Alternative Splicing Variants of IκBβ Establish Differential NF-κB Signal Responsiveness in Human Cells

Abstract

To release transcription factor NF-B into the nucleus, the mammalian IB molecules IB␣ and IB␤ are inactivated by phosphorylation and proteolytic degradation. Both proteins contain conserved signal-responsive phosphorylation sites and have conserved ankyrin repeats. To confer specific physiological functions to members of the NF-B/Rel family, the different IB molecules could vary in their specific NF-B/Rel factor binding activities and could respond differently to activation signals. We have demonstrated that both mechanisms apply to differential regulation of NF-B function by IB␤ relative to IB␣. Via alternative RNA processing, human IB␤ gives rise to different protein isoforms. IB␤1 and IB␤2, the major forms in human cells, differ in their carboxy-terminal PEST sequences. IB␤2 is the most abundant species in a number of human cell lines tested, whereas IB␤1 is the only form detected in murine cells. These isoforms are indistinguishable in their binding preferences to cellular NF-B/Rel homo-and heterodimers, which are distinct from those of IB␣, and both are constitutively phosphorylated. In unstimulated B cells, however, IB␤1, but not IB␤2, is found in the nucleus. Furthermore, the two forms differ markedly in their efficiency of proteolytic degradation after stimulation with several inducing agents tested. While IB␤1 is nearly as responsive as IB␣, indicative of a shared activation mechanism, IB␤2 is only weakly degraded and often not responsive at all. Alternative splicing of the IB␤ pre-mRNA may thus provide a means to selectively control the amount of IB␤-bound NF-B heteromers to be released under NF-B stimulating conditions.Transcriptional induction of genes driven by members of the NF-B/Rel family of activators plays a crucial role in many processes of the immune response and under a number of cellular stress conditions including viral infection (2,5,40,44). The activation of NF-B through different pathways is controlled primarily by its release from the interaction with inhibitory IB molecules. Five different mammalian NF-B/Rel proteins are known, p50, p52, p65, c-Rel, and RelB, which form homo-and heterodimers. These are sequestered by the IB molecules IB␣, IB␤, and IBε and by p105 and p100, the precursor molecules for p50 and p52, respectively (2, 42). Best understood is the activation of NF-B from complexes with IB␣ by diverse inducers like tumor necrosis factor alpha (TNF-␣), lipopolysaccharide (LPS), phorbol myristate acetate, (PMA) or human T-cell leukemia virus type 1 Tax (7,8,38,43). These lead to phosphorylation at Ser 32 and 36. Phosphorylated IB␣ is then polyubiquitinated and subsequently degraded by the proteasome (3, 9, 34). In addition to the aminoterminal signal response box containing Ser 32 and 36, signaldependent degradation is more efficient in the presence of the carboxy-terminal PEST sequence of IB␣ (7,8,38,43), although the mechanistic need of the latter is not understood. The high turnover of IB␣ in the resting state is also driven by the proteasome but is independent of the amino-...

High-level expression of IkB-b in the surface epithelium of the colon: in vitro evidence for an immunomodulatory role

“…3C). Although two hybridization signals of equal intensity appear due to alternative splicing of the I B-␤ pre-mRNA [39,52], neither band is induced by sodium butyrate. These results suggest that sodium butyrate may inhibit the nuclear translocation of NF-B by inducing the protein expression of I B-␤ via a mechanism independent of steady-state mRNA levels.…”

Analysis of SRrp86-regulated alternative splicing

“…Upon stimulation, IκBβ1 is rapidly degraded whereas IκBβ2 is only weakly degraded which serves to limit the activity of NFκB. 36 Thus, a balance between these two isoforms, in addition to the function of other IκB proteins, enables precise regulation of NFκB activity.…”

“…4A). 36 The two proteins differ in their C-termini, with the smaller protein, IκBβ2, being produced by an intron retention event. The SpliceArray indicated that overexpression of SRrp86 stimulated removal of this intron and therefore production of the larger protein, IκBβ1 (Fig.…”

“…These two isoforms differ in their stringency of NFκB inhibition and in their ability to be degraded in response to NFκB-activating stimuli. 36 Thus, SRrp86 levels should have profound effects on NFκB activity.…”

Nuclear IκBβ Maintains Persistent NF-κB Activation in HIV-1-infected Myeloid Cells

“…Hypophosphorylated IB␤ can bind NF-B without masking its nuclear localization signal (32), thus acting as a chaperone for NF-B nuclear entry and preventing its sequestration by IB␣. Recently, two isoforms of IB␤ that differ in their C-terminal PEST domain as a consequence of alternative splicing have been identified in human cells (34). The larger protein, approximately 43 kDa, is homologous to the murine IB␤, whereas the 41-kDa form is unique to human cells.…”