Two distinct signaling pathways participate in auxin-induced swelling of pea epidermal protoplasts

Protoplast swelling was used to investigate auxin signaling in the growth-limiting stem epidermis. The protoplasts of epidermal cells were isolated from elongating internodes of pea (Pisum sativum). These protoplasts swelled in response to auxin, providing the clearest evidence that the epidermis can directly perceive auxin. The swelling response to the natural auxin IAA showed a biphasic dose response curve but that to the synthetic auxin 1-naphthalene acetic acid (NAA) showed a simple bell-shaped dose response curve. The responses to IAA and NAA were further analyzed using antibodies raised against ABP1 (auxin-binding protein 1), and their dependency on extracellular ions was investigated. Two signaling pathways were resolved for IAA, an ABP1-dependent pathway and an ABP1-independent pathway that is much more sensitive to IAA than the former. The response by the ABP1 pathway was eliminated by anti-ABP1 antibodies, had a higher sensitivity to NAA, and did not depend on extracellular Ca(2+). In contrast, the response by the non-ABP1 pathway was not affected by anti-ABP1 antibodies, had no sensitivity to NAA, and depended on extracellular Ca(2+). The swelling by either pathway required extracellular K(+) and Cl(-). The auxin-induced growth of pea internode segments showed similar response patterns, including the occurrence of two peaks in the dose response curve for IAA and the difference in Ca(2+) requirements. It is suggested that two signaling pathways participate in auxin-induced internode growth and that the non-ABP1 pathway is more likely to be involved in the control of growth by constitutive concentrations of endogenous auxin.     

A Drosophila MAPKKK, D-MEKK1, mediates stress responses through activation of p38 MAPK

In cultured mammalian cells, the p38 mitogen-activated protein kinase (MAPK) pathway is activated in response to a variety of environmental stresses. How ever, there is little evidence from in vivo studies to demonstrate a role for this pathway in the stress response. We identified a Drosophila MAPK kinase kinase (MAPKKK), D-MEKK1, which can activate p38 MAPK. D-MEKK1 is structurally similar to the mammalian MEKK4/MTK1 MAPKKK. D-MEKK1 kinase activity was activated in animals under conditions of high osmolarity. Drosophila mutants lacking D-MEKK1 were hypersensitive to environmental stresses, including elevated temperature and increased osmolarity. In these D-MEKK1 mutants, activation of Drosophila p38 MAPK in response to stress was poor compared with activation in wild-type animals. These results suggest that D-MEKK1 regulation of the p38 MAPK pathway is critical for the response to environmental stresses in Drosophila.     

A role for the deubiquitinating enzyme USP28 in control of the DNA-damage response

The Chk2-p53-PUMA pathway is a major regulator of DNA-damage-induced apoptosis in response to double-strand breaks in vivo. Through analysis of 53BP1 complexes we have discovered a new ubiquitin protease, USP28, which regulates this pathway. Using a human cell line that faithfully recapitulated the Chk2-p53-PUMA pathway, we show that USP28 is required to stabilize Chk2 and 53BP1 in response to DNA damage. In this cell line, both USP28 and Chk2 are required for DNA-damage-induced apoptosis, and they accomplish this in part through regulation of the p53 induction of proapoptotic genes like PUMA. Our studies implicate DNA-damage-induced ubiquitination and deubiquitination as a major regulator of the DNA-damage response for Chk2, 53BP1, and a number of other proteins in the DNA-damage checkpoint pathway, including several mediators, such as Mdc1, Claspin, and TopBP1.     

Revisiting PD-1/PD-L pathway in T and B cell response: Beyond immunosuppression

The regulation of T cell response depends on co-inhibitory pathways that serve to control immune-mediated tissue damage and resolve inflammation by modulating the magnitude and duration of immune response. In this process, the axis of T-cell-expressed programmed death-1 (PD-1) and its ligands (PD-L1 and PD-L2) play a key role. While the PD-1/PD-L pathway has received considerable attention for its role in the maintenance of T cell exhaustion in cancer and chronic infection, the PD-1/PD-L pathway also plays diverse roles in regulating host immunity beyond T cell exhaustion. In this review, we will discuss emerging concepts in co-stimulatory functions of PD-1/PD-L pathway on T cell- and B cell response and explore the potential underlying mechanisms. In addition, based on the elevated expression of PD-1 and its ligands in local inflamed tissues, we further discussed the role of PD-1/PD-L pathway in autoimmune diseases.     

PKC-1 acts with the ERK MAPK signaling pathway to regulate Caenorhabditis elegans mechanosensory response

In most animals, multiple genes encode protein kinase C (PKC) proteins. Pharmacological studies have revealed numerous roles for this protein family, yet the in vivo roles of specific PKC proteins and the functional targets of PKC activation are poorly understood. We find that in Caenorhabditis elegans, two PKC genes, pkc-1 and tpa-1, are required for mechanosensory response; the role of the nPKCε/η ortholog, pkc-1, was examined in detail. pkc-1 function is required for response to nose touch in adult C. elegans and pkc-1 likely acts in the interneurons that regulate locomotion which are direct synaptic targets of mechanosensory neurons. Previous studies have suggested numerous possible targets of pkc-1; our analysis indicates that pkc-1 may act via the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway. We find that ERK/MAPK pathway function is required for mechanosensory response in C. elegans and that at least one component of this pathway, lin-45 Raf, acts in interneurons of the mechanosensory circuit. Genetic analysis indicates that lin-45 and pkc-1 act together to regulate nose touch response. Thus, these results functionally link two conserved signaling pathways in adult C. elegans neurons and define distinct roles for PKC genes in vivo.     

Distinct versus overlapping functions of MDC1 and 53BP1 in DNA damage response and tumorigenesis

The importance of the DNA damage response (DDR) pathway in development, genomic stability, and tumor suppression is well recognized. Although 53BP1 and MDC1 have been recently identified as critical upstream mediators in the cellular response to DNA double-strand breaks, their relative hierarchy in the ataxia telangiectasia mutated (ATM) signaling cascade remains controversial. To investigate the divergent and potentially overlapping functions of MDC1 and 53BP1 in the ATM response pathway, we generated mice deficient for both genes. Unexpectedly, the loss of both MDC1 and 53BP1 neither significantly increases the severity of defects in DDR nor increases tumor incidence compared with the loss of MDC1 alone. We additionally show that MDC1 regulates 53BP1 foci formation and phosphorylation in response to DNA damage. These results suggest that MDC1 functions as an upstream regulator of 53BP1 in the DDR pathway and in tumor suppression.     

The phospholipase B homolog Plb1 is a mediator of osmotic stress response and of nutrient-dependent repression of sexual differentiation in the fission yeast<Emphasis Type="Italic"> Schizosaccharomyces pombe</Emphasis>

Although phospholipase B (PLB) enzymes have been described in eukaryotes from yeasts to mammals, their biological functions are poorly understood. Here we describe the characterization of plb1, one of five genes predicted to encode PLB homologs in the fission yeast, Schizosaccharomyces pombe. The plb1 gene is dispensable under normal growth conditions but required for viability in high-osmolarity media and for normal osmotic stress-induced gene expression. Unlike mutants defective in function for the stress-activated MAP kinase Spc1, plb1Delta cells are not hypersensitive to oxidative or temperature stresses, nor do they undergo a G2-specific arrest in response to osmotic stress. In addition to defects in osmotic stress response, plb1Delta cells exhibit a cold-sensitive defect in nutrient-mediated mating repression, a phenotype reminiscent of mutants in the cyclic AMP (cAMP) pathway. We show that, like plb1Delta cells, mutants in the cAMP pathway are defective for growth in high-osmolarity media, demonstrating a previously unrecognized role for the cAMP pathway in osmotic stress response. Furthermore, we show that gain-of function in the cAMP pathway can rescue the osmosensitive growth defect of plb1Delta cells, suggesting that the cAMP pathway is a potential downstream target of the actions of Plb1 in S. pombe.     

Metabolic Respiration Induces AMPK- and Ire1p-Dependent Activation of the p38-Type HOG MAPK Pathway

Evolutionarily conserved mitogen activated protein kinase (MAPK) pathways regulate the response to stress as well as cell differentiation. In Saccharomyces cerevisiae, growth in non-preferred carbon sources (like galactose) induces differentiation to the filamentous cell type through an extracellular-signal regulated kinase (ERK)-type MAPK pathway. The filamentous growth MAPK pathway shares components with a p38-type High Osmolarity Glycerol response (HOG) pathway, which regulates the response to changes in osmolarity. To determine the extent of functional overlap between the MAPK pathways, comparative RNA sequencing was performed, which uncovered an unexpected role for the HOG pathway in regulating the response to growth in galactose. The HOG pathway was induced during growth in galactose, which required the nutrient regulatory AMP-dependent protein kinase (AMPK) Snf1p, an intact respiratory chain, and a functional tricarboxylic acid (TCA) cycle. The unfolded protein response (UPR) kinase Ire1p was also required for HOG pathway activation in this context. Thus, the filamentous growth and HOG pathways are both active during growth in galactose. The two pathways redundantly promoted growth in galactose, but paradoxically, they also inhibited each other''s activities. Such cross-modulation was critical to optimize the differentiation response. The human fungal pathogen Candida albicans showed a similar regulatory circuit. Thus, an evolutionarily conserved regulatory axis links metabolic respiration and AMPK to Ire1p, which regulates a differentiation response involving the modulated activity of ERK and p38 MAPK pathways.     

Genetic Analysis of Ethylene Signal Transduction in Arabidopsis Thaliana: Five Novel Mutant Loci Integrated into a Stress Response Pathway

The response of Arabidopsis thaliana etiolated seedlings to the plant hormone ethylene is a conspicuous phenotype known as the triple response. We have identified genes that are required for ethylene perception and response by isolating mutants that fail to display a triple response in the presence of exogenous ethylene. Five new complementation groups have been identified. Four of these loci, designated ein4, ein5, ein6 and ein7, are insensitive to ethylene. The fifth complementation group, eir1, is defined by a novel class of mutants that have agravitropic and ethylene-insensitive roots. Double-mutant phenotypes have allowed the positioning of these loci in a genetic pathway for ethylene signal transduction. The ethylene-response pathway is defined by the following loci: ETR1, EIN4, CTR1, EIN2, EIN3, EIN5, EIN6, EIN7, EIR1, AUX1 and HLS1. ctr1-1 is epistatic to etr1-3 and ein4, indicating that CTR1 acts after both ETR1 and EIN4 in the ethylene-response pathway. Mutations at the EIN2, EIN3, EIN5, EIN6 and EIN7 loci are all epistatic to the ctr1 seedling phenotype. The EIR1 and AUX1 loci define a root-specific ethylene response that does not require EIN3 or EIN5 gene activity. HLS1 appears to be required for differential cell growth in the apical hook. The EIR1, AUX1 and HLS1 genes may function in the interactions between ethylene and other plant hormones that occur late in the signaling pathway of this simple gas.     

Trex1 prevents cell-intrinsic initiation of autoimmunity

Detection of nucleic acids and induction of type I interferons (IFNs) are principal elements of antiviral defense but can cause autoimmunity if misregulated. Cytosolic DNA detection activates a potent, cell-intrinsic antiviral response through a poorly defined pathway. In a screen for proteins relevant to this IFN-stimulatory DNA (ISD) response, we identify 3' repair exonuclease 1 (Trex1). Mutations in the human trex1 gene cause Aicardi-Goutieres syndrome (AGS) and chilblain lupus, but the molecular basis of these diseases is unknown. We define Trex1 as an essential negative regulator of the ISD response and delineate the genetic pathway linking Trex1 deficiency to lethal autoimmunity. We show that single-stranded DNA derived from endogenous retroelements accumulates in Trex1-deficient cells, and that Trex1 can metabolize reverse-transcribed DNA. These findings reveal a cell-intrinsic mechanism for initiation of autoimmunity, implicate the ISD pathway as the cause of AGS, and suggest an unanticipated contribution of endogenous retroelements to autoimmunity.     

The DNA double-strand break response pathway: becoming more BRCAish than ever

Breast carcinoma is the leading cause of cancer incidence, and second in cancer mortality to lung cancer, in women of the Western hemisphere. Germ line mutations in the breast cancer susceptibility gene, BRCA1, is responsible for half of all cases of hereditary breast cancer, which constitutes about 5-10% of all cases of breast cancer. Current hypothesis has ascribed a role for Brca1 in maintaining genomic stability, through its involvement in cellular response pathway to the DNA double-strand breaks (DSB). DNA DSB, which are the most deleterious form of DNA damage, are repaired through a series of coordinated steps embedded in a signal transduction pathway that ultimately ensure the elimination of potentially harmful mutations to the genome. This pathway can be crudely divided into a primary and secondary phase. The primary response phase is initiated by sensor proteins that activate transducer protein kinases Atm and Atr, which target downstream effector proteins, such as Chk1 and Chk2, to elicit the secondary response phase. Brca1 has been intimately linked with various aspects of this signaling pathway. However, the precise role of Brca1 in this process remains unclear. In this review, we will provide a simple model in an attempt to clarify the role of Brca1 during cellular response to DNA DSB.     

Functional characterization of ARAKIN (ATMEKK1): a possible mediator in an osmotic stress response pathway in higher plants.

The Arabidopsis thaliana ARAKIN (ATMEKK1) gene shows strong homology to members of the (MAP) mitogen-activated protein kinase family, and was previously shown to functionally complement a mating defect in Saccharomyces cerevisiae at the level of the MEKK kinase ste11. The yeast STE11 is an integral component of two MAP kinase cascades: the mating pheromone pathway and the HOG (high osmolarity glycerol response) pathway. The HOG signal transduction pathway is activated by osmotic stress and causes increased glycerol synthesis. Here, we first demonstrate that ATMEKK1 encodes a protein with kinase activity, examine its properties in yeast MAP kinase cascades, then examine its expression under stress in A. thaliana. Yeast cells expressing the A. thaliana ATMEKK1 survive and grow under high salt (NaCl) stress, conditions that kill wild-type cells. Enhanced glycerol production, observed in non-stressed cells expressing ATMEKK1 is the probable cause of yeast survival. Downstream components of the HOG response pathway, HOG1 and PBS2, are required for ATMEKK1-mediated yeast survival. Because ATMEKK1 functionally complements the sho1/ssk2/ssk22 triple mutant, it appears to function at the level of the MEKK kinase step of the HOG response pathway. In A. thaliana, ATMEKK1 expression is rapidly (within 5 min) induced by osmotic (NaCl) stress. This is the same time frame for osmoticum-induced effects on the electrical properties of A. thaliana cells, both an immediate response and adaptation. Therefore, we propose that the A. thaliana ATMEKK1 may be a part of the signal transduction pathway involved in osmotic stress.     

A novel modulatory mechanism of transforming growth factor-beta signaling through decorin and LRP-1

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that signals to the nucleus through cell surface transmembrane receptors with serine/threonine kinase activity and cytoplasmic effectors, including Smad proteins. Here we describe two novel modulators of this pathway, lipoprotein-receptor related protein (LRP-1) and decorin. Decorin null (Dcn null) myoblasts showed a diminished TGF-beta response that is restored by decorin re-expression. Importantly, this reactivation occurs without changes in the binding to TGF-beta receptors, Smad protein phosphorylation, or Smad-4 nuclear translocation. In wild type myoblasts, inhibition of decorin binding to LRP-1 and depletion of LRP-1 inhibited TGF-beta response to levels similar to those observed in Dcn null myoblasts. Re-expression of decorin in Dcn null myoblasts cannot restore TGF-beta response if the Smad pathway or phosphatidylinositol 3-kinase activity is inhibited, suggesting that this LRP-1-decorin modulatory pathway requires activation of the Smad pathway by TGF-beta and involves phosphatidylinositol 3-kinase activity. This work unveils a new regulatory mechanism for TGF-beta signaling by decorin and LRP-1.