Proteolytic degradation of MAD3 (IϰBα) and enhanced processing of the NF-ϰB precursor p105 are obligatory steps in the activation of NF-ϰB

Abstract

We have studied the role of protein turnover in the induction of NF-kappa B DNA binding activity. Treatment of cells with tumour necrosis factor (TNF), double-stranded RNA (dsRNA), or phorbol esters is shown to be associated with an increase in the rate of p105 to p50 processing, and the loss of immunologically detectable MAD3/I kappa B alpha. Phosphate-labelling experiments indicate that these events are preceded by the phosphorylation of MAD3 and p105. The protease inhibitors TLCK (N alpha-p-Tosyl-L-Lysine Chloromethyl Ketone) and TPCK (N alpha-p-Tosyl-L-Phenylalanine Chloromethyl Ketone) inhibit both p105 to p50 processing and MAD3 degradation, and also cause a complete block to NF-kappa B activation. These data suggest a model for NF-kappa B activation in which phosphorylation destabilises the NF-kappa B/MAD3 complex but that, in vivo, this is insufficient to lead to activation in the absence of an obligatory mechanism that degrades MAD3.

Defective IκBα in Hodgkin cell lines with constitutively active NF-κB

“…Although dexamethasone alone has only a minor e ect on IkBa levels, the combination treatment results in a signi®cant increase in the total amount of IkBa protein ( Figure 4b). An identical result was obtained when the blots were probed with a previously described (Mellits et al, 1993) polyclonal antiserum raised against recombinant IkBa (data not shown). Stripping and reprobing the blot with an antibody to IkBb indicated that over the time scale of the experiment levels of this protein were not a ected by treatment with dexamethasone or ZLLLH (data not shown).…”

“…IkBa was not detected in KM-H2 cells ( Figure 3). Identical results were obtained when a polyclonal antibody raised against recombinant IkBa (Mellits et al, 1993) (7) or supernatant from cells expressing CD40 ligand (+) for the indicated periods of time prior to preparation of cytoplasmic extracts. 25 mg of cytoplasmic extract was analysed by Western blotting using the 10B monoclonal antibody speci®c for IkBa (upper panels).…”

“…The IkB family of proteins (a,b,g, are characterised by the presence of multiple ankyrin repeats and their ability to physically associate with NF-kB proteins (Baeuerle and Baltimore, 1996;Baldwin, 1996;Matthews and Hay, 1995;Roulston et al, 1995). Signal induced activation of NF-kB is preceded by phosphorylation and rapid degradation of IkBa (Beg and Baldwin, 1993;Brown et al, 1993;Henkel et al, 1993;Mellits et al, 1993) mediated by the ubiquitin proteasome pathway (Palombella et al, 1994). The target residues for signal induced modi®cation are located in the exposed N-terminus of the IkBa protein (Ja ray et al, 1995) with S32 and S36 being the sites of phosphoryla-tion Brown et al, 1995;DiDonato et al, 1996;Ro et al, 1996;Traenckner et al, 1995) and K21 and K22 being the principal sites of ubiquitination (Baldi et al, 1996;Chen et al, 1995;Rodriguez et al, 1996;Scherer et al, 1995).…”

A Role for NF-κB Activation in Perforin Expression of NK Cells Upon IL-2 Receptor Signaling

“…The antioxidant, PDTC, prevents de novo phosphorylation of I B␣ as well as its subsequent degradation via unknown primary cellular targets (30 -32). The protease inhibitor, TPCK, prevents the degradation of I B proteins (33,34). Both agents have been applied in numerous cell types to study NF-B-dependent events, suggesting that the observed block in the up-regulation of perforin in primary NK cells (Fig.…”

Fas activates NF-κB and induces apoptosis in T-cell lines by signaling pathways distinct from those induced by TNF-α

“…TNF-a induces IkB-a phosphorylation (Beg et al, 1993;Henkel et al, 1993), yet this modification is not sufficient to trigger release of NF-kB and may be required for targeting IkB-a for subsequent proteolytic degradation (Alkalay et al, 1995;Beg et al, 1993;DiDonato et al, 1995;Finco et al, 1994;Finco and Baldwin, 1995;Henkel et al, 1993;Lin et al, 1995;Miyamoto et al, 1994;Traeckner et al, 1994). As some protease inhibitors can also block the induction of IkB-a phosphorylation, there may be a role for proteases prior to this step Mellits et al, 1993). Finally, in some cells, NF-kB activation by TNF-a is inhibited by antioxidants, suggesting that the signaling pathways leading to IkB-a degradation also involve ROS or redox-sensitive processes .…”