Download Aucun titre de diapositive

J Biol Chem. 2001 May 18;276(20):17448-54. Epub 2001 Feb 22.
A-kinase-anchoring protein AKAP95 is targeted to the nuclear matrix
and associates with p68 RNA helicase.
Akileswaran L, Taraska JW, Sayer JA, Gettemy JM, Coghlan VM.
Neurological Sciences Institute, Oregon Health Sciences University, Beaverton, Oregon 97006, USA.
The cell nucleus is structurally and functionally organized by the nuclear matrix. We have examined
whether the nuclear cAMP-dependent protein kinase-anchoring protein AKAP95 contains specific signals for
targeting to the subnuclear compartment and for interaction with other proteins. AKAP95 was expressed in
mammalian cells and found to localize exclusively to the nuclear matrix. Mutational analysis was used to
identify determinants for nuclear localization and nuclear matrix targeting of AKAP95. These sites were found
to be distinct from previously identified DNA and protein kinase A binding domains. The nuclear matrixtargeting site is unique but conserved among members of the AKAP95 family. Direct binding of AKAP95 to
isolated nuclear matrix was demonstrated in situ and found to be dependent on the nuclear matrix-targeting
site. Moreover, Far Western blot analysis identified at least three AKAP95-binding proteins in nuclear matrix
isolated from rat brain. Yeast two-hybrid cloning identified one binding partner as p68 RNA helicase. The
helicase and AKAP95 co-localized in the nuclear matrix of mammalian cells, associated in vitro, and were
precipitated as a complex from solubilized cell extracts. The results define novel protein-protein interactions
among nuclear matrix proteins and suggest a potential role of AKAP95 as a scaffold for coordinating assembly
of hormonally responsive transcription complexes.
Mol Cell Biol. 1999 Aug;19(8):5363-72.
Purification and identification of p68 RNA helicase acting as a transcriptional
coactivator specific for the activation function 1 of human estrogen receptor alpha.
Endoh H, Maruyama K, Masuhiro Y, Kobayashi Y, Goto M, Tai H, Yanagisawa J, Metzger D, Hashimoto S, Kato S.
Molecular Medicine Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical, Tsukuba, Ibaraki
305-8585, Japan.
The estrogen receptor (ER) regulates the expression of target genes in a ligand-dependent manner. The ligand-dependent
activation function AF-2 of the ER is located in the ligand binding domain (LBD), while the N-terminal A/B domain (AF-1)
functions in a ligand-independent manner when isolated from the LBD. AF-1 and AF-2 exhibit cell type and promoter context
specificity. Furthermore, the AF-1 activity of the human ERalpha (hERalpha) is enhanced through phosphorylation of the
Ser(118) residue by mitogen-activated protein kinase (MAPK). From MCF-7 cells, we purified and cloned a 68-kDa protein
(p68) which interacted with the A/B domain but not with the LBD of hERalpha. Phosphorylation of hERalpha Ser(118)
potentiated the interaction with p68. We demonstrate that p68 enhanced the activity of AF-1 but not AF-2 and the estrogeninduced as well as the anti-estrogen-induced transcriptional activity of the full-length ERalpha in a cell-type-specific manner.
However, it did not potentiate AF-1 or AF-2 of ERbeta, androgen receptor, retinoic acid receptor alpha, or mineralocorticoid
receptor. We also show that the RNA helicase activity previously ascribed to p68 is dispensable for the ERalpha AF-1 coactivator
activity and that p68 binds to CBP in vitro. Furthermore, the interaction region for p68 in the ERalpha A/B domain was
essential for the full activity of hERalpha AF-1. Taken together, these findings show that p68 acts as a coactivator specific for the
ERalpha AF-1 and strongly suggest that the interaction between p68 and the hERalpha A/B domain is regulated by MAPKinduced phosphorylation of Ser(118).
EMBO J. 2002 Jul 1;21(13):3443-53.
Formation of an hER alpha-COUP-TFI complex enhances hER
alpha AF-1 through Ser118 phosphorylation by MAPK.
Metivier R, Gay FA, Hubner MR, Flouriot G, Salbert G, Gannon F, Kah O, Pakdel F.
Equipe d'Endocrinologie Moleculaire de la Reproduction, UMR CNRS 6026, Universite de
Rennes I, 35042 Rennes Cedex, France.
The enhancement of the human estrogen receptor alpha (hER alpha, NR3A1) activity by the
orphan nuclear receptor COUP-TFI is found to depend on the establishment of a tight hER alphaCOUP-TFI complex. Formation of this complex seems to involve dynamic mechanisms different
from those allowing hER alpha homodimerization. Although the hER alpha-COUP-TFI complex is
present in all cells tested, the transcriptional cooperation between the two nuclear receptors is
restricted to cell lines permissive to hER alpha activation function 1 (AF-1). In these cells, the
physical interaction between COUP-TFI and hER alpha increases the affinity of hER alpha for
ERK2/p42(MAPK), resulting in an enhanced phosphorylation state of the hER alpha Ser118. hER
alpha thus acquires a strengthened AF-1 activity due to its hyperphosphorylation. These data
indicate an alternative interaction process between nuclear receptors and demonstrate a novel
protein intercommunication pathway that modulates hER alpha AF-1.
Metivier et al. 2002
Phosphorylation of hER S118 by MAPK enhances its
physical interaction with the AF-1 co-activator, p68
RNA helicase (Endoh et al., 1999). One can then
propose that COUP-TFI modulates the interaction of
p68 RNA helicase with hER. COUP-TFI also
interacts with the CBP–p300 complex (Bailey et al.,
1998), which is a co-activator for hER AF-1 that is
known to bind SRC-1 and the p68 RNA helicase (Yao
et al., 1996; Endoh et al., 1999; Kobayashi et al.,
2000). Upon interaction with hER, COUP-TFI might
thus also recruit large protein complexes to modulate
hER actions.
Oncogene. 2003 Jan 9;22(1):151-6.
Synergism between p68 RNA helicase and the transcriptional coactivators
CBP and p300.
Rossow KL, Janknecht R.
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA.
p68 RNA helicase has been implicated in a variety of processes, including rearrangement of RNA secondary
structures, RNA splicing, gene transcription and tumor development, yet its mechanisms of action are not well
understood. In this study, we show that p68 is predominantly localized to the cell nucleus, where it partially colocalizes
with the transcriptional coactivator p300. Accordingly, p68 and p300, or the paralogous CREB-binding protein (CBP),
coimmunoprecipitate. Similarly, p68 and RNA polymerase II (Pol II) are able to interact in vivo. GST pull-down assays
confirmed these interactions in vitro, demonstrating that p68 can interact with several domains of CBP, while
CBP/p300 bind to amino acids 176-388 of p68 and RNA Pol II binds to the N-terminal 80 amino acids of p68.
Furthermore, p68 stimulates transcription mediated by the C-terminal transactivation domain of CBP. p68 is also able
to stimulate TPA oncogene responsive unit (TORU) promoter activity, and p300 acts in synergy with p68. On the other
hand, suppression of CBP/p300 function by the adenoviral protein E1A abolishes TORU promoter activation by p68.
Altogether, our results suggest the existence of a multiprotein complex in which p68 RNA helicase, CBP/p300 and RNA
Pol II jointly promote gene expression.
EMBO J. 2001 Mar 15;20(6):1341-52.
A subfamily of RNA-binding DEAD-box proteins acts as an estrogen receptor
alpha coactivator through the N-terminal activation domain (AF-1) with an RNA
coactivator, SRA.
Watanabe M, Yanagisawa J, Kitagawa H, Takeyama K, Ogawa S, Arao Y, Suzawa M, Kobayashi Y, Yano T, Yoshikawa H,
Masuhiro Y, Kato S.
Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0033, Japan.
One class of the nuclear receptor AF-2 coactivator complexes contains the SRC-1/TIF2 family, CBP/p300 and an RNA
coactivator, SRA. We identified a subfamily of RNA-binding DEAD-box proteins (p72/p68) as a human estrogen receptor
alpha (hER alpha) coactivator in the complex containing these factors. p72/p68 interacted with both the AD2 of any SRC1/TIF2 family protein and the hER alpha A/B domain, but not with any other nuclear receptor tested. p72/p68, TIF2 (SRC-1)
and SRA were co-immunoprecipitated with estrogen-bound hER alpha in MCF7 cells and in partially purified complexes
associated with hER alpha from HeLa nuclear extracts. Estrogen induced co-localization of p72 with hER alpha and TIF2 in
the nucleus. The presence of p72/p68 potentiated the estrogen-induced expression of the endogenous pS2 gene in MCF7 cells.
In a transient expression assay, a combination of p72/p68 with SRA and one TIF2 brought an ultimate synergism to the
estrogen-induced transactivation of hER alpha. These findings indicate that p72/p68 acts as an ER subtype-selective
coactivator through ER alpha AF-1 by associating with the coactivator complex to bind its AF-2 through direct binding with
SRA and the SRC-1/TIF2 family proteins.
1: Legagneux V, Cubizolles F, Watrin E.
Multiple roles of Condensins: a complex story.
Biol Cell. 2004 Apr;96(3):201-13.
PMID: 15182703 [PubMed - in process]
2: Dervyn E, Noirot-Gros MF, Mervelet P, McGovern S, Ehrlich SD, Polard P,
Noirot P.
The bacterial condensin/cohesin-like protein complex acts in DNA repair and
regulation of gene expression.
Mol Microbiol. 2004 Mar;51(6):1629-40.
PMID: 15009890 [PubMed - indexed for MEDLINE]
3: Machin F, Paschos K, Jarmuz A, Torres-Rosell J, Pade C, Aragon L.
Condensin regulates rDNA silencing by modulating nucleolar Sir2p.
Curr Biol. 2004 Jan 20;14(2):125-30.
PMID: 14738734 [PubMed - indexed for MEDLINE]
4: Timirbulatova ER, Kireev II, Kartavenko TV, Gulak PV, Poliakov VIu, Guellec
KL, Uzbekov RE.
[Intracellular localization of XCAP-E protein in XL2 (Xenopus laevis) cells
under normal conditions and during inhibition of pRNA transcription and
processing]
Tsitologiia. 2003;45(3):290-7. Russian.
PMID: 14520886 [PubMed - indexed for MEDLINE]
5: Beenders B, Watrin E, Legagneux V, Kireev I, Bellini M.
Distribution of XCAP-E and XCAP-D2 in the Xenopus oocyte nucleus.
Chromosome Res. 2003;11(6):549-64.
PMID: 14516064 [PubMed - indexed for MEDLINE]
condensin AND transcription
6: Uzbekov R, Timirbulatova E, Watrin E, Cubizolles F, Ogereau D, Gulak P,
Legagneux V, Polyakov VJ, Le Guellec K, Kireev I.
Nucleolar association of pEg7 and XCAP-E, two members of Xenopus laevis
condensin complex in interphase cells.
J Cell Sci. 2003 May 1;116(Pt 9):1667-78.
PMID: 12665548 [PubMed - indexed for MEDLINE]
7: MacCallum DE, Losada A, Kobayashi R, Hirano T.
ISWI remodeling complexes in Xenopus egg extracts: identification as major
chromosomal components that are regulated by INCENP-aurora B.
Mol Biol Cell. 2002 Jan;13(1):25-39.
PMID: 11809820 [PubMed - indexed for MEDLINE]
8: Cabello OA, Eliseeva E, He WG, Youssoufian H, Plon SE, Brinkley BR, Belmont
JW.
Cell cycle-dependent expression and nucleolar localization of hCAP-H.
Mol Biol Cell. 2001 Nov;12(11):3527-37.
PMID: 11694586 [PubMed - indexed for MEDLINE]
9: Neuwald AF, Hirano T.
HEAT repeats associated with condensins, cohesins, and other complexes involved
in chromosome-related functions.
Genome Res. 2000 Oct;10(10):1445-52.
PMID: 11042144 [PubMed - indexed for MEDLINE]
Mol Cell. 2001 Jan;7(1):127-36.
Drosophila chromosome condensation proteins Topoisomerase II and
Barren colocalize with Polycomb and maintain Fab-7 PRE silencing.
Lupo R, Breiling A, Bianchi ME, Orlando V.
DIBIT, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milano, Italy.
Mechanisms of cellular memory control the maintenance of cellular identity at the level of chromatin
structure. We have investigated whether the converse is true; namely, if functions responsible for
maintenance of chromosome structure play a role in epigenetic control of gene expression. We show that
Topoisomerase II (TOPOII) and Barren (BARR) interact in vivo with Polycomb group (PcG) target
sequences in the bithorax complex of Drosophila, including Polycomb response elements. In addition, we
find that the PcG protein Polyhomeotic (PH) interacts physically with TOPOII and BARR and that BARR
is required for Fab-7-regulated homeotic gene expression. Conversely, we find defects in chromosome
segregation associated with ph mutations. We propose that chromatin condensation proteins are involved
in mechanisms acting in interphase that regulate chromosome domain topology and are essential for the
maintenance of gene expression.
Mol Biol Cell. 2002 Feb;13(2):632-45.
Mutation of YCS4, a budding yeast condensin subunit, affects mitotic
and nonmitotic chromosome behavior.
Bhalla N, Biggins S, Murray AW.
Department of Cell Biology and Physiology, University of California, San Francisco, San Francisco,
California 94143, USA.
The budding yeast YCS4 gene encodes a conserved regulatory subunit of the condensin complex. We
isolated an allele of this gene in a screen for mutants defective in sister chromatid separation or
segregation. The phenotype of the ycs4-1 mutant is similar to topoisomerase II mutants and distinct from
the esp1-1 mutant: the topological resolution of sister chromatids is compromised in ycs4-1 despite normal
removal of cohesins from mitotic chromosomes. Consistent with a role in sister separation, YCS4 function
is required to localize DNA topoisomerase I and II to chromosomes. Unlike its homologs in Xenopus and
fission yeast, Ycs4p is associated with chromatin throughout the cell cycle; the only change in localization
occurs during anaphase when the protein is enriched at the nucleolus. This relocalization may reveal the
specific challenge that segregation of the transcriptionally hyperactive, repetitive array of rDNA genes can
present during mitosis. Indeed, segregation of the nucleolus is abnormal in ycs4-1 at the nonpermissive
temperature. Interrepeat recombination in the rDNA array is specifically elevated in ycs4-1 at the
permissive temperature, suggesting that the Ycs4p plays a role at the array aside from its segregation.
Furthermore, ycs4-1 is defective in silencing at the mating type loci at the permissive temperature. Taken
together, our data suggest that there are mitotic as well as nonmitotic chromosomal abnormalities
associated with loss of condensin function in budding yeast.
ER
AF1
AF2
COUP-TF
MAPK
CBP/p300
RNA Pol II
p68
AKAP95
CAP-D2
Condensin
Dev Biol. 2000 Feb 15;218(2):284-98.
Subcellular trafficking of the nuclear receptor COUP-TF in the
early embryonic cell cycle.
Vlahou A, Flytzanis CN.
Department of Cell Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030,
USA.
The nuclear receptor SpCOUP-TF is the highly conserved sea urchin homologue of the COUP
family of transcription factors. Previous results from our laboratory demonstrated that SpCOUPTF transcripts are localized in the egg and asymmetrically distributed in the early embryonic
blastomeres (A. Vlahou et al., 1996, Development 122, 521-526). To examine the subcellular
localization of SpCOUP-TF protein, polyclonal antibodies were separately raised against the
divergent N-terminus as well as the conserved DNA-binding and ligand-binding domains.
Immunohistochemical analyses suggest that SpCOUP-TF is a maternal protein residing in the
cytoplasm of the unfertilized egg. After fertilization, and as soon as the two-cell-stage embryo, most
of the receptor translocates from the cytoplasm to the cell nuclei. During the rapid embryonic cell
division, SpCOUP-TF was found to shuttle from the interphase nuclear periphery to the condensed
chromosomes in mitosis, in a cell-cycle-dependent manner. In an attempt to confirm these
observations, the subcellular localization of myc-tagged human COUP-TF I introduced into the sea
urchin embryo by RNA injection of fertilized eggs was examined. The pattern of human COUP-TF
I subcellular localization, detected with a monoclonal myc antibody, recapitulated the essential
features described for the endogenous SpCOUP-TF trafficking. Replacement of the N-terminus of
the human receptor with the unique sea urchin N-terminus enhanced its localization to the nuclear
rim during interphase. Deletion of the DNA-binding domain of human COUP-TF I resulted in loss
of all aspects of nuclear periphery and chromosomal localization. Taken together these data suggest
that SpCOUP-TF transcriptional activity is keyed on a cell-cycle-dependent mechanism that