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Nek2 phosphorylates and stabilizes β-catenin at mitotic centrosomes downstream of Plk1.

ABSTRACT: β-Catenin is a multifunctional protein with critical roles in cell-cell adhesion, Wnt signaling, and the centrosome cycle. Whereas the regulation of β-catenin in cell-cell adhesion and Wnt signaling are well understood, how β-catenin is regulated at the centrosome is not. NIMA-related protein kinase 2 (Nek2), which regulates centrosome disjunction/splitting, binds to and phosphorylates β-catenin. Using in vitro and cell-based assays, we show that Nek2 phosphorylates the same regulatory sites in the N-terminus of β-catenin as glycogen synthase kinase 3β (GSK3β), which are recognized by a specific phospho-S33/S37/T41 antibody, as well as additional sites. Nek2 binding to β-catenin appears to inhibit binding of the E3 ligase β-TrCP and prevents β-catenin ubiquitination and degradation. Thus β-catenin phosphorylated by Nek2 is stabilized and accumulates at centrosomes in mitosis. We further show that polo-like kinase 1 (Plk1) regulates Nek2 phosphorylation and stabilization of β-catenin. Taken together, these results identify a novel mechanism for regulating β-catenin stability that is independent of GSK3β and provide new insight into a pathway involving Plk1, Nek2, and β-catenin that regulates the centrosome cycle.

SUBMITTER: Mbom BC

PROVIDER: S-EPMC3967981 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Under pathological conditions like herpes simplex virus 1 (HSV-1) infection, host-pathogen interactions lead to major reconstruction of the host protein network, which contributes to the dysregulation of signaling pathways and disease onset. Of note is the upregulation of a multifunctional host protein, heparanase (HPSE), following infection, which serves as a mediator in HSV-1 replication. In this study, we identify a novel function of HPSE and highlight it as a key regulator of β-catenin signal transduction. The regulatory role of HPSE on the activation, nuclear translocation, and signal transduction of β-catenin disrupts cellular homeostasis and establishes a pathogenic environment that promotes viral replication. Under normal physiological conditions, β-catenin is bound to a group of proteins, referred to as the destruction complex, and targeted for ubiquitination and, ultimately, degradation. We show that virus-induced upregulation of HPSE leads to the activation of Akt and subsequent stabilization and activation of β-catenin through (i) the release of β-catenin from the destruction complex, and (ii) direct phosphorylation of β-catenin at Ser552. This study also provides an in-depth characterization of the proviral role of β-catenin signaling during HSV-1 replication using physiologically relevant cell lines and in vivo models of ocular infection. Furthermore, pharmacological inhibitors of this pathway generated a robust antiviral state against multiple laboratory and clinical strains of HSV-1. Collectively, our findings assign a novel regulatory role to HPSE as a driver of β-catenin signaling in HSV-1 infection. IMPORTANCE Heparanase (HPSE) and β-catenin have independently been implicated in regulating key pathophysiological processes, including neovascularization, angiogenesis, and inflammation; however, the relationship between the two proteins has remained elusive thus far. For that reason, characterizing this relationship is crucial and can lead to the development of novel therapeutics. For HSV-1 specifically, current antivirals are not able to abolish the virus from the host, leaving patients susceptible to episodes of viral reactivation. Identifying a host-based intervention can provide a better alternative with enhanced efficacy and sustained relief.

Project description:BackgroundSorafenib is the first-line treatment for advanced-stage hepatocellular carcinoma (HCC). Several studies have shown that the up-regulation of β-catenin plays a role in sorafenib resistance in HCC; however, the mechanism associated with this phenomenon remains elusive.MethodsWestern blotting, flow cytometry, and an evaluation of IC50 values were used to confirm the role of β-catenin in HCC sorafenib resistance. Immunoprecipitation and western blotting were then performed to identify regulatory interactions between β-catenin and Nek2. Further, western blotting, flow cytometry, and an in vivo xenograft model were used to evaluate the function of Nek2 in HCC sorafenib resistance, whereas rescue experiments were performed to confirm that Nek2 induces sorafenib resistance via β-catenin. Finally, western blotting and immunohistochemistry were used to evaluate the expression level of Nek2 in paired HCC and non-tumor tissues.ResultsWe showed that β-catenin could suppress sorafenib-induced apoptosis and cell growth inhibition in HCC cell lines. By screening β-catenin-interacting proteins, we found that Nek2 could bind β-catenin in sorafenib-treated HCC cell lines. Our results also showed that Nek2 stabilizes β-catenin and promotes its translocation to the nucleus, consequently activating the transcription of downstream target genes. We further confirmed that Nek2 could induce sorafenib resistance in HCC cell lines, and that β-catenin was the key element involved in this process. Further, a xenograft tumor model showed that Nek2 knockdown could improve the anti-tumor effect of sorafenib, whereas an analysis of tumor proteins showed that Nek2 regulates β-catenin protein levels and its nuclear translocation in vivo. In addition, Nek2 was found to be up-regulated in HCC tissue, and especially in advanced-stage disease.ConclusionsOur study proves that Nek2 induces HCC sorafenib resistance via β-catenin and suggests a novel therapeutic strategy to improve the anti-tumor effects of sorafenib in HCC.

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Nek2 phosphorylates and stabilizes β-catenin at mitotic centrosomes downstream of Plk1.

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