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J A DiDonato

Publications and source records attributed to J A DiDonato.

At least 19 recordsLinked to original sources
PubMed2001

IKK alpha on center stage.

Inhibitor of kappaB kinase alpha (IKK alpha) was originally identified as a component of a multiprotein kinase complex that regulates the activity of the transcription factor nuclear factor-kappaB (NF-kappaB) through phosphorylation of its inhibitor proteins, the IkappaBs. DiDonato discusses new roles that have been discovered for IKK alpha, focusing especially on its role in epidermal differentiation and on a new function of IKK alpha in B cell maturation. In epidermal differentiation, IKK alpha regulates the production of a secreted differentiation factor through a pathway that is independent of its role in activation of NF-kappaB. In B cell maturation, conventional NF-kappaB signal-induced activation of IKK alpha results in phosphorylation of p100 precursor proteins and increased proteolytic processing and constitutive NF-kappaB activation.

Animals↗
PubMed2001

B lymphocytes and plasma cells express functional E-selectin by constitutive activation of NF-kappaB.

E-selectin (CD62E), a cell adhesion molecule for most leukocytes, is known to be expressed exclusively on the cytokine-stimulated endothelial cells mainly by inductive activation of NF-kappaB. Using immunohistochemistry and in situ hybridization, we showed that B lymphocytes and plasma cells in the spleens and lymph nodes from nude mice (T-lymphocyte-deficient), but not from SCID mice (T- and B-lymphocyte-deficient), expressed E-selectin prior to cytokine stimulation. The expression of E-selectin was also confirmed on human B lymphocytes isolated from peripheral bloods. The mouse J774A.1 monocytes could adhere to the marginal zones of mouse spleens in an E-selectin Ab inhibitable manner, suggesting the functional activity of the expressed E-selectin. In addition, ARH-77 cells, a cell line derived from human plasma cells, were found to express E-selectin mRNA and protein and to have a NF-kappaB activity for an E-selectin promoter. NF-kappaB antagonists, such as TPCK (tosylsulfonyl phenylalanyl chloromethyl ketone), dexamethasone and a IkappaBalpha mutant plasmid could inhibit both the NF-kappaB activity and the expression of E-selectin. Transfection with an E-selectin promoter-driven reporter gene construct further verified the E-selectin promoter activity in ARH-77 cells. Again, TPCK, dexamethasone, and the IkappaBalpha mutant plasmid could neutralize this activity. These findings suggest that B lymphocytes and plasma cells can express E-selectin, which is functional for monocytic leukocytes, by a mechanism of constitutive activation of NF-kappaB.

Animals↗
PubMed2001

Activation of NF-kappa B is required for hypertrophic growth of primary rat neonatal ventricular cardiomyocytes.

The transcription factor NF-kappaB regulates expression of genes that are involved in inflammation, immune response, viral infection, cell survival, and division. However, the role of NF-kappaB in hypertrophic growth of terminally differentiated cardiomyocytes is unknown. Here we report that NF-kappaB activation is required for hypertrophic growth of cardiomyocytes. In cultured rat primary neonatal ventricular cardiomyocytes, the nuclear translocation of NF-kappaB and its transcriptional activity were stimulated by several hypertrophic agonists, including phenylephrine, endothelin-1, and angiotensin II. The activation of NF-kappaB was inhibited by expression of a "supersuppressor" IkappaBalpha mutant that is resistant to stimulation-induced degradation and a dominant negative IkappaB kinase (IKKbeta) mutant that can no longer be activated by phosphorylation. Furthermore, treatment with phenylephrine induced IkappaBalpha degradation in an IKK-dependent manner, suggesting that NF-kappaB is a downstream target of the hypertrophic agonists. Importantly, expression of the supersuppressor IkappaBalpha mutant or the dominant negative IKKbeta mutant blocked the hypertrophic agonist-induced expression of the embryonic gene atrial natriuretic factor and enlargement of cardiomyocytes. Conversely, overexpression of NF-kappaB itself induced atrial natriuretic factor expression and cardiomyocyte enlargement. These findings suggest that NF-kappaB plays a critical role in the hypertrophic growth of cardiomyocytes and may serve as a potential target for the intervention of heart disease.

Angiotensin II↗
PubMed2001

Activation of NF-kappaB by hepatitis B virus X protein through an IkappaB kinase-independent mechanism.

pX, the hepatitis B virus-encoded transcription coactivator, is involved in viral infection in vivo. pX stimulates the activity of several transcription factors including nuclear factor-kappaB (NF-kappaB), but the mechanism of activation is poorly understood. The IkappaB kinase complex (IKK) mediates activation of NF-kappaB in response to various extracellular stimuli, including inflammatory cytokines like tumor necrosis factor and interleukin 1, human T cell lymphoma virus 1 Tax protein, and tumor promoters like phorbol esters. It is not known whether IKK also mediates activation of NF-kappaB by pX. Here we report that IKK was not essential for activation of NF-kappaB by pX. Expression of pX resulted in the degradation of IkappaBalpha in the absence of its phosphorylation at Ser(32) and Ser(36) residues. Although pX stimulated the activity of cotransfected IKK-beta when it was overexpressed, it failed to activate endogenous IKK. Furthermore, expression of pX stimulated NF-kappaB nuclear translocation and transcriptional activity in IKK-gamma-null fibroblast 5R cells. Our data indicate that pX stimulates NF-kappaB activity through a mechanism that is dependent on IkappaBalpha degradation but not on IKK activation.

Cell Line↗
PubMed2000

NF-kappaB activation by double-stranded-RNA-activated protein kinase (PKR) is mediated through NF-kappaB-inducing kinase and IkappaB kinase.

The interferon (IFN)-inducible double-stranded-RNA (dsRNA)-activated serine-threonine protein kinase (PKR) is a major mediator of the antiviral and antiproliferative activities of IFNs. PKR has been implicated in different stress-induced signaling pathways including dsRNA signaling to nuclear factor kappa B (NF-kappaB). The mechanism by which PKR mediates activation of NF-kappaB is unknown. Here we show that in response to poly(rI). poly(rC) (pIC), PKR activates IkappaB kinase (IKK), leading to the degradation of the inhibitors IkappaBalpha and IkappaBbeta and the concomitant release of NF-kappaB. The results of kinetic studies revealed that pIC induced a slow and prolonged activation of IKK, which was preceded by PKR activation. In PKR null cell lines, pIC failed to stimulate IKK activity compared to cells from an isogenic background wild type for PKR in accord with the inability of PKR null cells to induce NF-kappaB in response to pIC. Moreover, PKR was required to establish a sustained response to tumor necrosis factor alpha (TNF-alpha) and to potentiate activation of NF-kappaB by cotreatment with TNF-alpha and IFN-gamma. By coimmunoprecipitation, PKR was shown to be physically associated with the IKK complex. Transient expression of a dominant negative mutant of IKKbeta or the NF-kappaB-inducing kinase (NIK) inhibited pIC-induced gene expression from an NF-kappaB-dependent reporter construct. Taken together, these results demonstrate that PKR-dependent dsRNA induction of NF-kappaB is mediated by NIK and IKK activation.

Animals↗
PubMed1999

NF-kappa B is a central regulator of the intestinal epithelial cell innate immune response induced by infection with enteroinvasive bacteria.

Human intestinal epithelial cells up-regulate the expression of an inflammatory gene program in response to infection with a spectrum of different strains of enteroinvasive bacteria. The conserved nature of this program suggested that diverse signals, which are activated by enteroinvasive bacteria, can be integrated into a common signaling pathway that activates a set of proinflammatory genes in infected host cells. Human intestinal epithelial cell lines, HT-29, Caco-2, and T84, were infected with invasive bacteria that use different strategies to induce their uptake and have different intracellular localizations (i.e., Salmonella dublin, enteroinvasive Escherichia coli, or Yersinia enterocolitica). Infection with each of these bacteria resulted in the activation of TNF receptor associated factors, two recently described serine kinases, I kappa B kinase (IKK) alpha and IKK beta, and increased NF-kappa B DNA binding activity. This was paralleled by partial degradation of I kappa B alpha and I kappa B epsilon in bacteria-infected Caco-2 cells. Mutant proteins that act as superrepressors of IKK beta and I kappa B alpha inhibited the up-regulated transcription and expression of downstream targets genes of NF-kappa B that are key components of the epithelial inflammatory gene program (i.e., IL-8, growth-related oncogene-alpha, monocyte chemoattractant protein-1, TNF-alpha, cyclooxygenase-2, nitric oxide synthase-2, ICAM-1) activated by those enteroinvasive bacteria. These studies position NF-kappa B as a central regulator of the epithelial cell innate immune response to infection with enteroinvasive bacteria.

Caco-2 Cells↗
PubMed1999

IKKgamma mediates the interaction of cellular IkappaB kinases with the tax transforming protein of human T cell leukemia virus type 1.

The Tax oncoprotein of human T cell leukemia virus type 1 constitutively activates transcription factor NF-kappaB by a mechanism involving Tax-induced phosphorylation of IkappaBalpha, a labile cytoplasmic inhibitor of NF-kappaB. To trigger this signaling cascade, Tax associates stably with and persistently activates a cellular IkappaB kinase (IKK) containing both catalytic (IKKalpha and IKKbeta) and noncatalytic (IKKgamma) subunits. We now demonstrate that IKKgamma enables Tax to dock with the IKKbeta catalytic subunit, resulting in chronic IkappaB kinase activation. Mutations in either IKKgamma or Tax that prevent formation of these higher order Tax.IKK complexes also interfere with the ability of Tax to induce IKKbeta catalytic function in vivo. Deletion mapping studies indicate that amino acids 1-100 of IKKgamma are required for this Tax targeting function. Together, these findings identify IKKgamma as an adaptor protein that directs the stable formation of pathologic Tax.IKK complexes in virally infected T cells.

Gene Expression Regulation↗
PubMed1998

Suppression of steady-state, but not stimulus-induced NF-kappaB activity inhibits alphavirus-induced apoptosis.

Recent studies have established cell type- specific, proapoptotic, or antiapoptotic functions for the transcription factor NF-kappaB. In each of these studies, inhibitors of NF-kappaB activity have been present before the apoptotic stimulus, and so the role of stimulus- induced NF-kappaB activation in enhancing or inhibiting survival could not be directly assessed. Sindbis virus, an alphavirus, induces NF-kappaB activation and apoptosis in cultured cell lines. To address whether Sindbis virus- induced NF-kappaB activation is required for apoptosis, we used a chimeric Sindbis virus that expresses a superrepressor of NF-kappaB activity. Complete suppression of virus-induced NF-kappaB activity neither prevents nor potentiates Sindbis virus-induced apoptosis. In contrast, inhibition of NF-kappaB activity before infection inhibits Sindbis virus-induced apoptosis. Our results demonstrate that suppression of steady-state, but not stimulus-induced NF-kappaB activity, regulates expression of gene products required for Sindbis virus-induced death. Furthermore, we show that in the same cell line, NF-kappaB can be proapoptotic or antiapoptotic depending on the death stimulus. We propose that the role of NF-kappaB in regulating apoptosis is determined by the death stimulus and by the timing of modulating NF-kappaB activity relative to the death stimulus.

3T3 Cells↗
PubMed1998

The tax oncoprotein of human T-cell leukemia virus type 1 associates with and persistently activates IkappaB kinases containing IKKalpha and IKKbeta.

The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV1) chronically activates transcription factor NF-kappaB by a mechanism involving degradation of IkappaBalpha, an NF-kappaB-associated cytoplasmic inhibitor. Tax-induced breakdown of IkappaBalpha requires phosphorylation of the inhibitor at Ser-32 and Ser-36, which is also a prerequisite for the transient activation of NF-kappaB in cytokine-treated T lymphocytes. However, it remained unclear how Tax interfaces with the cellular NF-kappaB/IkappaB signaling machinery to generate a chronic rather than a transient NF-kappaB response. We now demonstrate that Tax associates with cytokine-inducible IkappaB kinase (IKK) complexes containing catalytic subunits IKKalpha and IKKbeta, which mediate phosphorylation of IkappaBalpha at Ser-32 and Ser-36. Unlike their transiently activated counterparts in cytokine-treated cells, Tax-associated forms of IKK are constitutively active in either Tax transfectants or HTLV1-infected T lymphocytes. Moreover, point mutations in Tax that ablate its IKK-binding function also prevent Tax-mediated activation of IKK and NF-kappaB. Together, these findings suggest that the persistent activation of NF-kappaB in HTLV1-infected T-cells is mediated by a direct Tax/IKK coupling mechanism.

Cytoplasm↗
PubMed1998

Coordinate regulation of IkappaB kinases by mitogen-activated protein kinase kinase kinase 1 and NF-kappaB-inducing kinase.

IkappaB kinases (IKKalpha and IKKbeta) are key components of the IKK complex that mediates activation of the transcription factor NF-kappaB in response to extracellular stimuli such as inflammatory cytokines, viral and bacterial infection, and UV irradiation. Although NF-kappaB-inducing kinase (NIK) interacts with and activates the IKKs, the upstream kinases for the IKKs still remain obscure. We identified mitogen-activated protein kinase kinase kinase 1 (MEKK1) as an immediate upstream kinase of the IKK complex. MEKK1 is activated by tumor necrosis factor alpha (TNF-alpha) and interleukin-1 and can potentiate the stimulatory effect of TNF-alpha on IKK and NF-kappaB activation. The dominant negative mutant of MEKK1, on the other hand, partially blocks activation of IKK by TNF-alpha. MEKK1 interacts with and stimulates the activities of both IKKalpha and IKKbeta in transfected HeLa and COS-1 cells and directly phosphorylates the IKKs in vitro. Furthermore, MEKK1 appears to act in parallel to NIK, leading to synergistic activation of the IKK complex. The formation of the MEKK1-IKK complex versus the NIK-IKK complex may provide a molecular basis for regulation of the IKK complex by various extracellular signals.

Animals↗
PubMed1997

cAMP-induced NF-kappaB (p50/relB) binding to a c-myb intronic enhancer correlates with c-myb up-regulation and inhibition of erythroleukemia cell differentiation.

During hexamethylene bisactamide (HMBA)-induced differentiation of murine erythroleukemia (MEL) cells erythroid genes are transcriptionally activated while c-myb and several other nuclear proto-oncogenes are down-regulated. Differentiation is inhibited by cAMP analogues and the adenyl cyclase-stimulating agent forskolin. We found that these drugs prevented the late down-regulation of c-myb which is known to be critical for MEL cell differentiation. Since the increase in c-myb mRNA levels was due to increased mRNA transcription, we examined the transcription factors NF-kappaB and AP-1 which have been implicated in the regulation of c-myb expression. Binding of MEL cell nuclear proteins to a NF-kappaB recognition sequence in c-myb intron I was strongly induced by 8-Br-cAMP or forskolin; induction was delayed and correlated with the up-regulation of c-myb. The cAMP-induced NF-kappaB complex contained p50 and RelB; in co-transfection assays, p50 and RelB transactivated a reporter construct containing the c-myb intronic NF-kappaB site upstream of a minimal promoter. 8-Br-cAMP and forskolin also increased the DNA binding activity of AP-1 complexes containing JunB, JunD and c-Fos in MEL cells which could cooperate with NF-kappaB. We conclude that inhibition of MEL cell differentiation by pharmacological doses of cAMP can be explained by the up-regulation of c-myb which is mediated, at least in part, by NF-kappaB p50/RelB heterodimers.

Animals↗
PubMed1997

A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB.

Nuclear transcription factors of the NF-kappaB/Rel family are inhibited by IkappaB proteins, which inactivate NF-kappaB by trapping it in the cell cytoplasm. Phosphorylation of IkappaBs marks them out for destruction, thereby relieving their inhibitory effect on NF-kappaB. A cytokine-activated protein kinase complex, IKK (for IkappaB kinase), has now been purified that phosphorylates IkappaBs on the sites that trigger their degradation. A component of IKK was molecularly cloned and identified as a serine kinase. IKK turns out to be the long-sought-after protein kinase that mediates the critical regulatory step in NF-kappaB activation.

Amino Acid Sequence↗
PubMed1995

Immunosuppression by glucocorticoids: inhibition of NF-kappa B activity through induction of I kappa B synthesis.

Glucocorticoids are among the most potent anti-inflammatory and immunosuppressive agents. They inhibit synthesis of almost all known cytokines and of several cell surface molecules required for immune function, but the mechanism underlying this activity has been unclear. Here it is shown that glucocorticoids are potent inhibitors of nuclear factor kappa B (NF-kappa B) activation in mice and cultured cells. This inhibition is mediated by induction of the I kappa B alpha inhibitory protein, which traps activated NF-kappa B in inactive cytoplasmic complexes. Because NF-kappa B activates many immunoregulatory genes in response to pro-inflammatory stimuli, the inhibition of its activity can be a major component of the anti-inflammatory activity of glucocorticoids.

Animals↗
PubMed1995

Phosphorylation of I kappa B alpha precedes but is not sufficient for its dissociation from NF-kappa B.

NF-kappa B is an important activator of immune and inflammatory response genes. NF-kappa B is sequestered in the cytoplasm of nonstimulated cells through interaction with the I kappa B inhibitors. These inactive complexes are dissociated in response to a variety of extracellular signals, thereby allowing free NF-kappa B dimers to translocate to the nucleus and active transcription of specific target genes. The current dogma is that phosphorylation of the I kappa Bs is responsible for dissociation of the inactive complexes, an event that is rendered irreversible by rapid I kappa B degradation. Here, we show that inducers of NF-kappa B activity stimulate the hyperphosphorylation of one of the I kappa Bs, I kappa B alpha. However, contrary to the present dogma the hyperphosphorylated form of I kappa B alpha remains associated with NF-kappa B components such as RelA (p65). Thus, phosphorylation of I kappa B alpha is not sufficient to cause dissociation of the inactive NF-kappa B:I kappa B alpha complex. However, that complex is disrupted through the selective degradation of phosphorylated I kappa B alpha in response to extracellular signals. Using a variety of protease inhibitors, some of which have specificity towards the multicatalytic proteinase complex, we demonstrate that degradation of I kappa B alpha is required for NF-kappa B activation. The results of these experiments are more consistent with a new model according to which phosphorylation of I kappa B alpha associated with NF-kappa B marks it for proteolytic degradation. I kappa B alpha is degraded while bound to NF-kappa B. The selective degradation of I kappa B alpha releases active NF-kappa B dimers which can translocate to the nucleus to activate specific target genes.

Amino Acid Sequence↗
PubMed1993

NF-kappa B activation by ultraviolet light not dependent on a nuclear signal.

Exposure of mammalian cells to radiation triggers the ultraviolet (UV) response, which includes activation of activator protein-1 (AP-1) and nuclear factor kappa B (NF-kappa B). This was postulated to occur by induction of a nuclear signaling cascade by damaged DNA. Recently, induction of AP-1 by UV was shown to be mediated by a pathway involving Src tyrosine kinases and the Ha-Ras small guanosine triphosphate-binding protein, proteins located at the plasma membrane. It is demonstrated here that the same pathway mediates induction of NF-kappa B by UV. Because inactive NF-kappa B is stored in the cytosol, analysis of its activation directly tests the involvement of a nuclear-initiated signaling cascade. Enucleated cells are fully responsive to UV both in NF-kappa B induction and in activation of another key signaling event. Therefore, the UV response does not require a signal generated in the nucleus and is likely to be initiated at or near the plasma membrane.

Alleles↗
PubMed1993

p105 and p98 precursor proteins play an active role in NF-kappa B-mediated signal transduction.

The Rel/NF-kappa B family of transcription factors is composed of two distinct subgroups, proteins that undergo proteolytic processing and contain SWI6/ankyrin repeats in their carboxyl termini (p105, p98), and those without such repeats that do not require processing (p65, c-Rel, RelB, and Dorsal). We demonstrate that the p105 and p98 precursors share functional properties with the I kappa B proteins, which also contain SWI6/ankyrin repeats. Both p105 and p98 were found to form stable complexes with other Rel/NF-kappa B family members, including p65 and c-Rel. Association with the precursors is sufficient for cytoplasmic retention of either p65 or c-Rel, both of which are otherwise nuclear. These complexes undergo stimulus-responsive processing to produce active p50/c-Rel and p55/c-Rel complexes. These observations suggest a second pathway leading to NF-kappa B induction, in which processing of the precursors rather than phosphorylation of I kappa B plays a major role.

Antigen-Antibody Complex↗
PubMed1991

A predictive model for DNA recognition by the herpes simplex virus protein ICP4.

The herpes simplex virus (HSV) type 1 immediate early protein ICP4 is an essential regulatory enzyme that binds DNA directly in order to stimulate or repress gene expression. The degree of transaction is related to the locations and affinities of the ICP4 binding sites. A number of binding sites have been identified; some sites showed obvious homology to one another, and these were called consensus ICP4 binding sites. Other binding sites did not appear to be related, and these were termed non-consensus sites. We hypothesized, however, that a single model could describe all ICP4 binding sites, given the appropriate characterizations of sites. We performed statistical analyses on a set of ICP4 binding sites and found that the bases important for defining binding were located within a 13 base region. Missing contact analyses on several high-affinity binding sites revealed the same 13 base region as important for critical protein-DNA contacts. From these data we derived the consensus sequence RTCGTCNNYNYSG, where R is purine, Y is pyrimidine, S is C or G, and N is any base. In addition, we found that a better profile for ICP4 binding sites involves use of a matrix of base proportions from the binding site data; sites are analyzed by calculating the Matrix Mean score. We show that this Matrix Mean model could accurately predict the locations of novel ICP4 binding sites. Finally, we analyzed the entire HSV-1 genome for potential ICP4 binding sites and speculate about what these results suggest for the role of ICP4 in viral gene regulation.

Amino Acid Sequence↗