scholarly journals Distinct functional properties of IkappaB alpha and IkappaB beta.

1997 ◽  
Vol 17 (9) ◽  
pp. 5386-5399 ◽  
Author(s):  
K Tran ◽  
M Merika ◽  
D Thanos
Keyword(s):  
Transcriptional Activation ◽  
High Mobility ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
High Mobility Group Protein ◽  
Dna Binding Activity ◽  
Group Protein ◽  
Nf Kappab

The biological activity of the transcription factor NF-kappaB is controlled mainly by the IkappaB alpha and IkappaB beta proteins, which restrict NF-kappaB to the cytoplasm and inhibit its DNA binding activity. Here, we carried out experiments to determine and compare the mechanisms by which IkappaB alpha and IkappaB beta inhibit NF-kappaB-dependent transcriptional activation. First, we found that in vivo IkappaB alpha is a stronger inhibitor of NF-kappaB than is IkappaB beta. This difference is directly correlated with their abilities to inhibit NF-kappaB binding to DNA in vitro and in vivo. Moreover, IkappaB alpha, but not IkappaB beta, can remove NF-kappaB from functional preinitiation complexes in in vitro transcription experiments. Second, we showed that both IkappaBs function in vivo not only in the cytoplasm but also in the nucleus, where they inhibit NF-kappaB binding to DNA. Third, the inhibitory activity of IkappaB beta, but not that of IkappaB alpha, is facilitated by phosphorylation of the C-terminal PEST sequence by casein kinase II and/or by the interaction of NF-kappaB with high-mobility group protein I (HMG I) on selected promoters. The unphosphorylated form of IkappaB beta forms stable ternary complexes with NF-kappaB on the DNA either in vitro or in vivo. These experiments suggest that IkappaB alpha works as a postinduction repressor of NF-kappaB independently of HMG I, whereas IkappaB beta functions preferentially in promoters regulated by the NF-kappaB/HMG I complexes.

Rhinovirus stimulation of interleukin-8 in vivo and in vitro: role of NF-κB

1997 ◽  
Vol 273 (4) ◽  
pp. L814-L824 ◽  
Author(s):  
Zhou Zhu ◽  
Weiliang Tang ◽  
Jack M. Gwaltney ◽  
Yang Wu ◽  
Jack A. Elias
Keyword(s):  
Transcriptional Activation ◽  
Neutrophil Infiltration ◽  
Binding Activity ◽  
Early Event ◽  
Mrna Accumulation ◽  
Additional Increase ◽  
Dna Binding Activity ◽  
Infected Cells

Neutrophil infiltration is a well-documented early event in the pathogenesis of rhinovirus (RV) infections. To further understand the mechanisms responsible for this neutrophilia, we determined whether interleukin (IL)-8 was present at sites of experimental RV infection in vivo and characterized the mechanism(s) by which RV stimulates IL-8 production in vitro. IL-8 was readily detectable in the nasal washings of all normal volunteers and did not increase with sham nasal inoculation. In contrast, RV infection caused a significant additional increase in nasal IL-8, the levels of which peaked 48–72 h after virus inoculation. RV was a potent stimulator of IL-8 protein production by A549 epithelial-like cells, MRC-5 fibroblasts, and normal human bronchial epithelial cells in vitro. This induction was associated with a significant increase in IL-8 mRNA accumulation and gene transcription. RV also stimulated IL-8 promoter-driven luciferase activity. This stimulation was significantly decreased by mutation of the nuclear factor (NF)-IL-6 site and was completely abrogated by mutation of the NF-κB site in this promoter. In addition, NF-κB-DNA binding activity was rapidly induced in RV-infected cells. This inducible binding was made up of p65 and, to a lesser extent, p50 NF-κB moieties. These studies demonstrate that IL-8 is present in normal nasal secretions and that the levels of IL-8 are further increased after RV infection. They also demonstrate that RVs are potent stimulators of IL-8 production and that this induction is mediated, at least in part, by an NF-κB-dependent transcriptional activation pathway. IL-8 may contribute to the pathogenesis of RV infection, and NF-κB activation may be a central event in RV-induced pathologies.

scholarly journals Regulation of the DNA-binding and transcriptional activities of Xenopus laevis NFI-X by a novel C-terminal domain.

1995 ◽  
Vol 15 (10) ◽  
pp. 5552-5562 ◽  
Author(s):  
E Roulet ◽  
M T Armentero ◽  
G Krey ◽  
B Corthésy ◽  
C Dreyer ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Xenopus Laevis ◽  
Transcriptional Activation ◽  
Binding Activity ◽  
New Members ◽  
Binding Domains ◽  
Dna Binding Activity

The nuclear factor I (NFI) family consists of sequence-specific DNA-binding proteins that activate both transcription and adenovirus DNA replication. We have characterized three new members of the NFI family that belong to the Xenopus laevis NFI-X subtype and differ in their C-termini. We show that these polypeptides can activate transcription in HeLa and Drosophila Schneider line 2 cells, using an activation domain that is subdivided into adjacent variable and subtype-specific domains each having independent activation properties in chimeric proteins. Together, these two domains constitute the full NFI-X transactivation potential. In addition, we find that the X. laevis NFI-X proteins are capable of activating adenovirus DNA replication through their conserved N-terminal DNA-binding domains. Surprisingly, their in vitro DNA-binding activities are specifically inhibited by a novel repressor domain contained within the C-terminal part, while the dimerization and replication functions per se are not affected. However, inhibition of DNA-binding activity in vitro is relieved within the cell, as transcriptional activation occurs irrespective of the presence of the repressor domain. Moreover, the region comprising the repressor domain participates in transactivation. Mechanisms that may allow the relief of DNA-binding inhibition in vivo and trigger transcriptional activation are discussed.

scholarly journals Jun is phosphorylated by several protein kinases at the same sites that are modified in serum-stimulated fibroblasts.

1992 ◽  
Vol 12 (10) ◽  
pp. 4694-4705 ◽  
Author(s):  
S J Baker ◽  
T K Kerppola ◽  
D Luk ◽  
M T Vandenberg ◽  
D R Marshak ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dna Binding ◽  
Glycogen Synthase ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Serum Stimulation ◽  
Dependent Protein

c-jun is a member of the family of immediate-early genes whose expression is induced by factors such as serum stimulation, phorbol ester, and differentiation signals. Here we show that increased Jun synthesis after serum stimulation is accompanied by a concomitant increase in phosphorylation. Several serine-threonine kinases were evaluated for their ability to phosphorylate Jun in vitro. p34cdc2, protein kinase C, casein kinase II, and pp44mapk phosphorylated Jun efficiently, whereas cyclic AMP-dependent protein kinase and glycogen synthase kinase III did not. The sites phosphorylated by p34cdc2 were similar to those phosphorylated in vivo after serum induction. The major sites of phosphorylation were mapped to serines 63, 73, and 246. Phosphorylation of full-length Jun with several kinases did not affect the DNA-binding activity of Jun homodimers or Fos-Jun heterodimers. Comparison of the DNA binding and in vitro transcription properties of wild-type and mutated proteins containing either alanine or aspartic acid residues in place of Ser-63, -73, and -246 revealed only minor differences among homodimeric complexes and no differences among Fos-Jun heterodimers. Thus, phosphorylation of Jun did not produce a significant change in dimerization, DNA-binding, or in vitro transcription activity. The regulatory role of phosphorylation in the modulation of Jun function is likely to be considerably more complex than previously suggested.

scholarly journals Knockdown of MSI2 inhibits metastasis by interacting with caveolin-1 and inhibiting its ubiquitylation in human NF1-MPNST cells

2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Kang Yang ◽  
Jianwei Du ◽  
Dai Shi ◽  
Feng Ji ◽  
Yong Ji ◽  
...  
Keyword(s):  
Epithelial Mesenchymal Transition ◽  
High Mobility ◽  
Neurofibromatosis Type ◽  
Migration And Invasion ◽  
High Mobility Group Protein ◽  
Mesenchymal Transition ◽  
Caveolin 1 ◽  
Group Protein

AbstractMalignant peripheral nerve sheath tumours (MPNSTs) are highly aggressive Schwann cell-derived sarcomas, and they are either associated with neurofibromatosis type 1 (NF1) or sporadic. Our previous study found that high mobility group protein A2 (HMGA2) regulates NF1-MPNST growth through Musashi-2 (MSI2); however, whether MSI2 regulates MPNST metastasis and what the mechanism is remain unclear. Here, we demonstrated that the protein caveolin-1 (CAV1) directly interacts with MSI2 in human NF1-MPNST cells. Moreover, we discovered that knockdown of MSI2 induces CAV1 protein expression by inhibiting its ubiquitylation level in NF1-MPNSTs. In addition, CAV1 mediates the suppressive function of MSI2 in epithelial-mesenchymal transition, migration and invasion in vitro and metastasis in vivo. These results help to reveal the potential mechanisms of MSI2 as a target of antimetastatic treatment for human NF1-MPNST.

scholarly journals Cooperative interactions between HOX and PBX proteins mediated by a conserved peptide motif.

1995 ◽  
Vol 15 (8) ◽  
pp. 3989-3997 ◽  
Author(s):  
M L Phelan ◽  
I Rambaldi ◽  
M S Featherstone
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Binding Activity ◽  
Cooperative Interaction ◽  
Transcriptional Activation Domain ◽  
Conserved Sequence ◽  
Dna Binding Activity ◽  
Group Protein

Homeoprotein products of the Hox/HOM gene family pattern the animal embryo through the transcriptional regulation of target genes. We have previously shown that the labial group protein HOXA-1 has intrinsically weak DNA-binding activity due to residues in the N-terminal arm of its homeodomain (M. L. Phelan, R. Sadoul, and M. S. Featherstone, Mol. Cell. Biol. 14:5066-5075, 1994). This observation, among others, suggests that HOX and HOM proteins require cofactors for stable interactions with DNA. We have demonstrated that a putative HOX cofactor, PBX1A, participates in cooperative DNA binding with HOXA-1 and the Deformed group protein HOXD-4. Three Abdominal-B class HOX proteins failed to cooperate with PBX1A. We mapped the interacting domain of HOXD-4 to the YPWMK pentapeptide motif, a conserved sequence found N terminal to the homeodomain of HOXA-1 and many other homeoproteins but absent from the Abdominal-B class. The naturally occurring fusion of the transcriptional activation domain of E2A with PBX1 creates an oncoprotein implicated in human pre-B-cell leukemias (M. P. Kamps, C. Murre, X.-H. Sun, and D. Baltimore, Cell 60:547-555, 1990; J. Nourse, J. D. Mellentin, N. Galili, J. Wilkinson, E. Starbridge, S. D. Smith, and M. L. Cleary, Cell 60:535-545, 1990). A pentapeptide mutation that abolished cooperative interaction with PBX1A in vitro also abrogated synergistic transcriptional activation with the E2A/PBX oncoprotein. The direct contact of PBX family members by the HOX pentapeptide is likely to play an important role in developmental and oncogenic processes.

Preferential Phosphorylation of High Mobility Group Protein 17 in vitro by a Nuclear Protein Kinase

1981 ◽  
Vol 36 (3-4) ◽  
pp. 319-322 ◽  
Keyword(s):  
Nuclear Protein ◽  
High Mobility ◽  
High Specificity ◽  
High Mobility Group ◽  
Hmg Proteins ◽  
High Mobility Group Protein ◽  
High Preference ◽  
Group Protein

The ability of the high mobility group proteins (HMG-1,2,14 and 17) to serve as substrate for protein kinases was investigated by incubating them with a cytoplasmic and nuclear kinase. In both cases phosphate was incorporated into all four HMG proteins. The amount of phosphate incorporated and the specificity for the four proteins was quite different for the two kinases. Whereas the cytoplasmic kinase phosphorylated the HMG-1 and 2 to a higher degree than HMG-14 and 17, the nuclear kinase exhibited a high specificity for the HMG -17, leaving the other three proteins with only a small amount. The high preference of a nuclear kinase for HMG-17 may be indicative of a specific phosphorylation occuring also in vivo

scholarly journals Effects of prednisolone on glomerular signal transduction cascades in experimental glomerulonephritis.

1998 ◽  
Vol 9 (8) ◽  
pp. 1367-1376
Author(s):  
M Seto ◽  
S Kim ◽  
H Yoshifusa ◽  
Y Nakamura ◽  
T Masuda ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Activity ◽  
Kinase Assay ◽  
Mobility Shift ◽  
Glomerular Cell ◽  
Beneficial Effects ◽  
Dna Binding Activity ◽  
Nf Kappab

In vitro data support that activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) regulate the gene expression of numerous growth factors and cytokines involved in the development of glomerulonephritis (GN). However, the in vivo activation and role of these transcription factors are poorly understood. This study examines whether these transcription factors are activated in antithymocyte serum (ATS)-induced GN in vivo and whether prednisolone suppresses activation of them. As assessed by gel mobility shift assay, glomerular DNA binding activity of AP-1 containing both c-Jun and c-Fos and NF-kappaB composed of P-50 and P-65 subunits was significantly increased after ATS injection. Furthermore, as estimated by in-gel kinase assay, glomerular activity of extracellular signal-regulated kinases (ERK) and c-jun NH2-terminal kinases (JNK), which are mitogen-activated protein kinases (MAPK) known to activate AP-1 and NF-kappaB in vitro, was significantly increased after ATS injection, preceding the increase in AP-1 activity. Prednisolone treatment significantly prevented the increase in urinary protein and albumin excretion and glomerular cell proliferation in ATS-induced GN, indicating the beneficial effects of prednisolone on this GN. Prednisolone significantly suppressed the increased glomerular ERK and JNK activities and AP-1 binding activity, but not glomerular NF-kappa binding activity. This study provides the first evidence of the marked increase in glomerular MAPK activities, and AP-1 and NF-kappa binding activities in ATS-induced GN. The beneficial effect of prednisolone on this GN may be partially mediated by the suppression of MAPK, followed by the suppression of AP-1.

scholarly journals Acetylation-Mediated Transcriptional Activation of the ETS Protein ER81 by p300, P/CAF, and HER2/Neu

2003 ◽  
Vol 23 (17) ◽  
pp. 6243-6254 ◽  
Author(s):  
Apollina Goel ◽  
Ralf Janknecht
Keyword(s):  
Protein Kinase ◽  
Transcriptional Activation ◽  
Binding Activity ◽  
Tumor Formation ◽  
Mitogen Activated Protein Kinase ◽  
Transactivation Domain ◽  
Dna Binding Activity ◽  
Mitogen Activated Protein

ABSTRACT The regulated expression of the ETS transcription factor ER81 is a prerequisite for normal development, and its dysregulation contributes to neoplasia. Here, we demonstrate that ER81 is acetylated by two coactivators/acetyltransferases, p300 and p300- and CBP-associated factor (P/CAF) in vitro and in vivo. Whereas p300 acetylates two lysine residues (K33 and K116) within the ER81 N-terminal transactivation domain, P/CAF targets only K116. Acetylation of ER81 not only enhances its ability to transactivate but also increases its DNA binding activity and in vivo half-life. Furthermore, oncogenic HER2/Neu, which induces phosphorylation and thereby activation of ER81, was less able to activate acetylation-deficient ER81 mutants, indicating that both acetyltransferase and protein kinase-specific regulatory mechanisms control ER81 activity. Importantly, HER2/Neu overexpression stimulates the ability of p300 to acetylate ER81, likely by inducing phosphorylation of p300 through the Ras→Raf→mitogen-activated protein kinase pathway. This represents a novel mechanism by which oncogenic HER2/Neu, Ras, or Raf may promote tumor formation by enhancing acetylation not only of ER81 but also of other downstream effector transcription factors as well as histones.

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