S-adenosyl methionine regulates ubiquitin-conjugating enzyme 9 protein expression and sumoylation in murine liver and human cancers

Abstract

Background and rationale Ubiquitin-conjugating enzyme 9 (Ubc9) is required for sumoylation and overexpressed in several malignancies but its expression in hepatocellular carcinoma (HCC) is unknown. Hepatic S-adenosylmethionine (SAMe) level falls in methionine adenosyltransferase 1A (Mat1a) knockout (KO) mice, which develop HCC, and in ethanol fed mice. Here we examined regulation of Ubc9 by SAMe in murine liver and human HCC, breast and colon carcinoma cell lines and specimens. Methods Real-time PCR and Western blotting measured gene and protein expression, respectively. Immunoprecipitation followed by Western blotting examined protein-protein interactions. Results Ubc9 expression is increased in HCC and when hepatic SAMe level falls. SAMe treatment in Mat1a KO mice reduced Ubc9 protein but not mRNA level and lowered sumoylation. Similarly, treatment of liver cancer cell lines HepG2 and Huh-7, colon cancer cell line RKO and breast cancer cell line MCF-7 with SAMe or its metabolite methylthioadenosine (MTA) reduced only Ubc9 protein level. Ubc9 post-translational regulation is unknown. Ubc9 sequence predicts a possible phosphorylation site by cell division cycle 2 (Cdc2), which directly phosphorylated recombinant Ubc9. Mat1a KO mice have higher phospho-Ubc9 level, which normalized after SAMe treatment. SAMe and MTA treatment lowered Cdc2 mRNA and protein levels, phospho-Ubc9 and protein sumoylation in liver, colon and breast cancer cells. Serine 71 of Ubc9 is required for phosphorylation, interaction with Cdc2 and protein stability. Cdc2, Ubc9 and phospho-Ubc9 levels are increased in human liver, breast and colon cancers. Conclusions Cdc2 expression is increased and Ubc9 is hyperphosphorylated in several cancers and this represents a novel mechanism to maintain high Ubc9 protein expression that can be inhibited by SAMe and MTA.

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

“…We have previously demonstrated that recombinant UBC9 is phosphorylated by CDK1. 13 Herein, we show that LPS stimulation raises the activity of CDK1 in Kupffer cells but not hepatocytes by lowering phosphorylation at the inhibitory phosphorylation site, Tyr-15 of CDK1 19 ( Figure 4C) and concomitantly enhancing the Thr-161 phosphorylation that is known to activate CDK1 19 ( Figure 4C). Hence, during LPS stimulation, enhanced CDK1 activation was associated with increased UBC9 phosphorylation specifically in Kupffer cells.…”

“…We previously reported that CDK1 could phosphorylate recombinant UBC9 and UBC9 phosphorylation enhanced its stability. 13 Blocking dephosphorylation of UBC9 in cells by phosphatase inhibitors also enhanced the total and phospho-level of UBC9 and stabilized it. 13 Our results that both the activity of CDK1 and UBC9 phosphorylation are up-regulated in LPS-treated Kupffer cells are in sync with our previously published work.…”

“…After the above treatment periods, mice were sacrificed and livers were collected and preserved for RNA or protein isolation, as described previously. 13 The core services provided by the Liver Center at Cedars-Sinai were used for the isolation of hepatocytes and Kupffer cells from LPS-treated or control mice, as previously described. 15 Simultaneous isolation of mouse hepatocytes and Kupffer cells is described briefly below.…”

“…Equal amounts of phosphorylated or unphosphorylated protein fractions were immunoprecipitated with two micrograms of UBC9 antibody, according to previously published protocols, 13,17 and then immunoblotted with Ikba antibody.…”

“…We recently published that UBC9 was a substrate for phosphorylation mediated by cyclin-dependent kinase 1 (CDK1) and phospho-UBC9 exhibited enhanced protein stability. 13 Herein, we describe the role of UBC9 in controlling inflammation. We show that lack of UBC9 potentiates LPS toxicity, whereas forced expression of UBC9 suppresses it.…”