Eek Hoon Jho

Methylation by protein arginine methyltransferase 1 increases stability of Axin, a negative regulator of Wnt signaling

Axin, a negative regulator of Wnt signaling, is a key scaffold protein for the β-catenin destruction complex. It has been previously shown that multiple post-translational modification enzymes regulate the level of Axin. Here, we provide evidence that protein arginine methyltransferase 1 (PRMT1) directly interacts with and methylates the 378th arginine residue of Axin both in vitro and in vivo. We found that the transient expression of PRMT1 led to an increased level of Axin and that knockdown of endogenous PRMT1 by short hairpin RNA reduced the level of Axin. These results suggest that methylation by PRMT1 enhanced the stability of Axin. Methylation of Axin by PRMT1 also seemingly enhanced the interaction between Axin and glycogen synthase kinase 3β, leading to decreased ubiquitination of Axin. Consistent with the role of PRMT1 in the regulation of Axin, knockdown of PRMT1 enhanced the level of cytoplasmic β-catenin as well as β-catenin-dependent transcription activity. In summary, we show that the methylation of Axin occurred in vivo and controlled the stability of Axin. Therefore, methylation of Axin by PRMT1 may serve as a finely tuned regulation mechanism for Wnt/β-catenin signaling.

Axin localizes to mitotic spindles and centrosomes in mitotic cells.

Wnt signaling plays critical roles in cell proliferation and carcinogenesis. In addition, numerous recent studies have shown that various Wnt signaling components are involved in mitosis and chromosomal instability. However, the role of Axin, a negative regulator of Wnt signaling, in mitosis has remained unclear. Using monoclonal antibodies against Axin, we found that Axin localizes to the centrosome and along mitotic spindles. This localization was suppressed by siRNA specific for Aurora A kinase and by Aurora kinase inhibitor. Interestingly, Axin over-expression altered the subcellular distribution of Plk1 and of phosphorylated glycogen synthase kinase (GSK3beta) without producing any notable changes in cellular phenotype. In the presence of Aurora kinase inhibitor, Axin over-expression induced the formation of cleavage furrow-like structures and of prominent astral microtubules lacking midbody formation in a subset of cells. Our results suggest that Axin modulates distribution of Axin-associated proteins such as Plk1 and GSK3beta in an expression level-dependent manner and these interactions affect the mitotic process, including cytokinesis under certain conditions, such as in the presence of Aurora kinase inhibitor.

Hippo signaling interactions with Wnt/β-catenin and Notch signaling repress liver tumorigenesis

Malignant tumors develop through multiple steps of initiation and progression, and tumor initiation is of singular importance in tumor prevention, diagnosis, and treatment. However, the molecular mechanism whereby a signaling network of interacting pathways restrains proliferation in normal cells and prevents tumor initiation is still poorly understood. Here, we have reported that the Hippo, Wnt/&bgr;-catenin, and Notch pathways form an interacting network to maintain liver size and suppress hepatocellular carcinoma (HCC). Ablation of the mammalian Hippo kinases Mst1 and Mst2 in liver led to rapid HCC formation and activated Yes-associated protein/WW domain containing transcription regulator 1 (YAP/TAZ), STAT3, Wnt/&bgr;-catenin, and Notch signaling. Previous work has shown that abnormal activation of these downstream pathways can lead to HCC. Rigorous genetic experiments revealed that Notch signaling forms a positive feedback loop with the Hippo signaling effector YAP/TAZ to promote severe hepatomegaly and rapid HCC initiation and progression. Surprisingly, we found that Wnt/&bgr;-catenin signaling activation suppressed HCC formation by inhibiting the positive feedback loop between YAP/TAZ and Notch signaling. Furthermore, we found that STAT3 in hepatocytes is dispensable for HCC formation when mammalian sterile 20–like kinase 1 and 2 (Mst1 and Mst2) were removed. The molecular network we have identified provides insights into HCC molecular classifications and therapeutic developments for the treatment of liver tumors caused by distinct genetic mutations.

Dual functions of DP1 promote biphasic Wnt-on and Wnt-off states during anteroposterior neural patterning

DP1, a dimerization partner protein of the transcription factor E2F, is known to inhibit Wnt/β‐catenin signalling along with E2F, although the function of DP1 itself was not well characterized. Here, we present a novel dual regulatory mechanism of Wnt/β‐catenin signalling by DP1 independent from E2F. DP1 negatively regulates Wnt/β‐catenin signalling by inhibiting Dvl–Axin interaction and by enhancing poly‐ubiquitination of β‐catenin. In contrast, DP1 positively modulates the signalling upon Wnt stimulation, via increasing cytosolic β‐catenin and antagonizing the kinase activity of NLK. In Xenopus embryos, DP1 exerts both positive and negative roles in Wnt/β‐catenin signalling during anteroposterior neural patterning. From subcellular localization analyses, we suggest that the dual roles of DP1 in Wnt/β‐catenin signalling are endowed by differential nucleocytoplasmic localizations. We propose that these dual functions of DP1 can promote and stabilize biphasic Wnt‐on and Wnt‐off states in response to a gradual gradient of Wnt/β‐catenin signalling to determine differential cell fates.

Axin expression enhances herpes simplex virus type 1 replication by inhibiting virus-mediated cell death in L929 cells

Herpes simplex virus type 1 (HSV-1) replicates in various cell types and induces early cell death, which limits viral replication in certain cell types. Axin is a scaffolding protein that regulates Wnt signalling and participates in various cellular events, including cellular proliferation and cell death. The effects of axin expression on HSV-1 infection were investigated based on our initial observation that Wnt3a treatment or axin knockdown reduced HSV-1 replication. L929 cells expressed the axin protein in a doxycycline-inducible manner (L-axin) and enhanced HSV-1 replication in comparison to control cells (L-EV). HSV-1 infection induced cell death as early as 6 h after infection through the necrotic pathway and required de novo protein synthesis in L929 cells. Subsequent analysis of viral protein expression suggested that axin expression led to suppression of HSV-1-induced premature cell death, resulting in increased late gene expression. In analysis of axin deletion mutants, the regulators of the G-protein signalling (RGS) domain were involved in the axin-mediated enhancement of viral replication and reduction in cell death. These results suggest that viral replication enhancement might be mediated by the axin RGS domain.

High prevalence of amantadine-resistant influenza A virus isolated in Gyeonggi Province, South Korea, during 2005-2010

Amantadine resistance among influenza A viruses was investigated in South Korea in 2005–2010. Of 308 influenza A viruses examined, 229 had the S31N substitution in the M2 protein. The frequency of amantadine resistance was 30xa0%, 100xa0%, and 76xa0% in influenza A/H1N1, pandemic A/H1N1 2009(A/H1N1pdm), and A/H3N2 subtypes, respectively. The amantadine-resistant influenza A/H1N1pdm and A/H3N2 viruses were circulating continuously from 2008 to 2009 and from 2005 to 2006, respectively. Amantadine resistance among influenza A viruses increased dramatically during the 5-year study period, and this has diminished the usefulness of this class of drugs.

Wip1 directly dephosphorylates NLK and increases Wnt activity during germ cell development

Mice null for wild-type p53-induced phosphatase 1 (WIP1) display defects in testis development and spermatogenesis, resulting in reduced fertility. However, the molecular mechanism underlying these abnormalities in the testis remains uncharacterized. We report that the phosphatase activity of WIP1 increases Wnt activity through Nemo-like kinase (NLK). WIP1 directly interacted with NLK, which is highly homologous to p38 MAPK, a WIP1 substrate, and dephosphorylated its activation site. The WIP1-mediated inhibition of NLK activity markedly decreased the phosphorylation of lymphoid enhancer-binding factor 1 (LEF1), enhancing its interaction with β-catenin. Additionally, WIP1 depletion impaired germ cell development, as evidenced by the expression of Oct4 and the germ cell-specific markers Ddx4, Nanos3 and Dnd1 during the development of germ cells from Oct4-GFP transgenic (OG2) mouse embryonic stem cells (mESCs). The expression of WIP1, whose level was significantly lower after the differentiation of germ cells from mESCs, occurred in parallel with the expression of germ cell development markers and SRY-box 17 (Sox17), a downstream target of Wnt. These results indicate that WIP1 is essential for germ cell development, which is known to require Wnt activity.

Ubiquitylation and degradation of adenomatous polyposis coli by MKRN1 enhances Wnt/β-catenin signaling

The adenomatous polyposis coli (APC) protein has a tumor-suppressor function by acting as a negative regulator of the Wnt signaling pathway. While its role as a tumor suppressor is well-defined, the post-translational modifications that regulate APC stability are not fully understood. Here we showed that MKRN1, an E3 ligase, could directly interact with and ubiquitylate APC, promoting its proteasomal degradation. In contrast, an E3 ligase-defective MKRN1 mutant was no longer capable of regulating APC, indicating that its E3 ligase activity is required for APC regulation by MKRN1. Strengthening these results, MKRN1 ablation resulted in reduced β-catenin activity and decreased expression of Wnt target genes. The ability of the Wnt-dependent pathway to induce cancer cell proliferation, migration, and invasion was impaired by MKRN1 depletion, but restored by simultaneous APC knockdown. Taken together, these results demonstrate that MKRN1 functions as a novel E3 ligase of APC that positively regulates Wnt/β-catenin-mediated biological processes.

Oseltamivir-resistant influenza viruses isolated in South Korea from 2005 to 2010

South Korean isolates of oseltamivir-resistant influenza viruses from 2005–2010 were investigated with a total 491 influenza viruses identified from 1702 specimens. Neuraminidase genes from 342 influenza viruses (71 A/H1N1, 74 pandemic A/H1N1 2009, 117 A/H3N2, and 80 B) were analyzed by RT-PCR with molecular markers for oseltamivir resistance. The H274Y mutation in the NA protein was identified in 100xa0% (n=40) of A/H1N1 viruses circulating in 2008–2009. Influenza A/H1N1 viruses harboring the H274Y substitution exhibited, on average, a 626-fold reduction in oseltamivir susceptibility and clustered with the A/Norway/1736/2007 strain. Close and timely monitoring for resistance to clinically available influenza antivirals should be consistently performed.