scholarly journals PhoP-PhoP Interaction at Adjacent PhoP Binding Sites Is Influenced by Protein Phosphorylation

2007 ◽  
Vol 190 (4) ◽  
pp. 1317-1328 ◽  
Author(s):  
Akesh Sinha ◽  
Sankalp Gupta ◽  
Shweta Bhutani ◽  
Anuj Pathak ◽  
Dibyendu Sarkar
Keyword(s):  
Dna Binding ◽  
Protein Phosphorylation ◽  
Critical Role ◽  
Structural Effect ◽  
Transactivation Domain ◽  
Virulence Determinants ◽  
Protein Interface ◽  
Amino Terminal ◽  
Carboxy Terminal

ABSTRACT Mycobacterium tuberculosis PhoP regulates the expression of unknown virulence determinants and the biosynthesis of complex lipids. PhoP, like other members of the OmpR family, comprises a phosphorylation domain at the amino-terminal half and a DNA-binding domain at the carboxy-terminal half of the protein. To explore structural effect of protein phosphorylation and to examine effect of phosphorylation on DNA binding, purified PhoP was phosphorylated by acetyl phosphate in a reaction that was dependent on Mg2+ and Asp-71. Protein phosphorylation was not required for DNA binding; however, phosphorylation enhanced in vitro DNA binding through protein-protein interaction(s). Evidence is presented here that the protein-protein interface is different in the unphosphorylated and phosphorylated forms of PhoP and that specific DNA binding plays a critical role in changing the nature of the protein-protein interface. We show that phosphorylation switches the transactivation domain to a different conformation, which specifies additional protein-protein contacts between PhoP protomers bound to adjacent cognate sites. Together, our observations raise the possibility that PhoP, in the unphosphorylated and phosphorylated forms, may be capable of adopting different orientations as it binds to a vast array of genes to activate or repress transcription.

Functional and Genetic Characterization of the Oligomerization and DNA Binding Properties of the Drosophila Doublesex Proteins

Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1639-1652 ◽  
Author(s):  
Scott E Erdman ◽  
Hui-Ju Chen ◽  
Kenneth C Burtis
Keyword(s):  
Dna Binding ◽  
Critical Role ◽  
Target Sequence ◽  
Yeast Two Hybrid ◽  
Specific Expression ◽  
Amino Terminal ◽  
Normal Differentiation ◽  
Carboxy Terminal ◽  
Two Hybrid

The doublesex (dsx) gene of Drosophila melanogaster encodes both male-specific (DSXM) and female-specific (DSXF) polypeptides, which are required for normal differentiation of numerous sexually dimorphic somatic traits. The DSX polypeptides are transcription factors and have been shown previously to bind through a zinc finger-like domain to specific sites in an enhancer regulating sex-specific expression of yolk protein genes. We have determined the consensus target sequence for this DNA binding domain to be a palindromic sequence NNACTAAGAATGTNNTC composed of two half-sites around a central (A/T) base pair. As predicted by the symmetric nature of this site, we have found that the DSX proteins exist as dimers in vivo and have mapped two independent dimerization domains by the yeast two-hybrid method; one in the non-sex-specific amino-terminal region of the protein and one that includes the partially sex-specific carboxy-terminal domains of both the male and female polypeptides. We have further identified a missense mutation that eliminates dsx function in female flies, and shown that the same mutation prevents dimerization of DSXF in the yeast two-hybrid system, indicating a critical role for dimerization in dsx function in vivo.

scholarly journals Residues near the Amino Terminus of Rns Are Essential for Positive Autoregulation and DNA Binding

2008 ◽  
Vol 190 (7) ◽  
pp. 2279-2285 ◽  
Author(s):  
Georgeta N. Basturea ◽  
Maria D. Bodero ◽  
Mario E. Moreno ◽  
George P. Munson
Keyword(s):  
Dna Binding ◽  
Amino Terminus ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Amino Terminal Domain ◽  
Regulated Promoters

ABSTRACT Most members of the AraC/XylS family contain a conserved carboxy-terminal DNA binding domain and a less conserved amino-terminal domain involved in binding small-molecule effectors and dimerization. However, there is no evidence that Rns, a regulator of enterotoxigenic Escherichia coli virulence genes, responds to an effector ligand, and in this study we found that the amino-terminal domain of Rns does not form homodimers in vivo. Exposure of Rns to the chemical cross-linker glutaraldehyde revealed that the full-length protein is also a monomer in vitro. Nevertheless, deletion analysis of Rns demonstrated that the first 60 amino acids of the protein are essential for the activation and repression of Rns-regulated promoters in vivo. Amino-terminal truncation of Rns abolished DNA binding in vitro, and two randomly generated mutations, I14T and N16D, that independently abolished Rns autoregulation were isolated. Further analysis of these mutations revealed that they have disparate effects at other Rns-regulated promoters and suggest that they may be involved in an interaction with the carboxy-terminal domain of Rns. Thus, evolution may have preserved the amino terminus of Rns because it is essential for the regulator's activity even though it apparently lacks the two functions, dimerization and ligand binding, usually associated with the amino-terminal domains of AraC/XylS family members.

scholarly journals Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil Differentiation

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4154-4166 ◽  
Author(s):  
Robert L. Ilaria ◽  
Robert G. Hawley ◽  
Richard A. Van Etten
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cytokine Signaling ◽  
Transactivation Domain ◽  
Negative Effects ◽  
Murine Bone Marrow ◽  
Negative Effect

Abstract STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR→AAA and R290QQ→AAA), two in the DNA-binding domain (E437E→AA and V466VV→AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/▵53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, ▵53C. Stable expression of either the WKR or ▵53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3–dependent proliferation and granulocyte colony-stimulating factor (G-CSF)–dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF–dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.

scholarly journals Intra-domain communication between the N-terminal and DNA-binding domains of the androgen receptor: modulation of androgen response element DNA binding

2005 ◽  
Vol 34 (3) ◽  
pp. 603-615 ◽  
Author(s):  
Jacqueline Brodie ◽  
Iain J McEwan
Keyword(s):  
Androgen Receptor ◽  
Dna Binding ◽  
Fluorescence Emission ◽  
Progesterone Receptors ◽  
Fluorescence Emission Spectrum ◽  
Response Element ◽  
Amino Terminal ◽  
Binding Domains ◽  
Receptor Modulation

The androgen receptor (AR) is a ligand-activated transcription factor that recognises and binds to specific DNA response elements upon activation by the steroids testosterone or dihydrotestosterone. In vitro, two types of response element have been characterised - non-selective elements that bind the androgen, glucocorticoid and progesterone receptors, and androgen receptor-selective sequences. In the present study, the allosteric effects of DNA binding on the receptor amino-terminal domain (NTD) were studied. Binding to both types of DNA response element resulted in changes in the intrinsic fluorescence emission spectrum for four tryptophan residues within the AR-NTD and resulted in a more protease-resistant conformation. In binding experiments, it was observed that the presence of the AR-NTD reduced the affinity of receptor polypeptides for binding to both selective and non-selective DNA elements derived from the probasin, PEM and prostatin C3 genes respectively, without significantly altering the protein–base pair contacts. Taken together, these results highlight the role of intra-domain communications between the AR-NTD and the DNA binding domain in receptor structure and function.

scholarly journals Stability of the Human Papillomavirus Type 18 E2 Protein Is Regulated by a Proteasome Degradation Pathway through Its Amino-Terminal Transactivation Domain

2001 ◽  
Vol 75 (16) ◽  
pp. 7244-7251 ◽  
Author(s):  
Sophie Bellanger ◽  
Caroline Demeret ◽  
Sylvain Goyat ◽  
Françoise Thierry
Keyword(s):  
Human Papillomavirus ◽  
Hela Cells ◽  
Half Life ◽  
Fluorescent Protein ◽  
Growth Arrest ◽  
Transactivation Domain ◽  
Mcf7 Cells ◽  
E2 Protein ◽  
Amino Terminal

ABSTRACT The E2 proteins of papillomaviruses regulate both viral transcription and DNA replication. The human papillomavirus type 18 (HPV18) E2 protein has been shown to repress transcription of the oncogenic E6 and E7 genes, inducing growth arrest in HeLa cells. Using HPV18 E2 fused to the green fluorescent protein (GFP), we showed that this protein was short-lived in transfected HeLa cells. Real-time microscopy experiments indicated that the E2-dependent signal increased for roughly 24 h after transfection and then rapidly disappeared, indicating that E2 was unstable in HeLa cells and could confer instability to GFP. Similar studies done with a protein lacking the transactivation domain indicated that this truncation strongly stabilizes the E2 protein. In vitro, full-length E2 or the transactivation domain alone was efficiently ubiquitinated, whereas deletion of the transactivation domain strongly decreased the ubiquitination of the E2 protein. Proteasome inhibition in cells expressing E2 increased its half-life about sevenfold, which was comparable to the half-life of the amino-terminally truncated protein. These characteristics of E2 instability were independent of the E2-mediated G1 growth arrest in HeLa cells, as they were reproduced in MCF7 cells, where E2 does not affect the cell cycle. Altogether, these experiments showed that the HPV18 E2 protein was degraded by the ubiquitin-proteasome pathway through its amino-terminal transactivation domain. Tight regulation of the stability of the HPV 18 E2 protein may be essential to avoid accumulation of a potent transcriptional repressor and antiproliferative agent during the viral vegetative cycle.

scholarly journals Effects of point mutations in the cytidine deaminase domains of APOBEC3B on replication and hypermutation of hepatitis B virus in vitro

2007 ◽  
Vol 88 (12) ◽  
pp. 3270-3274 ◽  
Author(s):  
Marianne Bonvin ◽  
Jobst Greeve
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Point Mutations ◽  
Alternative Mechanism ◽  
Amino Terminal ◽  
Hbv Replication ◽  
B Virus ◽  
Carboxy Terminal ◽  
Deaminase Domain

APOBEC3 cytidine deaminases hypermutate hepatitis B virus (HBV) and inhibit its replication in vitro. Whether this inhibition is due to the generation of hypermutations or to an alternative mechanism is controversial. A series of APOBEC3B (A3B) point mutants was analysed in vitro for hypermutational activity on HBV DNA and for inhibitory effects on HBV replication. Point mutations inactivating the carboxy-terminal deaminase domain abolished the hypermutational activity and reduced the inhibitory activity on HBV replication to approximately 40 %. In contrast, the point mutation H66R, inactivating the amino-terminal deaminase domain, did not affect hypermutations, but reduced the inhibition activity to 63 %, whilst the mutant C97S had no effect in either assay. Thus, only the carboxy-terminal deaminase domain of A3B catalyses cytidine deaminations leading to HBV hypermutations, but induction of hypermutations is not sufficient for full inhibition of HBV replication, for which both domains of A3B must be intact.

Cell transformation by avian defective leukaemia viruses

1980 ◽  
Vol 210 (1180) ◽  
pp. 397-409 ◽  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Cell Transformation ◽  
Nucleic Acid Hybridization ◽  
Phenotypic Analysis ◽  
Amino Terminal ◽  
Cell Specificity ◽  
Core Proteins ◽  
Carboxy Terminal

A comparative study of seven independently isolated defective leukaemia viruses has been carried out. Phenotypic analysis of the chicken bone marrow cells transformed in vitro allowed the separation of these seven viruses into three groups based on the differentiation phenotype of the transformed cell. Nucleic acid hybridization studies revealed that these seven viruses had acquired cellular sequences. Interestingly, these studies also showed that the viruses within the same biological grouping had acquired related sequences. This indicates that viruses that have acquired the same or similar cellular sequences have very similar oncogenic capabilities. Analysis of proteins expressed in cells transformed by these viruses demonstrated that the cellular sequences were usually inserted within the gene for the viral core proteins, gag . Therefore the cellular sequences are expressed as a gag -related fusion protein which has an amino-terminal region derived from the gag gene and a carboxy-terminal half derived from the cellular sequences. Two exceptions to this are discussed. The general conclusion from these studies is that defective leukaemia viruses transform cells by virtue of acquired host cellular sequences. The ability of these viruses to transform cells and the target cell specificity of the transformation depends on these cellular sequences.

Structural requirements for enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck

1992 ◽  
Vol 12 (6) ◽  
pp. 2720-2729
Author(s):  
L Caron ◽  
N Abraham ◽  
T Pawson ◽  
A Veillette
Keyword(s):  
T Cell ◽  
Protein Phosphorylation ◽  
Phospholipase C ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Amino Terminal ◽  
Src Homology ◽  
Phospholipase C Gamma ◽  
Domain 3

To understand the mechanism(s) by which p56lck participates in T-cell receptor (TCR) signalling, we have examined the effects of mutations in known regulatory domains of p56lck on the ability of F505 p56lck to enhance the responsiveness of an antigen-specific murine T-cell hybridoma. A mutation of the amino-terminal site of myristylation (glycine 2), which prevents stable association of p56lck with the plasma membrane, completely abolished the ability of F505 p56lck to enhance TCR-induced tyrosine protein phosphorylation. Alteration of the major site of in vitro autophosphorylation, tyrosine 394, to phenylalanine diminished the enhancement of TCR-induced tyrosine protein phosphorylation by F505 p56lck. Such a finding is consistent with the previous demonstration that this site is required for full activation of p56lck by mutation of tyrosine 505. Strikingly, deletion of the noncatalytic Src homology domain 2, but not of the Src homology domain 3, markedly reduced the improvement of TCR-induced tyrosine protein phosphorylation by F505 Lck. Additional studies revealed that all the mutations tested, including deletion of the Src homology 3 region, abrogated the enhancement of antigen-triggered interleukin-2 production by F505 p56lck, thus implying more stringent requirements for augmentation of antigen responsiveness by F505 Lck. Finally, it was also observed that expression of F505 p56lck greatly increased TCR-induced tyrosine phosphorylation of phospholipase C-gamma 1, raising the possibility that phospholipase C-gamma 1 may be a substrate for p56lck in T lymphocytes. Our results indicate that p56lck regulates T-cell antigen receptor signalling through a complex process requiring multiple distinct structural domains of the protein.

Control of AMP deaminase 1 binding to myosin heavy chain

1998 ◽  
Vol 275 (3) ◽  
pp. C870-C881 ◽  
Author(s):  
Ichiro Hisatome ◽  
Takayuki Morisaki ◽  
Hiroshi Kamma ◽  
Takako Sugama ◽  
Hiroko Morisaki ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Sequence Similarity ◽  
Critical Role ◽  
Energy Charge ◽  
Adenylate Energy Charge ◽  
Amp Deaminase ◽  
Amino Terminal

AMP deaminase (AMPD) plays a central role in preserving the adenylate energy charge in myocytes following exercise and in producing intermediates for the citric acid cycle in muscle. Prior studies have demonstrated that AMPD1 binds to myosin heavy chain (MHC) in vitro; binding to the myofibril varies with the state of muscle contraction in vivo, and binding of AMPD1 to MHC is required for activation of this enzyme in myocytes. The present study has identified three domains in AMPD1 that influence binding of this enzyme to MHC using a cotransfection model that permits assessment of mutations introduced into the AMPD1 peptide. One domain that encompasses residues 178–333 of this 727-amino acid peptide is essential for binding of AMPD1 to MHC. This region of AMPD1 shares sequence similarity with several regions of titin, another MHC binding protein. Two additional domains regulate binding of this peptide to MHC in response to intracellular and extracellular signals. A nucleotide binding site, which is located at residues 660–674, controls binding of AMPD1 to MHC in response to changes in intracellular ATP concentration. Deletion analyses demonstrate that the amino-terminal 65 residues of AMPD1 play a critical role in modulating the sensitivity to ATP-induced inhibition of MHC binding. Alternative splicing of the AMPD1 gene product, which alters the sequence of residues 8–12, produces two AMPD1 isoforms that exhibit different MHC binding properties in the presence of ATP. These findings are discussed in the context of the various roles proposed for AMPD in energy production in the myocyte.

scholarly journals Phosphorylation of Ser158 regulates inflammatory redox-dependent hepatocyte nuclear factor-4α transcriptional activity

2006 ◽  
Vol 394 (2) ◽  
pp. 379-387 ◽  
Author(s):  
Hongtao Guo ◽  
Chengjiang Gao ◽  
Zhiyong Mi ◽  
Philip Y. Wai ◽  
Paul C. Kuo
Keyword(s):  
Dna Binding ◽  
Kinase Inhibitor ◽  
Critical Role ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
P38 Kinase ◽  
Mobility Shift ◽  
Transactivation Potential ◽  
Hepatocyte Nuclear Factor 4Α

In IL-1β (interleukin 1β)-stimulated rat hepatocytes exposed to superoxide, we have previously identified an IRX (inflammatory redox)-sensitive DR1 [direct repeat of RG(G/T)TCA with one base spacing] cis-acting activator element (nt –1327 to –1315) in the iNOS (inducible nitric oxide synthase) promoter: AGGTCAGGGGACA. The corresponding transcription factor was identified to be HNF4α (hepatocyte nuclear factor-4α). HNF4α DNA binding activity and transactivation potential are tightly regulated by its state of phosphorylation. However, the functional consequences of IRX-mediated post-translational phosphorylation of HNF4α have not been well characterized. In the setting of IL-1β+H2O2, HNF4α functional activity is associated with a unique serine/threonine phosphorylation pattern. This indicates that an IRX-sensitive serine/threonine kinase pathway targets HNF4α to augment hepatocyte iNOS transcription. In the present study, following identification of phosphorylated residues in HNF4α, serial mutations were performed to render the target residues phosphorylation-resistant. Electrophoretic mobility-shift assays and transient transfection studies utilizing the iNOS promoter showed that the S158A mutation ablates IRX-mediated HNF4α DNA binding and transactivation. Gain-of-function mutation with the S158D phosphomimetic HNF4α vector supports a critical role for Ser158 phosphorylation. In vitro phosphorylation and kinase inhibitor studies implicate p38 kinase activity. Our results indicate that p38 kinase-mediated Ser158 phosphorylation is essential for augmentation of the DNA binding and transactivation potential of HNF4α in the presence of IL-1β+H2O2. This pathway results in enhanced iNOS expression in hepatocytes exposed to pro-inflammatory cytokines and oxidative stress.

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