Hsp90 inhibition results in autophagy-mediated proteasome-independent degradation of IκB kinase （IKK）

Autophagic and proteasomal proteolysis are two major pathways for degradation of cellular constituents. Current models suggest that autophagy is responsible for the nonselective bulk degradation of long-lived proteins and organelles while the proteasome specifically degrades short-lived proteins including misfolded proteins caused by the absence of Hsp90 function. Here, we show that the IκB kinase （IKK）, an essential activator of NF-κB, is selectively degraded by autophagy when Hsp90 is inhibited by geldanamycin （GA）, a specific Hsp90 inhibitor showing highly effective anti-tumor activity. We find that in this case inactivation of ubiquitination or proteasome fails to block IKK degradation. However, inhibition of autophagy by an autophagy inhibitor or knockout of Atg5, a key component of the autophagy pathway, significantly rescues IKK from GA-induced degradation. These findings provide the first evidence that an Hsp90 client may be degraded by a mechanism different from the proteasome pathway and establish a molecular link among Hsp90, NF-κB and autophagy     

Cells in G_2/M phase increased in human nasopharyngeal carcinoma cell line by EBV-LMP1 through activation of NF-кB and AP-1

Although previous studies showed that the principal oncoprotein encoded by Epstein-Barr virus, latentmembrane protein 1 (LMP1) 5 could induce the nasopharyngeal carcinoma cells in G_2/M phase increased, littleis known about the target molecules and mechanisms. The present study demonstrated that LMP1 couldinduce the accumulation of p53 protein and upregulate its transactivity in a dose dependent manner, whichresulted in the decrease of the kinase activity of cdc2/cyclin B complex and inducing arrest at G2/M phasethrough the activation of NF-κB and AP-1 signaling pathways, and the effect of NF-κB was more obviousthan that of AP-1. This study provided some significant evidence for further elucidating the molecularmechanisms that LMP1 had effects on the surveillance mechanism of cell cycle and promoting the survivalof transformed cells and tumorigenesis.     

EGCG attenuates pro-inflammatory cytokines and chemokines production in LPS-stimulated L02 hepatocyte

Endotoxin lipopolysaccharide （LPS） plays an important role in the acceleration of inflammatory reaction of hepa- titis as the second attack. Compounds that can prevent in- flammation by targeting LPS have potential therapeutic clinical application. Epigallocatechin-3-gallate （EGCG） has potent hepatocyte-protective effect and mild anti-hepatitis virus function. Here, we investigated whether EGCG attenuated the severity of inflammatory response in LPS-stimulated L02 hepatocytes. L02 hepatocytes were pretreated with EGCG for 2 h, then stimulated by LPS at 250 ng/ml. The expression levels of chemokine regulated upon activation normal T-cell expressed and secreted （Rantes） and monocyte chemotactic protein-1 （MCP-1）, pro-inflammatory cytokines tumor necrosis factor-α （TNF-α and interferon-% adhesion molecule intercellular adhesion molecule-1 （ICAM-1）, oxidant stress molecules nitric oxide （NO）, vascular endothelial growth factor （VEGF）, and matrix metaHoproteinase-2 （MMP-2） were tested by enzyme-linked immunosorbent assay. The expression of total extracellular signal-regulated kinase 1/2 （ERK1/2）, phospho-ERK1/2 （p-ERK1/2）, p-AKT, total p38, phospho-p38 （p-p38）, total p65 and phospho-p65 （p-p65）, IκBα, phospho-IκBα（p-IκBα and TNF receptor associated factor 2 were tested by western blot analysis. Our results showed that pre-treatment with EGCG could significantly reduce the production of TNF-α, Rantes, MCP-1, ICAM-1, NO, VEGF, and MMP-2 in LPS-stimulated L02 hepatocytes in a dose-dependent manner. The effect of EGCG may be related to the inhibition of nuclear factor-κB （NF-κB） and mitogen-activated protein kinase （MAPK） signaling pathways by down-regulation of p-IκBα, p65, p-p65, p-p38, p-ERK1/2, and p-AKT. These results indicate that EGCG suppresses LPS-induced inflammatory response and oxidant stress and exerts its hepatocyte-protective activity partially by inhibiting NF-κB and MAPK pathways.     

Promoting effect of IFN-γ on the expression of LPS-induced IL-12 p40 and p35 mRNA in murine suppressor macrophages

We detected the expression of IL-12 p40/p35 mRNA by semi-quantitative RT-PCR and silver staining, and studied the molecular interaction between the IL-12 expression and the NF-κB activation induced by LPS and IFN-γ/LPS in murine peritoneal suppressor macrophages (MPSMs). It was found that IFN-γ strongly enhanced the LPS-induced IL-12 p40 and p35 mRNA expression. Both p40 and p35 mRNA levels were approximately equal. IFN-γ also greatly promoted the LPS-induced secretion of IL-12 p70 in MPSMs. The Proteasome Inhibitor I (PSI) could block the expression of IL-12 p40 and p35 mRNA, and the degradation of κBα induced by LPS or LPS/IFN-γ. EMSA showed that LPS could augment the NF-κB binding activity to p40 promoter DNA. However, IFN-γ could neither enhance the LPS-induced NF-κB activity nor promote the degradation of kBa. Taken together, the data suggest: (i) IFN-γ/LPS could strongly induce the expression of IL-12 p40 and p35 mRNA; both the expression levels were equal; this phenomenon coincided wit     

Selective degradation of the IκB kinase （IKK） by autophagy

Proteasome-mediated degradation and autophagy are the two major pathways mediating the turnover of cellular proteins. The proteasomal pathway is known to be a highly specific and regulated process mediating the degradation of short-lived proteins such as many important factors involved in cellular signaling. In contrast, it is generally thought that autophagy is rather nonselective as it is responsible for the bulk degradation of long-lived proteins and organelles. Challenging this general view, in this issue of Cell Research, Qing et al. report that selective degradation of the IκB kinase （IKK） triggered by the loss of Hsp90 function is mediated by autophagy [1].     

Regulation of RelA Subcellular Localization by a Putative Nuclear Export Signal and p50

Nuclear factor kappaB (NF-kappaB) represents a family of dimeric DNA binding proteins, the pleotropic form of which is a heterodimer composed of RelA and p50 subunits. The biological activity of NF-kappaB is controlled through its subcellular localization. Inactive NF-kappaB is sequestered in the cytoplasm by physical interaction with an inhibitor, IkappaBalpha. Signal-mediated IkappaBalpha degradation triggers the release and subsequent nuclear translocation of NF-kappaB. It remains unknown whether the NF-kappaB shuttling between the cytoplasm and nucleus is subjected to additional steps of regulation. In this study, we demonstrated that the RelA subunit of NF-kappaB exhibits strong cytoplasmic localization activity even in the absence of IkappaBalpha inhibition. The cytoplasmic distribution of RelA is largely mediated by a leucine-rich sequence homologous to the recently characterized nuclear export signal (NES). This putative NES is both required and sufficient to mediate cytoplasmic localization of RelA as well as that of heterologous proteins. Furthermore, the cytoplasmic distribution of RelA is sensitive to a nuclear export inhibitor, leptomycin B, suggesting that RelA undergoes continuous nuclear export. Interestingly, expression of p50 prevents the cytoplasmic expression of RelA, leading to the nuclear accumulation of both RelA and p50. Together, these results suggest that the nuclear and cytoplasmic shuttling of RelA is regulated by both an intrinsic NES-like sequence and the p50 subunit of NF-kappaB.     

Characterization of the nuclear import and export functions of Ikappa B(epsilon)

Control over the nuclear localization of nuclear factor kappaB/Rel proteins is accomplished in large part through association with members of the inhibitor of kappaB (IkappaB) protein family. For example, the well studied IkappaBalpha protein actively shuttles between the nucleus and the cytoplasm and both inhibits nuclear import and mediates nuclear export of NF-kappaB/Rel proteins. In contrast, the IkappaBbeta protein can inhibit nuclear import of NF-kappaB/Rel proteins but does not remove NF-kappaB/Rel proteins from the nucleus. To further understand how the IkappaB proteins control the nuclear-cytoplasmic distribution of NF-kappaB/Rel proteins, we have characterized the nuclear import and nuclear export functions of IkappaBepsilon. Our results indicate that the IkappaBepsilon protein, like the IkappaBalpha protein, actively shuttles between the nucleus and the cytoplasm. Similar to IkappaBalpha, nuclear import of IkappaBepsilon is mediated by its ankyrin repeat domain and is not blocked by the dominant-negative RanQ69L protein. However, the nuclear import function of the IkappaBepsilon ankyrin repeat domain is markedly less efficient than that of IkappaBalpha, with the result that nuclear shuttling of IkappaBepsilon between the nucleus and the cytoplasm is significantly slower than IkappaBalpha. Nuclear export of IkappaBepsilon is mediated by a short leucine-rich nuclear export sequence (NES)-like sequence ((343)VLLPFDDLKI(352)), located between amino acids 343 and 352. This NES-like sequence is required for RanGTP-dependent binding of IkappaBepsilon to CRM1. Nuclear accumulation of IkappaB(epsilon) is increased by either leptomycin B treatment or alanine substitutions within the IkappaBepsilon-derived NES. A functional NES is required for both efficient cytoplasmic retention and post-induction control of c-Rel by IkappaBepsilon, consistent with the notion that IkappaBepsilon-mediated nuclear export contributes to control over the nucleocytoplasmic distribution of NF-kappaB/Rel proteins.     

p105.Ikappa Bgamma and prototypical Ikappa Bs use a similar mechanism to bind but a different mechanism to regulate the subcellular localization of NF-kappa B

p105, also known as NF-kappaB1, is an atypical IkappaB molecule with a multi-domain organization distinct from other prototypical IkappaBs, like IkappaBalpha and IkappaBbeta. To understand the mechanism by which p105 binds and inhibits NF-kappaB, we have used both p105 and its C-terminal inhibitory segment known as IkappaBgamma for our study. We show here that one IkappaBgamma molecule binds to NF-kappaB dimers wherein at least one NF-kappaB subunit is p50. We suggest that the obligatory p50 subunit in IkappaBgamma.NF-kappaB complexes is equivalent to the N-terminal p50 segment in all p105.NF-kappaB complexes. The nuclear localization signal (NLS) of the obligatory p50 subunit is masked by IkappaBgamma, whereas the NLS of the nonobligatory NF-kappaB subunit is exposed. Thus, the global binding mode of all IkappaB.NF-kappaB complexes seems to be similar where one obligatory (or specific) NF-kappaB subunit makes intimate contact with IkappaB and the nonobligatory (or nonspecific) subunit is bound primarily through its ability to dimerize. In the case of IkappaBalpha and IkappaBbeta, the specific NF-kappaB subunit in the complex is p65. In contrast to IkappaBalpha.NF-kappaB complexes, where the exposed NLS of the nonspecific subunit imports the complex to the nucleus, p105.NF-kappaB and IkappaBgamma.NF-kappaB complexes are cytoplasmic. We show that the death domain of p105 (also of IkappaBgamma) is essential for the cytoplasmic sequestration of NF-kappaB by p105 and IkappaBgamma. However, the death domain does not mask the exposed NLS of the complex. We also demonstrate that the death domain alone is not sufficient for cytoplasmic retention and instead functions only in conjunction with other parts in the three-dimensional scaffold formed by the association of the ankyrin repeat domain (ARD) and NF-kappaB dimer. We speculate that additional cytoplasmic protein(s) may sequester the entire p105.NF-kappaB complex by binding through the death domain and other segments, including the exposed NLS.