Plant root architecture dynamically adapts to various environmental conditions, such as salt-containing soil. The phytohormone abscisic acid (ABA) is involved among others also in these developmental adaptations, but the underlying molecular mechanism remains elusive. Here, a novel branch of the ABA signaling pathway in Arabidopsis involving PYR/PYL/ RCAR (abbreviated as PYLs) receptor-protein phosphatase 2A (PP2A) complex that acts in parallel to the canonical PYLs-protein phosphatase 2C (PP2C) mechanism is identified. The PYLs-PP2A signaling modulates root gravitropism and lateral root formation through regulating phytohormone auxin transport. In optimal conditions, PYLs ABA receptor interacts with the catalytic subunits of PP2A, increasing their phosphatase activity and thus counteracting PINOID (PID) kinase-mediated phosphorylation of PIN-FORMED (PIN) auxin transporters. By contrast, in salt and osmotic stress conditions, ABA binds to PYLs, inhibiting the PP2A activity, which leads to increased PIN phosphorylation and consequently modulated directional auxin transport leading to adapted root architecture. This work reveals an adaptive mechanism that may flexibly adjust plant root growth to withstand saline and osmotic stresses. It occurs via the cross-talk between the stress hormone ABA and the versatile developmental regulator auxin.
The hematopoietic protein tyrosine phosphatase (HePTP) is implicated in the development of blood cancers through its ability to negatively regulate the mitogen-activated protein kinases (MAPKs) ERK1/2 and p38. Small-molecule modulators of HePTP activity may become valuable in treating hematopoietic malignancies such as T cell acute lymphoblastic leukemia (T-ALL) and acute myelogenous leukemia (AML). Moreover, such compounds will further elucidate the regulation of MAPKs in hematopoietic cells. Although transient activation of MAPKs is crucial for growth and proliferation, prolonged activation of these important signaling molecules induces differentiation, cell cycle arrest, cell senescence, and apoptosis. Specific HePTP inhibitors may promote the latter and thereby may halt the growth of cancer cells. Here, we report the development of a small molecule that augments ERK1/2 and p38 activation in human T cells, specifically by inhibiting HePTP. Structure-activity relationship analysis, in silico docking studies, and mutagenesis experiments reveal how the inhibitor achieves selectivity for HePTP over related phosphatases by interacting with unique amino acid residues in the periphery of the highly conserved catalytic pocket. Importantly, we utilize this compound to show that pharmacological inhibition of HePTP not only augments, but also prolongs activation of ERK1/2 and, especially, p38. Moreover, we present similar effects in leukocytes from mice intraperitoneally injected with the inhibitor at doses as low as 3 mg/kg. Our results warrant future studies with this probe compound that may establish HePTP as a new drug target for acute leukemic conditions.
The pheromone response pathway of the yeast S. cerevisiae is a well-established model for the study of G proteins and mitogen-activated protein kinase (MAPK) cascades. Our longstanding ability to combine sophisticated genetic approaches with established functional assays has provided a thorough understanding of signaling mechanisms and regulation. In this report we compare new and established methods used to quantify pheromone-dependent MAPK phosphorylation, transcriptional induction, mating morphogenesis, and gradient tracking. These include both single-cell and population-based assays of activity. We describe several technical advances, provide example data for benchmark mutants, highlight important differences between newer and established methodologies, and compare the advantages and disadvantages of each as applied to the yeast model. Quantitative measurements of pathway activity have been used to develop mathematical models and reveal new regulatory mechanisms in yeast. It is our expectation that experimental and computational approaches developed in yeast may eventually be adapted to human systems biology and pharmacology.
Background: Ras proteins are important molecular switches. RasGAP is an essential negative regulator of Ras, and its activity is controlled by ubiquitination. Results: RasGAP interacts with a deubiquitinating enzyme, Ubp3. Disrupting Ubp3 activity leads to accumulation of ubiquitinated RasGAP and hyperactivation of Ras signaling. Conclusion: Appropriate deubiquitination of RasGAP by Ubp3 is important for Ras signaling. Significance: This study reveals a new layer of mechanism that regulates Ras.
Sumoylation, a post-translational protein modification by small ubiquitin-like modifier (SUMO), has been implicated in many stress responses. Here we analyzed the potential role of sumoylation in osmo-response in yeast. We find that osmotic stress induces rapid accumulation of sumoylated species in normal yeast cells. Interestingly, disruption of MAP kinase Hog1 leads to a much higher level of accumulation of sumoylated conjugates that are independent of new protein synthesis. We also find that the accumulation of sumoylated species is dependent on a SUMO ligase Siz1. Notably, overexpression of SIZ1 in HOG1-disruption mutants (hog1Δ) but not in wild type cells leads to a markedly increased and prolonged accumulation of sumoylated species. Examination of osmo-tolerance of yeast mutants that display either an increase or a decrease in the global sumoylation level revealed an inverse relationship between accumulation of sumoylated conjugates and osmo-tolerance. Further investigation has shown that many of the sumoylated species induced by hyperosmotic stress are actually poly-sumoylated. Together, these findings indicate that abnormal accumulation of poly-sumoylated conjugates is harmful for osmo-tolerance in yeast, and suggest that Hog1 promotes adaptation to hyperosmotic stress partially via regulation of global sumoylation level.
Ras1 is a small GTPase in the budding yeast Saccharomyces cerevisiae that regulates nutrient signaling. It has been shown that Ras1 undergoes phosphorylation, but the functional consequences and regulation of Ras1 phosphorylation remain unknown. Here we identify Ser-226 as an important residue for Ras1 phosphorylation, as mutating this residue to an alanine drastically diminishes the level of Ras1 phosphorylation. Notably, phosphorylated Ras1 accumulates as the cells approach the stationary phase of growth. Likewise, subjecting cells to nitrogen starvation also elevates the level of Ras1 phosphorylation. Interestingly, blocking Ras1 phosphorylation diminishes the level of autophagy and also renders the cells more sensitive to heat shock. Together, these data suggest a role of Ras1 phosphorylation in modulating nutrient signaling and stress response.
Chromatin instability and protein homeostasis (proteostasis) stress are two well-established hallmarks of aging, which have been considered largely independent of each other. Using microfluidics and single-cell imaging approaches, we observed that, during the replicative aging of S. cerevisiae, a challenge to proteostasis occurs specifically in the fraction of cells with decreased stability within the ribosomal DNA (rDNA). A screen of 170 yeast RNA-binding proteins identified ribosomal RNA (rRNA)-binding proteins as the most enriched group that aggregate upon a decrease in rDNA stability induced by inhibition of a conserved lysine deacetylase Sir2. Further, loss of rDNA stability induces age-dependent aggregation of rRNA-binding proteins through aberrant overproduction of rRNAs. These aggregates contribute to age-induced proteostasis decline and limit cellular lifespan. Our findings reveal a mechanism underlying the interconnection between chromatin instability and proteostasis stress and highlight the importance of cell-to-cell variability in aging processes.
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