The Viral SUMO-Targeted Ubiquitin Ligase ICP0 is Phosphorylated and Activated by Host Kinase Chk2.

When the herpes simplex virus (HSV) genome enters the nucleus for replication and transcription, phase-segregated nuclear protein bodies called Promyelocytic leukemia protein nuclear bodies (PML NBs) colocalize with the genome and repress it. HSV encodes a small ubiquitin-like modifier (SUMO)-targeted ubiquitin ligase (STUbL) infected cell polypeptide 0 (ICP0) that degrades PML NBs to alleviate the repression. The molecular details of the mechanism used by ICP0 to target PML NBs are unclear. Here, we identify a bona fide SUMO-interacting motif in ICP0 (SIM-like sequence [SLS] 4) that is essential and sufficient to target SUMOylated proteins in PML NBs such as the PML and Sp100. We shown that phosphorylation of SLS4 creates new salt bridges between SUMO and SLS4, increases the SUMO/SLS4 affinity, and switches ICP0 into a potent STUbL. HSV activates the Ataxia-telangiectasia-mutated kinase-Checkpoint kinase 2 (ATM-Chk2) pathway to regulate the cell cycle of the host. We report that the activated Chk2 also phosphorylates ICP0 at SLS4 and enhances its STUbL activity. Our results uncover that a viral STUbL counters antiviral response by exploiting an unprecedented cross-talk of three post-translational modifications: ubiquitination, SUMOylation, and phosphorylation.

Demonstration of helicase activity in the nonstructural protein, NSs, of the negative-sense RNA virus, groundnut bud necrosis virus.

The nonstructural protein NSs, encoded by the S RNA of groundnut bud necrosis virus (GBNV) (genus Tospovirus, family Bunyaviridae) has earlier been shown to possess nucleic-acid-stimulated NTPase and 5' α phosphatase activity. ATP hydrolysis is an essential function of a true helicase. Therefore, NSs was tested for DNA helicase activity. The results demonstrated that GBNV NSs possesses bidirectional DNA helicase activity. An alanine mutation in the Walker A motif (K189A rNSs) decreased DNA helicase activity substantially, whereas a mutation in the Walker B motif resulted in a marginal decrease in this activity. The parallel loss of the helicase and ATPase activity in the K189A mutant confirms that NSs acts as a non-canonical DNA helicase. Furthermore, both the wild-type and K189A NSs could function as RNA silencing suppressors, demonstrating that the suppressor activity of NSs is independent of its helicase or ATPase activity. This is the first report of a true helicase from a negative-sense RNA virus.

RhoC mediates epidermal growth factor-stimulated migration and invasion in head and neck squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) is overexpressed in head and neck squamous cell carcinoma (HNSCC) where it has been shown to promote tumor cell invasion upon phosphorylation. One mechanism by which EGFR promotes tumor progression is by activating signal cascades that lead to loss of E-cadherin, a transmembrane glycoprotein of the cell-cell adherence junctions; however mediators of these signaling cascades are not fully understood. One such mediator, RhoC, is activated upon a number of external stimuli, such as epidermal growth factor (EGF), but its role as a mediator of EGF-stimulated migration and invasion has not been elucidated in HNSCC. In the present study, we investigate the role of RhoC as a mediator of EGF-stimulated migration and invasion in HNSCC. We show that upon EGF stimulation, EGFR and RhoC were strongly activated in HNSCC. This resulted in activation of the phosphatidylinositol 3-Kinase Akt pathway (PI3K-Akt), phosphorylation of GSK-3ß at the Ser(9) residue, and subsequent down regulation of E-cadherin cell surface expression resulting in increased tumor cell invasion. Knockdown of RhoC restored E-cadherin expression and inhibited EGF-stimulated migration and invasion. This is the first report in HNSCC demonstrating the role RhoC plays in mediating EGF-stimulated migration and invasion by down-regulating the PI3K-Akt pathway and E-cadherin expression. RhoC may serve as a treatment target for HNSCC.

Modulation of liver-intestine cadherin (Cadherin 17) expression, ERK phosphorylation and WNT signaling in EPHB6 receptor-expressing MDA-MB-231 cells.

Aberrant expression of erythropoietin-producing hepatocellular carcinoma cell (EPH) receptors has been reported in a variety of human cancer types. In addition to modulating cell proliferation and migration, EPH receptors are also involved in tumor progression. The transcriptional activation and silencing of EPH receptors are also associated with tumorigenesis. However, the mechanisms underlying the involvement of EPH receptors in tumorigenesis have not been completely deciphered. We have investigated and described the role of EPHB6, a kinase-deficient receptor, in modulating the abundance of cadherin 17 and activation of other intracellular signaling proteins. We previously showed that EPHB6 alters the tumor phenotype of breast carcinoma cells. However, the mechanisms underlying these phenotypic changes had not previously been investigated. Herein we demonstrated the downstream effects of EPHB6 expression on the abundance of cadherin 17, mitogen-activated protein kinase (MEK2), extracellular signal-regulated kinase (ERK), phospho-ERK, ß-catenin, phospho- glycogen synthase kinase 3 beta (GSK3ß) (ser21/9), cell morphology and actin cytoskeleton. These comparisons were made between EPHB6-deficient MDA-MB-231 cells transfected with an empty pcDNA3 vector and cells stably transfected with an expression construct of EPHB6. The results indicate elevated levels of MEK2 and phospho-ERK. While there was no change in the amount of ERK, the abundance of cadherin 17, ß-catenin and phospho-GSK3ß was significantly reduced in EPHB6-transfected cells. These studies clearly demonstrate an inverse relationship between the levels of phospho-ERK and the abundance of cadherin 17, ß-catenin and phospho-GSK3ß in EPHB6-expressing MDA-MB-231 cells. From these data we conclude that EPHB6-mediated alterations arise due to changes in abundance and localization of cadherin 17 and activation of WNT signaling pathway. Transcriptional silencing of EPHB6 in native MDA-MB-231 cells and consequent effects on cadherin 17 and WNT pathway may, thus, be responsible for the invasive behavior of these cells.

EphB6 receptor modulates micro RNA profile of breast carcinoma cells.

Breast carcinoma cells have a specific pattern of expression for Eph receptors and ephrin ligands. EphB6 has previously been characterized as a signature molecule for invasive breast carcinoma cells. The transcription of EphB6 is silenced in breast carcinoma cells and its re-expression leads to decreased invasiveness of MDA-MB-231 cells. Such differences in phenotypes of native and EphB6 expressing MDA-MB-231 cells relate to an altered profile of micro RNAs. Comparative hybridization of total RNA to slides containing all known miRNAs by using locked nucleic acid (LNA) miRCURY platform yielded a significantly altered profile of miRNAs in MDA-MB-231 cells stably transfected with EphB6. After applying a threshold of change and a p-value of <0.001, the list of significantly altered miRNAs included miR-16, miR-23a, miR-24, miR-26a, miR-29a, miR-100, miRPlus-E1172 and miRPlus-E1258. The array-based changes were validated by real-time qPCR of miR-16, miR-23a, miR-24 and miR-100. Except miRPlus-E1172 and miRPlus-E1258, the remaining six miRNAs have been observed in a variety of cancers. The biological relevance of target mRNAs was predicted by using a common-target selection approach that allowed the identification of SMARCA5, SMARCC1, eIF2C2, eIF2C4, eIF4EBP2, FKABP5, FKBP1A, TRIB1, TRIB2, TRIB3, BMPR2, BMPR1A and BMPR1B as important targets of a subset of significantly altered miRNAs. Quantitative PCR revealed that the levels of SMARCC1, eIFC4, eIF4EB2, FKBP1a, FKBP5, TRIB1, TRIB3, BMPR1a and BMPR2 transcripts were significantly decreased in MDA-MB-231 cells transfected with EphB6. These observations confirm targeting of specific mRNAs by miR-100, miR-23a, miR-16 and miR-24, and suggest that the kinase-deficient EphB6 receptor is capable of initiating signal transduction from the cell surface to the nucleus resulting in the altered expression of a variety of genes involved in tumorigenesis and invasion. The alterations in miRNAs and their target mRNAs also suggest indirect involvement of EphB6 in PI3K/Akt/mTOR pathways.