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Significance Proper elaboration of the plant body plan requires that cell division patterns are coordinated during development in complex tissues. Activation of cell cycle machinery is critical for this process, but it is not clear how or if this links to cell-to-cell communication networks that are important during development. Here we show that key cell divisions that generate the plant root are controlled by cell-to-cell signaling peptides which act through plant-specific receptor kinases to control expression of a specific cyclinD cell cycle regulatory gene. We show that cyclinD gene expression depends on both receptor signaling and the SHORT-ROOT transcription factor to ensure timely and robust cell division patterns., Cell division is often regulated by extracellular signaling networks to ensure correct patterning during development. In Arabidopsis, the SHORT-ROOT (SHR)/SCARECROW (SCR) transcription factor dimer activates CYCLIND6;1 (CYCD6;1) to drive formative divisions during root ground tissue development. Here, we show plasma-membrane-localized BARELY ANY MERISTEM1/2 (BAM1/2) family receptor kinases are required for SHR-dependent formative divisions and CYCD6;1 expression, but not SHR-dependent ground tissue specification. Root-enriched CLE ligands bind the BAM1 extracellular domain and are necessary and sufficient to activate SHR-mediated divisions and CYCD6;1 expression. Correspondingly, BAM-CLE signaling contributes to the restriction of formative divisions to the distal root region. Additionally, genetic analysis reveals that BAM-CLE and SHR converge to regulate additional cell divisions outside of the ground tissues. Our work identifies an extracellular signaling pathway regulating formative root divisions and provides a framework to explore this pathway in patterning and evolution.

The bromodomain and extra-terminal domain (BET) proteins are promising drug targets for cancer and immune diseases. However, BET inhibition effects have been studied more in the context of bromodomain-containing protein 4 (BRD4) than BRD2, and the BET protein association to histone H4-hyperacetylated chromatin is not understood at the genome-wide level. Here, we report transcription start site (TSS)-resolution integrative analyses of ChIP-seq and transcriptome profiles in human non-small cell lung cancer (NSCLC) cell line H23. We show that di-acetylation at K5 and K8 of histone H4 (H4K5acK8ac) co-localizes with H3K27ac and BRD2 in the majority of active enhancers and promoters, where BRD2 has a stronger association with H4K5acK8ac than H3K27ac. Although BET inhibition by JQ1 led to complete reduction of BRD2 binding to chromatin, only local changes of H4K5acK8ac levels were observed, suggesting that recruitment of BRD2 does not influence global histone H4 hyperacetylation levels. This finding supports a model in which recruitment of BET proteins via histone H4 hyperacetylation is predominant over hyperacetylation of histone H4 by BET protein-associated acetyltransferases. In addition, we found that a remarkable number of BRD2-bound genes, including MYC and its downstream target genes, were transcriptionally upregulated upon JQ1 treatment. Using BRD2-enriched sites and transcriptional activity analysis, we identified candidate transcription factors potentially involved in the JQ1 response in BRD2-dependent and -independent manner.

The connection between signaling pathways activating cancer-associated fibroblasts (CAFs) remains to be determined. Metabolic alterations linked to autophagy have also been implicated in CAF activation. CSL/RBPJ, a transcriptional repressor that mediates Notch signaling, suppresses the gene expression program(s), leading to stromal senescence and CAF activation. Deregulated GLI signaling can also contribute to CAF conversion. Here, we report that compromised CSL function depends on GLI activation for conversion of human dermal fibroblasts into CAFs, separately from cellular senescence. Decreased CSL upregulates the expression of the ULK3 kinase, which binds and activates GLI2. Increased ULK3 also induces autophagy, which is unlinked from GLI and CAF activation. ULK3 upregulation occurs in the CAFs of several tumor types, and ULK3 silencing suppresses the tumor-enhancing properties of these cells. Thus, ULK3 links two key signaling pathways involved in CAF conversion and is an attractive target for stroma-focused anti-cancer intervention.

Loss of epithelial differentiation and extracellular matrix (ECM) remodeling are known to facilitate cancer progression and are associated with poor prognosis in patients with lung cancer. We have identified Receptor-interacting serine/threonine protein kinase 4 (RIP4) as a regulator of tumor differentiation in lung adenocarcinoma (AC). Bioinformatics analyses of human lung AC samples showed that poorly differentiated tumors express low levels of RIP4, whereas high levels are associated with better overall survival. In vitro, lung tumor cells expressing reduced RIP4 levels showed enhanced activation of STAT3 signaling and had a greater ability to invade through collagen. In contrast, overexpression of RIP4 inhibited STAT3 activation, which abrogated interleukin-6-dependent induction of lysyl oxidase, a collagen cross-linking enzyme. In an autochthonous mouse model of lung AC initiated by Kras(G12D) expression with loss of p53, Rip4 knockdown tumors progressed to a poorly differentiated state marked by an increase in Hmga2, reduced Ttf1, and enrichment of genes regulating extracellular remodeling and Jak-Stat signaling. Tail vein injections of cells overexpressing Rip4 showed a reduced potential to invade and form tumors, which was restored by co-expression of Stat3. Altogether, our work has identified that loss of RIP4 enhances STAT3 signaling in lung cancer cells, promoting the expression of ECM remodeling genes and cancer dedifferentiation.

The mineralocorticoid hormone aldosterone plays a crucial role in the control of Na + and K + balance, blood volume, and arterial blood pressure, by acting in the aldosterone-sensitive distal nephron (ASDN) and stimulating a complex transcriptional, translational, and cellular program. Because the complexity of the aldosterone response is still not fully appreciated, we aimed at identifying new elements in this pathway. Here, we demonstrate that the expression of the proto-oncogene PIM3 (Proviral Integration Site of Moloney Murine Leukemia Virus 3), a serine/threonine kinase belonging to the calcium/calmodulin-regulated group of kinases, is stimulated by aldosterone in vitro (mCCD cl1 cells), ex vivo (mouse kidney slices), and in vivo in mice. Characterizing a germline Pim3 - / - mouse model, we found that these mice have an upregulated Renin-Angiotensin-Aldosterone System (RAAS), with high circulating aldosterone and plasma renin activity levels on both standard or Na + -deficient diet. Surprisingly, we did not observe any obvious salt-losing phenotype in Pim3 KO mice as shown by normal blood pressure, plasma and urinary electrolytes, as well as unchanged expression levels of the major Na + transport proteins. These observations suggest that the potential effects of the loss of the Pim3 gene are physiologically compensated. Indeed, the 2 other family members of the PIM kinase family, PIM1 and PIM2 are upregulated in the kidney of Pim3 - / - mice, and may therefore be involved in such compensation. In conclusion, our data demonstrate that the PIM3 kinase is a novel aldosterone-induced protein, but its precise role in aldosterone-dependent renal homeostasis remains to be determined.

Protein concentration gradients pattern developing organisms and single cells. In Schizosaccharomyces pombe rod-shaped cells, Pom1 kinase forms gradients with maxima at cell poles. Pom1 controls the timing of mitotic entry by inhibiting Cdr2, which forms stable membrane-associated nodes at mid-cell. Pom1 gradients rely on membrane association regulated by a phosphorylation-dephosphorylation cycle and lateral diffusion modulated by clustering. Using quantitative PALM imaging, we find individual Pom1 molecules bind the membrane too transiently to diffuse from pole to mid-cell. Instead, we propose they exchange within longer lived clusters forming the functional gradient unit. An allelic series blocking auto-phosphorylation shows that multi-phosphorylation shapes and buffers the gradient to control mid-cell levels, which represent the critical Cdr2-regulating pool. TIRF imaging of this cortical pool demonstrates more Pom1 overlaps with Cdr2 in short than long cells, consistent with Pom1 inhibition of Cdr2 decreasing with cell growth. Thus, the gradients modulate Pom1 mid-cell levels according to cell size., eLife digest All organisms need to know how to arrange different cell types during the development of their organs and tissues. This information is provided by protein concentration patterns, or gradients, that tell cells how to behave based on where they are positioned. The same fundamental principles also work on a smaller scale. For example, although the rod-shaped yeast Schizosaccharomyces pombe is a single-celled organism, it uses protein concentration gradients to control its growth and timing of division. Before S. pombe cells divide, they need to check that they have reached the right size. Several mechanisms contribute to this information. One of them involves a concentration gradient of a protein known as Pom1, which is found on the cell membrane, with more protein at the cell extremities and less towards the middle. Pom1 serves to block the activity of Cdr2 – an enzyme that localizes to the cell middle and controls cell division. An open question has been whether Pom1 levels at the center drop as the cell grows, coordinating growth and division. One explanation for how the Pom1 gradient could be regulated is by the removal and addition of phosphate groups. At the cell’s tip, an enzyme removes phosphate groups from Pom1, causing it to bind to the membrane. As Pom1 diffuses along the membrane, it continuously ‘re-phosphorylates’ itself. This promotes Pom1 to gradually detach, restricting it from spreading along the membrane towards the cell middle. Another explanation is that clusters of Pom1, formed at the membrane, help establish a gradient by moving along the membrane at different rates: larger clusters, formed in high concentration areas, move slower than smaller clusters, causing levels of Pom1 to be higher at the tip, and lower towards the middle. Now, Gerganova et al. set out to find which of these two processes contributes more to shaping the Pom1 gradient, and determine where Pom1 acts on Cdr2. Gerganova et al. used super resolution microscopy to track individual Pom1 molecules inside yeast cells. This revealed two findings. First, that individual Pom1 molecules do not travel all the way from the cell tip to the center, but ‘hop’ between clusters as they move towards the middle. Second, in longer cells levels of Pom1 on the membrane drop at the center, where Pom1 encounters Cdr2. As a result, Cdr2 will come across higher levels of Pom1 in short cells, but low levels of Pom1 in long cells. This allows Pom1 to act as a measure of cell size, preventing short cells from dividing too soon. The role of clusters in creating gradients is not only relevant for yeast cell division. It could potentially apply to the gradients that organize cells and tissues in different organisms. Future work could examine whether similar principles apply in more complex systems.

The onset and/or growth of breast tumor are controlled, at least in part, by estrogen. Therefore, to prevent the development of breast tumor, estrogen-dependent proliferation of mammary epithelial cells during pregnancy needs to be suppressed once the mammary gland is fully developed to enable lactation. However, the underlying molecular mechanisms remain unknown. Nrk is an X-linked protein serine/threonine kinase in the germinal center kinase family. Herein, we demonstrate a frequent occurrence of breast tumors in homozygous and heterozygous Nrk mutant mice that have experienced pregnancy/parturition. The tumors never developed in the mutant mice without a history of pregnancy/parturition. They exhibited histopathological features of noninvasive tubular adenocarcinoma, and expressed estrogen receptor α. At late gestation when estrogen receptor α expression was significantly reduced in the wild-type mammary gland, grossly normal mammary glands in the pregnant Nrk mutant mice occasionally contained hyperplastic foci continuously expressing the receptor. Consistently, Nrk expression was induced in the wild-type mammary gland at this period of pregnancy. On the other hand, the pregnant Nrk mutant mice also showed elevated blood estrogen levels at late gestation. We suggest that Nrk suppresses the excessive proliferation of mammary epithelial cells during pregnancy, and the impairment of this regulatory system leads to frequent occurrence of breast tumor in Nrk mutant mice.

The NaCl cotransporter (NCC) is essential for electrolyte homeostasis and control of blood pressure. The human SLC12A3 gene, which encodes NCC, gives rise to 3 isoforms, of which only the shortest isoform [NaCl cotransporter isoform 3 (NCC3)] has been studied extensively. All NCC isoforms share key phosphorylation sites at T55 and T60 that are essential mediators of NCC function. Recently, a novel phosphorylation site at S811 was identified in isoforms 1 and 2 [NaCl cotransporter splice variant (NCCSV)], which are only present in humans and higher primates. The aim of the current study, therefore, is to investigate the role of S811 phosphorylation in the regulation of NCC by a combination of biochemical and fluorescent microscopy analyses. We demonstrate that hypotonic low-chloride buffer increases S811 phosphorylation, whereas phosphorylation-deficient S811A mutant hinders phosphorylation at T55 and T60 in NCCSV and NCC3. NCCSV S811A impairs NCC3 activity in a dominant-negative fashion, although it does not affect plasma membrane abundance. This effect may be explained by the heterodimerization of NCCSV with NCC3. Taken together, our study highlights the dominant-negative effect of NCCSV on T55 and T60 phosphorylation and NCC activity. Here, we reveal a new function of NCCSV in humans that broadens the understanding on NCC regulation in blood pressure control.-Tutakhel, O. A. Z., Bianchi, F., Smits, D. A., Bindels, R. J. M., Hoenderop, J. G. J., van der Wijst, J. Dominant functional role of the novel phosphorylation site S811 in the human renal NaCl cotransporter.

Expression of the neuronal kinase DCLK3 is reduced in Huntington’s disease. Galvan et al. report that DCLK3 silencing in the mouse striatum exacerbates the toxicity of mutant huntingtin, whereas DCLK3 overexpression is neuroprotective, and show that DCLK3 regulates the expression of many genes involved in transcription regulation and chromatin remodelling., The neurobiological functions of a number of kinases expressed in the brain are unknown. Here, we report new findings on DCLK3 (doublecortin like kinase 3), which is preferentially expressed in neurons in the striatum and dentate gyrus. Its function has never been investigated. DCLK3 expression is markedly reduced in Huntington’s disease. Recent data obtained in studies related to cancer suggest DCLK3 could have an anti-apoptotic effect. Thus, we hypothesized that early loss of DCLK3 in Huntington’s disease may render striatal neurons more susceptible to mutant huntingtin (mHtt). We discovered that DCLK3 silencing in the striatum of mice exacerbated the toxicity of an N-terminal fragment of mHtt. Conversely, overexpression of DCLK3 reduced neurodegeneration produced by mHtt. DCLK3 also produced beneficial effects on motor symptoms in a knock-in mouse model of Huntington’s disease. Using different mutants of DCLK3, we found that the kinase activity of the protein plays a key role in neuroprotection. To investigate the potential mechanisms underlying DCLK3 effects, we studied the transcriptional changes produced by the kinase domain in human striatal neurons in culture. Results show that DCLK3 regulates in a kinase-dependent manner the expression of many genes involved in transcription regulation and nucleosome/chromatin remodelling. Consistent with this, histological evaluation showed DCLK3 is present in the nucleus of striatal neurons and, protein-protein interaction experiments suggested that the kinase domain interacts with zinc finger proteins, including the transcriptional activator adaptor TADA3, a core component of the Spt-ada-Gcn5 acetyltransferase (SAGA) complex which links histone acetylation to the transcription machinery. Our novel findings suggest that the presence of DCLK3 in striatal neurons may play a key role in transcription regulation and chromatin remodelling in these brain cells, and show that reduced expression of the kinase in Huntington’s disease could render the striatum highly vulnerable to neurodegeneration.

The Angiotensin II type 2 receptor (AT 2R) promotes vasodilation by nitric oxide (NO) release from endothelial cells. However, the mechanisms underlying the AT 2R-induced stimulation of endothelial NO synthase (eNOS) is still not completely understood. Therefore, we investigated whether in addition to the known AT 2R-mediated phosphorylation of eNOS at Ser 1177, activation of phosphatases and dephosphorylation of eNOS at Tyr 657 and Thr 495 are also involved. Human aortic endothelial cells (HAEC) were stimulated with the AT 2R-agonist Compound 21 (C21) (1 μM) in the presence or absence of either PD123319 (10 μM; AT 2R antagonist), L-NG-Nitroarginine methyl ester (L-NAME) (10 μM; eNOS inhibitor), MK-2206 (100 nM; protein kinase B (Akt) inhibitor) sodium fluoride (NaF) (1 nM; serine/threonine phosphatase inhibitor) or sodium orthovanadate (Na 3VO 4) (10 nM; tyrosine phosphatase inhibitor). NO release was estimated by quantifying 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) fluorescence. The phosphorylation status of activating (eNOS-Ser 1177) or inhibitory eNOS residues (eNOS-Tyr 657, eNOS-Thr 495) was determined by Western blotting. Phosphorylation of Akt at Ser 473 was measured to estimate Akt activity. AT 2R stimulation significantly increased NO release from HAEC, which was blocked by PD123319, L-NAME and both phosphatase inhibitors. Intracellular calcium transients were not changed by C21. AT 2R stimulation resulted in phosphorylation of eNOS-Ser 1177 and dephosphorylation of eNOS-Tyr 657 and eNOS-Thr 495. Phosphorylation at eNOS-Ser 1177 was prevented by inhibition of Akt with MK-2206. From these data, we conclude that AT 2R stimulation in human endothelial cells increases eNOS activity through phosphorylation of activating eNOS residues (eNOS-Ser 1177) by Akt, and through dephosphorylation of inactivating eNOS residues (eNOS-Tyr 657, eNOS-Thr 495) by serine/threonine and tyrosine phosphatases, thus increasing NO release.

CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptides are secreted endogenous plant ligands that are sensed by receptor kinases (RKs) to convey environmental and developmental inputs. Typically, this involves an RK with narrow ligand specificity that signals together with a more promiscuous co-receptor. For most CLEs, biologically relevant (co-)receptors are unknown. The dimer of the receptor-like protein CLAVATA 2 (CLV2) and the pseudokinase CORYNE (CRN) conditions perception of so-called root-active CLE peptides, the exogenous application of which suppresses root growth by preventing protophloem formation in the meristem. clv2 as well as crn null mutants are resistant to root-active CLE peptides, possibly because CLV2-CRN promotes expression of their cognate receptors. Here, we have identified the CLE-RESISTANT RECEPTOR KINASE ( CLERK ) gene, which is required for full sensing of root-active CLE peptides in early developing protophloem. CLERK protein can be replaced by its close homologs, SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE (SARK) and NSP-INTERACTING KINASE 1 (NIK1). Yet neither CLERK nor NIK1 ectodomains interact biochemically with described CLE receptor ectodomains. Consistently, CLERK also acts genetically independently of CLV2-CRN We, thus, have discovered a novel hub for redundant CLE sensing in the root.

In terrestrial ecosystems most plant species live in mutualistic symbioses with nutrient-delivering arbuscular mycorrhizal (AM) fungi. Establishment of AM symbioses includes transient, intracellular formation of fungal feeding structures, the arbuscules. A plant-derived peri-arbuscular membrane (PAM) surrounds the arbuscules, mediating reciprocal nutrient exchange. Signaling at the PAM must be well coordinated to achieve this dynamic cellular intimacy. Here, we identify the PAM-specific Arbuscular Receptor-like Kinase 1 (ARK1) from maize and rice to condition sustained AM symbiosis. Mutation of rice ARK1 causes a significant reduction in vesicles, the fungal storage structures, and a concomitant reduction in overall root colonization by the AM fungus Rhizophagus irregularis. Arbuscules, although less frequent in the ark1 mutant, are morphologically normal. Co-cultivation with wild-type plants restores vesicle and spore formation, suggesting ARK1 function is required for the completion of the fungal life-cycle, thereby defining a functional stage, post arbuscule development., Nature Communications, 9 (1), ISSN:2041-1723

Phosphorylation of SPAT-1/Bora by Cdk1 enhances Plk1 phosphorylation by Aurora A and promotes entry into mitosis in C. elegans., The molecular mechanisms governing mitotic entry during animal development are incompletely understood. Here, we show that the mitotic kinase CDK-1 phosphorylates Suppressor of Par-Two 1 (SPAT-1)/Bora to regulate its interaction with PLK-1 and to trigger mitotic entry in early Caenorhabditis elegans embryos. Embryos expressing a SPAT-1 version that is nonphosphorylatable by CDK-1 and that is defective in PLK-1 binding in vitro present delays in mitotic entry, mimicking embryos lacking SPAT-1 or PLK-1 functions. We further show that phospho–SPAT-1 activates PLK-1 by triggering phosphorylation on its activator T loop in vitro by Aurora A. Likewise, we show that phosphorylation of human Bora by Cdk1 promotes phosphorylation of human Plk1 by Aurora A, suggesting that this mechanism is conserved in humans. Our results suggest that CDK-1 activates PLK-1 via SPAT-1 phosphorylation to promote entry into mitosis. We propose the existence of a positive feedback loop that connects Cdk1 and Plk1 activation to ensure a robust control of mitotic entry and cell division timing.

The Hippo signaling pathway has recently moved to center stage in cardiac research because of its key role in cardiomyocyte proliferation and regeneration of the embryonic and newborn heart. However, its role in the adult heart is incompletely understood. We investigate here the role of mammalian Ste20-like kinase 2 (Mst2), one of the central regulators of this pathway. Mst2(-/-) mice showed no alteration in cardiomyocyte proliferation. However, Mst2(-/-) mice exhibited a significant reduction of hypertrophy and fibrosis in response to pressure overload. Consistently, overexpression of MST2 in neonatal rat cardiomyocytes significantly enhanced phenylephrine-induced cellular hypertrophy. Mechanistically, Mst2 positively modulated the prohypertrophic Raf1-ERK1/2 pathway. However, activation of the downstream effectors of the Hippo pathway (Yes-associated protein) was not affected by Mst2 ablation. An initial genetic study in mitral valve prolapse patients revealed an association between a polymorphism in the human MST2 gene and adverse cardiac remodeling. These results reveal a novel role of Mst2 in stress-dependent cardiac hypertrophy and remodeling in the adult mouse and likely human heart.

Purpose: To investigate the applicability of serine/arginine-rich protein kinase (SRPK)-specific inhibitor, SRPIN340, for attenuation of choroidal neovascularization (CNV) formation using a mouse model. Methods: Laser photocoagulation was performed to induce CNV in C57BL/6J mice, followed by intravitreal injection of SRPIN340 or vehicle. Seven days after the treatment, the CNV size was evaluated using a flatmount technique. Protein levels of vascular endothelial growth factor (VEGF) and inflammation-associated molecules, such as monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1, in the retinal pigment epithelium-choroid complex were measured with enzyme-linked immunosorbent assay. Expression levels of total Vegf, exon 8a-containing Vegf isoforms, and F4/80 (a specific marker for macrophage) were assessed using real-time PCR. Results: SRPIN340 inhibited CNV formation in a dose-dependent manner. Compared with the vehicle, SRPIN340 significantly decreased the protein levels of VEGF, MCP-1, ICAM-1, and consequently inhibited macrophage infiltration. Furthermore, SRPIN340 suppressed the gene expression levels of total Vegf and exon 8a-containing Vegf isoforms. Conclusions: SRPIN340, a specific inhibitor of SRPK, suppressed Vegf expression and attenuated CNV formation. Our data suggest the possibility that SRPIN340 is applicable for neovascular age-related macular degeneration as a novel chemical therapeutics.

Division site positioning is critical for both symmetric and asymmetric cell divisions. In many organisms, positive and negative signals cooperate to position the contractile actin ring for cytokinesis. In rod-shaped fission yeast Schizosaccharomyces pombe cells, division at midcell is achieved through positive Mid1/anillin-dependent signaling emanating from the central nucleus and negative signals from the dual-specificity tyrosine phosphorylation-regulated kinase family kinase Pom1 at the cell poles. In this study, we show that Pom1 directly phosphorylates the F-BAR protein Cdc15, a central component of the cytokinetic ring. Pom1-dependent phosphorylation blocks Cdc15 binding to paxillin Pxl1 and C2 domain protein Fic1 and enhances Cdc15 dynamics. This promotes ring sliding from cell poles, which prevents septum assembly at the ends of cells with a displaced nucleus or lacking Mid1. Pom1 also slows down ring constriction. These results indicate that a strong negative signal from the Pom1 kinase at cell poles converts Cdc15 to its closed state, destabilizes the actomyosin ring, and thus promotes medial septation.

Regulated protein-protein interactions are critical for cell signaling, differentiation, and development. For the study of dynamic regulation of protein interactions in vivo, there is a need for techniques that can yield time-resolved information and probe multiple protein binding partners simultaneously, using small amounts of starting material. Here we describe a single-cell protein interaction assay. Single-cell lysates are generated at defined time points and analyzed using single-molecule pull-down, yielding information about dynamic protein complex regulation in vivo. We established the utility of this approach by studying PAR polarity proteins, which mediate polarization of many animal cell types. We uncovered striking regulation of PAR complex composition and stoichiometry during Caenorhabditis elegans zygote polarization, which takes place in less than 20 min. PAR complex dynamics are linked to the cell cycle by Polo-like kinase 1 and govern the movement of PAR proteins to establish polarity. Our results demonstrate an approach to study dynamic biochemical events in vivo.

[EN] The proper maintenance of potassium homeostasis is crucial for cell viability. Among the major determinants of potassium uptake in the model organism Saccharomyces cerevisiae are the Trk1 high affinity potassium transporter and the functionally redundant Hal4 (Sat4) and Hal5 protein kinases. These kinases are required for the plasma membrane accumulation of not only Trk1, but also several nutrient permeases. Here, we show that overexpression of the Target of Rapamycin Complex 1 (TORC1) effector NPR1 improves hal4 hal5 growth defects by stabilizing nutrient permeases at the plasma membrane. We subsequently found that internal potassium levels and TORC1 activity are linked. Specifically, growth under limiting potassium alters the activities of Npr1 and another TORC1 effector kinase, Sch9; hal4 hal5 and trk1 trk2 mutants display hypersensitivity to rapamycin; and, reciprocally, TORC1 inhibition reduces potassium accumulation. Our results demonstrate that in addition to carbon and nitrogen, TORC1 also responds to and regulates potassium fluxes., This work was supported by Spanish Ministry of Science and Innovation (Madrid, Spain) Grant BFU2011-30197-C03-03 (to L. Y.). The authors declare that they have no conflicts of interest with the contents of this article. Supported by a pre-doctoral fellowship from the Spanish Research Council (Consejo Superior de Investigaciones Cientificas). Supported by the Swiss National Science Foundation, the European Research Council, and the Canton of Geneva.

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit. We thank Geert Kops (UMC, Utrecht, The Netherlands), Thomas Maresca (University of Massachu-setts Amherst, USA) and Christian Lehner (University of Zurich, Switzerland) for antibodies, con-structs and fly stocks. We thank Carla Sofia Lopes (i3S, IBMC, University of Porto) and all the members of the Sunkel laboratory for critical and helpful discussions. This article is a result of the project Norte-01-0145-FEDER-000029 - Advancing Cancer Research: From basic knowledge to application, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). MO is supported by a fellowship from the GABBA PhD program from the University of Porto, PD/BD/105746/2014. JB is supported by an FCT PhD grant SFRH/BD/87871/2012. CC is sup-ported by an FCT investigator position and funding (IF/01755/2014). HM is funded by PRECISE and CODECHECK grants from the European Research Council, FLAD Life Science 2020, and the Louis-Jeantet Young Investigator Career Award.

International audience; V(D)J recombination is essential for generating a diverse array of B and T cell receptors that can recognize and combat foreign antigens. As with any recombination event, tight control is essential to prevent the occurrence of genetic anomalies that drive cellular transformation. One important aspect of regulation is directed targeting of the RAG recombinase. Indeed, RAG accumulates at the 3' end of individual antigen receptor loci poised for rearrangement; however, it is not known whether focal binding is involved in regulating cleavage, and what mechanisms lead to enrichment of RAG in this region. Here, we show that monoallelic looping out of the 3' end of the T cell receptor α (Tcra) locus, coupled with transcription and increased chromatin/nuclear accessibility, is linked to focal RAG binding and ATM-mediated regulation of monoallelic cleavage on looped-out 3' regions. Our data identify higher-order loop formation as a key determinant of directed RAG targeting and the maintenance of genome stability.

UBXN-2, a substrate adaptor of the AAA ATPase CDC-48/p97, is required to coordinate centrosome maturation timing with mitosis., Coordination of cell cycle events in space and time is crucial to achieve a successful cell division. Here, we demonstrate that UBXN-2, a substrate adaptor of the AAA ATPase Cdc48/p97, is required to coordinate centrosome maturation timing with mitosis. In UBXN-2–depleted Caenorhabditis elegans embryos, centrosomes recruited more AIR-1 (Aurora A), matured precociously, and alignment of the mitotic spindle with the axis of polarity was impaired. UBXN-2 and CDC-48 coimmunoprecipitated with AIR-1 and the spindle alignment defect was partially rescued by co-depleting AIR-1, indicating that UBXN-2 controls these processes via AIR-1. Similarly, depletion in human cells of the UBXN-2 orthologues p37/p47 resulted in an accumulation of Aurora A at centrosomes and a delay in centrosome separation. The latter defect was also rescued by inhibiting Aurora A. We therefore postulate that the role of this adaptor in cell cycle regulation is conserved.

The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45‐MCM‐GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin‐dependent kinase (CDK) and Dbf4‐dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK‐dependent manner. Sld3 binds specifically to DDK‐phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho‐MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK‐independent replication. Thus, Sld3 is an essential “reader” of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase.

Glucose-induced insulin secretion from pancreatic β-cells depends on mitochondrial activation. In the organelle, glucose-derived pyruvate is metabolised along the oxidative and anaplerotic pathway to generate downstream signals leading to insulin granule exocytosis. Entry into the oxidative pathway is catalysed by pyruvate dehydrogenase (PDH) and controlled in part by phosphorylation of the PDH E1α subunit blocking enzyme activity. We find that glucose but not other nutrient secretagogues induce PDH E1α phosphorylation in INS-1E cells and rat islets. INS-1E cells and primary β-cells express pyruvate dehydrogenase kinase (PDK) 1, 2 and 3, which mediate the observed phosphorylation. In INS-1E cells, suppression of the two main isoforms, PDK1 and PDK3, almost completely prevented PDH E1α phosphorylation. Under basal glucose conditions, phosphorylation was barely detectable and therefore the enzyme almost fully active (90% of maximal). During glucose stimulation, PDH is only partially inhibited (to 78% of maximal). Preventing PDH phosphorylation in situ after suppression of PDK1, 2 and 3 neither enhanced pyruvate oxidation nor insulin secretion. In conclusion, although glucose stimulates E1α phosphorylation and therefore inhibits PDH activity, this control mechanism by itself does not alter metabolism-secretion coupling in INS-1E clonal β-cells.

OBJECTIVE Type 2 diabetes is characterized by insulin resistance with a relative deficiency in insulin secretion. This study explored the potential communication between insulin-resistant human skeletal muscle and primary (human and rat) β-cells. RESEARCH DESIGN AND METHODS Human skeletal muscle cells were cultured for up to 24 h with tumor necrosis factor (TNF)-α to induce insulin resistance, and mRNA expression for cytokines was analyzed and compared with controls (without TNF-α). Conditioned media were collected and candidate cytokines were measured by antibody array. Human and rat primary β-cells were used to explore the impact of exposure to conditioned media for 24 h on apoptosis, proliferation, short-term insulin secretion, and key signaling protein phosphorylation and expression. RESULTS Human myotubes express and release a different panel of myokines depending on their insulin sensitivity, with each panel exerting differential effects on β-cells. Conditioned medium from control myotubes increased proliferation and glucose-stimulated insulin secretion (GSIS) from primary β-cells, whereas conditioned medium from TNF-α–treated insulin-resistant myotubes (TMs) exerted detrimental effects that were either independent (increased apoptosis and decreased proliferation) or dependent on the presence of TNF-α in TM (blunted GSIS). Knockdown of β-cell mitogen-activated protein 4 kinase 4 prevented these effects. Glucagon-like peptide 1 protected β-cells against decreased proliferation and apoptosis evoked by TMs, while interleukin-1 receptor antagonist only prevented the latter. CONCLUSIONS Taken together, these data suggest a possible new route of communication between skeletal muscle and β-cells that is modulated by insulin resistance and could contribute to normal β-cell functional mass in healthy subjects, as well as the decrease seen in type 2 diabetes.

Location, Location, Location Cell division is orchestrated by a complex signaling pathway that ensures the correct segregation of newly replicated chromosomes to the two daughter cells. The pathway is controlled in part by restricting the activity of critical regulators to specific subcellular locations. For example, the chromosomal passenger complex (CPC) is recruited to chromosomes during mitosis where it oversees kinetochore activity and cytokinesis (see Perspective by Musacchio ). Wang et al. (p. 231 , published online 12 August), Kelly et al. (p. 235 , published online 12 August), and Yamagishi et al. (p. 239 ) now show that the phosphorylation of the chromatin protein, histone H3, acts to bring the CPC to chromosomes, thereby activating its aurora B kinase subunit. The Survivin subunit of CPC binds specifically to phosphorylated H3, with the phosphorylation at centromeres being carried out by the mitosis-specific kinase, haspin. Furthermore, Bub1 phosphorylation of histone H2A recruits shugoshin, a centromeric CPC adapter. Thus, these two histone marks in combination define the inner centromere.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor that, by virtue of its resistance to chemotherapy and radiotherapy, is currently incurable. Identification of molecules whose targeting may eliminate GBM cells and/or sensitize glioblastoma cells to cytotoxic drugs is therefore urgently needed. CD44 is a major cell surface hyaluronan receptor and cancer stem cell marker that has been implicated in the progression of a variety of cancer types. However, the major downstream signaling pathways that mediate its protumor effects and the role of CD44 in the progression and chemoresponse of GBM have not been established. Here we show that CD44 is upregulated in GBM and that its depletion blocks GBM growth and sensitizes GBM cells to cytotoxic drugs in vivo. Consistent with this observation, CD44 antagonists potently inhibit glioma growth in preclinical mouse models. We provide the first evidence that CD44 functions upstream of the mammalian Hippo signaling pathway and that CD44 promotes tumor cell resistance to reactive oxygen species–induced and cytotoxic agent–induced stress by attenuating activation of the Hippo signaling pathway. Together, our results identify CD44 as a prime therapeutic target for GBM, establish potent antiglioma efficacy of CD44 antagonists, uncover a novel CD44 signaling pathway, and provide a first mechanistic explanation as to how upregulation of CD44 may constitute a key event in leading to cancer cell resistance to stresses of different origins. Finally, our results provide a rational explanation for the observation that functional inhibition of CD44 augments the efficacy of chemotherapy and radiation therapy. Cancer Res; 70(6); 2455–64

Narcolepsy is a sleep disorder characterized by excessive daytime sleepiness and attacks of muscle atonia triggered by strong emotions (cataplexy). Narcolepsy is caused by hypocretin (orexin) deficiency, paralleled by a dramatic loss in hypothalamic hypocretin-producing neurons. It is believed that narcolepsy is an autoimmune disorder, although definitive proof of this, such as the presence of autoantibodies, is still lacking. We engineered a transgenic mouse model to identify peptides enriched within hypocretin-producing neurons that could serve as potential autoimmune targets. Initial analysis indicated that the transcript encoding Tribbles homolog 2 (Trib2), previously identified as an autoantigen in autoimmune uveitis, was enriched in hypocretin neurons in these mice. ELISA analysis showed that sera from narcolepsy patients with cataplexy had higher Trib2-specific antibody titers compared with either normal controls or patients with idiopathic hypersomnia, multiple sclerosis, or other inflammatory neurological disorders. Trib2-specific antibody titers were highest early after narcolepsy onset, sharply decreased within 2-3 years, and then stabilized at levels substantially higher than that of controls for up to 30 years. High Trib2-specific antibody titers correlated with the severity of cataptexy. Serum of a patient showed specific immunoreactivity with over 86% of hypocretin neurons in the mouse hypothalamus. Thus, we have identified reactive autoantibodies in human narcolepsy, providing evidence that narcolepsy is an autoimmune disorder.

Heterochromatin protein 1 (HP1) has an essential role in heterochromatin formation and mitotic progression through its interaction with various proteins. We have identified a unique HP1alpha-binding protein, POGZ (pogo transposable element-derived protein with zinc finger domain), using an advanced proteomics approach. Proteins generally interact with HP1 through a PxVxL (where x is any amino-acid residue) motif; however, POGZ was found to bind to HP1alpha through a zinc-finger-like motif. Binding by POGZ, mediated through its zinc-finger-like motif, competed with PxVxL proteins and destabilized the HP1alpha-chromatin interaction. Depletion experiments confirmed that the POGZ HP1-binding domain is essential for normal mitotic progression and dissociation of HP1alpha from mitotic chromosome arms. Furthermore, POGZ is required for the correct activation and dissociation of Aurora B kinase from chromosome arms during M phase. These results reveal POGZ as an essential protein that links HP1alpha dissociation with Aurora B kinase activation during mitosis.

Obesity and type 2 diabetes present partially overlapping phenotypes with systemic inflammation as a common feature, raising the hypothesis that elevated cytokine levels may contribute to peripheral insulin resistance as well as the decreased beta cell functional mass observed in type 2 diabetes. In healthy humans, TNF-alpha infusion induces skeletal muscle insulin resistance. We now explore the impact of TNF-alpha on primary beta cell function and the underlying signaling pathways. Human and rat primary beta cells were sorted by FACS and cultured for 24 h +/- 20 ng/ml TNF-alpha to explore the impact on apoptosis, proliferation, and short-term insulin secretion (1 h, 2.8 mm glucose followed by 1 h, 16.7 mm glucose at the end of the 24-h culture period) as well as key signaling protein phosphorylation and expression. Prior exposure to TNF-alpha for 24 h inhibits glucose-stimulated insulin secretion from primary beta cells. This is associated with a decrease in glucose-stimulated phosphorylation of key proteins in the insulin signaling pathway including Akt, AS160, and other Akt substrates, ERK as well as the insulin receptor. Strikingly, TNF-alpha treatment decreased IRS-2 protein level by 46 +/- 7% versus control, although mRNA expression was unchanged. While TNF-alpha treatment increased MAP4K4 mRNA expression by 33 +/- 5%, knockdown of MAP4K4 by siRNA-protected beta cells against the detrimental effects of TNF-alpha on both insulin secretion and signaling. We thus identify MAP4K4 as a key upstream mediator of TNF-alpha action on the beta cell, making it a potential therapeutic target for preservation of beta cell function in type 2 diabetes.

Diverse cargo molecules (i.e., receptors and ligand/receptor complexes) are taken into the cell by clathrin-mediated endocytosis (CME) utilizing a core machinery consisting of cargo-specific adaptors, clathrin and the GTPase dynamin. Numerous endocytic accessory proteins are also required, but their differential roles and functional hierarchy during CME are not yet understood. Here, we used a combination of quantitative live-cell imaging by total internal reflection fluorescence microscopy (TIR-FM), and decomposition of the lifetime distributions of clathrin-coated pits (CCPs) to measure independent aspects of CCP dynamics, including the turnover of abortive and productive CCP species and their relative contributions. Capitalizing on the sensitivity of this assay, we have examined the effects of specific siRNA-mediated depletion of endocytic accessory proteins on CME progression. Of the 12 endocytic accessory proteins examined, we observed seven qualitatively different phenotypes upon protein depletion. From this data we derive a temporal hierarchy of protein function during early steps of CME. Our results support the idea that a subset of accessory proteins, which mediate coat assembly, membrane curvature, and cargo selection, can provide input into an endocytic restriction point/checkpoint mechanism that monitors CCP maturation.

Ribosome biogenesis drives cell growth, and the large transcriptional output underlying this process is tightly regulated. The Target of Rapamycin (TOR) kinase is part of a highly conserved signaling pathway linking nutritional and stress signals to regulation of ribosomal protein (RP) and ribosome biogenesis (Ribi) gene transcription. In Saccharomyces cerevisiae, one of the downstream effectors of TOR is Sfp1, a transcriptional activator that regulates both RP and Ribi genes. Here, we report that Sfp1 interacts directly with TOR complex 1 (TORC1) in a rapamycin-regulated manner, and that phosphorylation of Sfp1 by this kinase complex regulates its function. Sfp1, in turn, negatively regulates TORC1 phosphorylation of Sch9, another key TORC1 target that acts in parallel with Sfp1, revealing a feedback mechanism controlling the activity of these proteins. Finally, we show that the Sfp1-interacting protein Mrs6, a Rab escort protein involved in membrane trafficking, regulates both Sfp1 nuclear localization and TORC1 signaling.

The BRCA1-associated ring domain protein 1 (BARD1) interacts with BRCA1 via its RING finger domain. The BARD1-BRCA1 complex participates in DNA repair, cell cycle control, genomic stability, and mitotic spindle formation through its E3 ubiquitin ligase activity. Cancer cells express several BARD1 protein isoforms, including the RING finger–deficient variant BARD1β. Here, we show that BARD1 has BRCA1-dependent and BRCA1-independent functions in mitosis. BARD1, but not BRCA1, localizes to the midbody at telophase and cytokinesis, where it colocalizes with Aurora B. The 97-kDa full-length (FL) BARD1 coimmunoprecipates with BRCA1, but the 82-kDa BARD1β coimmunoprecipitates with Aurora B and BRCA2. We used selective small interfering RNAs to distinguish the functions of FL BARD1 and BARD1β. Depletion of FL BARD1 had only minor effects on cell growth and did not abolish midbody localization of BARD1 staining, but resulted in massive up-regulation of Aurora B. In contrast, suppression of FL BARD1 and BARD1β led to growth arrest and correlated with various mitotic defects and disappearance of midbody localization of BARD1 staining. Our data suggest a novel function of FL BARD1 in Aurora B ubiquitination and degradation, opposing a proproliferative function of BARD1β in scaffolding Aurora B and BRCA2. Thus, loss of FL BARD1 and up-regulation of Aurora B, as observed in cancer cells, can be explained by an imbalance of FL BARD1 and BARD1β. [Cancer Res 2009;69(3):1125–34]

A new technique illuminates differential H3S10 phosphorylation dynamics in normal and cancer cells; spatial and temporal regulation of this process by aurora B kinase is required for accurate chromosome segregation., Posttranslational histone modifications regulate both gene expression and genome integrity. Despite the dynamic nature of these modifications, appropriate real-time monitoring systems are lacking. In this study, we developed a method to visualize histone modifications in living somatic cells and preimplantation embryos by loading fluorescently labeled specific Fab antibody fragments. The technique was used to study histone H3 Ser10 (H3S10) phosphorylation, which occurs during chromosome condensation in mitosis mediated by the aurora B kinase. In aneuploid cancer cells that frequently missegregate chromosomes, H3S10 is phosphorylated just before the chromosomes condense, whereas aurora B already accumulates in nuclei during S phase. In contrast, in nontransformed cells, phosphorylated H3S10 foci appear for a few hours during interphase, and transient exposure to an aurora B–selective inhibitor during this period induces chromosome missegregation. These results suggest that, during interphase, moderate aurora B activity or H3S10 phosphorylation is required for accurate chromosome segregation. Visualizing histone modifications in living cells will facilitate future epigenetic and cell regulation studies.

SR proteins are non-snRNP splicing factors harbouring a domain rich in Arg-Ser repeats, which are extensively phosphorylated by several kinases. We performed a comparative study of different SR kinases, including SRPK, Clk, PRP4 and DYRK, and found that only Clks efficiently altered 5' splice site selection of Adenovirus E1A. The phosphorylation state of SR proteins was examined using a phospho-SR specific antibody mAb1H4 and a 75 kDa protein was most evidently hyperphosphorylated by Clks. Administration of TG003, a specific inhibitor for the Clk family members, specifically and rapidly induced dephosphorylation of 75 kDa SR protein. Imaging with mRFP-SRp75 in living cells revealed that its nuclear distribution was rapidly altered upon inhibition of the Clk activity by TG003. Co-transfection experiments demonstrated that HA-tagged SRp75 was hyperphosphorylated by Clk family members, but not by other SR kinases. These results indicate that Clks specifically hyperphosphorylate SRp75. Furthermore, SRp75 over-expression promoted the selection of 12S 5' splice site in E1A pre-mRNA, which is stimulated by co-expression of Clks. These results suggest that the specific combination of SR protein and SR kinase plays a distinct role in alternative splicing through dynamic balance of phosphorylation.

The stimulation of postprandial K+ clearance involves aldosterone-independent and -dependent mechanisms. In this context, serum- and glucocorticoid-induced kinase (SGK)1, a ubiquitously expressed kinase, is one of the primary aldosterone-induced proteins in the aldosterone-sensitive distal nephron. Germline inactivation of SGK1 suggests that this kinase is fundamental for K+ excretion under conditions of K+ load, but the specific role of renal SGK1 remains elusive. To avoid compensatory mechanisms that may occur during nephrogenesis, we used inducible, nephron-specific Sgk1 Pax8/ LC1 mice to assess the role of renal tubular SGK1 in K+ regulation. Under a standard diet, these animals exhibited normal K+ handling. When challenged by a high-K+ diet, they developed severe hyperkalemia accompanied by a defect in K+ excretion. Molecular analysis revealed reduced neural precursor cell expressed developmentally downregulated protein (NEDD)4-2 phosphorylation and total expression. γ-Epithelial Na+ channel (ENaC) expression and α/γENaC proteolytic processing were also decreased in mutant mice. Moreover, with no lysine kinase (WNK)1, which displayed in control mice punctuate staining in the distal convoluted tubule and diffuse distribution in the connecting tubule/cortical colleting duct, was diffused in the distal convoluted tubule and less expressed in the connecting tubule/collecting duct of Sgk Pax8/ LC1 mice. Moreover, Ste20-related proline/alanine-rich kinase phosphorylation, and Na+-Cl− cotransporter phosphorylation/apical localization were reduced in mutant mice. Consistent with the altered WNK1 expression, increased renal outer medullary K+ channel apical localization was observed. In conclusion, our data suggest that renal tubular SGK1 is important in the regulation of K+ excretion via the control of NEDD4-2, WNK1, and ENaC.

International audience; Polo-like kinase 1 (Plk1) is an important mitotic kinase that is crucial for entry into mitosis after recovery from DNA damage-induced cell cycle arrest. Plk1 activation is promoted by the conserved protein Bora (SPAT-1 in C. elegans), which stimulates the phosphorylation of a conserved residue in the activation loop by the Aurora A kinase. In a recent article published in Cell Reports, we show that the master mitotic kinase Cdk1 contributes to Plk1 activation through SPAT-1/Bora phosphorylation. We identified 3 conserved Sp/Tp residues that are located in the N-terminal, most conserved part, of SPAT-1/Bora. Phosphorylation of these sites by Cdk1 is essential for Plk1 function in mitotic entry in C. elegans embryos and during DNA damage checkpoint recovery in mammalian cells. Here, using an untargeted Förster Resonance Energy Transfer (FRET) biosensor to monitor Plk1 activation, we provide additional experimental evidence supporting the importance of these phosphorylation sites for Plk1 activation and subsequent mitotic entry after DNA damage. We also briefly discuss the mechanism of Plk1 activation and the potential role of Bora phosphorylation by Cdk1 in this process. As Plk1 is overexpressed in cancer cells and this correlates with poor prognosis, understanding how Bora contributes to Plk1 activation is paramount for the development of innovative therapeutical approaches.

During division of metazoan cells, the nucleus disassembles to allow chromosome segregation, and then reforms in each daughter cell. Reformation of the nucleus involves chromatin decondensation and assembly of the double- membrane nuclear envelope around the chromatin; however, regulation of the process is still poorly understood(1,2). In vitro, nucleus formation requires p97 ( ref. 3), a hexameric ATPase implicated in membrane fusion and ubiquitin- dependent processes(4,5). However, the role and relevance of p97 in nucleus formation have remained controversial. Here we show that p97 stimulates nucleus reformation by inactivating the chromatin- associated kinase Aurora B. During mitosis, Aurora B inhibits nucleus reformation by preventing chromosome decondensation and formation of the nuclear envelope membrane. During exit from mitosis, p97 binds to Aurora B after its ubiquitylation and extracts it from chromatin. This leads to inactivation of Aurora B on chromatin, thus allowing chromatin decondensation and nuclear envelope formation. These data reveal an essential pathway that regulates reformation of the nucleus after mitosis and defines ubiquitin- dependent protein extraction as a common mechanism of Cdc48/p97 activity also during nucleus formation.

The Target of Rapamycin (TOR) protein is a Ser/Thr kinase that functions in two distinct multiprotein complexes: TORC1 and TORC2. These conserved complexes regulate many different aspects of cell growth in response to intracellular and extracellular cues. Here we report that the AGC kinase Sch9 is a substrate of yeast TORC1. Six amino acids in the C terminus of Sch9 are directly phosphorylated by TORC1. Phosphorylation of these residues is lost upon rapamycin treatment as well as carbon or nitrogen starvation and transiently reduced following application of osmotic, oxidative, or thermal stress. TORC1-dependent phosphorylation is required for Sch9 activity, and replacement of residues phosphorylated by TORC1 with Asp/Glu renders Sch9 activity TORC1 independent. Sch9 is required for TORC1 to properly regulate ribosome biogenesis, translation initiation, and entry into G0 phase, but not expression of Gln3-dependent genes. Our results suggest that Sch9 functions analogously to the mammalian TORC1 substrate S6K1 rather than the mTORC2 substrate PKB/Akt.

Phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome downstream of anabolic signals has long been assumed to promote protein synthesis. Both target of rapamycin complexes regulate this modification in yeast, but the use of ribosome profiling shows no role for Rps6 phosphorylation in mRNA translation., Nutrient-sensitive phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome is highly conserved. However, despite four decades of research, the functional consequences of this modification remain unknown. Revisiting this enigma in Saccharomyces cerevisiae, we found that the regulation of Rps6 phosphorylation on Ser-232 and Ser-233 is mediated by both TOR complex 1 (TORC1) and TORC2. TORC1 regulates phosphorylation of both sites via the poorly characterized AGC-family kinase Ypk3 and the PP1 phosphatase Glc7, whereas TORC2 regulates phosphorylation of only the N-terminal phosphosite via Ypk1. Cells expressing a nonphosphorylatable variant of Rps6 display a reduced growth rate and a 40S biogenesis defect, but these phenotypes are not observed in cells in which Rps6 kinase activity is compromised. Furthermore, using polysome profiling and ribosome profiling, we failed to uncover a role of Rps6 phosphorylation in either global translation or translation of individual mRNAs. Taking the results together, this work depicts the signaling cascades orchestrating Rps6 phosphorylation in budding yeast, challenges the notion that Rps6 phosphorylation plays a role in translation, and demonstrates that observations made with Rps6 knock-ins must be interpreted cautiously.

Primary cultures of bovine microvascular endothelial cells (BME) isolated from the adrenal cortex, are commonly used to study vascular endothelium, but have a limited life span. To circumvent these limitations, we have immortalized BME cells with either simian virus 40 (SV40) or with a retrovirus containing the coding region of human telomerase reverse transcriptase (hTERT), and have investigated whether the clonal populations obtained, maintain differentiated properties characteristic of microvascular endothelium. Immortalized cells were characterized for maintenance of typical endothelial morphology, marker expression, and functional characteristics including uptake of Acetylated low-density lipoprotein (Ac-LDL), capillary-like tube formation in three-dimensional collagen gels, as well as metalloproteinase (MMP) and plasminogen activator (PA)-mediated extracellular proteolysis. Whilst immortalization of BME cells with SV40 was associated with loss of endothelial-specific properties, hTERT-BME exhibited an endothelial phenotype similar to that of wild-type endothelial cells. Specifically, they showed a typical cobblestone morphology, were contact-inhibited, expressed endothelial cell-specific markers (e.g., CD31, vWF) and both fibroblast growth factor receptor 1 (FGFR-1) and vascular endothelial growth factor receptor-2 (VEGFR-2). In addition, they expressed receptors for LDL. Importantly, when grown on collagen gels, hTERT-BME cells underwent MMP-dependent tube-like structure formation in response to VEGFR-2 activation. In a collagen gel sandwich assay, hTERT-BME formed tubular structures in the absence of exogenously added angiogenic cytokines. Sustained tube formation was induced by VEGF-A alone or in combination with FGF-2. From 17 sub-clones that displayed a non-transformed phenotype, a high proliferative capacity and tubulogenic properties in three-dimensional collagen gels, we isolated two distinct subpopulations that display a highly specific response to VEGF-A or to FGF-2. We have generated hTERT-BME cells that maintain endothelial-specific properties and function and have isolated clones that respond differentially to VEGF-A or FGF-2. These immortalized cell lines will facilitate the study of endothelial cell biology.

The drugs troglitazone and metformin are used to reduce the degree of insulin resistance in type 2 diabetes. Both compounds act through different mechanisms which might include opposing effects on the production of adiponectin, an insulin-sensitizer released by adipocytes. This study compared the effects of troglitazone and metformin on adiponectin production by 3T3-L1 adipocytes during 48 h culture. Troglitazone increased adiponectin mRNA and protein expression as well as release, whereas metformin did not affect transcription but reduced protein expression and release. The effect of metformin was also seen with phenformin, and with low-glucose culture, all conditions with a reduced mitochondrial activity and an activated AMP activated protein kinase (AMPK). Addition of the AMPK activator 5-aminoimidazole-4-carboxamide-riboside (AICAR) also caused a decrease in adiponectin protein expression. These data indicate that metformin and troglitazone exert opposing effects on adiponectin expression and release by differentiated 3T3-L1 adipocytes. The metformin-induced suppression involves an activation of AMP activated protein kinase.

Peutz-Jeghers syndrome (PJS) is caused by germline mutations in the LKB1 gene, which encodes a serine-threonine kinase that regulates cell proliferation and polarity. This autosomal dominant disorder is characterized by mucocutaneous melanin pigmentation, multiple gastrointestinal hamartomatous polyposis and an increased risk of developing various neoplasms. To understand the molecular pathogenesis of PJS phenotypes, we used microarrays to analyze gene expression profiles in proliferating HeLa cells transduced with lentiviral vectors expressing wild type or mutant LKB1 proteins. We show that gene expression is differentially affected by mutations that impair the kinase activity (K78I) or alter the cellular localization of the LKB1 protein. However, both mutations abrogate the ability of LKB1 to up-regulate the transcription of several genes involved in Wnt signaling, including DKK3, WNT5B and FZD2. In addition-and in contrast to the wild type protein-these LKB1 mutants fail to activate the GSK-3beta kinase, which otherwise phosphorylates beta-catenin. The increase in beta-catenin phosphorylation that occurs upon expression of wild-type LKB1 results in transcriptional inhibition of a canonical Wnt reporter gene. This suggests that pathogenic LKB1 mutations that lead to activation of the Wnt/beta-catenin pathway could contribute to the cancer predisposition of PJS patients.

In an attempt to identify phytochemicals contributing to the well-documented preventive effect of plant-based diets on cancer incidence and mortality, we have previously shown that certain flavonoids inhibit in vitro angiogenesis. Here, we show that the flavonoid luteolin inhibited tumor growth and angiogenesis in a murine xenograft model. Furthermore, luteolin inhibited vascular endothelial growth factor (VEGF)-induced in vivo angiogenesis in the rabbit corneal assay. In agreement, luteolin inhibited both VEGF-induced survival and proliferation of human umbilical vein endothelial cells (HUVECs) with an IC50 of about 5 μmol/L. Luteolin inhibited VEGF-induced phosphatidylinositol 3′-kinase (PI3K) activity in HUVECs, and this inhibition was critical for both the antisurvival and antimitotic affects of the compound. Indeed, luteolin abolished VEGF-induced activation of Akt, a downstream target of PI3K conveying both survival and mitotic downstream signals. Because overexpression of a constitutively active form of Akt rescued HUVECs only from the antisurvival effects of luteolin, the result indicated that luteolin targeted mainly the survival signals of the PI3K/Akt pathway. With regard to its antimitotic activity, luteolin inhibited VEGF-induced phosphorylation of p70 S6 kinase (S6K), a downstream effector of PI3K responsible for G1 progression. Indeed, VEGF-induced proliferation of HUVECs was sensitive to rapamycin, an inhibitor of p70 S6K activation. Surprisingly, luteolin did not affect VEGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases, a pathway that is considered important for the mitotic effects of VEGF. Thus, blockade of PI3K by luteolin was responsible for the inhibitory effects of the compound on VEGF-induced survival and proliferation of HUVECs. The antisurvival effects of luteolin were mediated via blockage of PI3K/Akt-dependent pathways, whereas inhibition of the PI3K/p70 S6K pathway mediated the antimitotic effects of the compound.

Brassinosteroids (BRs) are plant steroid hormones that control many aspects of plant growth and development. BRs are perceived at the cell-surface by the plasma membrane-localized receptor complex composed of the receptor kinase BRI1 and its co-receptor BAK1. Here we show that BRI1 is post-translationally modified by K63 polyubiquitin chains in vivo. Artificially ubiquitinated BRI1 is recognized at the trans-Golgi Network/Early Endosomes (TGN/EE) and rapidly routed for vacuolar degradation. Mass spectrometry analyses identified residue K866 as an in vivo ubiquitination target in BRI1 involved in the negative regulation of BRI1. Model prediction revealed several redundant ubiquitination sites required for the endosomal sorting and vacuolar targeting of BRI1. Using total internal reflection fluorescence microscopy (TIRF), we also uncovered a role for BRI1 ubiquitination in promoting internalization from the cell-surface. Finally, we demonstrate that the control of BRI1 protein dynamics by ubiquitination is a fundamental control mechanism for BR responses in plants. Altogether, our results identify K63-linked polyubiquitin chain formation as a dual targeting signal for BRI1 internalization and sorting along the endocytic pathway, and highlight its role in hormonally controlled plant development.

Pom1 regulation of Cdr2 membrane association and interaction with Mid1 prevents Cdr2 assembly into stable nodes in the cell tip region, which ensures proper positioning of cytokinetic ring precursors and accurate division plane positioning in fission yeast., Proper division plane positioning is essential to achieve faithful DNA segregation and to control daughter cell size, positioning, or fate within tissues. In Schizosaccharomyces pombe, division plane positioning is controlled positively by export of the division plane positioning factor Mid1/anillin from the nucleus and negatively by the Pom1/DYRK (dual-specificity tyrosine-regulated kinase) gradients emanating from cell tips. Pom1 restricts to the cell middle cortical cytokinetic ring precursor nodes organized by the SAD-like kinase Cdr2 and Mid1/anillin through an unknown mechanism. In this study, we show that Pom1 modulates Cdr2 association with membranes by phosphorylation of a basic region cooperating with the lipid-binding KA-1 domain. Pom1 also inhibits Cdr2 interaction with Mid1, reducing its clustering ability, possibly by down-regulation of Cdr2 kinase activity. We propose that the dual regulation exerted by Pom1 on Cdr2 prevents Cdr2 assembly into stable nodes in the cell tip region where Pom1 concentration is high, which ensures proper positioning of cytokinetic ring precursors at the cell geometrical center and robust and accurate division plane positioning.

Background During a viral infection, the intracellular RIG-I-like receptors (RLRs) sense viral RNA and signal through the mitochondrial antiviral signaling adaptor MAVS (also known as IPS-1, Cardif and VISA) whose activation triggers a rapid production of type I interferons (IFN) and of pro-inflammatory cytokines through the transcription factors IRF3/IRF7 and NF-κB, respectively. While MAVS is essential for this signaling and known to operate through the scaffold protein NEMO and the protein kinase TBK1 that phosphorylates IRF3, its mechanism of action and regulation remain unclear. Results We report here that RLR activation triggers MAVS ubiquitination on lysine 7 and 10 by the E3 ubiquitin ligase TRIM25 and marks it for proteasomal degradation concomitantly with downstream signaling. Inhibition of this MAVS degradation with a proteasome inhibitor does not affect NF-κB signaling but it hampers IRF3 activation, and NEMO and TBK1, two essential mediators in type I IFN production, are retained at the mitochondria. Conclusions These results suggest that MAVS functions as a recruitment platform that assembles a signaling complex involving NEMO and TBK1, and that the proteasome-mediated MAVS degradation is required to release the signaling complex into the cytosol, allowing IRF3 phosphorylation by TBK1.

Item does not contain fulltext Down syndrome or trisomy 21 is the most common genetic disorder leading to mental retardation. One feature is impaired short- and long-term spatial memory, which has been linked to altered brain-derived neurotrophic factor (BDNF) levels. Mouse models of Down syndrome have been used to assess neurotrophin levels, and reduced BDNF has been demonstrated in brains of adult transgenic mice overexpressing Dyrk1a, a candidate gene for Down syndrome phenotypes. Given the link between DYRK1A overexpression and BDNF reduction in mice, we sought to assess a similar association in humans with Down syndrome. To determine the effect of DYRK1A overexpression on BDNF in the genomic context of both complete trisomy 21 and partial trisomy 21, we used lymphoblastoid cell lines from patients with complete aneuploidy of human chromosome 21 (three copies of DYRK1A) and from patients with partial aneuploidy having either two or three copies of DYRK1A. Decreased BDNF levels were found in lymphoblastoid cell lines from individuals with complete aneuploidy as well as those with partial aneuploidies conferring three DYRK1A alleles. In contrast, lymphoblastoid cell lines from individuals with partial trisomy 21 having only two DYRK1A copies displayed increased BDNF levels. A negative correlation was also detected between BDNF and DYRK1A levels in lymphoblastoid cell lines with complete aneuploidy of human chromosome 21. This finding indicates an upward regulatory role of DYRK1A expression on BDNF levels in lymphoblastoid cell lines and emphasizes the role of genetic variants associated with psychiatric disorders.

Background: Endoplasmic reticulum (ER) stress has been implicated in causing hepatic insulin resistance. Results: Fructose-fed XBP1 knock-out mice were protected from hepatic insulin resistance despite increased hepatic ER stress and JNK activation. Conclusion: ER stress and hepatic JNK activation can be disassociated from hepatic insulin resistance. Significance: Hepatic ER stress is not a direct causal factor in hepatic insulin resistance., Hepatic insulin resistance has been attributed to both increased endoplasmic reticulum (ER) stress and accumulation of intracellular lipids, specifically diacylglycerol (DAG). The ER stress response protein, X-box-binding protein-1 (XBP1), was recently shown to regulate hepatic lipogenesis, suggesting that hepatic insulin resistance in models of ER stress may result from defective lipid storage, as opposed to ER-specific stress signals. Studies were designed to dissociate liver lipid accumulation and activation of ER stress signaling pathways, which would allow us to delineate the individual contributions of ER stress and hepatic lipid content to the pathogenesis of hepatic insulin resistance. Conditional XBP1 knock-out (XBP1Δ) and control mice were fed fructose chow for 1 week. Determinants of whole-body energy balance, weight, and composition were determined. Hepatic lipids including triglyceride, DAGs, and ceramide were measured, alongside markers of ER stress. Whole-body and tissue-specific insulin sensitivity were determined by hyperinsulinemic-euglycemic clamp studies. Hepatic ER stress signaling was increased in fructose chow-fed XBP1Δ mice as reflected by increased phosphorylated eIF2α, HSPA5 mRNA, and a 2-fold increase in hepatic JNK activity. Despite JNK activation, XBP1Δ displayed increased hepatic insulin sensitivity during hyperinsulinemic-euglycemic clamp studies, which was associated with increased insulin-stimulated IRS2 tyrosine phosphorylation, reduced hepatic DAG content, and reduced PKCϵ activity. These studies demonstrate that ER stress and IRE1α-mediated JNK activation can be disassociated from hepatic insulin resistance and support the hypothesis that hepatic insulin resistance in models of ER stress may be secondary to ER stress modulation of hepatic lipogenesis.

The spindle assembly checkpoint links the onset of anaphase to completion of chromosome-microtubule attachment and is mediated by the binding of Mad and Bub proteins to kinetochores of unattached or maloriented chromosomes. Mad2 and BubR1 traffic between kinetochores and the cytosol, thereby transmitting a “wait anaphase” signal to the anaphase-promoting complex. It is generally assumed that this signal dissipates automatically upon kinetochore-microtubule binding, but it has been shown that under conditions of nocodazole-induced arrest p31comet, a Mad2-binding protein, is required for mitotic progression. In this article we investigate the localization and function of p31comet during normal, unperturbed mitosis in human and marsupial cells. We find that, like Mad2, p31comet traffics on and off kinetochores and is also present in the cytosol. Cells depleted of p31comet arrest in metaphase with mature bipolar kinetochore-microtubule attachments, a satisfied checkpoint, and high cyclin B levels. Thus p31comet is required for timely mitotic exit. We propose that p31comet is an essential component of the machinery that silences the checkpoint during each cell cycle., Molecular Biology of the Cell, 22 (22), ISSN:1939-4586, ISSN:1059-1524

Aurora A kinase is a key regulator of cell division, whose functions were attributed to its ability to phosphorylate diverse substrates. Aurora A is now shown to have a kinase-independent role in the regulation of chromatin-mediated microtubule assembly.