Post‐translational activation of the higher eukaryotic transcription factor NF‐kappa B requires both phosphorylation and proteolytic degradation of the inhibitory subunit I kappa B‐alpha. Inhibition of proteasome activity can stabilize an inducibly phosphorylated form of I kappa B‐alpha in intact cells, suggesting that phosphorylation targets the protein for degradation. In this study, we have identified serines 32 and 36 in human I kappa B‐alpha as essential for the control of I kappa B‐alpha stability and the activation of NF‐kappa B in HeLa cells. A point mutant substituting serines 32 and 36 by alanine residues was no longer phosphorylated in response to okadaic acid (OA) stimulation. This and various other Ser32 and Ser36 mutants behaved as potent dominant negative I kappa B proteins attenuating kappa B‐dependent transactivation in response to OA, phorbol 12‐myristate 13‐acetate (PMA) and tumor necrosis factor‐alpha (TNF). While both endogenous and transiently expressed wild‐type I kappa B‐alpha were proteolytically degraded in response to PMA and TNF stimulation of cells, the S32/36A mutant of I kappa B‐alpha remained largely intact under these conditions. Our data suggest that such diverse stimuli as OA, TNF and PMA use the same kinase system to phosphorylate and thereby destabilize I kappa B‐alpha, leading to NF‐kappa B activation.

The transcription factor NF-kappa B regulates genes participating in immune and inflammatory responses. In T lymphocytes, NF-kappa B is sequestered in the cytosol by the inhibitor I kappa B-alpha and released after serine phosphorylation of I kappa B-alpha that regulates its ubiquitin-dependent degradation. We report an alternative mechanism of NF-kappa B activation. Stimulation of Jurkat T cells with the protein tyrosine phosphatase inhibitor and T cell activator pervanadate led to NF-kappa B activation through tyrosine phosphorylation but not degradation of I kappa B-alpha. Pervanadate-induced I kappa B-alpha phosphorylation and NF-kappa B activation required expression of the T cell tyrosine kinase p56ick. Reoxygenation of hypoxic cells appeared as a physiological effector of I kappa B-alpha tyrosine phosphorylation. Tyrosine phosphorylation of I kappa B-alpha represents a proteolysis-independent mechanism of NF-kappa B activation that directly couples NF-kappa B to cellular tyrosine kinase.

Activation of the inducible transcription factor NF‐kappa B involves removal of the inhibitory subunit I kappa B‐alpha from a latent cytoplasmic complex. It has been reported that I kappa B‐alpha is subject to both phosphorylation and proteolysis in the process of NF‐kappa B activation. In this study, we present evidence that the multicatalytic cytosolic protease (proteasome) is involved in the degradation of I kappa B‐alpha. Micromolar amounts of the peptide Cbz‐Ile‐Glu(O‐t‐Bu)‐Ala‐leucinal (PSI), a specific inhibitor of the chymotrypsin‐like activity of the proteasome, prevented activation of NF‐kappa B in response to tumor necrosis factor‐alpha (TNF) and okadaic acid (OA) through inhibition of I kappa B‐alpha degradation. The m‐calpain inhibitor Cbz‐Leu‐leucinal was ineffective. In the presence of PSI, a newly phosphorylated form of I kappa B‐alpha accumulated in TNF‐ and OA‐stimulated cells. However, the covalent modification of I kappa B‐alpha was not sufficient for activation of NF‐kappa B: no substantial NF‐kappa B DNA binding activity appeared in cells because the newly phosphorylated form of I kappa B‐alpha was still tightly bound to p65 NF‐kappa B. Pyrrolidinedithiocarbamate, an antioxidant inhibitor of NF‐kappa B activation which did not interfere with proteasome activities, prevented de novo phosphorylation of I kappa B‐alpha as well as its subsequent degradation. This suggests that phosphorylation of I kappa B‐alpha is equally necessary for the activation of NF‐kappa B.(ABSTRACT TRUNCATED AT 250 WORDS)

SummaryOpportunistic infections, such as aspergillosis, are among the most serious complications suffered by immunocompromised patients. Aspergillus fumigatus and other pathogenic fungi synthesize a toxic epipolythiodioxopiperazine metabolite called gliotoxin. Gliotoxin exhibits profound immunosuppressive activity in vivo. It induces apoptosis in thymocytes, splenocytes, and mesenteric lymph node cells and can selectively deplete bone marrow of mature lymphocytes. The molecular mechanism by which gliotoxin exerts these effects remains unknown. Here, we report that nanomolar concentrations of gliotoxin inhibited the activation of transcription factor NF-~B in response to a variety of stimuli in T and B cells. The effect ofgliotoxin was specific because, at the same concentrations, the toxin did not affect activation of the transcription factor NF-AT or of interferon-responsive signal transducers and activators of transcription. Likewise, the activity of the constitutively DNA-binding transcription factors Oct-1 and cyclic AMP response element binding protein (CREB), as well as the activation of protein tyrosine kinases p56 lck and p59 fyn, was not altered by gliotoxin. Very high concentrations of gliotoxin prevented NF-~B DNA binding in vitro. However, in intact cells, inhibition of NF-~13 did not occur at the level of DNA binding; rather, the toxin appeared to prevent degradation of IKB-ci, NF-KB's inhibitory subunit. Our data raise the possibility that the immunosuppression observed during aspergillosis results in part from gliotoxin-mediated NF-KB inhibition.

The widely used phosphatase 1 and 2A inhibitor okadaic acid is one of the many stimuli activating transcription factor NF-B in cultured cells. Phosphorylation of IB-␣, one of NF-B's inhibitory subunits, is a prerequisite for IB degradation and the subsequent liberation of transcriptionally active NF-B. This observation suggested that the phosphorylation status of IB is influenced by an okadaic acid-sensitive phosphatase. In this study, we provide evidence that the effect of okadaic acid on NF-B activation is indirect and dependent on the production of reactive oxygen intermediates rather than the inhibition of an IB-␣ phosphatase. Okadaic acid was found to be a strong inducer of cellular H 2 O 2 and superoxide production in two distinct cell lines. The structurally unrelated phosphatase inhibitor calyculin A also induced oxidative stress. The delayed onset of reactive oxygen production in response to okadaic acid correlated with the delayed activation of NF-B. Moreover, NF-B induction was optimal at the same okadaic acid concentration that caused optimal H 2 O 2 production. Both reactive oxygen intermediates production and NF-B activation were inhibited by the antioxidant pyrrolidine dithiocarbamate and 8-(diethylamino)octyl-3,4,5-trimethyoxybenzoate, a Ca 2؉ chelator. Future experiments using phosphatase inhibitors in intact cells must consider that the compounds can act as strong inducers of oxidative stress, which provides one explanation for their tumor-promoting activity.

The genomes of human adenoviruses encode several regulatory proteins, including the two differentially spliced gene products E1A and E1B. Here, we show that the 13S but not the 12S splice variant of E1A of adenovirus type 5 can activate the human transcription factor NF-B in a bimodal fashion. One mode is the activation of NF-B containing the p65 subunit from the cytoplasmic NF-B-IB complex. This activation required reactive oxygen intermediates and the phosphorylation of IB␣ at serines 32 and 36, followed by IB␣ degradation and the nuclear uptake of NF-B. In addition, 13S E1A stimulated the transcriptional activity of the C-terminal 80 amino acids of p65 at a core promoter with either a TATA box or an initiator (INR) element. The C-terminal 80 amino acids of p65 were found to associate with E1A in vitro. The activation of NF-Bdependent reporter gene transcription by E1A was potently suppressed upon coexpression of the E1B 19-kDa protein (19K). E1B 19K prevented both the activation of NF-B and the E1A-mediated transcriptional enhancement of p65. These inhibitory effects were not found for the 55-kDa splice variant of the E1B protein.We suggest that the inductive effect of E1A 13S on the host factor NF-B, whose activation is important for the transcription of various adenovirus genes, must be counteracted by the suppressive effect of E1B 19K so that the adenovirus-infected cell can escape the immune-stimulatory and apoptotic effects of NF-B.

SUMMARYNF-kB is a dimeric protein that serves to initiate gene tran scription in higher eukaryotic cells in response to mainly pathogenic stimuli. Its activity is controlled by a third inhibitory subunit, called IkB. When IkB is bound, NF-kB cannot bind to DNA or enter the nucleus but is stored in a latent cytoplasmic form. Upon stimulation of cells IkB is released, which allows the activation of NF-kB. We have analyzed the molecular mechanism underlying the removal of IkB-oc. Distinct extracellular stimuli lead to a phospho rylation of IkB-oc on serines 32 and 36 by a yet unidentified kinase. These modifications do not directly dissociate IkB from NF-kB but render the inhibitor highly susceptible for proteolytic degradation by, presumably, the proteasome. In this paper, we report for the first time that higher molecular mass forms of IkB-oc occur under conditions that lead to a phosphorylation of IkB-oc and activation of NFkB. These IkB-oc variants had discrete molecular masses and were most prominent in cells overexpressing IkB-oc, suggesting the covalent modification of IkB-oc by ubiquitin conjugation. The proteasome inhibitor Cbz-Ile-Glu(0-tBu)-Ala-leucinal (PSI), which stabilizes the phospho form of IkB-oc, only slightly increased the amount of conjugates indicating that the conjugation of IkB-oc with ubiquitin was the rate-limiting step in IkB-oc degradation, and not its phosphorylation or proteolysis. Our data suggest that con jugation of IkB-oc with ubiquitin is an intermediate reaction in the phosphorylation-controlled degradation of IkB-oc and the subsequent activation of NF-kB.