Osmoregulation is important for plant growth, development and response to environmental changes. SNF1-related protein kinase 2s (SnRK2s) are quickly activated by osmotic stress and are central components in osmotic stress and abscisic acid (ABA) signaling pathways; however, the upstream components required for SnRK2 activation and early osmotic stress signaling are still unknown. Here, we report a critical role for B2, B3 and B4 subfamilies of Raflike kinases (RAFs) in early osmotic stress as well as ABA signaling in Arabidopsis thaliana. B2, B3 and B4 RAFs are quickly activated by osmotic stress and are required for phosphorylation and activation of SnRK2s. Analyses of high-order mutants of RAFs reveal critical roles of the RAFs in osmotic stress tolerance and ABA responses as well as in growth and development. Our findings uncover a kinase cascade mediating osmoregulation in higher plants.

bPrevious studies revealed that the potential tumor suppressor EAF2 binds to and stabilizes pVHL, suggesting that EAF2 may function by disturbing the hypoxia signaling pathway. However, the extent to which EAF2 affects hypoxia and the mechanisms underlying this activity remain largely unknown. In this study, we found that EAF2 is a hypoxia response gene harboring the hypoxia response element (HRE) in its promoter. By taking advantage of the pVHL-null cell lines RCC4 and 786-O, we demonstrated that hypoxia-induced factor 1␣ (HIF-1␣), but not HIF-2␣, induced EAF2 under hypoxia. Subsequent experiments showed that EAF2 bound to and suppressed HIF-1␣ but not HIF-2␣ transactivity. In addition, we observed that EAF2 inhibition of HIF-1␣ activity resulted from the disruption of p300 recruitment and that this occurred independently of FIH-1 (factor inhibiting HIF-1) and Sirt1. Furthermore, we found that EAF2 protected cells against hypoxia-induced cell death and inhibited cellular uptake of glucose under hypoxic conditions, suggesting that EAF2 indeed may act by modulating the hypoxia-signaling pathway. Our findings not only uncover a unique feedback regulation loop between EAF2 and HIF-1␣ but also provide a novel insight into the mechanism of EAF2 tumor suppression.

Dorsal organizer formation is one of the most critical steps in early embryonic development. Several genes and signaling pathways that positively regulate the dorsal organizer development have been identified; however, little is known about the factor(s) that negatively regulates the organizer formation. Here, we show that Setdb2, a SET domain-containing protein possessing potential histone H3K9 methyltransferase activity, restricts dorsal organizer development and regulates left-right asymmetry by suppressing fibroblast growth factor 8 (fgf8) expression. Knockdown of Setdb2 results in a massive expansion of dorsal organizer markers floating head (flh), goosecoid (gsc), and chordin (chd), as well as a significant increase of fgf8, but not fgf4 mRNAs. Consequently, disrupted midline patterning and resultant randomization of left-right asymmetry are observed in Setdb2-deficient embryos. These characteristic changes induced by Setdb2 deficiency can be nearly corrected by either overexpression of a dominant-negative fgf receptor or knockdown of fgf8, suggesting an essential role for Setdb2-Fgf8 signaling in restricting dorsal organizer territory and regulating left-right asymmetry. These results provide unique evidence that a SET domain-containing protein potentially involved in the epigenetic control negatively regulates dorsal organizer formation during early embryonic development.nder bilateral external symmetry, vertebrates conserve a leftright asymmetry placement of internal organs and nervous system. By using mouse, chicken, Xenopus, and zebrafish as animal models, the developmental and molecular mechanisms of how left-right asymmetry is established during embryogenesis have been well characterized in the past few years: two embryonic structures-the node [also known as Kupffer's vesicle (KV) in zebrafish] and the midline, mainly consisting of notochord and floorplate-are considered essential for maintaining the left-right asymmetry (1, 2). Inside the KV, cilia are specifically organized to generate a counterclockwise fluid flow, by which the first laterality signal is propagated to the left lateral plate mesoderm (LPM), resulting in an asymmetric gene expression on the left side (3, 4). The southpaw (member of nodal-related subfamily of TGF-β superfamily) is the earliest known gene that is asymmetrically expressed in the left LPM at 10-12 somite stage to initiate the left-right signaling through inducing its downstream targets lefty2 and pitx2 in the left LPM, and lefty1 in the axial midline that acts as a molecular barrier to prevent the left-sided signals from leaking to the other side. This signaling cascade is highly conserved during vertebrate evolution (5).It has been shown that the KV is originated from the dorsal forerunner cells (DFCs), a group of noninvoluting cells at the leading edge of the embryonic dorsal organizer or shield, which also produces the midline/notochord (6, 7). Eighty-five years ago Spemann and Mangold originally identified the dorsal organizer in amphibian through transplanting the...

The TAO (for thousand-and-one amino acids) protein kinases activate p38 mitogen-activated protein (MAP) kinase cascades in vitro and in cells by phosphorylating the MAP/ERK kinases (MEKs) 3 and 6. We found that TAO2 activity was increased by carbachol and that carbachol and the heterotrimeric G protein G␣ o could activate p38 in 293 cells. Using dominant interfering kinase mutants, we found that MEKs 3 and 6 and TAOs were required for p38 activation by carbachol or the constitutively active mutant G␣ o Q205L. To explore events downstream of TAOs, the effects of TAO2 on ternary complex factors (TCFs) were investigated. Transfection studies demonstrated that TAO2 stimulates phosphorylation of the TCF Elk1 on the major activating site, Ser 383 , and that TAO2 stimulates transactivation of Elk1 and the related TCF, Sap1. Reporter activity was reduced by the p38-selective inhibitor SB203580. Taken together, these studies suggest that TAO protein kinases relay signals from carbachol through heterotrimeric G proteins to the p38 MAP kinase, which then activates TCFs in the nucleus. TAO11 (thousand-and-one amino acids 1) and TAO2 are protein kinases that were originally identified based on their similarity to the yeast p21-activated protein kinase Ste20p, a protein kinase upstream in a mitogen-activated protein kinase (MAPK) pathway in yeast (1, 2). JIK is a third TAO-like kinase (3). The Ste20p family contains a diverse array of protein kinases, several of which have been shown to act upstream of MAPKs (4). TAO1 and TAO2 each contain over 1000 residues with catalytic domains at their N termini. TAOs activate MAPK pathways because they have MAP kinase kinase kinase (MAP3K) activity; they phosphorylate the p38-activating kinases called MAP/ERK kinases (MEKs, also known as MKKs or MAP2Ks) 3 and 6, which then phosphorylate p38 (1, 2, 5). The preferential phosphorylation of these two MEKs arises in part because TAOs dock to these MEKs through a region Cterminal to the TAO kinase domain (2, 5).Ternary complex factors (TCFs) are among the transcription factors under the control of MAPKs (6 -12). Upon phosphorylation by MAPKs, TCFs form a complex with the serum response factor at the serum response element, thereby stimulating the transcription of c-fos and other genes containing this element. The TCFs, Elk1, Sap1, and Sap2, contain a conserved C-terminal transactivation domain with multiple (S/T)P motifs, which are minimal consensus sites for MAPK phosphorylation. Elk1 can be phosphorylated by at least three MAPK subgroups, ERK1/2, c-Jun-N-terminal kinases/stress-activated protein kinases (JNK/SAPKs), and p38. In contrast, Sap1 can be phosphorylated by ERK1/2, ERK5, and p38, but not JNK/ SAPKs (7, 12, 13). We explored downstream actions of TAO2 by examining its effects on transactivation of TCFs. Cotransfection studies and reporter assays demonstrated that TAO2 enhances phosphorylation and transactivation of TCFs in 293 cells. TAO2-dependent transactivation of TCFs was inhibited by the p38 selective inhibitor SB203580, suggest...

RLR‐mediated type I IFN production plays a pivotal role in innate antiviral immune responses, where the signaling adaptor MAVS is a critical determinant. Here, we show that MAVS is a physiological substrate of SIRT5. Moreover, MAVS is succinylated upon viral challenge, and SIRT5 catalyzes desuccinylation of MAVS. Mass spectrometric analysis indicated that Lysine 7 of MAVS is succinylated. SIRT5‐catalyzed desuccinylation of MAVS at Lysine 7 diminishes the formation of MAVS aggregation after viral infection, resulting in the inhibition of MAVS activation and leading to the impairment of type I IFN production and antiviral gene expression. However, the enzyme‐deficient mutant of SIRT5 (SIRT5‐H158Y) loses its suppressive role on MAVS activation. Furthermore, we show that Sirt5‐deficient mice are resistant to viral infection. Our study reveals the critical role of SIRT5 in limiting RLR signaling through desuccinylating MAVS.

No abstract

A nucleosome is made up of DNA and histones, and acetylation of histones perturbs the interaction of DNA and histones and thus affects the chromatin conformation and function. However, whether or how acetylation induces DNA conformation changes is still elusive. In this work, we applied FT-IR spectroscopy to monitor the DNA signals in cells as the histone acetylation was regulated by trichostatin A (TSA), a reversible inhibitor to histone deacetylases (HDACs). Our results unambiguously demonstrate the significant transformation of B-DNA to Z-DNA upon histone acetylation in the TSA treated HeLa cells. This is the first report providing the explicit experimental evidence for such a B-Z transformation of DNA in the epigenetic states of cells.

Low-temperature plasma may induce oxidative stress and result in different modes of cell death, such as necrosis, apoptosis and necroptosis, which can be monitored by Raman micro-spectroscopy based on the change of cellular cytochrome c redox state.