ABSTRACT
The ethylene-responsive element binding factor (ERF) family is a large family of transcription factors involved in plant development and environmental stress responses. We previously reported the identification of 29 putative substrates of Mitogen-activated Protein Kinase3 (AtMPK3), AtMPK4 and AtMPK6, based on a solid-phase phosphorylation screening using a lambda phage expression library in Arabidopsis thaliana. In this study, a putative MPK substrate, AtERF72 (At3g16770), was strongly phosphorylated by AtMPK6 on the serine residue at position 151 (Ser151). AtERF72 binds to the GCC box (AGCCGCC) in the promoters of several pathogenesis-related (PR) genes and activates their transcription. We also show that the DNA-binding activity of AtERF72 is enhanced upon phosphorylation by AtMPK6 in vitro. In addition, transient co-expression experiments in Arabidopsis protoplasts revealed that effector constructs expressing a mutant variant of AtERF72, AtERF72S151D (carrying a Ser to aspartic acid [Asp] substitution at amino acid position 151) showed higher expression of the ß-glucuronidase (GUS) reporter gene driven by the GCC box element than effector constructs expressing the wild-type AtERF72. Furthermore, yeast two-hybrid assays revealed that the interaction between AtERF72S151D and TGA4/OBF4 was stronger than that between wild-type AtERF72 and TGA4/OBF4. Since AtERF72S151D is equivalent to AtERF72 phosphorylated by AtMPK6 at Ser151, these results suggest that the phosphorylation of AtERF72 by AtMPK6 triggers an event of transcriptional regulation from defence signalling in Arabidopsis.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Basic-Leucine Zipper Transcription Factors/metabolism , DNA-Binding Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Transcription Factors/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , DNA , DNA-Binding Proteins/genetics , Gene Expression Regulation, Plant , Mitogen-Activated Protein Kinases/genetics , Phosphorylation , Transcription Factors/geneticsABSTRACT
Reactive oxygen species (ROS) are produced in growth factor-signaling pathways leading to cell proliferation, but the mechanisms leading to ROS generation and the targets of ROS signals are not well understood. Using a focused siRNA screen to identify redox-related proteins required for growth factor-induced cell cycle entry, we show that two ROS-generating proteins, the NADPH oxidases NOX4 and DUOX2, are required for platelet-derived growth factor (PDGF) induced retinoblastoma protein (Rb) phosphorylation in normal human fibroblasts. Unexpectedly, NOX4 and DUOX2 knockdown did not inhibit the early signaling pathways leading to cyclin D1 upregulation. However, hours after growth factor stimulation, NOX4 and DUOX2 knockdown reduced ERK1 phosphorylation and increased levels of the tumor suppressor protein p53 and a cell cycle inhibitor protein p21 (Waf1/Cip1) that is transcriptionally regulated by p53. Co-knockdown of NOX4 or DUOX2 with either p53 or with p21 overcame the inhibition of Rb phosphorylation that occurred with NOX4 or DUOX2 knockdown alone. Our results argue that rather than primarily affecting growth factor receptor signaling, NOX4 and DUOX2 regulate cell cycle entry as part of a p53-dependent checkpoint for proliferation.
Subject(s)
Cell Cycle/genetics , NADPH Oxidases/physiology , Tumor Suppressor Protein p53/physiology , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Dual Oxidases , Gene Knockdown Techniques , Humans , Mitogen-Activated Protein Kinase 3/metabolism , Models, Biological , NADPH Oxidase 4 , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Phosphorylation/drug effects , Platelet-Derived Growth Factor/pharmacology , RNA, Small Interfering/pharmacology , Retinoblastoma Protein/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/physiology , Validation Studies as TopicABSTRACT
To study the effects of positive acceleration on the plasma atrial natriuretic peptide (ANP) and renin concentration (PRC), students of the Korean Air Force Academy and pilots were exposed to +6 Gz for 30 s in a human centrifuge without anti-G suits. An acceleration to +6 Gz caused a significant increase (p < 0.01) in the heart rate of both the student and pilot groups. We performed blood collection within 2 min after terminating centrifugation. Systolic blood pressure was increased significantly (p < 0.01), and heart rates were elevated and did not return to control levels by 2 min after centrifugation. Plasma ANP decreased significantly in both groups (p < 0.05). The difference in the percentage of ANP decrement was not significant between student and pilot groups. PRC increased significantly (p < 0.05) in both groups. This study demonstrated that high +Gz exposure in man caused a significant reduction of plasma ANP concentration, although PRC, heart rate, and blood pressure increase.
