scholarly journals Spatial Interplay between PIASy and FIP200 in the Regulation of Signal Transduction and Transcriptional Activity

2008 ◽  
Vol 28 (8) ◽  
pp. 2771-2781 ◽  
Author(s):  
Nadine Martin ◽  
Klaus Schwamborn ◽  
Henning Urlaub ◽  
Boyi Gan ◽  
Jun-Lin Guan ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Ring Finger ◽  
Subcellular Fractionation ◽  
Interacting Protein ◽  
Cellular Processes ◽  
Bona Fide ◽  
Independent Effects ◽  
P21 Promoter

ABSTRACT The members of the protein inhibitor of activated STAT (PIAS) family of proteins are implicated in fundamental cellular processes, including transcriptional regulation, either through action as E3 SUMO ligases or through SUMO-independent effects. We report here the identification of FIP200 (focal adhesion kinase family-interacting protein of 200 kDa) as a new PIASy-interacting protein. We show that the interaction depends on the integrity of the RING finger of PIASy and the carboxy terminus of FIP200. Both in vitro and in vivo sumoylation assays failed to reveal any sumoylation of FIP200, suggesting that FIP200 is not a bona fide SUMO substrate. Immunofluorescence microscopy and subcellular fractionation, either upon forced PIASy expression or in the absence of PIASy, revealed that interaction with PIASy redistributes FIP200 from the cytoplasm to the nucleus, correlating with abrogation of FIP200 regulation of TSC/S6K signaling. Conversely, FIP200 enhances the transcriptional activation of the p21 promoter by PIASy whereas PIASy transcription activity is severely reduced upon FIP200 depletion by RNA interference. Chromatin immunoprecipitation analysis demonstrates that endogenous PIASy and FIP200 are corecruited to the p21 promoter. Altogether, these results provide the first evidence for the existence of a close—spatially controlled—mode of regulation of FIP200 and PIASy nucleocytoplasmic functions.

scholarly journals Arabidopsis ALIX is required for the endosomal localization of the deubiquitinating enzyme AMSH3

2015 ◽  
Vol 112 (40) ◽  
pp. E5543-E5551 ◽  
Author(s):  
Kamila Kalinowska ◽  
Marie-Kristin Nagel ◽  
Kaija Goodman ◽  
Laura Cuyas ◽  
Franziska Anzenberger ◽  
...  
Keyword(s):  
Cellular Level ◽  
Deubiquitinating Enzymes ◽  
Interacting Protein ◽  
Endosomal Sorting ◽  
Cellular Processes ◽  
Late Endosomes ◽  
Knockout Mutants ◽  
Important Regulator

Ubiquitination is a signal for various cellular processes, including for endocytic degradation of plasma membrane cargos. Ubiquitinating as well as deubiquitinating enzymes (DUBs) can regulate these processes by modifying the ubiquitination status of target protein. Although accumulating evidence points to the important regulatory role of DUBs, the molecular basis of their regulation is still not well understood. Associated molecule with the SH3 domain of signal transduction adaptor molecule (STAM) (AMSH) is a conserved metalloprotease DUB in eukaryotes. AMSH proteins interact with components of the endosomal sorting complex required for transport (ESCRT) and are implicated in intracellular trafficking. To investigate how the function of AMSH is regulated at the cellular level, we carried out an interaction screen for the Arabidopsis AMSH proteins and identified the Arabidopsis homolog of apoptosis-linked gene-2 interacting protein X (ALIX) as a protein interacting with AMSH3 in vitro and in vivo. Analysis of alix knockout mutants in Arabidopsis showed that ALIX is essential for plant growth and development and that ALIX is important for the biogenesis of the vacuole and multivesicular bodies (MVBs). Cell biological analysis revealed that ALIX and AMSH3 colocalize on late endosomes. Although ALIX did not stimulate AMSH3 activity in vitro, in the absence of ALIX, AMSH3 localization on endosomes was abolished. Taken together, our data indicate that ALIX could function as an important regulator for AMSH3 function at the late endosomes.

scholarly journals CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation

2008 ◽  
Vol 181 (6) ◽  
pp. 959-972 ◽  
Author(s):  
Xueni Li ◽  
Mei Huang ◽  
Huiling Zheng ◽  
Yinyin Wang ◽  
Fangli Ren ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Osteoblast Differentiation ◽  
Degradation Mechanism ◽  
Interacting Protein ◽  
E3 Ligases ◽  
Protein Levels ◽  
C Terminus ◽  
Osteoblast Lineage

Runx2, an essential transactivator for osteoblast differentiation, is tightly regulated at both the transcriptional and posttranslational levels. In this paper, we report that CHIP (C terminus of Hsc70-interacting protein)/STUB1 regulates Runx2 protein stability via a ubiquitination-degradation mechanism. CHIP interacts with Runx2 in vitro and in vivo. In the presence of increased Runx2 protein levels, CHIP expression decreases, whereas the expression of other E3 ligases involved in Runx2 degradation, such as Smurf1 or WWP1, remains constant or increases during osteoblast differentiation. Depletion of CHIP results in the stabilization of Runx2, enhances Runx2-mediated transcriptional activation, and promotes osteoblast differentiation in primary calvarial cells. In contrast, CHIP overexpression in preosteoblasts causes Runx2 degradation, inhibits osteoblast differentiation, and instead enhances adipogenesis. Our data suggest that negative regulation of the Runx2 protein by CHIP is critical in the commitment of precursor cells to differentiate into the osteoblast lineage.

scholarly journals Cooperative Interaction between GATA-4 and GATA-6 Regulates Myocardial Gene Expression

1999 ◽  
Vol 19 (6) ◽  
pp. 4355-4365 ◽  
Author(s):  
Frédéric Charron ◽  
Pierre Paradis ◽  
Odile Bronchain ◽  
Georges Nemer ◽  
Mona Nemer
Keyword(s):  
Cooperative Interaction ◽  
Gata Factors ◽  
Cellular Processes ◽  
Two Factors ◽  
Bona Fide ◽  
Transcriptional Pathway ◽  
Combinatorial Interactions ◽  
Myocardial Gene Expression

ABSTRACT Two members of the GATA family of transcription factors, GATA-4 and GATA-6, are expressed in the developing and postnatal myocardium and are equally potent transactivators of several cardiac promoters. However, several in vitro and in vivo lines of evidence suggest distinct roles for the two factors in the heart. Since identification of the endogenous downstream targets of GATA factors would greatly help to elucidate their exact functions, we have developed an adenovirus-mediated antisense strategy to specifically inhibit GATA-4 and GATA-6 protein production in postnatal cardiomyocytes. Expression of several endogenous cardiac genes was significantly down-regulated in cells lacking GATA-4 or GATA-6, indicating that these factors are required for the maintenance of the cardiac genetic program. Interestingly, transcription of some genes like the α- and β-myosin heavy-chain (α- and β-MHC) genes was preferentially regulated by GATA-4 due, in part, to higher affinity of GATA-4 for their promoter GATA element. However, transcription of several other genes, including the atrial natriuretic factor and B-type natriuretic peptide (ANF and BNP) genes, was similarly down-regulated in cardiomyocytes lacking one or both GATA factors, suggesting that GATA-4 and GATA-6 could act through the same transcriptional pathway. Consistent with this, GATA-4 and GATA-6 were found to colocalize in postnatal cardiomyocytes and to interact functionally and physically to provide cooperative activation of the ANF and BNP promoters. The results identify for the first time bona fide in vivo targets for GATA-4 and GATA-6 in the myocardium. The data also show that GATA factors act in concert to regulate distinct subsets of genes, suggesting that combinatorial interactions among GATA factors may differentially control various cellular processes.

Characterization Of In Vitro Hdm2 E3 Ubiquitin Ligase-Mediated p53 Ubiquitination

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4903-4903
Author(s):  
Bradley B Brasher ◽  
Eduardo Guillen ◽  
Ivan Tomasic ◽  
Carsten Schwerdtfeger ◽  
Francesco D Melandri
Keyword(s):  
Transcriptional Activation ◽  
Cell Function ◽  
Ring Finger ◽  
Ubiquitin Ligases ◽  
Wild Type ◽  
Western Blots ◽  
Hematopoietic Stem ◽  
In Vivo Experiments

Abstract Double minute 2 protein (Mdm2, Hdm2 in humans) is a RING-finger Ubiquitin E3 Ligase that acts as a major regulator of the tumor suppressor protein p53. Mdm2 inhibits p53 -mediated cell cycle arrest and apoptosis by binding its transcriptional activation domain. The ligase activity of Mdm2 is responsible for the ubiquitination and subsequent proteasomal degradation of p53. Mdm2 also regulates its own intracellular levels by auto-ubiquitination, and can be SUMOylated, which reportedly decreases autoubiquitination activity but increases activity toward p53. Imbalances in the p53 pathway are frequently associated with hematologic disease states. Loss of p53 function is a driving force in leukemia and lymphoma in humans and mice, while increased p53 activity can inhibit hematopoietic stem cell function and contribute to myelodysplasia. Thus, careful control of p53 activity is critical for homeostasis. Most of our understanding of p53 function in hematopoiesis is derived from in vivo experiments using genetically modified mice (Pant V., et al, Blood. 2012; 120:5118-27). While this is a powerful system for elucidating genetic pathways that influence p53 activity, there is still much to learn about the mechanisms of p53 regulation at the enzymatic level. To facilitate studies in this area, we purified recombinant Hdm2 and p53 from E.coli and developed gel-based assays to monitor both autoubiquitination and ubiquitination of protein substrates. We observed rapid autoubiquitination of Hdm2 using both wild-type and lysine-less (K0) ubiquitin, though reactions containing the former generated significantly higher molecular weight Hdm2-ubiquitin adducts. Hdm2 ubiquitination of p53 produced a discrete, ladder-like banding pattern on Western Blots regardless of whether wild-type or K0 ubiquitin was included in the reaction. This suggests that the principal product of this defined Hdm2-p53 reaction is multi-monoubiquitinated p53, as opposed to p53 modified with polyubiquitin chains. Reactions using an alternative substrate yielded different results. Hdm2 ubiquitination of Angiocidin/S5a protein generated a large smeary pattern on Western Blots instead of discrete bands. This is consistent with the Hdm2-catalyzed polyubiquitination of S5a, demonstrating that ubiquitin ligases are capable of generating different in vitro ubiquitination patterns that are dependent on the substrate utilized in the assay. These results suggest that care must be taken in experimental designs, particularly with respect to substrate and assay read out. Finally, recombinant UBE4B was included in Mdm2/p53 reactions to test the recently reported E4-ligase activity of this enzyme. Ultimately these reagents should prove useful for fully defined, in vitro studies investigating the interactions between p53 and the ubiquitin ligases and deubiquitinases that modify it in normal and diseased cellular states. Disclosures: Brasher: Boston Biochem Inc: Employment. Guillen:Boston Biochem Inc: Employment. Tomasic:Boston Biochem Inc: Employment. Schwerdtfeger:Boston Biochem Inc: Employment. Melandri:Boston Biochem Inc: Employment.

scholarly journals Enhancement of Actin-depolymerizing Factor/Cofilin-dependent Actin Disassembly by Actin-interacting Protein 1 Is Required for Organized Actin Filament Assembly in the Caenorhabditis elegans Body Wall Muscle

2006 ◽  
Vol 17 (5) ◽  
pp. 2190-2199 ◽  
Author(s):  
Kurato Mohri ◽  
Kanako Ono ◽  
Robinson Yu ◽  
Sawako Yamashiro ◽  
Shoichiro Ono
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Muscle Cells ◽  
Actin Depolymerizing Factor ◽  
Interacting Protein ◽  
Cellular Processes ◽  
Cytoskeletal Dynamics ◽  
End Capping

Regulated disassembly of actin filaments is involved in several cellular processes that require dynamic rearrangement of the actin cytoskeleton. Actin-interacting protein (AIP) 1 specifically enhances disassembly of actin-depolymerizing factor (ADF)/cofilin-bound actin filaments. In vitro, AIP1 actively disassembles filaments, caps barbed ends, and binds to the side of filaments. However, how AIP1 functions in the cellular actin cytoskeletal dynamics is not understood. We compared biochemical and in vivo activities of mutant UNC-78 proteins and found that impaired activity of mutant UNC-78 proteins to enhance disassembly of ADF/cofilin-bound actin filaments is associated with inability to regulate striated organization of actin filaments in muscle cells. Six functionally important residues are present in the N-terminal β-propeller, whereas one residue is located in the C-terminal β-propeller, suggesting the presence of two separate sites for interaction with ADF/cofilin and actin. In vitro, these mutant UNC-78 proteins exhibited variable alterations in actin disassembly and/or barbed end-capping activities, suggesting that both activities are important for its in vivo function. These results indicate that the actin-regulating activity of AIP1 in cooperation with ADF/cofilin is essential for its in vivo function to regulate actin filament organization in muscle cells.

Androgen-receptor-interacting nuclear proteins

2000 ◽  
Vol 28 (4) ◽  
pp. 401-405 ◽  
Author(s):  
O. A. Jänne ◽  
A.-M. Moilanen ◽  
H. Poukka ◽  
N. Rouleau ◽  
U. Karvonen ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Protein Kinase ◽  
Molecular Mechanisms ◽  
Hormone Receptors ◽  
Cultured Cells ◽  
Ring Finger ◽  
Interacting Protein ◽  
Transcription Activators

Androgen receptor (AR) belongs to the super-family of nuclear hormone receptors that employ complex molecular mechanisms to guide the development and physiological functions of their target tissues. Our recent work has led to the identification of four novel proteins that recognize AR zinc-finger region (ZFR) both in vivo and in vitro. One is a small nuclear RING-finger protein that possesses separate interaction interfaces for AR and for other transcription activators such as Spl. The second is a nuclear serine/threonine protein kinase (androgen-receptor-interacting nuclear protein kinase; ANPK); however, the receptor itself does not seem to be a substrate for this kinase. The third one is dubbed androgen-receptor-interacting protein 3 (ARIP3) and is a novel member of the PIAS (protein inhibitor of activated STAT) protein family. The fourth protein, termed ARIP4, is a nuclear ATPase that belongs to the SNF2-like family of chromatin remodelling proteins. All four proteins exhibit a punctate nuclear pattern when expressed in cultured cells. Each protein modulates AR-dependent transactivation in co-transfection experiments; their activating functions are not restricted to AR. Current work is aimed at elucidating the biochemical and functional properties of these AR-interacting proteins and at finding the partner proteins that form complexes with them in vivo.

scholarly journals PDIP38 is a novel adaptor-like modulator of the mitochondrial AAA+ protease CLPXP

2020 ◽  
Author(s):  
Philip R. Strack ◽  
Erica J. Brodie ◽  
Hanmiao Zhan ◽  
Verena J. Schuenemann ◽  
Liz J. Valente ◽  
...  
Keyword(s):  
Adaptor Protein ◽  
Dna Polymerase Delta ◽  
Interacting Protein ◽  
The Matrix ◽  
Steady State Levels ◽  
Bona Fide ◽  
Matrix Compartment ◽  
Zinc Binding Domain

SummaryPolymerase δ interacting protein of 38 kDa (PDIP38) was originally identified in a yeast two hybrid screen as an interacting protein of DNA polymerase delta, more than a decade ago. Since this time several subcellular locations have been reported and hence its function remains controversial. Our current understanding of PDIP38 function has also been hampered by a lack of detailed biochemical or structural analysis of this protein. Here we show, that human PDIP38 is directed to the mitochondrion, where it resides in the matrix compartment, together with its partner protein CLPX. PDIP38 is a bifunctional protein, composed of two conserved domains separated by an α-helical hinge region (or middle domain). The N-terminal (YccV-like) domain of PDIP38 forms an SH3-like β-barrel, which interacts specifically with CLPX, via the adaptor docking loop within the N-terminal Zinc binding domain (ZBD) of CLPX. In contrast, the C-terminal (DUF525) domain forms an Immunoglobin-like β-sandwich fold, which contains a highly conserved hydrophobic groove. Based on the physicochemical properties of this groove, we propose that PDIP38 is required for the recognition (and delivery to CLPXP) of proteins bearing specific hydrophobic degrons, potentially located at the termini of the target protein. Significantly, interaction with PDIP38 stabilizes the steady state levels of CLPX in vivo. Consistent with these data, PDIP38 inhibits the LONM-mediated turnover of CLPX in vitro. Collectively, our findings shed new light on the mechanistic and functional significance of PDIP38, indicating that in contrast to its initial identification as a nuclear protein, PIDP38 is a bona fide mitochondrial adaptor protein for the CLPXP protease.

scholarly journals Human Histone Chaperone Nucleophosmin Enhances Acetylation-Dependent Chromatin Transcription

2005 ◽  
Vol 25 (17) ◽  
pp. 7534-7545 ◽  
Author(s):  
V. Swaminathan ◽  
A. Hari Kishore ◽  
K. K. Febitha ◽  
Tapas K. Kundu
Keyword(s):  
Transcriptional Activation ◽  
Histone Chaperone ◽  
Dependent Manner ◽  
Histone Chaperones ◽  
Cellular Processes ◽  
The Core ◽  
Core Histones ◽  
Nucleosomal Structure

ABSTRACT Histone chaperones are a group of proteins that aid in the dynamic chromatin organization during different cellular processes. Here, we report that the human histone chaperone nucleophosmin interacts with the core histones H3, H2B, and H4 but that this histone interaction is not sufficient to confer the chaperone activity. Significantly, nucleophosmin enhances the acetylation-dependent chromatin transcription and it becomes acetylated both in vitro and in vivo. Acetylation of nucleophosmin and the core histones was found to be essential for the enhancement of chromatin transcription. The acetylated NPM1 not only shows an increased affinity toward acetylated histones but also shows enhanced histone transfer ability. Presumably, nucleophosmin disrupts the nucleosomal structure in an acetylation-dependent manner, resulting in the transcriptional activation. These results establish nucleophosmin (NPM1) as a human histone chaperone that becomes acetylated, resulting in the enhancement of chromatin transcription.

Promoter-associated small double-stranded RNA interacts with heterogeneous nuclear ribonucleoprotein A2/B1 to induce transcriptional activation

2012 ◽  
Vol 447 (3) ◽  
pp. 407-416 ◽  
Author(s):  
Jia Hu ◽  
Zhong Chen ◽  
Ding Xia ◽  
Jia Wu ◽  
Hua Xu ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Kinase Inhibitor ◽  
Suppressor Gene ◽  
Tumour Suppressor Gene ◽  
Double Stranded Rna ◽  
P21 Promoter

Several recent reports have demonstrated that small activating dsRNA [double-stranded RNA; saRNA (small activating dsRNA)] complementary to promoter regions can up-regulate gene expression in mammalian cells, a phenomenon termed RNAa (RNA activation). However, the mechanism of RNAa remains obscure with regard to what is the target molecule for promoter-targeted saRNA and what are the proteins involved in this process. p21Waf1/Cip1 (p21) [CDKN1A (cyclin-dependent kinase inhibitor 1A)], an important tumour suppressor gene, is among the genes that can be activated by RNAa in tumour cells. In the present study, we provide direct evidence that p21 promoter-targeted saRNA interact with its intended target on the p21 promoter to activate p21 expression. This process is associated with recruitment of RNA polymerase II and AGO2 (argonaute 2) protein to the saRNA-target site. Additionally, we found that several hnRNPs (heterogeneous nuclear ribonucleoproteins) (A1, A2/B1 and C1/C2) are associated with saRNA. Further studies show that hnRNPA2/B1 interacts with the saRNA in vivo and in vitro and is required for RNAa activity. These findings indicate that RNAa results from specific targeting of promoters and reveals additional mechanistic details of RNAa.

Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

2005 ◽  
Vol 83 (4) ◽  
pp. 535-547 ◽  
Author(s):  
Gareth N Corry ◽  
D Alan Underhill
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Current Knowledge ◽  
Nuclear Architecture ◽  
Subnuclear Localization ◽  
Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

Sign in / Sign up

Export Citation Format

Share Document