTIF IS ASSOCIATED WITH ELEVATED Hb F.

Publication Date: 2008 Apr 28 PMID: 18443038
Authors: Chen, Z. - Luo, H. Y. - Basran, R. K. - Hsu, T. H. - Mang, D. W. - Nuntakam, L. - Rosenfield, C. G. - Patrinos, G. P. - Hardison, R. C. - Steinberg, M. H. - Chui, D. H.
Journal: Mol Cell Biol

Increased fetal hemoglobin (Hb F; alpha2gamma2) production in adult can ameliorate clinical severity of sickle cell disease and beta-thalassemia major. Thus, understanding the regulation of gamma-globin gene expression and its silencing in adult has potential therapeutic implications. We studied the father and one of his sons in an Iranian American family who had elevated Hb F levels and found a novel T>G transversion at NT (nucleotide) -567 of the HBG2 promoter. This mutation alters a GATA-1 binding motif to a GAGA sequence located within a previously identified silencing element. DNA-protein binding assays showed that the GATA motif of interest is capable of binding GATA-1 transcription factor in vitro and in vivo. Truncation analyses of HBG2 promoter linked to a luciferase reporter gene revealed a negative regulatory activity present between NT -675 and -526. In addition, the T>G mutation at the GATA motif increases the promoter activity by 2 to 3 fold in transiently transfected erythroid cell lines. The binding motif is uniquely conserved in simian primates with a fetal pattern of gamma-globin gene expression. These results suggest that the GATA motif under study has a functional role in silencing gamma-globin gene expression in adult. The T>G mutation in this motif disrupts GATA-1 binding and the associated repressor complex, abolishing its silencing effect and resulting in the up-regulation of gamma-globin gene expression in adult.

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Nucleolar localization and dynamic roles of flap endonuclease 1 in ribosomal DNA replication and damage repair.

Publication Date: 2008 Apr 28 PMID: 18443037
Authors: Guo, Z. - Qian, L. - Liu, R. - Dai, H. - Zhou, M. - Zheng, L. - Shen, B.
Journal: Mol Cell Biol

Despite the wealth of information available on the biochemical functions and our recent findings of its roles in genome stability and cancer avoidance of the structure specific flap endonuclease-1 (FEN1), its cellular compartmentalization and dynamics corresponding to its involvement in various DNA metabolic pathways, are not yet elucidated. Several years ago we demonstrated that FEN1 migrates into the nucleus in response to DNA damage and under certain cell cycle conditions. In the current manuscript, we found that FEN1 is super-accumulated in the nucleolus and plays a role in resolution of stalled DNA replication forks formed at the sites of natural replication fork barriers. In response to UV irradiation and upon phosphorylation, FEN1 migrates to nuclear plasma to participate in resolution of UV cross-links on DNA, most likely employing its concerted action of exonuclease (EXO) and gap-dependent endonuclease (GEN) activities. Based on the yeast complementation experiments, mutation of Ser(187)Asp, mimicking constant phosphorylation, excludes FEN1 from nucleolar accumulation. Replacement of the Ser187 by Ala, eliminating the only phosphorylation site, retains FEN1 in nucleoli. Both of the mutations cause UV sensitivity, impair cellular UV damage repair capacity, and decline overall cellular survivorship.

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How antigen quantity and quality determine T cell decisions in lymphoid tissue.

Publication Date: 2008 Apr 21 PMID: 18426917
Authors: Zheng, H. - Jin, B. - Henrickson, S. - Perelson, A. S. - von Andrian, U. H. - Chakraborty, A. K.
Journal: Mol Cell Biol

T lymphocytes (T cells) express T cell receptor (TCR) molecules on their surface that can recognize peptides (p) derived from antigenic proteins bound to products of the major histocompatibility (MHC) genes. The pMHC molecules are expressed on the surface of antigen presenting cells, such as dendritic cells (DC). T cells first encounter antigen on DCs in lymph nodes (LN). Intravital microscopy experiments show that, upon entering the LN containing antigen, CD8+ T cells first move rapidly. After a few hours, they stop and make extended contacts with DCs. The factors that determine when and how this transition occurs are not well understood. We report results from computer simulations that suggest that the duration of phase one is related to the low probability of productive interactions between T cells and DCs. This is demonstrated by our finding that antigen dose and type determine when such a transition occurs. These results are in agreement with experimental observations. TCR-pMHC binding characteristics and antigen dose determine the time required for a productive T cell-DC encounter (resulting in sustained contact). We find that the ratio of this time scale and the half-life of the pMHC complex itself provides a consolidated measure of antigen quantity and type. Results obtained upon varying different measures of antigen quantity and type fall on one curve when graphed against this ratio of time scales. Thus, we provide a mechanism for how the effects of varying one set of parameters is influenced by other prevailing conditions. This understanding should help guide future experimentation.

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The anaphase-promoting complex/cyclosome (APC/C) controls repair and recombination by ubiquitylating Rhp54 in fission yeast.

Publication Date: 2008 Apr 21 PMID: 18426916
Authors: Trickey, M. - Grimaldi, M. - Yamano, H.
Journal: Mol Cell Biol

Homologous recombination (HR) is important for maintaining genome integrity and for the process of meiotic chromosome segregation and the generation of variation. HR is regulated throughout the cell cycle, being prevalent in S and G2 phase and suppressed in G1 phase. Here we show that the anaphase-promoting complex/cyclosome (APC/C) regulates homologous recombination in the fission yeast S. pombe by ubiquitylating Rhp54 (an ortholog of Rad54). We show that Rhp54 is a novel APC/C substrate that is destroyed in G1 phase in a KEN-box and Ste9/Fizzy-related manner. The biological consequences of failing to temporally regulate HR via Rhp54 degradation are seen in haploid only in the absence of anti-recombinase, Srs2 function, and are more extensive in diploid cells which become sensitive to a range of DNA damaging agents, including hydroxyurea (HU), methyl methane sulfonate (MMS), bleomycin and UV. During meiosis, expression of non-degradable Rhp54 inhibits interhomolog recombination and stimulates sister chromatid recombination. We thus propose that it is critical to control levels of Rhp54 in G1 to suppress HR repair of double strand breaks (DSBs) and during meiosis to coordinate interhomolog recombination.

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FANCJ Helicase Defective in Fanconia Anemia and Breast Cancer Unwinds G-Quadruplex DNA to Defend Genomic Stability.

Publication Date: 2008 Apr 21 PMID: 18426915
Authors: Wu, Y. - Shin-Ya, K. - Brosh, R. M. Jr
Journal: Mol Cell Biol

FANCJ mutations are associated with breast cancer and genetically linked to the bone marrow disease Fanconi Anemia (FA). Genomic instability of FA-J mutant cells suggests FANCJ helicase functions in the replicational stress response. Putative helicases with sequence similarity to FANCJ in C. elegans (DOG-1) and mouse (RTEL) are required for polyG-tract maintenance, suggesting their involvement in resolution of alternate DNA structures that impede replication. Under physiological conditions, guanine-rich sequences spontaneously assemble into four-stranded structures (G4) that influence genomic stability. FANCJ unwound G4 DNA substrates in an ATPase-dependent manner. FANCJ G4 unwinding is specific since another Superfamily 2 helicase, RECQ1, failed to unwind all G4 substrates tested under conditions the helicase unwound duplex DNA. RPA stimulated FANCJ G4 unwinding, whereas the mismatch repair complex MSH2/6 inhibited this activity. FANCJ-depleted cells treated with the G4 interactive compound telomestatin displayed impaired proliferation, elevated apoptosis and DNA damage compared to siRNA control cells, suggesting that G4 DNA represents a physiological substrate of FANCJ. Although the FA pathway has been classically described in terms of interstrand cross-link (ICL) repair, the cellular defects associated with FANCJ mutation extend beyond the reduced ability to repair ICLs and involve other types of DNA structural roadblocks to replication.

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Study of the functional interaction between the Fab-7 and Fab-8 boundaries and the upstream promoter region in Drosophila Abd-B gene.

Publication Date: 2008 Apr 21 PMID: 18426914
Authors: Kyrchanova, O. - Toshchakov, S. - Podstreshnaya, Y. - Parshikov, A. - Georgiev, P.
Journal: Mol Cell Biol

Boundary elements have been found in the regulatory region of the Drosophila Abd-B gene, which is subdivided into a series of iab domains. The best studied Fab-7 and Fab-8 boundaries flank the iab-7 enhancer and isolate it from the four promoters regulating Abd-B expression. Recently, binding sites for the Drosophila homolog of the vertebrate insulator protein CTCF have been identified in the Fab-8 boundary and upstream of the Abd-B promoter A, with no CTCF binding to the Fab-7 boundary being detected either in vivo or in vitro. Taking into account the inability of the yeast GAL4 activator to stimulate white promoter when its binding sites are separated by a 5 kb yellow gene, we have tested the functional interactions between the Fab-7 and Fab-8 boundaries and between these boundaries and the upstream promoter A region containing dCTCF binding site. It has been found that dCTCF binding sites are essential for pairing between two Fab-8 insulators. However, a strong functional interaction between the Fab-7 and Fab-8 boundaries suggest that additional, as yet unidentified proteins are involved in long-distance interactions between them. We have also shown that Fab-7 and Fab-8 boundaries effectively interact with the upstream region of the Abd-B promoter.

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Sphingosine Kinases and Sphingosine-1-Phosphate are Critical for TGF{beta}-Induced ERK1/2 Activation and Promotion of Migration and Invasion of Esophageal Cancer Cells.

Publication Date: 2008 Apr 21 PMID: 18426913
Authors: Miller, A. V. - Alvarez, S. E. - Spiegel, S. - Lebman, D. A.
Journal: Mol Cell Biol

TGFbeta plays a dual role in oncogenesis acting as both a tumor suppressor and a tumor promoter. These disparate processes are mediated primarily by Smad and non-Smad signaling, respectively. A central issue in understanding the role of TGFbeta in the progression of epithelial cancers is elucidation of the mechanisms underlying activation of non-Smad signaling cascades. Because the potent lipid mediator sphingosine-1-phosphate has been shown to transactivate the TGFbeta receptor and activate Smad3, we examined its role in TGFbeta activation of ERK1/2 and promotion of migration and invasion of esophageal cancer cells. Both S1P and TGFbeta activate ERK1/2, but only TGFbeta activates Smad3. Both ligands promoted ERK1/2-dependent migration and invasion. Furthermore, TGFbeta rapidly increased S1P, which was required for TGFbeta induced ERK1/2 activation as well as migration and invasion since downregulation of sphingosine kinases, the enzymes that produce S1P, inhibited these responses. Finally, our data demonstrate that TGFbeta activation of ERK1/2 as well as induction of migration and invasion is mediated at least in part by ligation of the S1P receptor, S1P2. Thus, these studies provide the first evidence that TGFbeta activation of sphingosine kinases and formation of S1P contribute to non-Smad signaling and could be important for progression of esophageal cancer.

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Hepatocyte Nuclear Factor 4{alpha} contributes to thyroid hormone homeostasis by cooperatively regulating the type1 Iodothyronine Deiodinase gene with GATA4 and Kruppel-like transcription factor 9.

Publication Date: 2008 Apr 21 PMID: 18426912
Authors: Ohguchi, H. - Tanaka, T. - Uchida, A. - Magoori, K. - Kudo, H. - Kim, I. - Daigo, K. - Sakakibara, I. - Okamura, M. - Harigae, H. - Sasaki, T. - Osborne, T. F. - Gonzalez, F. J. - Hamakubo, T. - Kodama, T. - Sakai, J.
Journal: Mol Cell Biol

Type1 iodothyronine deiodinase (Dio1), a selenoenzyme catalyzing the bioactivation of thyroid hormone, is highly expressed in the liver. Dio1 mRNA and enzyme activity are markedly reduced in the livers of hepatic hepatocyte nuclear factor 4alpha (HNF4alpha)-null mice, thus accounting for its liver-specific expression. Consistent with this deficiency, serum T4 and rT3 concentrations are elevated in these mice compared with those of HNF4alpha-floxed control littermates, however serum T3 levels are unchanged. Promoter analysis of the mouse Dio1 gene demonstrated that HNF4alpha plays a key role in the trans-activation of the mouse Dio1 gene. Deletion and substitution mutation analyses demonstrated that a proximal HNF4alpha site (Direct Repeat-1: TGGACA A AGGTGC; HNF4alpha-RE) is crucial for trans-activation of the mouse Dio1 gene by HNF4alpha. Mouse Dio1 is also stimulated by thyroid hormone signaling but a direct role for thyroid hormone receptor action has not been reported. We also showed that the thyroid hormone inducible Kruppel-like factor 9 (KLF9) stimulates mouse Dio1 promoter very efficiently through two CACCC sequences that are located on either side of the HNF4alpha-RE. Furthermore, KLF9 functions together with HNF4alpha and GATA4 to synergistically activate the mouse Dio1 promoter, suggesting that Dio1 regulation by thyroid hormone in the mouse is through an indirect mechanism requiring prior KLF9 induction. In addition, we showed that physical interactions between the C-terminal zinc finger domain (Cf) of GATA4 and activation function -2 of HNF4alpha and between the basic domain adjacent to Cf of GATA4 and a C-terminal domain of KLF9 are both required for this synergistic response. Taken together, these results suggest that HNF4alpha regulates thyroid hormone homeostasis through transcriptional regulation of the mouse Dio1 gene with GATA4 and KLF9.

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A Rictor-Myo1c complex participates in dynamic cortical actin events in 3T3-L1 adipocytes.

Publication Date: 2008 Apr 21 PMID: 18426911
Authors: Hagan, G. N. - Lin, Y. - Magnuson, M. A. - Avruch, J. - Czech, M. P.
Journal: Mol Cell Biol

Insulin signaling through PI 3-kinase activates the protein kinase Akt through phosphorylation of its threonine 308 and serine 473 residues by the PDK1 protein kinase and the Rictor-mTOR complex (mTORC2), respectively. Remarkably, we show here that the protein Rictor is also present in cultured adipocytes in complexes containing Myo1c, a molecular motor that promotes cortical actin remodeling. Interestingly, the Rictor-Myo1c complex is biochemically distinct from the previously reported Rictor-mTOR complex (mTORC2), and can be immunoprecipitated independently of mTORC2. Furthermore, while RNAi-directed silencing of Rictor results in the expected attenuation of Akt phosphorylation at serine 473, depletion of Myo1c is without effect. In contrast, loss of either Rictor or Myo1c inhibits phosphorylation of the actin filament regulatory protein paxillin at tyrosine 118. Furthermore, Myo1c-induced membrane ruffling of 3T3-L1 adipocytes is also compromised following Rictor knockdown. Interestingly, neither the mTORC2 inhibitor rapamycin nor the PI 3-kinase inhibitor wortmannin affects paxillin tyrosine 118 phosphorylation. Taken together, our findings suggest the Rictor-Myo1c complex is distinct from the Rictor-mTOR complex and that Myo1c, in conjunction with Rictor, participates in cortical actin remodeling events.

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Caspase-2 cleavage of BID is a critical apoptotic signal downstream of endoplasmic reticulum stress.

Publication Date: 2008 Apr 21 PMID: 18426910
Authors: Upton, J. P. - Austgen, K. - Nishino, M. - Coakley, K. M. - Hagen, A. - Han, D. - Papa, F. R. - Oakes, S. A.
Journal: Mol Cell Biol

Accumulation of misfolded proteins stresses the endoplasmic reticulum (ER) and triggers cell death through activation of the multidomain pro-apoptotic BCL-2 proteins BAX and BAK at the outer mitochondrial membrane. The signaling events that connect ER stress with the mitochondrial apoptotic machinery remain unclear, despite evidence that deregulation of this pathway contributes to cell loss in many human degenerative diseases. In order to "trap" and identify the apoptotic signals upstream of mitochondrial permeabilization, we challenged Bax(-/-)Bak(-/-) mouse embryonic fibroblasts (MEFs) with pharmacological inducers of ER stress. We found that ER stress induces proteolytic activation of the BH3-only protein BID as a critical apoptotic switch. Moreover, we identified Caspase-2 as the pre-mitochondrial protease that cleaves BID in response to ER stress, and show that resistance to ER stress-induced apoptosis can be conferred by inhibiting Caspase-2 activity. Our work defines a novel signaling pathway that couples the ER and mitochondria, and establishes a principal apoptotic effector downstream of ER stress.

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PROPERTIES OF AN INTERGENIC TERMINATOR AND START SITE SWITCHING THAT REGULATES IMD2 TRANSCRIPTION IN YEAST.

Publication Date: 2008 Apr 21 PMID: 18426909
Authors: Jenks, M. H. - O'Rourke, T. W. - Reines, D.
Journal: Mol Cell Biol

The IMD2 gene in Saccharomyces cerevisiae is regulated by intracellular guanine nucleotides. Regulation is exerted through the choice of alternative transcription start sites that results in synthesis of either an unstable short transcript terminating upstream of the start codon or a full length productive IMD2 mRNA. Start site selection is dictated by the intracellular guanine nucleotide levels. Here we have mapped the polyadenylation sites of the upstream, unstable short transcripts that form a heterogeneous family of RNAs of approximately 200 nt. The switch from the upstream to downstream start sites required the Rpb9 subunit of RNA polymerase II. The enzyme's ability to locate the downstream initiation site decreased exponentially as the start was moved downstream from the TATA box. This suggests that RNA polymerase II's pincer grip is important as it slides on DNA in search of a start site. Exosome degradation of the upstream transcripts was highly dependent upon the distance between the terminator and the promoter. Similarly, termination was dependent upon the Sen1 helicase when close to the promoter. These findings extend the emerging concept that distinct modes of termination by RNA polymerase II exist and that the distance of the terminator from the promoter, as well as its sequence, is important for the pathway chosen.

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Targeting the GABP{beta}1L isoform does not perturb lymphocyte development and function.

Publication Date: 2008 Apr 21 PMID: 18426908
Authors: Xue, H. H. - Jing, X. - Bollenbacher-Reilley, J. - Zhao, D. M. - Haring, J. S. - Yang, B. - Liu, C. Y. - Bishop, G. A. - Harty, J. T. - Leonard, W. J.
Journal: Mol Cell Biol

GA binding protein (GABP) is a ubiquitously expressed Ets family transcription factor that consists of two subunits, GABPalpha and GABPbeta. GABPalpha binds to DNA, and GABPbeta heterodimerizes with GABPalpha and possesses the ability to transactivate target genes. Our previous studies using GABPalpha-deficient mice revealed that GABPalpha is required for the development of both T and B cells. Two splice variants of GABPbeta are generated from the Gabpb1 locus, and differ in their carboxy-terminal lengths and sequences. The longer isoform (GABPbeta1L) can homodimerize and thus form alpha2beta2 tetramers depending on the gene context, whereas the shorter isoform (GABPbeta1S) cannot. In this study, we generated mice that are deficient in GABPbeta1L but retain expression of GABPbeta1S. Surprisingly, GABPbeta1L(-/-) mice had normal T and B cell development, and mature T and B cells showed normal responses to various stimuli. In contrast, targeting both GABPbeta1L and GABPbeta1S resulted in early embryonic lethality. Because of its incapability of forming homodimers, GABPbeta1S has been suspected to have a dominant negative role in regulating GABP target genes. Our findings argue against such a possibility, and rather suggest that GABPbeta1S has a critical role in maintaining the transcriptional activity of the GABPalpha/beta complex.

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Aberrant expression of nucleostemin activates p53 and induces cell cycle arrest via inhibition of MDM2.

Publication Date: 2008 Apr 21 PMID: 18426907
Authors: Dai, M. S. - Sun, X. X. - Lu, H.
Journal: Mol Cell Biol

The nucleolar protein nucleostemin (NS) is essential for cell proliferation and early embryogenesis. Both depletion and overexpression of NS reduce cell proliferation. However, the mechanisms underlying this regulation are still unclear. Here we show that NS regulates p53 activity through inhibition of MDM2. NS binds to the central acidic domain of MDM2 and inhibits MDM2-mediated p53 ubiquitylation and degradation. Consequently, ectopic overexpression of NS activates p53, induces G1 cell cycle arrest, and inhibits cell proliferation. Interestingly, knockdown of NS by siRNA also activates p53 and induces G1 arrest. These effects require the ribosomal proteins L5 and L11, since depletion of NS enhanced their interaction with MDM2 and knockdown L5 or L11 abrogated the NS-depletion-induced p53 activation and cell cycle arrest. These results suggest that a p53-dependent cell cycle checkpoint monitors changes of cellular NS levels via the impediment of MDM2 function.

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Residues of Tim44 Involved in Both Association with the Translocon of the Inner Mitochondrial Membrane and Regulation of mtHsp70 Tethering.

Publication Date: 2008 Apr 21 PMID: 18426906
Authors: Schiller, D. - Cheng, Y. C. - Liu, Q. - Walter, W. - Craig, E. A.
Journal: Mol Cell Biol

Translocation of proteins from the cytosol across the mitochondrial inner membrane is driven by the action of the import motor, which is associated with the translocon on the matrix side of the membrane. It is well-established that an essential peripheral membrane protein Tim44 tethers mitochondrial Hsp70 (mtHsp70), the core of the import motor, to the translocon. This Tim44:mtHsp70 interaction, which can be recapitulated in vitro, is destabilized by binding of mtHsp70 to a substrate polypeptide. Here we report that the N-terminal 167 amino acid segment of mature Tim44 is sufficient for both interaction with mtHsp70 and destabilization of a Tim44:mtHsp70 complex caused by client protein binding. Amino acid alterations within a 30 amino acid segment affected both the release of mtHsp70 upon peptide binding and the interaction of Tim44 with the translocon. Our results support the idea that Tim44 plays multiple roles in mitochondrial protein import by recruiting Ssc1 and its J-protein co-chaperone to the translocon and coordinating their interactions to promote efficient protein translocation in vivo.

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Down regulation of vertebrate Tel (ETV6) and Drosophila Yan is facilitated by an evolutionarily conserved mechanism of F-box-mediated ubiquitination.

Publication Date: 2008 Apr 21 PMID: 18426905
Authors: Roukens, M. G. - Alloul-Ramdhani, M. - Moghadasi, S. - Op Den Brouw, M. - Baker, D. A.
Journal: Mol Cell Biol

The vertebrate Ets transcriptional repressor Tel (ETV6), and its invertebrate orthologue Yan, are each indispensable for development, and orchestrate cell growth and differentiation by binding to DNA thus inhibiting gene expression. To trigger cell differentiation this barrier to transcriptional activation must be relieved and it is established that post-translational modifications such as phosphorylation and sumoylation can specifically impair their repressive function and are crucial for modulating their transcriptional activity. To date, however, relatively little is known about the control of Tel and Yan protein degradation. In recent years there has been a concentrated effort to assign functions to the large numbers of F-box proteins encoded by both vertebrate and invertebrate genomes. Here we report the identification and characterization of a previously unreported, evolutionarily conserved F-box protein named Fbl6. We isolated both human and Drosophila fbl6 cDNA and show that the encoded Fbl6 protein binds to both Tel and Yan via their SAM domains. We demonstrate that both Tel and Yan are ubiquitinated, which is stimulated by Fbl6, and leads to their proteasomal degradation. We recently established that sumoylation of Tel lysine 11 negatively regulates its repressive function and that sumoylation of Tel monomers but not Tel oligomers may sensitize Tel for proteasomal degradation. Here we found that Fbl6 regulates Tel/Yan protein stability and allows appropriate spatio-temporal control of gene expression by these repressors.

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p23/Sba1p protects against Hsp90 inhibitors independently of its intrinsic chaperone activity.

Publication Date: 2008 May PMID: 18362168
Authors: Forafonov, F. - Toogun, O. A. - Grad, I. - Suslova, E. - Freeman, B. C. - Picard, D.
Journal: Mol Cell Biol

The molecular chaperone Hsp90 assists a subset of cellular proteins and is essential in eukaryotes. A cohort of cochaperones contributes to and regulates the multicomponent Hsp90 machine. Unlike the biochemical activities of the cochaperone p23, its in vivo functions and the structure-function relationship remain poorly understood, even in the genetically tractable model organism Saccharomyces cerevisiae. The SBA1 gene that encodes the p23 ortholog in this species is not an essential gene. We found that in the absence of p23/Sba1p, yeast and mammalian cells are hypersensitive to Hsp90 inhibitors. This protective function of Sba1p depends on its abilities to bind Hsp90 and to block the Hsp90 ATPase and inhibitor binding. In contrast, the protective function of Sba1p does not require the Hsp90-independent molecular chaperone activity of Sba1p. The structure-function analysis suggests that Sba1p undergoes considerable structural rearrangements upon binding Hsp90 and that the large size of the p23/Sba1p-Hsp90 interaction surface facilitates maintenance of high affinity despite sequence divergence during evolution. The large interface may also contribute to preserving a protective function in an environment in which Hsp90 inhibitory compounds can be produced by various microorganisms.

MeSH Categories: Amino Acid Sequence, Animals, Base Sequence, Cells, Cultured, DNA Primers/genetics, DNA, Fungal/genetics, Genes, Fungal, Genetic Complementation Test, HSP90 Heat-Shock Proteins/antagonists & inhibitors/genetics/*metabolism, Intramolecular Oxidoreductases/deficiency/genetics/metabolism, Mice, Models, Molecular, Molecular Chaperones/chemistry/genetics/*metabolism, Mutagenesis, Site-Directed, Phenotype, Point Mutation, Protein Binding, Recombinant Proteins/chemistry/genetics/metabolism, Saccharomyces cerevisiae/genetics/*metabolism, Saccharomyces cerevisiae Proteins/antagonists &, inhibitors/chemistry/genetics/*metabolism, Sequence Deletion, Sequence Homology, Amino Acid

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Cell-type-specific function of BCL9 involves a transcriptional activation domain that synergizes with beta-catenin.

Publication Date: 2008 May PMID: 18347063
Authors: Sustmann, C. - Flach, H. - Ebert, H. - Eastman, Q. - Grosschedl, R.
Journal: Mol Cell Biol

Transcriptional regulation by the canonical Wnt pathway involves the stabilization and nuclear accumulation of beta-catenin, which assembles with LEF1/TCF transcription factors and cofactors to activate Wnt target genes. Recently, the nuclear beta-catenin complex has been shown to contain BCL9, which interacts with beta-catenin and recruits Pygopus as a transcriptional coactivator. However, the presumed general functions of Pygopus and BCL9, which has been proposed to act as a scaffolding protein for Pygopus, have been challenged by the rather specific and modest developmental defects of targeted inactivations of both the Pygo1 and the Pygo2 genes. Here, we analyze the function of BCL9 in transcriptional activation by beta-catenin. We find that BCL9 acts in a cell-type-specific manner and, in part, independent of Pygopus. We show that BCL9 itself contains a transcriptional activation domain in the C terminus, which functionally synergizes in lymphoid cells with the C-terminal transactivation domain of beta-catenin. Finally, we identify amino acids in the transactivation domain of beta-catenin that are important for its function and association with the histone acetyltransferases CBP/p300 and TRRAP/GCN5. Thus, BCL9 may serve to modulate and diversify the transcriptional responses to Wnt signaling in a cell-type-specific manner.

MeSH Categories: Amino Acid Sequence, Animals, Base Sequence, Cell Line, DNA Primers/genetics, Hela Cells, Humans, Intracellular Signaling Peptides and Proteins/genetics, Jurkat Cells, Molecular Sequence Data, Mutation, Neoplasm Proteins/antagonists & inhibitors/chemistry/genetics/*metabolism, Promoter Regions (Genetics), Protein Structure, Tertiary, RNA, Small Interfering/genetics, Recombinant Proteins/chemistry/genetics/metabolism, Signal Transduction, *Trans-Activation (Genetics), Transfection, Wnt Proteins/metabolism, beta Catenin/genetics/*metabolism

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Requirement of the tissue-restricted homeodomain transcription factor Nkx6.3 in differentiation of gastrin-producing G cells in the stomach antrum.

Publication Date: 2008 May PMID: 18347062
Authors: Choi, M. Y. - Romer, A. I. - Wang, Y. - Wu, M. P. - Ito, S. - Leiter, A. B. - Shivdasani, R. A.
Journal: Mol Cell Biol

Many homeodomain transcription factors function in organogenesis and cell differentiation. The Nkx family illustrates these functions especially well, and the Nkx6 subfamily controls differentiation in the central nervous system and pancreas. Nkx6.3, a recent addition to this subfamily, overlaps Nkx6.1 and Nkx6.2 in expression in the hindbrain and stomach. Nkx6.3 transcripts localize in the epithelium of the most distal stomach region, the antrum and pylorus; expression in the adult intestine is lower and confined to the proximal duodenum. Nkx6.3(-)(/)(-) mice develop and grow normally, with a grossly intact stomach and duodenum. These mice show markedly reduced gastrin mRNA, many fewer gastrin-producing (G) cells in the stomach antrum, hypogastrinemia, and increased stomach luminal pH, with a corresponding increase in somatostatin mRNA levels and antral somatostatin-producing (D) cells. They express normal levels of other transcription factors required for gastric endocrine cell differentiation, Pdx1, Pax6, and Ngn3; conversely, Ngn3(-)(/)(-) mice, which also show reduced gastrin levels, express Nkx6.3 normally. These studies implicate Nkx6.3 as a selective regulator of G- and D-cell lineages, which are believed to derive from a common progenitor, and suggest that it operates in parallel with Ngn3.

MeSH Categories: Animals, Base Sequence, Cell Differentiation, DNA Primers/genetics, Gastrin-Secreting Cells/*cytology/*metabolism, Gene Expression, Gene Targeting, Homeodomain Proteins/genetics/*metabolism, Mice, Mice, Inbred C57BL, Mice, Knockout, Pyloric Antrum/cytology/metabolism, RNA, Messenger/genetics/metabolism, Transcription Factors/genetics/*metabolism

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p110 CUX1 cooperates with E2F transcription factors in the transcriptional activation of cell cycle-regulated genes.

Publication Date: 2008 May PMID: 18347061
Authors: Truscott, M. - Harada, R. - Vadnais, C. - Robert, F. - Nepveu, A.
Journal: Mol Cell Biol

The transcription factor p110 CUX1 was shown to stimulate cell proliferation by accelerating entry into S phase. As p110 CUX1 can function as a transcriptional repressor or activator depending on promoter context, we investigated its mechanism of transcriptional activation using the DNA polymerase alpha gene promoter as a model system. Linker-scanning analysis revealed that a low-affinity E2F binding site is required for transcriptional activation. Moreover, coexpression with a dominant-negative mutant of DP-1 suggested that endogenous E2F factors are indeed needed for p110-mediated activation. Tandem affinity purification, coimmunoprecipitation, chromatin immunoprecipitation, and reporter assays indicated that p110 CUX1 can engage in weak protein-protein interactions with E2F1 and E2F2, stimulate their recruitment to the DNA polymerase alpha gene promoter, and cooperate with these factors in transcriptional activation. On the other hand, in vitro assays suggested that the interaction between CUX1 and E2F1 either is not direct or is regulated by posttranslational modifications. Genome-wide location analysis revealed that targets common to p110 CUX1 and E2F1 included many genes involved in cell cycle, DNA replication, and DNA repair. Comparison of the degree of enrichment for various E2F factors suggested that binding of p110 CUX1 to a promoter will favor the specific recruitment of E2F1, and to a lesser extent E2F2, over E2F3 and E2F4. Reporter assays on a subset of common targets confirmed that p110 CUX1 and E2F1 cooperate in their transcriptional activation. Overall, our results show that p110 CUX1 and E2F1 cooperate in the regulation of many cell cycle genes.

MeSH Categories: Base Sequence, Binding Sites/genetics, Cell Cycle/*genetics, Cell Line, Chromatin Immunoprecipitation, DNA/genetics/metabolism, DNA Polymerase I/genetics, E2F Transcription Factors/*metabolism, E2F1 Transcription Factor/metabolism, E2F2 Transcription Factor/metabolism, Homeodomain Proteins/*metabolism, Humans, Nuclear Proteins/*metabolism, Oligonucleotide Array Sequence Analysis, Plasmids/genetics, Promoter Regions (Genetics), Repressor Proteins/*metabolism, Trans-Activation (Genetics), Transfection

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The LIM protein AJUBA recruits protein arginine methyltransferase 5 to mediate SNAIL-dependent transcriptional repression.

Publication Date: 2008 May PMID: 18347060
Authors: Hou, Z. - Peng, H. - Ayyanathan, K. - Yan, K. P. - Langer, E. M. - Longmore, G. D. - Rauscher, F. J. 3rd
Journal: Mol Cell Biol

The SNAIL transcription factor contains C-terminal tandem zinc finger motifs and an N-terminal SNAG repression domain. The members of the SNAIL family have recently emerged as major contributors to the processes of development and metastasis via the regulation of epithelial-mesenchymal transition events during embryonic development and tumor progression. However, the mechanisms by which SNAIL represses gene expression are largely undefined. Previously we demonstrated that the AJUBA family of LIM proteins function as corepressors for SNAIL and, as such, may serve as a platform for the assembly of chromatin-modifying factors. Here, we describe the identification of the protein arginine methyltransferase 5 (PRMT5) as an effector recruited to SNAIL through an interaction with AJUBA that functions to repress the SNAIL target gene, E-cadherin. PRMT5 binds to the non-LIM region of AJUBA and is translocated into the nucleus in a SNAIL- and AJUBA-dependent manner. The depletion of PRMT5 in p19 cells stimulates E-cadherin expression, and the SNAIL, AJUBA, and PRMT5 ternary complex can be found at the proximal promoter region of the E-cadherin gene, concomitant with increased arginine methylation of histones at the locus. Together, these data suggest that PRMT5 is an effector of SNAIL-dependent gene repression.

MeSH Categories: Active Transport, Cell Nucleus, Amino Acid Sequence, Animals, Base Sequence, Cadherins/genetics/metabolism, Cell Line, DNA Primers/genetics, Homeodomain Proteins/genetics/*metabolism, Humans, Mice, Models, Biological, Molecular Sequence Data, Multiprotein Complexes, Promoter Regions (Genetics), Protein Binding, Protein Methyltransferases/*genetics/*metabolism, RNA, Small Interfering/genetics, Recombinant Proteins/genetics/metabolism, Transcription Factors/genetics/*metabolism, Transfection

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Integration of protein kinases mTOR and extracellular signal-regulated kinase 5 in regulating nucleocytoplasmic localization of NFATc4.

Publication Date: 2008 May PMID: 18347059
Authors: Yang, T. T. - Yu, R. Y. - Agadir, A. - Gao, G. J. - Campos-Gonzalez, R. - Tournier, C. - Chow, C. W.
Journal: Mol Cell Biol

The target of rapamycin (TOR) signaling regulates the nucleocytoplasmic shuttling of transcription factors in yeast. Whether the mammalian counterpart of TOR (mTOR) also regulates nucleocytoplasmic shuttling is not known. Using a phospho-specific monoclonal antibody, we demonstrate that mTOR phosphorylates Ser(168,170) of endogenous NFATc4, which are conserved gate-keeping Ser residues that control NFAT subcellular distribution. The mTOR acts as a basal kinase during the resting state to maintain NFATc4 in the cytosol. Inactivation and nuclear export of NFATc4 are mediated by rephosphorylation of Ser(168,170), which can be a nuclear event. Kinetic analyses demonstrate that rephosphorylation of Ser(168,170) of endogenous NFATc4 is mediated by mTOR and, surprisingly, by extracellular signal-regulated kinase 5 (ERK5) mitogen-activated protein kinase as well. Ablation of ERK5 in the Erk5(-/-) cells ascertains defects in NFATc4 rephosphorylation and nucleocytoplasmic shuttling. In addition, phosphorylation of NFATc4 by ERK5 primes subsequent phosphorylation mediated by CK1alpha. These results demonstrate that distinct protein kinases are integrated to phosphorylate the gate-keeping residues Ser(168,170) of NFATc4, to regulate subcellular distribution. These data also expand the repertoire of physiological substrates of mTOR and ERK5.

MeSH Categories: Active Transport, Cell Nucleus, Animals, Antibodies, Monoclonal, Antibody Specificity, COS Cells, Cell Line, Cells, Cultured, Cercopithecus aethiops, Cricetinae, Mice, Mice, Knockout, Mitogen-Activated Protein Kinase 7/deficiency/genetics/*metabolism, Models, Biological, NFATC Transcription, Factors/chemistry/deficiency/genetics/immunology/*metabolism, Phosphorylation, Protein Kinases/*metabolism, Recombinant Proteins/genetics/metabolism, Serine/chemistry, p38 Mitogen-Activated Protein Kinases/metabolism

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The RASSF1A tumor suppressor restrains anaphase-promoting complex/cyclosome activity during the G1/S phase transition to promote cell cycle progression in human epithelial cells.

Publication Date: 2008 May PMID: 18347058
Authors: Whitehurst, A. W. - Ram, R. - Shivakumar, L. - Gao, B. - Minna, J. D. - White, M. A.
Journal: Mol Cell Biol

Multiple molecular lesions in human cancers directly collaborate to deregulate proliferation and suppress apoptosis to promote tumorigenesis. The candidate tumor suppressor RASSF1A is commonly inactivated in a broad spectrum of human tumors and has been implicated as a pivotal gatekeeper of cell cycle progression. However, a mechanistic account of the role of RASSF1A gene inactivation in tumor initiation is lacking. Here we have employed loss-of-function analysis in human epithelial cells for a detailed investigation of the contribution of RASSF1 to cell cycle progression. We found that RASSF1A has dual opposing regulatory connections to G(1)/S phase cell cycle transit. RASSF1A associates with the Ewing sarcoma breakpoint protein, EWS, to limit accumulation of cyclin D1 and restrict exit from G(1). Surprisingly, we found that RASSF1A is also required to restrict SCF(betaTrCP) activity to allow G/S phase transition. This restriction is required for accumulation of the anaphase-promoting complex/cyclosome (APC/C) inhibitor Emi1 and the concomitant block of APC/C-dependent cyclin A turnover. The consequence of this relationship is inhibition of cell cycle progression in normal epithelial cells upon RASSF1A depletion despite elevated cyclin D1 concentrations. Progression to tumorigenicity upon RASSF1A gene inactivation should therefore require collaborating genetic aberrations that bypass the consequences of impaired APC/C regulation at the G(1)/S phase cell cycle transition.

MeSH Categories: Base Sequence, Cell Cycle/*physiology, Cell Cycle Proteins/genetics/metabolism, DNA Primers/genetics, Epithelial Cells/cytology/metabolism, F-Box Proteins/genetics/metabolism, G1 Phase, Hela Cells, Humans, RNA, Small Interfering/genetics, RNA-Binding Protein EWS/genetics/metabolism, Recombinant Fusion Proteins/genetics/metabolism, S Phase, Transfection, Tumor Suppressor Proteins/antagonists & inhibitors/genetics/*physiology, Two-Hybrid System Techniques, Ubiquitin-Protein Ligase Complexes/*metabolism, beta-Transducin Repeat-Containing Proteins/antagonists &, inhibitors/genetics/metabolism

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Mutation of the PDK1 PH domain inhibits protein kinase B/Akt, leading to small size and insulin resistance.

Publication Date: 2008 May PMID: 18347057
Authors: Bayascas, J. R. - Wullschleger, S. - Sakamoto, K. - Garcia-Martinez, J. M. - Clacher, C. - Komander, D. - van Aalten, D. M. - Boini, K. M. - Lang, F. - Lipina, C. - Logie, L. - Sutherland, C. - Chudek, J. A. - van Diepen, J. A. - Voshol, P. J. - Lucocq, J. M. - Alessi, D. R.
Journal: Mol Cell Biol

PDK1 activates a group of kinases, including protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), and serum and glucocorticoid-induced protein kinase (SGK), that mediate many of the effects of insulin as well as other agonists. PDK1 interacts with phosphoinositides through a pleckstrin homology (PH) domain. To study the role of this interaction, we generated knock-in mice expressing a mutant of PDK1 incapable of binding phosphoinositides. The knock-in mice are significantly small, insulin resistant, and hyperinsulinemic. Activation of PKB is markedly reduced in knock-in mice as a result of lower phosphorylation of PKB at Thr308, the residue phosphorylated by PDK1. This results in the inhibition of the downstream mTOR complex 1 and S6K1 signaling pathways. In contrast, activation of SGK1 or p90 ribosomal S6 kinase or stimulation of S6K1 induced by feeding is unaffected by the PDK1 PH domain mutation. These observations establish the importance of the PDK1-phosphoinositide interaction in enabling PKB to be efficiently activated with an animal model. Our findings reveal how reduced activation of PKB isoforms impinges on downstream signaling pathways, causing diminution of size as well as insulin resistance.

MeSH Categories: Amino Acid Substitution, Animals, Body Size/*genetics/physiology, Female, Insulin Resistance/*genetics/physiology, Male, Mice, Mice, Inbred C57BL, Mice, Mutant Strains, Models, Molecular, Mutagenesis, Site-Directed, *Mutation, Phenotype, Prediabetic State/genetics/metabolism, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases/chemistry/*genetics/*metabolism, Proto-Oncogene Proteins c-akt/*antagonists & inhibitors

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Transcription-coupled methylation of histone H3 at lysine 36 regulates dosage compensation by enhancing recruitment of the MSL complex in Drosophila melanogaster.

Publication Date: 2008 May PMID: 18347056
Authors: Bell, O. - Conrad, T. - Kind, J. - Wirbelauer, C. - Akhtar, A. - Schubeler, D.
Journal: Mol Cell Biol

In Drosophila melanogaster, dosage compensation relies on the targeting of the male-specific lethal (MSL) complex to hundreds of sites along the male X chromosome. Transcription-coupled methylation of histone H3 lysine 36 is enriched toward the 3' end of active genes, similar to the MSL proteins. Here, we have studied the link between histone H3 methylation and MSL complex targeting using RNA interference and chromatin immunoprecipitation. We show that trimethylation of histone H3 at lysine 36 (H3K36me3) relies on the histone methyltransferase Hypb and is localized promoter distal at dosage-compensated genes, similar to active genes on autosomes. However, H3K36me3 has an X-specific function, as reduction specifically decreases acetylation of histone H4 lysine 16 on the male X chromosome. This hypoacetylation is caused by compromised MSL binding and results in a failure to increase expression twofold. Thus, H3K36me3 marks the body of all active genes yet is utilized in a chromosome-specific manner to enhance histone acetylation at sites of dosage compensation.

MeSH Categories: Animals, Binding Sites/genetics, Cell Line, Chromatin Immunoprecipitation, Dosage Compensation, Genetic, Drosophila Proteins/chemistry/genetics/*metabolism, Drosophila melanogaster/*genetics/*metabolism, Female, Histones/chemistry/genetics/*metabolism, Lysine/chemistry, Male, Methylation, Models, Biological, Multiprotein Complexes, RNA Interference, Sex Determination (Genetics), Transcription Factors/chemistry/genetics/*metabolism, Transcription, Genetic, X Chromosome/genetics/metabolism

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Lys63-linked polyubiquitination of IRAK-1 is required for interleukin-1 receptor- and toll-like receptor-mediated NF-kappaB activation.

Publication Date: 2008 May PMID: 18347055
Authors: Conze, D. B. - Wu, C. J. - Thomas, J. A. - Landstrom, A. - Ashwell, J. D.
Journal: Mol Cell Biol

Stimulation through the interleukin-1 receptor (IL-1R) and some Toll-like receptors (TLRs) induces ubiquitination of TRAF6 and IRAK-1, signaling components required for NF-kappaB and mitogen-activated protein kinase activation. Here we show that although TRAF6 and IRAK-1 acquired Lys63 (K63)-linked polyubiquitin chains upon IL-1 stimulation, only ubiquitinated IRAK-1 bound NEMO, the regulatory subunit of IkappaB kinase (IKK). The sites of IRAK-1 ubiquitination were mapped to Lys134 and Lys180, and arginine substitution of these residues impaired IL-1R/TLR-mediated IRAK-1 ubiquitination, NEMO binding, and NF-kappaB activation. K63-linked ubiquitination of IRAK-1 required enzymatically active TRAF6, indicating that it is the physiologically relevant E3. Thus, K63-linked polyubiquitination of proximal signaling proteins is a common mechanism used by diverse innate immune receptors for recruiting IKK and activating NF-kappaB.

MeSH Categories: Animals, Base Sequence, Binding Sites, Cell Line, Cells, Cultured, DNA Primers/genetics, Humans, I-kappa B Kinase/chemistry/genetics/metabolism, Interleukin-1 Receptor-Associated Kinases/*chemistry/genetics/*metabolism, Lysine/chemistry, Mice, NF-kappa B/*metabolism, Receptors, Interleukin-1/genetics/*metabolism, Recombinant Proteins/chemistry/genetics/metabolism, TNF Receptor-Associated Factor 6/genetics/metabolism, Toll-Like Receptor 4/genetics/metabolism, Toll-Like Receptors/genetics/*metabolism, Transfection, Ubiquitination

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Stimulation of human and rat islet beta-cell proliferation with retention of function by the homeodomain transcription factor Nkx6.1.

Publication Date: 2008 May PMID: 18347054
Authors: Schisler, J. C. - Fueger, P. T. - Babu, D. A. - Hohmeier, H. E. - Tessem, J. S. - Lu, D. - Becker, T. C. - Naziruddin, B. - Levy, M. - Mirmira, R. G. - Newgard, C. B.
Journal: Mol Cell Biol

The homeodomain transcription factor Nkx6.1 plays an important role in pancreatic islet beta-cell development, but its effects on adult beta-cell function, survival, and proliferation are not well understood. In the present study, we demonstrated that treatment of primary rat pancreatic islets with a cytomegalovirus promoter-driven recombinant adenovirus containing the Nkx6.1 cDNA (AdCMV-Nkx6.1) causes dramatic increases in [methyl-(3)H] thymidine and 5-bromo-2'-deoxyuridine (BrdU) incorporation and in the number of cells per islet relative to islets treated with a control adenovirus (AdCMV-betaGAL), whereas suppression of Nkx6.1 expression reduces thymidine incorporation. Immunocytochemical studies reveal that >80% of BrdU-positive cells in AdCMV-Nkx6.1-treated islets are beta cells. Microarray, real-time PCR, and immunoblot analyses reveal that overexpression of Nkx6.1 in rat islets causes concerted upregulation of a cadre of cell cycle control genes, including those encoding cyclins A, B, and E, and several regulatory kinases. Cyclin E is upregulated earlier than the other cyclins, and adenovirus-mediated overexpression of cyclin E is shown to be sufficient to activate islet cell proliferation. Moreover, chromatin immunoprecipitation assays demonstrate direct interaction of Nkx6.1 with the cyclin A2 and B1 genes. Overexpression of Nkx6.1 in rat islets caused a clear enhancement of glucose-stimulated insulin secretion (GSIS), whereas overexpression of Nkx6.1 in human islets caused an increase in the level of [(3)H]thymidine incorporation that was twice the control level, along with complete retention of GSIS. We conclude that Nkx6.1 is among the very rare factors capable of stimulating beta-cell replication with retention or enhancement of function, properties that may be exploitable for expansion of beta-cell mass in treatment of both major forms of diabetes.

MeSH Categories: Adenoviridae/genetics, Animals, Base Sequence, Cell Cycle/genetics, Cell Proliferation, Cyclin A/genetics, Cyclin B/genetics, Cyclins/metabolism, DNA Primers/genetics, Gene Expression, Glucose/pharmacology, Homeodomain Proteins/antagonists & inhibitors/genetics/*metabolism, Humans, Insulin/secretion, Insulin-Secreting Cells/*cytology/drug effects/*metabolism/secretion, Kinetics, Models, Biological, Oligonucleotide Array Sequence Analysis, Promoter Regions (Genetics), RNA, Messenger/genetics/metabolism, RNA, Small Interfering/genetics, Rats, Recombinant Proteins/genetics/metabolism, Tissue Culture Techniques

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Silencing of Agamma-globin gene expression during adult definitive erythropoiesis mediated by GATA-1-FOG-1-Mi2 complex binding at the -566 GATA site.

Publication Date: 2008 May PMID: 18347053
Authors: Harju-Baker, S. - Costa, F. C. - Fedosyuk, H. - Neades, R. - Peterson, K. R.
Journal: Mol Cell Biol

Autonomous silencing of gamma-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the (A)gamma-globin gene was identified between -730 and -378 relative to the mRNA start site. A marked copy of the (A)gamma-globin gene inserted between locus control region 5' DNase I-hypersensitive site 1 and the epsilon-globin gene was transcriptionally silenced in adult beta-globin locus yeast artificial chromosome (beta-YAC) transgenic mice, but deletion of the 352-bp region restored expression. This fragment reduced reporter gene expression in K562 cells, and GATA-1 was shown to bind within this sequence at the -566 GATA site. Further, the Mi2 protein, a component of the NuRD complex, was observed in erythroid cells with low gamma-globin levels, whereas only a weak signal was detected when gamma-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from beta-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the (A)gamma-globin -566 or (G)gamma-globin -567 GATA site when gamma-globin expression was low (day 18) but not when gamma-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, gamma-globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the -566/-567 GATA sites of the proximal gamma-globin promoters.

MeSH Categories: Animals, Base Sequence, Binding Sites/genetics, Chromosomes, Artificial, Yeast/genetics, DNA Primers/genetics, DNA-Binding Proteins/metabolism, Erythropoiesis/*genetics, GATA Transcription Factors/metabolism, GATA1 Transcription Factor/*metabolism, Gene Expression, Gene Silencing, Globins/*genetics, Histone Deacetylases/metabolism, Humans, K562 Cells, Macromolecular Substances, Mice, Mice, Inbred C57BL, Mice, Transgenic, Nuclear Proteins/*metabolism, Promoter Regions (Genetics), RNA, Messenger/genetics/metabolism, Transcription Factors/*metabolism

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The U11-48K protein contacts the 5' splice site of U12-type introns and the U11-59K protein.

Publication Date: 2008 May PMID: 18347052
Authors: Turunen, J. J. - Will, C. L. - Grote, M. - Luhrmann, R. - Frilander, M. J.
Journal: Mol Cell Biol

Little is currently known about proteins that make contact with the pre-mRNA in the U12-dependent spliceosome and thereby contribute to intron recognition. Using site-specific cross-linking, we detected an interaction between the U11-48K protein and U12-type 5' splice sites (5'ss). This interaction did not require branch point recognition and was sensitive to 5'ss mutations, suggesting that 48K interacts with the 5'ss during the first steps of prespliceosome assembly in a sequence-dependent manner. RNA interference-induced knockdown of 48K in HeLa cells led to reduced cell growth and the inhibition of U12-type splicing, as well as the activation of cryptic, U2-type splice sites, suggesting that 48K plays a critical role in U12-type intron recognition. 48K knockdown also led to reduced levels of U11/U12 di-snRNP, indicating that 48K contributes to the stability and/or formation of this complex. In addition to making contact with the 5'ss, 48K interacts with the U11-59K protein, a protein at the interface of the U11/U12 di-snRNP. These studies provide important insights into the protein-mediated recognition of the U12-type 5'ss, as well as functionally important interactions within the U11/U12 di-snRNP.

MeSH Categories: Animals, Apoptosis Regulatory Proteins/*genetics/*metabolism, Base Sequence, Binding Sites/genetics, DNA Primers/genetics, Hela Cells, Humans, Introns, Mice, RNA Interference, *RNA Splice Sites, RNA, Small Nuclear/genetics/metabolism, Ribonucleoproteins, Small Nuclear/antagonists &, inhibitors/*genetics/*metabolism, Spliceosomes/metabolism, Transcription Factors/*genetics/*metabolism, Two-Hybrid System Techniques, Vesicular Transport Proteins/genetics

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Regulation of the Drosophila hypoxia-inducible factor alpha Sima by CRM1-dependent nuclear export.

Publication Date: 2008 May PMID: 18332128
Authors: Romero, N. M. - Irisarri, M. - Roth, P. - Cauerhff, A. - Samakovlis, C. - Wappner, P.
Journal: Mol Cell Biol

Hypoxia-inducible factor alpha (HIF-alpha) proteins are regulated by oxygen levels through several different mechanisms that include protein stability, transcriptional coactivator recruitment, and subcellular localization. It was previously reported that these transcription factors are mainly nuclear in hypoxia and cytoplasmic in normoxia, but so far the molecular basis of this regulation is unclear. We show here that the Drosophila melanogaster HIF-alpha protein Sima shuttles continuously between the nucleus and the cytoplasm. We identified the relevant nuclear localization signal and two functional nuclear export signals (NESs). These NESs are in the Sima basic helix-loop-helix (bHLH) domain and promote CRM1-dependent nuclear export. Site-directed mutagenesis of either NES provoked Sima nuclear retention and increased transcriptional activity, suggesting that nuclear export contributes to Sima regulation. The identified NESs are conserved and probably functional in the bHLH domains of several bHLH-PAS proteins. We propose that rapid nuclear export of Sima regulates the duration of cellular responses to hypoxia.

MeSH Categories: Active Transport, Cell Nucleus, Amino Acid Sequence, Animals, Animals, Genetically Modified, Conserved Sequence, DNA-Binding Proteins/chemistry/genetics/*metabolism, Drosophila Proteins/chemistry/genetics/*metabolism, Drosophila melanogaster/genetics/*metabolism, Evolution, Molecular, Hypoxia-Inducible Factor 1, alpha Subunit/chemistry/genetics/*metabolism, Karyopherins/genetics/*metabolism, Models, Biological, Molecular Sequence Data, Nuclear Export Signals/genetics, Nuclear Localization Signals/chemistry/genetics, Protein Processing, Post-Translational, Receptors, Cytoplasmic and Nuclear/genetics/*metabolism, Sequence Homology, Amino Acid, Transcription, Genetic

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Redundant roles of Tead1 and Tead2 in notochord development and the regulation of cell proliferation and survival.

Publication Date: 2008 May PMID: 18332127
Authors: Sawada, A. - Kiyonari, H. - Ukita, K. - Nishioka, N. - Imuta, Y. - Sasaki, H.
Journal: Mol Cell Biol

Four members of the TEAD/TEF family of transcription factors are expressed widely in mouse embryos and adult tissues. Although in vitro studies have suggested various roles for TEAD proteins, their in vivo functions remain poorly understood. Here we examined the role of Tead genes by generating mouse mutants for Tead1 and Tead2. Tead2(-/-) mice appeared normal, but Tead1(-/-); Tead2(-/-) embryos died at embryonic day 9.5 (E9.5) with severe growth defects and morphological abnormalities. At E8.5, Tead1(-/-); Tead2(-/-) embryos were already small and lacked characteristic structures such as a closed neural tube, a notochord, and somites. Despite these overt abnormalities, differentiation and patterning of the neural plate and endoderm were relatively normal. In contrast, the paraxial mesoderm and lateral plate mesoderm were displaced laterally, and a differentiated notochord was not maintained. These abnormalities and defects in yolk sac vasculature organization resemble those of mutants for Yap, which encodes a coactivator of TEAD proteins. Moreover, we demonstrated genetic interactions between Tead1 and Tead2 and Yap. Finally, Tead1(-/-); Tead2(-/-) embryos showed reduced cell proliferation and increased apoptosis. These results suggest that Tead1 and Tead2 are functionally redundant, use YAP as a major coactivator, and support notochord maintenance as well as cell proliferation and survival in mouse development.

MeSH Categories: Adaptor Proteins, Signal Transducing/genetics/physiology, Animals, Apoptosis, Base Sequence, Body Patterning, Cell Proliferation, Cell Survival, DNA Primers/genetics, DNA-Binding Proteins/deficiency/genetics/*physiology, Endoderm/abnormalities/embryology, Gene Expression Regulation, Developmental, Gene Targeting, Mesoderm/abnormalities/embryology, Mice, Mice, Knockout, Mice, Mutant Strains, Mutation, Notochord/abnormalities/cytology/*embryology, Phosphoproteins/genetics/physiology, Transcription Factors/deficiency/genetics/*physiology

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Signaling through ShcA is required for transforming growth factor beta- and Neu/ErbB-2-induced breast cancer cell motility and invasion.

Publication Date: 2008 May PMID: 18332126
Authors: Northey, J. J. - Chmielecki, J. - Ngan, E. - Russo, C. - Annis, M. G. - Muller, W. J. - Siegel, P. M.
Journal: Mol Cell Biol

Cooperation between the Neu/ErbB-2 and transforming growth factor beta (TGF-beta) signaling pathways enhances the invasive and metastatic capabilities of breast cancer cells; however, the underlying mechanisms mediating this synergy have yet to be fully explained. We demonstrate that TGF-beta induces the migration and invasion of mammary tumor explants expressing an activated Neu/ErbB-2 receptor, which requires signaling from autophosphorylation sites located in the C terminus. A systematic analysis of mammary tumor explants expressing Neu/ErbB-2 add-back receptors that couple to distinct signaling molecules has mapped the synergistic effect of TGF-beta-induced motility and invasion to signals emanating from tyrosine residues 1226/1227 and 1253 of Neu/ErbB-2. Given that the ShcA adaptor protein is known to interact with Neu/ErbB-2 through these residues, we investigated the importance of this signaling molecule in TGF-beta-induced cell motility and invasion. The reduction of ShcA expression rendered cells expressing activated Neu/ErbB-2, or add-back receptors signaling specifically through tyrosines 1226/1227 or 1253, unresponsive to TGF-beta-induced motility and invasion. In addition, a dominant-negative form of ShcA, lacking its three known tyrosine phosphorylation sites, completely abrogates the TGF-beta-induced migration and invasion of breast cancer cells expressing activated Neu/ErbB-2. Our results implicate signaling through the ShcA adaptor as a key component in the synergistic interaction between these pathways.

MeSH Categories: Adaptor Proteins, Signal Transducing/antagonists &, inhibitors/genetics/*metabolism, Animals, Cell Movement/drug effects, Female, Focal Adhesions, Gene Expression, Lung Neoplasms/secondary, Mammary Neoplasms,, Experimental/genetics/*metabolism/pathology/physiopathology, Mice, Mice, Nude, Neoplasm Invasiveness, Phosphotyrosine/metabolism, Point Mutation, RNA, Small Interfering/genetics, Receptor, erbB-2/chemistry/genetics/*metabolism, Signal Transduction, Transfection, Transforming Growth Factor beta/*pharmacology

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PTEN nuclear localization is regulated by oxidative stress and mediates p53-dependent tumor suppression.

Publication Date: 2008 May PMID: 18332125
Authors: Chang, C. J. - Mulholland, D. J. - Valamehr, B. - Mosessian, S. - Sellers, W. R. - Wu, H.
Journal: Mol Cell Biol

The tumor suppressor gene PTEN (phosphatase and tensin homologue deleted on chromosome 10) is frequently mutated or deleted in various human cancers. PTEN localizes predominantly to the cytoplasm and functions as a lipid phosphatase, thereby negatively regulating the phosphatidylinositol 3-kinase-AKT signaling pathway. PTEN can also localize to the nucleus, where it binds and regulates p53 protein level and transcription activity. However, the precise function of nuclear PTEN and the factors that control PTEN nuclear localization are still largely unknown. In this study, we identified oxidative stress as one of the physiological stimuli that regulate the accumulation of nuclear PTEN. Specifically, oxidative stress inhibits PTEN nuclear export, a process depending on phosphorylation of its amino acid residue Ser-380. Nuclear PTEN, independent of its phosphatase activity, leads to p53-mediated G(1) growth arrest, cell death, and reduction of reactive oxygen species production. Using xenografts propagated from human prostate cancer cell lines, we reveal that nuclear PTEN is sufficient to reduce tumor progression in vivo in a p53-dependent manner. The data outlined in this study suggest a unique role of nuclear PTEN to arrest and protect cells upon oxidative damage and to regulate tumorigenesis. Since tumor cells are constantly exposed to oxidative stress, our study elucidates the cooperative roles of nuclear PTEN with p53 in tumor suppression.

MeSH Categories: Active Transport, Cell Nucleus, Animals, Apoptosis, Base Sequence, Cell Cycle, Cell Line, Tumor, Cell Nucleus/metabolism, Cells, Cultured, DNA Primers/genetics, Genes, p53, Humans, Male, Mice, Mice, Knockout, Mice, SCID, Models, Biological, Neoplasm Transplantation, Oxidative Stress, PTEN Phosphohydrolase/deficiency/genetics/*metabolism, Prostatic Neoplasms/genetics/metabolism/pathology/prevention & control, Transplantation, Heterologous, Tumor Suppressor Protein p53/*metabolism

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