Identification of Nedd4 E3 Ubiquitin Ligase as a Binding Partner and Regulator of MAK-V Protein Kinase

MAK-V/Hunk is a scantily characterized AMPK-like protein kinase. Recent findings identified MAK-V as a pro-survival and anti-apoptotic protein and revealed its role in embryonic development as well as in tumorigenesis and metastasis. However molecular mechanisms of MAK-V action and regulation of its activity remain largely unknown. We identified Nedd4 as an interaction partner for MAK-V protein kinase. However, this HECT-type E3 ubiquitin ligase is not involved in the control of MAK-V degradation by the ubiquitin-proteasome system that regulates MAK-V abundance in cells. However, Nedd4 in an ubiquitin ligase-independent manner rescued developmental defects in Xenopus embryos induced by MAK-V overexpression, suggesting physiological relevance of interaction between MAK-V and Nedd4. This identifies Nedd4 as the first known regulator of MAK-V function

Hunk/Mak-v is a negative regulator of intestinal cell proliferation

BACKGROUND: Conditional deletion of the tumour suppressor gene Apc within the murine intestine results in acute Wnt signalling activation. The associated over-expression of a myriad of Wnt signalling target genes yields phenotypic alterations that encompass many of the hallmarks of neoplasia. Previous transcriptomic analysis aimed at identifying genes that potentially play an important role in this process, inferred the Hormonally upregulated Neu-associated kinase (HUNK/Mak-v/Bstk1) gene as a possible candidate. Hunk is a SNF1 (sucrose non fermenting 1)-related serine/threonine kinase with a proposed association with many different tumour types, including colorectal cancer. METHODS: Here we describe the generation of a novel Hunk kinase deficient mouse which has been used to investigate the involvement of Hunk-kinase activity in intestinal homeostasis and tumourigenesis. RESULTS: We show that in the morphologically normal intestine, Hunk-kinase negatively regulates epithelial cell proliferation. However, the increase in cell proliferation observed in the Hunk kinase deficient intestine is counteracted by increased cell migration, thereby maintaining intestinal homeostasis. Using qRT-PCR, we further demonstrate that Hunk is significantly over-expressed in Apc deficient / Wnt-signalling activated intestinal tissue. Using the classical intestinal tumourigenesis Apc(Min) mouse model we show that loss of Hunk-kinase activity significantly reduced tumour initiation rates in the small intestine. However, an accompanying increase in the size of the tumours counteracts the impact this has on overall tumour burden or subsequently survival. CONCLUSIONS: In the intestinal setting we demonstrate that Hunk has a role in normal intestinal proliferation and homeostasis and, although it does not alter overall survival rates, activity of this kinase does impact on tumour initiation rates during the early stages in tumourigenesis in the small intestine. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12885-015-1087-2) contains supplementary material, which is available to authorized users

Non-resonant operation of microcavity Brillouin lasers

We present theoretical framework to describe Brillouin lasing in microcavities in the case of a significant mismatch between the Brillouin shift and the cavity intermode spacing. We show that despite an increase of the lasing threshold a significant increase of the Brillouin power in comparison with the resonance case is achievable. A necessary condition for this effect is the optimal value of the pump frequency detuning from the cavity mode frequency. An increase of the Brillouin threshold is accompanied by narrowing of the spectrum range where the Brillouin signal could be generated in non-resonant case. Besides, with the optimal pump frequency detuning the Brillouin signal noise level is reduced. Analytical results are in quantitative agreement with the results of numerical simulations

Genome-wide association studies of autoimmune vitiligo identify 23 new risk loci and highlight key pathways and regulatory variants

Vitiligo is an autoimmune disease in which depigmented skin results from the destruction of melanocytes1, with epidemiological association with other autoimmune diseases2. In previous linkage and genome-wide association studies (GWAS1 and GWAS2), we identified 27 vitiligo susceptibility loci in patients of European ancestry. We carried out a third GWAS (GWAS3) in European-ancestry subjects, with augmented GWAS1 and GWAS2 controls, genome-wide imputation, and meta-analysis of all three GWAS, followed by an independent replication. The combined analyses, with 4,680 cases and 39,586 controls, identified 23 new significantly associated loci and 7 suggestive loci. Most encode immune and apoptotic regulators, with some also associated with other autoimmune diseases, as well as several melanocyte regulators. Bioinformatic analyses indicate a predominance of causal regulatory variation, some of which corresponds to expression quantitative trait loci (eQTLs) at these loci. Together, the identified genes provide a framework for the genetic architecture and pathobiology of vitiligo, highlight relationships with other autoimmune diseases and melanoma, and offer potential targets for treatment

Rho family GTPase Chp/RhoV induces PC12 apoptotic cell death via JNK activation

Rho GTPases regulate numerous cellular processes including apoptosis. Chp/RhoV is an atypical Rho GTPase which functions are poorly understood. Here we investigated the role of Chp in regulation of cell viability using PC12 cells with inducible expression of Chp as a model. We found that expression of Chp results in apoptosis in PC12 cells. Chp-induced apoptosis was accompanied by activation of JNK signaling and both death receptor-mediated and mitochondrial apoptotic pathways as justified by caspase-8 and caspase-9 activation, respectively. Moreover, inhibition of JNK by SP600125 rescued PC12 cells from Chp-triggered cell death and attenuated activation of caspases-9 and -3/7 suggesting that activation of JNK mediates pro-apoptotic effect of Chp. Expression of Chp resulted in increased phosphorylation of c-Jun in PC12 cells, and Chp expression in HE K293 cells upregulated AP-1-dependent transcription in a JNK-dependent manner. Together results of our study reveal the role of Chp GTPase as a putative regulator of JNK-dependent apoptotic death in PC12 cells, similarly to previously described pro-apoptotic activity of the related Cdc42 and Rac1 GTPases

Rho family GTPase Chp/RhoV induces PC12 apoptotic cell death via JNK activation

Rho GTPases regulate numerous cellular processes including apoptosis. Chp/RhoV is an atypical Rho GTPase which functions are poorly understood. Here we investigated the role of Chp in regulation of cell viability using PC12 cells with inducible expression of Chp as a model. We found that expression of Chp results in apoptosis in PC12 cells. Chp-induced apoptosis was accompanied by activation of JNK signaling and both death receptor-mediated and mitochondrial apoptotic pathways as justified by caspase-8 and caspase-9 activation, respectively. Moreover, inhibition of JNK by SP600125 rescued PC12 cells from Chp-triggered cell death and attenuated activation of caspases-9 and -3/7 suggesting that activation of JNK mediates pro-apoptotic effect of Chp. Expression of Chp resulted in increased phosphorylation of c-Jun in PC12 cells, and Chp expression in HE K293 cells upregulated AP-1-dependent transcription in a JNK-dependent manner. Together results of our study reveal the role of Chp GTPase as a putative regulator of JNK-dependent apoptotic death in PC12 cells, similarly to previously described pro-apoptotic activity of the related Cdc42 and Rac1 GTPases

LY294002 Enhances Expression of Proteins Encoded by Recombinant Replication-Defective Adenoviruses via mTOR- and Non-mTOR-Dependent Mechanisms

Adenovirus-based drugs are efficient when combined with other anticancer treatments. Here we show that treatment with LY294002 and LY303511 upregulates expression of recombinant proteins encoded by replication-defective adenoviruses, including expression of therapeutically valuable combination of herpes simplex virus thymidine kinase controlled by human telomerase reverse transcriptase promoter (Ad-hTERT-HSVtk). In line with this, treatment with LY294002 synergized with Ad-hTERT-HSVtk infection in the presence of gancyclovir prodrug on Calu-I lung cancer cell death. The effect of LY294002 and LY303511 on adenovirus-delivered transgene expression was demonstrated in 4 human lung cancer cell lines. LY294002-induced upregulation of adenovirally delivered transgene is mediated in part by direct inhibition of mTOR protein kinase in mTORC2 signaling complex thus suggesting that anticancer drugs targeting mTOR will also enhance expression of transgenes delivered with adenoviral vectors. As both LY294002 and LY303511 are candidate prototypic anticancer drugs, and many mTOR inhibitors for cancer treatment are under development, our results have important implication for development of future therapeutic strategies with adenoviral gene delivery

Nedd4 regulates MAK-V activity.

<p>Nedd4 in ubiquitin ligase-independent manner rescue axis extension defects in <i>Xenopus</i> development induced by MAK-V overexpression. Two ng each of <i>MAK-V</i>, <i>Nedd4</i>, <i>Nedd4(CS)</i> mRNA was injected alone or together as indicated into two dorso-animal blastomeres at 4–8 cell stage. Embryos were fixed at stage 38.</p

MAK-V is subjected to ubiquitin-dependent proteasomal degradation.

<p>(<b>A</b>) Doxycycline-treated (<i>DOX +</i>) or untreated (<i>DOX</i> -) PC12TetOn MAK-V-FLAG cells were incubated with 100 µM of ALLN (<i>ALLN</i> +) or vehicle (<i>ALLN</i> -) for 8 hrs. Results of Western blot analysis of total cell lysates with anti-FLAG antibodies are shown. MAK-V-FLAG protein marked with arrow, ubiquitinated higher molecular weight MAK-V-FLAG species marked by asterisk. To monitor total protein loading, membrane was re- probed with anti-α-tubulin antibodies. (<b>B</b>) HEK293 cells were transiently transfected with plasmid for MAK-V-FLAG protein expression and treated with 10 µM MG132 (<i>MG+</i>) for indicated time prior to lysis or left untreated (<i>MG-</i>). Lysates were blotted with anti-FLAG antibodies to detect MAK-V-FLAG protein (<i>anti-FLAG</i>). To monitor total protein loading, membrane was re-probed with anti-α-tubulin antibodies. (<b>C</b>) HEK293 cells were transfected with plasmid for HA-tagged ubiquitin expression alone (<i>MAK-V-FLAG -</i>) or together with plasmid for MAK-V-FLAG protein expression (<i>MAK-V-FLAG +</i>). Cells were treated with MG132 prior to lysis. MAK-V-FLAG protein was precipitated from lysates with anti-FLAG M2 affinity gel as described for PC12TetOn cells. Anti-FLAG antibodies were used to detect MAK-V-FLAG protein (<i>anti-FLAG</i>) in immunoprecipitates (<i>IP: anti-FLAG</i>), and ubiquitin was detected with anti-HA antibodies (<i>anti-HA</i>). To monitor HA-tagged ubiquitin and MAK-V-FLAG protein expression, aliquots of lysates were blotted as described above. To monitor total protein loading, membrane was re-probed with anti-α-tubulin antibodies. (<b>D</b>) HEK293 cells were co-transfected with MAK-V-FLAG expression plasmid and plasmid for expression of FLAG-tagged wild-type ubiquitin (<i>wt</i>), its K48R (<i>K48R</i>) or K63R (<i>K63R</i>) mutant. Results of Western blot analysis of total cell lysates with anti-MAK-V antibodies are shown. To monitor total protein loading, membrane was re-probed with anti-α-tubulin antibodies. To control variations in basal level of MAK-V-FLAG protein expression, samples were also probed with antibodies against neomycin phosphotransferase II (<i>anti-NPT</i>) which is encoded by MAK-V-FLAG expression vector.</p