The Serine/Threonine Phosphatase Inhibitor Calyculin A Induces Rapid Degradation of IκBβ: REQUIREMENT OF BOTH THE N- AND C-TERMINAL SEQUENCES

Edward W Harhaj,Shao-Cong Sun

doi:10.1074/jbc.272.9.5409

Edward W Harhaj, Shao-Cong Sun

Open Access

https://doi.org/10.1074/jbc.272.9.5409

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Abstract

Signal-initiated activation of the transcription factor NF-kappaB is mediated through proteolysis of its cytoplasmic inhibitory proteins IkappaBalpha and IkappaBbeta. While most NF-kappaB inducers trigger the degradation of IkappaBalpha, only certain stimuli are able to induce the degradation of IkappaBbeta. The degradation of IkappaBalpha is targeted by its site-specific phosphorylations, although the mechanism underlying the degradation of IkappaBbeta remains elusive. In the present study, we have analyzed the effect of phosphatase inhibitors on the proteolysis of IkappaBbeta. We show that the serine/threonine phosphatase inhibitor calyculin A induces the hyperphosphorylation and subsequent degradation of IkappaBbeta in both human Jurkat T cells and the murine 70Z-3 preB cells, which is associated with the nuclear expression of active NF-kappaB. The calyculin A-mediated degradation of IkappaBbeta is further enhanced by the cytokine tumor necrosis factor-alpha (TNF-alpha), although TNF-alpha alone is unable to induce the degradation of IkappaBbeta. Mutational analyses have revealed that the inducible degradation of IkappaBbeta induced by calyculin A, and TNF-alpha requires two N-terminal serines (serines 19 and 23) that are homologous to the inducible phosphorylation sites present in IkappaBalpha. Furthermore, the C-terminal 51 amino acid residues, which are rich in serines and aspartic acids, are also required for the inducible degradation of IkappaBbeta. These results suggest that the degradation signal of IkappaBbeta may be controlled by the opposing actions of protein kinases and phosphatases and that both the N- and C-terminal sequences of IkappaBbeta are required for the inducible degradation of this NF-kappaB inhibitor.

Highlights

Signal-initiated activation of the transcription factor NF-␬B is mediated through proteolysis of its cytoplasmic inhibitory proteins I␬B␣ and I␬B␤
We show that the serine/threonine phosphatase inhibitor calyculin A induces the hyperphosphorylation and subsequent degradation of I␬B␤ in both human Jurkat T cells and the murine 70Z-3 preB cells, which is associated with the nuclear expression of active NF-␬B
The calyculin A-mediated degradation of I␬B␤ is further enhanced by the cytokine tumor necrosis factor-␣ (TNF-␣), TNF-␣ alone is unable to induce the degradation of I␬B␤

Summary

Introduction

Signal-initiated activation of the transcription factor NF-␬B is mediated through proteolysis of its cytoplasmic inhibitory proteins I␬B␣ and I␬B␤. The NF-␬B/Rel family of transcription factors play a pivotal role in the regulation of various cellular genes involved in the immediate early processes of immune, acute phase, and inflammatory responses [1, 2]. The latent cytoplasmic NF-␬B/Rel complexes can be activated by a variety of cellular stimuli, including the mitogen phorbol esters, cytokines such as tumor necrosis factor-␣ (TNF␣), and interleukin-1, the bacterial component lipopolysaccharide, serine/threonine phosphatase inhibitors such as okadaic acid and calyculin A, and the Tax protein from the type I human T cell leukemia virus (HTLV-I) [8, 17]. We have further investigated the role of phosphorylation in the inducible degradation of I␬B␤ by examining the effect of a serine/threonine phosphatase inhibitor, calyculin A, on the fate of I␬B␤. We demonstrate that incubation of Jurkat T cells or 70Z/3 pre-B cells with calyculin A is sufficient to induce the hyperphosphorylation and subsequent degradation of I␬B␤

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