The Serine/Threonine Phosphatase Inhibitor Calyculin A Induces Rapid Degradation of IκBβ

Abstract

Signal-initiated activation of the transcription factor NF-B is mediated through proteolysis of its cytoplasmic inhibitory proteins IB␣ and IB␤. While most NF-B inducers trigger the degradation of IB␣, only certain stimuli are able to induce the degradation of IB␤. The degradation of IB␣ is targeted by its sitespecific phosphorylations, although the mechanism underlying the degradation of IB␤ remains elusive. In the present study, we have analyzed the effect of phosphatase inhibitors on the proteolysis of IB␤. We show that the serine/threonine phosphatase inhibitor calyculin A induces the hyperphosphorylation and subsequent degradation of IB␤ 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 IB␤ is further enhanced by the cytokine tumor necrosis factor-␣ (TNF-␣), although TNF-␣ alone is unable to induce the degradation of IB␤. Mutational analyses have revealed that the inducible degradation of IB␤ induced by calyculin A, and TNF-␣ requires two N-terminal serines (serines 19 and 23) that are homologous to the inducible phosphorylation sites present in IB␣. Furthermore, the C-terminal 51 amino acid residues, which are rich in serines and aspartic acids, are also required for the inducible degradation of IB␤. These results suggest that the degradation signal of IB␤ may be controlled by the opposing actions of protein kinases and phosphatases and that both the Nand C-terminal sequences of IB␤ are required for the inducible degradation of this NF-B inhibitor.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). In addition, these cellular factors have also been implicated in the transcriptional activation of certain human viruses, most notably the type 1 human immune deficiency virus (3-7). The mammalian NF-B/Rel family is composed of at least five structurally related DNA-binding proteins, including p50, p52, RelA, RelB, and c-Rel, which bind to a target DNA sequence (B) as various heterodimers or homodimers (reviewed in Siebenlist et al. (8)). In most cell types, including resting T cells, the NF-B/Rel proteins are sequestered in the cytoplasmic compartment by physical association with inhibitory proteins that are characteristic of the presence of various numbers of ankyrin-like repeats (reviewed in Verma et al. (9)). The major cytoplasmic inhibitors include IB␣ (10, 11), IB␤ (12), and the precursor proteins of p50 and p52 (9). The IB molecules appear to bind to and mask the nuclear localization signal of NF-B/Rel, thereby preventing the nuclear translocation of these transcription factors (13-16).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-␣), 1 and interleukin-1, the bacterial component lipopolysaccharide, serine/...

Regulation of IκB Kinase Complex by Phosphorylation of γ-Binding Domain of IκB Kinase β by Polo-like Kinase 1

“…Data presented here provide experimental evidence to support our hypothesis. This mode of regulation for IKK is also supported by the reports that phosphatase inhibitors activate NF-B through IKK complex (5,(45)(46)(47). Furthermore, the recent crystal structure of the ␥BD of IKK␤ with IKK␥ reveals a critical intramolecular hydrogen bond within ␥BD between serine 740 and aspartate 738 (22).…”

Characterization of the IκB-kinase NEMO Binding Domain

“…Whereas none of these mechanisms have been tested, it is tempting to speculate that a possible negative regulator may be an IKK phosphatase that is recruited to NEMO to dephosphorylate and inactivate the complex. In support of this, previous workers have demonstrated that the IKK complex can be activated by treatment with specific phosphatase inhibitors (42,43). Why then is IKK not constitutively active in NEMO Ϫ/Ϫ cells?…”

Effects of protein phosphorylation on ubiquitination and stability of the translational inhibitor protein 4E-BP1

“…It is therefore of interest that calyculin A is also antiapoptotic (Song and Lavin, 1993). Clearly, this protein phosphatase inhibitor is likely to affect the phosphorylation of a wide range of additional target proteins that may have regulatory effects on apoptosis (for example, IkB; Harhaj and Sun, 1997). Nevertheless, the regulation of 4E-BP1 may contribute to the inhibition of cell death by calyculin A.…”