Ubiquitin-Conjugating Enzyme 9 Phosphorylation as a Novel Mechanism for Potentiation of the Inflammatory Response

Abstract

Lipopolysaccharide (LPS), a bacterial endotoxin, induces inflammation in macrophages via activation of NF-kB signaling. Sumoylation is a post-translational modification mediated by the small ubiquitin-like modifier, SUMO. Ubiquitin-conjugating enzyme 9 (UBC9) is the only known SUMO conjugating enzyme. LPS treatment lowers SUMO-1 and UBC9 mRNA levels in primary astrocytes. UBC9 can degrade NF-kB inhibitor a (Ikba) via a SUMO2/3-ubiquitin pathway. However, UBC9 may also promote Ikba stability by SUMO-1 conjugation that further regulates NF-kB signaling. The role of UBC9 in liver inflammation is unknown. We reported that CDK1-mediated phosphorylation of UBC9 enhanced its stability. Herein, we describe an anti-inflammatory role of UBC9 that is lost when it is phosphorylated during inflammation. LPS exposure caused induction in UBC9 phosphorylation and CDK1 activation specifically in Kupffer cells in vivo and in RAW264.7 macrophages in vitro. Silencing or overexpression experiments in vitro and in vivo showed that UBC9 was required to blunt the proinflammatory response elicited by LPS. LPS stimulation raised the binding of phospho-UBC9 but not the unphosphorylated counterpart, to Ikba in RAW264.7 macrophages. Hence, phospho-UBC9 may promote NF-kB signaling by regulating Ikba and this may be a novel mechanism that deregulates liver inflammatory signaling. (Am J Pathol 2016 http://dx

SUMO conjugation – a mechanistic view

“…N-terminal Ubc9 sumoylation in mammalian cells can enhance the affinity to substrates with a SIM in close distance to the SCM, as we have shown for the transcriptional regulator Sp100 (69). A related mechanism was recently proposed for Ubc9 phosphorylation, although the molecular details remain to be shown (118). Interestingly, Ubc9 acetylation regulates substrate selection by exclusions, as it removes the positive charge on Ubc9 required for the interaction with NDSM-containing SUMO substrates (119).…”

“…In clear contrast to general regulators of Ubc9 function, the above-mentioned E2 modifications that interfere with specific substrate interactions like acetylation, N-terminal sumoylation in mammals (currently, there is no evidence that C-terminal Ubc9 sumoylation can bind to substrates) and phosphorylation display a more selective form of regulation, as these modifications only affect a subgroup of SUMO substrates (50,72,118,119).…”

SUMOylation and phosphorylation cross-talk in hepatocellular carcinoma

“…Serine 71 phosphorylation of UBC9 appears to favor its stability in cancer cell lines (12). Phosphorylation of UBC9 also promotes inflammatory signaling in Kupffer cells of the liver (54). Since SUMOylation is also higher in HCC, it appears that phosphorylation of UBC9 may drive enhanced SUMOylation in this disease (12).…”

“…The overall data imply a correlation between enhanced PYK2 autophosphorylation, SUMOylation and C-Src signaling as well as enhanced ERK activation in HCC. The E2 enzyme UBC9 is also phosphorylated in human HCC and during inflammatory signaling in Kupffer cells of the liver (12,54). Serine 71 phosphorylation of UBC9 appears to favor its stability in cancer cell lines (12).…”

“…p65 has been described as a critical key regulatory protein on NF-κB transcriptional activity and is associated with liver cancer development. When p65 is localized into the nucleus appears to be under its active form, that controls a large number of genes transcription in response to inflammatory stimuli (53,54). Accordingly, p65 protein stability regulation plays an important role in hepatitis-related HCC development.…”