XCtBP is a XTcf-3 co-repressor with roles throughout Xenopus development

Development ◽  
1999 ◽  
Vol 126 (14) ◽  
pp. 3159-3170 ◽  
Author(s):  
M. Brannon ◽  
J.D. Brown ◽  
R. Bates ◽  
D. Kimelman ◽  
R.T. Moon
Keyword(s):  
Binding Sites ◽  
Transcriptional Repression ◽  
Wnt Pathway ◽  
Ectopic Expression ◽  
Axis Formation ◽  
Beta Catenin ◽  
Carboxy Terminus ◽  
Xenopus Development ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

XTcf-3 is an HMG box transcription factor that mediates Xenopus dorsal-ventral axis formation. As a Wnt pathway effector, XTcf-3 interacts with beta-catenin and activates the expression of the dorsal organizing gene siamois, while in the absence of beta-catenin, XTcf-3 functions as a transcriptional repressor. We show that XTcf-3 contains amino- and carboxy-terminal repressor domains and have identified a Xenopus member of the C-terminal Binding Protein family of transcriptional co-repressors (XCtBP) as the C-terminal co-repressor. We show that two XCtBP binding sites near the XTcf-3 carboxy-terminus are required for the interaction of XTcf-3 and XCtBP and for the transcriptional repression mediated by the XTcf-3 carboxy-terminal domain. By fusing the GAL4 activation domain to XCtBP we have generated an antimorphic protein, XCtBP/G4A, that activates siamois transcription through an interaction with endogenous XTcf-3. Ectopic expression of XCtBP/G4A demonstrates that XCtBP functions in the regulation of head and notochord development. Our data support a role for XCtBP as a co-repressor throughout Xenopus development and indicate that XCtBP/G4A will be a useful tool in determining how XCtBP functions in various developmental processes.

Endogenous patterns of TGFbeta superfamily signaling during early Xenopus development

Development ◽  
2000 ◽  
Vol 127 (13) ◽  
pp. 2917-2931 ◽  
Author(s):  
S. Faure ◽  
M.A. Lee ◽  
T. Keller ◽  
P. ten Dijke ◽  
M. Whitman
Keyword(s):  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Ectopic Expression ◽  
Xenopus Embryo ◽  
Bmp Signaling ◽  
Type I ◽  
Embryonic Cells ◽  
Xenopus Development ◽  
Zygotic Transcription ◽  
Carboxy Terminal

Transforming growth factor beta (TGFbeta) superfamily signaling has been implicated in patterning of the early Xenopus embryo. Upon ligand stimulation, TGFbeta receptors phosphorylate Smad proteins at carboxy-terminal SS(V/M)S consensus motifs. Smads 1/5/8, activated by bone morphogenetic protein (BMP) signaling, induce ventral mesoderm whereas Smad2, activated by activin-like ligands, induces dorsal mesoderm. Although ectopic expression studies are consistent with roles for TGFbeta signals in early Xenopus embryogenesis, when and where BMP and activin-like signaling pathways are active endogenously has not been directly examined. In this study, we investigate the temporal and spatial activation of TGFbeta superfamily signaling in early Xenopus development by using antibodies specific for the type I receptor-phosphorylated forms of Smad1/5/8 and Smad2. We find that Smad1/5/8 and two distinct isoforms of Smad2, full-length Smad2 and Smad2(delta)exon3, are phosphorylated in early embryos. Both Smad1/5/8 and Smad2/Smad2(delta)exon3 are activated after, but not before, the mid-blastula transition (MBT). Endogenous activation of Smad2/Smad2(delta)exon3 requires zygotic transcription, while Smad1/5/8 activation at MBT appears to involve transcription-independent regulation. We also find that the competence of embryonic cells to respond to TGF(delta) superfamily ligands is temporally regulated and may be a determinant of early patterning. Levels of phospho-Smad1/5/8 and of phospho-Smad2/Smad2(delta)exon3 are asymmetrically distributed across both the animal-vegetal and dorsoventral axes. The timing of the development of these asymmetries differs for phospho-Smad1/5/8 and for phospho-Smad2/Smad2(delta)exon3, and the spatial distribution of phosphorylation of each Smad changes dramatically as gastrulation begins. We discuss the implications of our results for endogenous functions of BMP and activin-like signals as candidate morphogens regulating primary germ layer formation and dorsoventral patterning of the early Xenopus embryo.

scholarly journals Oct2 transactivation from a remote enhancer position requires a B-cell-restricted activity.

1992 ◽  
Vol 12 (7) ◽  
pp. 3107-3116 ◽  
Author(s):  
A Annweiler ◽  
M Müller-Immerglück ◽  
T Wirth
Keyword(s):  
B Cell ◽  
Ectopic Expression ◽  
Terminal Portion ◽  
Enhancer Element ◽  
Amino Terminal ◽  
Pou Domain ◽  
Carboxy Terminal Domain ◽  
Octamer Motif ◽  
Restricted Activity ◽  
Carboxy Terminal

Previous cotransfection experiments had demonstrated that ectopic expression of the lymphocyte-specific transcription factor Oct2 could efficiently activate a promoter containing an octamer motif. Oct2 expression was unable to stimulate a multimerized octamer enhancer element in HeLa cells, however. We have tested a variety of Oct2 isoforms generated by alternative splicing for the capability to activate an octamer enhancer in nonlymphoid cells and a B-cell line. Our analyses show that several Oct2 isoforms can stimulate from a remote position but that this stimulation is restricted to B cells. This result indicates the involvement of either a B-cell-specific cofactor or a specific modification of a cofactor or the Oct2 protein in Oct2-mediated enhancer activation. Mutational analyses indicate that the carboxy-terminal domain of Oct2 is critical for enhancer activation. Moreover, this domain conferred enhancing activity when fused to the Oct1 protein, which by itself was unable to stimulate from a remote position. The glutamine-rich activation domain present in the amino-terminal portion of Oct2 and the POU domain contribute only marginally to the transactivation function from a distal position.

scholarly journals Nuclear localization of the PEP protein tyrosine phosphatase.

1994 ◽  
Vol 14 (7) ◽  
pp. 4938-4946 ◽  
Author(s):  
E Flores ◽  
G Roy ◽  
D Patel ◽  
A Shaw ◽  
M L Thomas
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Lymphoid Tissues ◽  
Carboxy Terminus ◽  
Protein Tyrosine ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

PEP is an intracellular protein tyrosine phosphatase expressed primarily by cells of hematopoietic origin that can be divided structurally into a catalytic domain and a large carboxy-terminal domain. The carboxy-terminal domain is enriched in proline, glutamic acid, serine, and threonine residues (PEST sequences) and contains a nonperfect tandem repeat sequence enriched in proline residues and a carboxy terminus enriched in basic amino acids. Here we show that PEP is diffusely expressed in lymphoid tissues, consistent with expression by many different cell types. Analysis of the PEP protein identifies a nuclear localization sequence within the extreme carboxy terminus. Transfer of 18 amino acids from the carboxy terminus of PEP to beta-galactosidase conferred nuclear localization, indicating that this sequence was sufficient for nuclear localization. Proteins enriched in PEST sequences are often rapidly degraded. However, pulse-chase analysis indicates that PEP has a half-life of greater than 5 h.

scholarly journals The Carboxy-Terminal Domain of Hsc70 Provides Binding Sites for a Distinct Set of Chaperone Cofactors

1998 ◽  
Vol 18 (4) ◽  
pp. 2023-2028 ◽  
Author(s):  
Jens Demand ◽  
Jens Lüders ◽  
Jörg Höhfeld
Keyword(s):  
Binding Sites ◽  
Mammalian Cells ◽  
Chaperone Activity ◽  
Structural Basis ◽  
Atpase Domain ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Chaperone Protein ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

ABSTRACT The modulation of the chaperone activity of the heat shock cognate Hsc70 protein in mammalian cells involves cooperation with chaperone cofactors, such as Hsp40; BAG-1; the Hsc70-interacting protein, Hip; and the Hsc70-Hsp90-organizing protein, Hop. By employing the yeast two-hybrid system and in vitro interaction assays, we have provided insight into the structural basis that underlies Hsc70’s cooperation with different cofactors. The carboxy-terminal domain of Hsc70, previously shown to form a lid over the peptide binding pocket of the chaperone protein, mediates the interaction of Hsc70 with Hsp40 and Hop. Remarkably, the two cofactors bind to the carboxy terminus of Hsc70 in a noncompetitive manner, revealing the existence of distinct binding sites for Hsp40 and Hop within this domain. In contrast, Hip interacts exclusively with the amino-terminal ATPase domain of Hsc70. Hence, Hsc70 possesses separate nonoverlapping binding sites for Hsp40, Hip, and Hop. This appears to enable the chaperone protein to cooperate simultaneously with multiple cofactors. On the other hand, BAG-1 and Hip have recently been shown to compete in binding to the ATPase domain. Our data thus establish the existence of a network of cooperating and competing cofactors regulating the chaperone activity of Hsc70 in the mammalian cell.

scholarly journals Functional Relationships of Srb10-Srb11 Kinase, Carboxy-Terminal Domain Kinase CTDK-I, and Transcriptional Corepressor Ssn6-Tup1

1998 ◽  
Vol 18 (3) ◽  
pp. 1163-1171 ◽  
Author(s):  
Sergei Kuchin ◽  
Marian Carlson
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Repression ◽  
Functional Link ◽  
Synthetic Promoters ◽  
Functional Relationships ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Rna Polymerase Ii Holoenzyme

ABSTRACT The Srb10-Srb11 protein kinase of Saccharomyces cerevisiae is a cyclin-dependent kinase (cdk)-cyclin pair which has been found associated with the carboxy-terminal domain (CTD) of RNA polymerase II holoenzyme forms. Previous genetic findings implicated the Srb10-Srb11 kinase in transcriptional repression. Here we use synthetic promoters and LexA fusion proteins to test the requirement for Srb10-Srb11 in repression by Ssn6-Tup1, a global corepressor. We show that srb10Δ and srb11Δ mutations reduce repression by DNA-bound LexA-Ssn6 and LexA-Tup1. A point mutation in a conserved subdomain of the kinase similarly reduced repression, indicating that the catalytic activity is required. These findings establish a functional link between Ssn6-Tup1 and the Srb10-Srb11 kinase in vivo. We also explored the relationship between Srb10-Srb11 and CTD kinase I (CTDK-I), another member of the cdk-cyclin family that has been implicated in CTD phosphorylation. We show that mutation of CTK1, encoding the cdk subunit, causes defects in transcriptional repression by LexA-Tup1 and in transcriptional activation. Analysis of the mutant phenotypes and the genetic interactions of srb10Δ and ctk1Δ suggests that the two kinases have related but distinct roles in transcriptional control. These genetic findings, together with previous biochemical evidence, suggest that one mechanism of repression by Ssn6-Tup1 involves functional interaction with RNA polymerase II holoenzyme.

Nuclear localization of the PEP protein tyrosine phosphatase

1994 ◽  
Vol 14 (7) ◽  
pp. 4938-4946
Author(s):  
E Flores ◽  
G Roy ◽  
D Patel ◽  
A Shaw ◽  
M L Thomas
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Lymphoid Tissues ◽  
Carboxy Terminus ◽  
Protein Tyrosine ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

PEP is an intracellular protein tyrosine phosphatase expressed primarily by cells of hematopoietic origin that can be divided structurally into a catalytic domain and a large carboxy-terminal domain. The carboxy-terminal domain is enriched in proline, glutamic acid, serine, and threonine residues (PEST sequences) and contains a nonperfect tandem repeat sequence enriched in proline residues and a carboxy terminus enriched in basic amino acids. Here we show that PEP is diffusely expressed in lymphoid tissues, consistent with expression by many different cell types. Analysis of the PEP protein identifies a nuclear localization sequence within the extreme carboxy terminus. Transfer of 18 amino acids from the carboxy terminus of PEP to beta-galactosidase conferred nuclear localization, indicating that this sequence was sufficient for nuclear localization. Proteins enriched in PEST sequences are often rapidly degraded. However, pulse-chase analysis indicates that PEP has a half-life of greater than 5 h.

Oct2 transactivation from a remote enhancer position requires a B-cell-restricted activity

1992 ◽  
Vol 12 (7) ◽  
pp. 3107-3116
Author(s):  
A Annweiler ◽  
M Müller-Immerglück ◽  
T Wirth
Keyword(s):  
B Cell ◽  
Ectopic Expression ◽  
Terminal Portion ◽  
Enhancer Element ◽  
Amino Terminal ◽  
Pou Domain ◽  
Carboxy Terminal Domain ◽  
Octamer Motif ◽  
Restricted Activity ◽  
Carboxy Terminal

Previous cotransfection experiments had demonstrated that ectopic expression of the lymphocyte-specific transcription factor Oct2 could efficiently activate a promoter containing an octamer motif. Oct2 expression was unable to stimulate a multimerized octamer enhancer element in HeLa cells, however. We have tested a variety of Oct2 isoforms generated by alternative splicing for the capability to activate an octamer enhancer in nonlymphoid cells and a B-cell line. Our analyses show that several Oct2 isoforms can stimulate from a remote position but that this stimulation is restricted to B cells. This result indicates the involvement of either a B-cell-specific cofactor or a specific modification of a cofactor or the Oct2 protein in Oct2-mediated enhancer activation. Mutational analyses indicate that the carboxy-terminal domain of Oct2 is critical for enhancer activation. Moreover, this domain conferred enhancing activity when fused to the Oct1 protein, which by itself was unable to stimulate from a remote position. The glutamine-rich activation domain present in the amino-terminal portion of Oct2 and the POU domain contribute only marginally to the transactivation function from a distal position.

scholarly journals Interference between Proteins Hap46 and Hop/p60, Which Bind to Different Domains of the Molecular Chaperone hsp70/hsc70

1998 ◽  
Vol 18 (11) ◽  
pp. 6238-6244 ◽  
Author(s):  
Mathias Gebauer ◽  
Matthias Zeiner ◽  
Ulrich Gehring
Keyword(s):  
Molecular Chaperones ◽  
Binding Sites ◽  
Direct Interaction ◽  
Atp Binding ◽  
Amino Terminal ◽  
Hsp70 Family ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Substrate Proteins ◽  
Chaperone Hsp70

ABSTRACT Several structurally divergent proteins associate with molecular chaperones of the 70-kDa heat shock protein (hsp70) family and modulate their activities. We investigated the cofactors Hap46 and Hop/p60 and the effects of their binding to mammalian hsp70 and the cognate form hsc70. Hap46 associates with the amino-terminal ATP binding domain and stimulates ATP binding two- to threefold but inhibits binding of misfolded protein substrate to hsc70 and reactivation of thermally denatured luciferase in an hsc70-dependent refolding system. By contrast, Hop/p60 interacts with a portion of the carboxy-terminal domain of hsp70s, which is distinct from that involved in the binding of misfolded proteins. Thus, Hop/p60 and substrate proteins can form ternary complexes with hsc70. Hop/p60 exerts no effect on ATP and substrate binding but nevertheless interferes with protein refolding. Even though there is no direct interaction between these accessory proteins, Hap46 inhibits the binding of Hop/p60 to hsc70 but Hop/p60 does not inhibit the binding of Hap46 to hsc70. As judged from respective deletions, the amino-terminal portions of Hap46 and Hop/p60 are involved in this interference. These data suggest steric hindrance between Hap46 and Hop/p60 during interaction with distantly located binding sites on hsp70s. Thus, not only do the major domains of hsp70 chaperones communicate with each other, but cofactors interacting with these domains affect each other as well.

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