Brassinosteroids (BRs) are essential plant hormones that regulate multiple aspects of plant growth and development and require two receptor kinases, Brassinosteroid Insensitive 1 (BRI1) and BRI1-Associated Receptor Kinase 1 (BAK1), for hormone perception and signal transduction. The principal investigators identified specific BR-dependent phosphorylation sites of Arabidopsis BRI1 and BAK1 in planta and isolated a putative cytoplasmic substrate of BRI1 with homology to the mammalian TGF-beta receptor interacting protein (TRIP-1). TRIP-1 (also known as eIF3i) is a dual function protein that regulates TGF-beta signaling in mammals and also plays a critical role in the eIF3 protein translation initiation complex in animals, yeast and plants. Arabidopsis BRI1 interacts with TRIP-1 in planta and phosphorylates TRIP-1 on three specific residues in vitro. Initiation is the rate-limiting step in eukaryotic protein translation and is often regulated by phosphorylation of specific initiation factor subunits in response to various signals. A proteomic screen for novel BRI1 and BAK1 interactors identified an additional four eIF subunits; eIF2B, eIF3g, eIF4A and eIF5, as putative kinase domain substrates for BRI1 and/or BAK1. Taken together, the preliminary evidence suggests that BR-dependent phosphorylation of TRIP-1 (and other eIF subunits) by BRI1 may affect initiation factor activity and/or assembly and thus impact the global cellular phenomenon of protein translation, providing a novel mechanism for BR regulation of plant growth. The research will examine the intersection of BR signal transduction and protein translation initiation by focusing on three objectives. Objective 1. Characterization of Arabidopsis TRIP-1 in vivo phosphorylation sites by a variety of mass spectrometry approaches and analysis of their functional significance with respect to BR signaling and eIF3 activity. Objective 2. Detailed in vivo and in vitro characterization of the putative interaction of eIF3g and eIF5 with BRI1 and BAK1. Objective 3.Generation of an in vivo phosphorylation site database of multiple eIF subunits followed by quantitative studies of BR-dependent phosphorylation in these proteins using label-free mass spectrometry methodologies.
Intellectual merit: A great deal is known about genomic effects of BR signaling and BR regulated gene expression, but little is known about non-genomic pathways through which BRs might regulate cellular physiology directly, e.g. by phosphorylating cytoplasmic proteins such as translation initiation factor subunits. Identification of specific in vivo phosphorylation sites in eIF subunits coupled with their functional characterization will enhance our understanding of the molecular mechanisms regulating protein translation in plants.
Broader impacts: The proposed research will provide excellent training in biochemistry, molecular biology and mass spectrometry at all levels, including postdoctoral scientists, graduate and undergraduate students and high school student summer interns. A database of eIF phosphorylation sites will be made publicly available and vector constructs and transgenic plants useful to the research community will be distributed via the Arabidopsis Biological Resource Center. BRs are now firmly established as essential regulators of plant growth and development affecting a broad spectrum of developmental processes. The identification of BR biosynthetic and insensitive mutants in tomato, rice, barley and pea, clearly extends the importance of these compounds from the experimental plant Arabidopsis thaliana to crop plants and recent field experiments have shown slight alterations in rice BRI1 expression can alter rice yields by up to 30%. Understanding the molecular details of BR signal transduction can thus have practical application in regulating the growth of agricultural plants.
Brassinosteroids (BRs) are endogenous plant growth-promoting hormones found throughout the plant kingdom in seeds, pollen and young vegetative tissues. BRs influence cellular expansion and proliferation, and the phenotype of mutants affected in BR biosynthesis or signaling clearly show that these plant steroids are essential for normal organ elongation, vascular differentiation, male fertility, timing of senescence, and leaf development. The emerging picture of BR signal transduction reveals that plant steroids are perceived at the cell surface by BRASSINOSTEROID INSENSITIVE 1 (BRI1), a member of the large family of leucine-rich repeat receptor-like kinases (LRR RLKs) found in plants. Receptor kinases are membrane-bound proteins that recognize an extracellular signal and use ATP to transfer a phosphate group to specific amino acids in cellular proteins in a process called phosphorylation, which alters the function of proteins in significant ways. Understanding the mechanism of BRI1 action requires discovery of protein targets of phosphorylation. Characterizing BR-dependent phosphorylation sites in ceullar targets of BRI1 is essential for a complete understanding of BR action. In mammals, the TGF-beta family of polypeptides modulate numerous aspects of development and are perceived at the cell surface by a complex of Type I (RI) and Type II (RII) TGF-beta receptor kinases. TGF-beta Receptor Interacting Protein (TRIP-1) is a cytoplasmic substrate of the TGF-beta Type II receptor kinase and plays a role in TGF-beta signaling. We previously cloned TRIP-1 homologs from bean and Arabidopsis and found that transgenic Arabidopsis plants with reduced TRIP-1 RNA exhibited a broad range of developmental defects including some morphological characteristics that resemble the phenotype of BR-deficient and –insensitive mutants. We also found that the BRI1 kinase domain phosphorylates Arabidopsis TRIP-1 on at least three specific residues and that TRIP-1 and BRI1 also interact directly in the plant. These findings support a role for TRIP-1 in the molecular mechanisms of BR-regulated plant growth and development, most likely as a cellular substrate of the BRI1 receptor kinase. TRIP-1 (also known as eIF3i) is a dual function protein that regulates TGF-beta signaling in mammals and also plays a critical role in the eIF3 protein translation initiation complex in animals, yeast and plants. As part of our NSF funded project, we used advanced mass spectrometry methods to identify eight additional sites in Arabidopsis TRIP-1 that are phosphorylated by BRI1, bringing the total to 11. Furthermore, a large-scale screen for novel BRI1 interactors identified an additional four eIF subunits; eIF2B, eIF3g, eIF4A and eIF5, as putative kinase domain substrates for BRI1. We used genetic and biochemical approaches to confirm that BRI1 interacted with all of these translation initiation factors in the plant cell. We also used mass spectrometry to identify multiple sites of phosphorylation by BRI1 in eIF2B, eIF3g, eIF4A and eIF5. Because the regulation of BRI1 kinase activity is critical for downstream signaling and control of plant growth and development, we also studied the activation of the BRI1 kinase domain and discovered several phosphorylation sites that were essential for the overall regulation of plant development. The critical role of phosphorylation in regulating the activity of multiple eIF subunits in translation initiation has been demonstrated in many plant and animal species suggesting regulatory phosphorylation of specific subunits of translation initiation factors occurs widely in eukaryotes. This raises the intriguing possibility that BR-dependent phosphorylation of TRIP-1 by BRI1 may affect eIF3 activity and/or assembly and thus impact the global cellular phenomenon of protein translation, providing a novel mechanism for BR regulation of plant growth. BRs are firmly established as essential regulators of plant growth and development affecting a broad spectrum of developmental processes in both model plants such as Arabidopsis and in crop plants. Understanding the molecular details of BR signal transduction can thus have practical application in regulating the growth of agricultural plants. Besides advances in basic research, our project also had a strong training component. During the project we had two senior research scientists, two PhD graduate research assistants, two high school summer interns, and one technician working on project objectives. Advanced training in plant biology, mass spectrometry, proteomic techniques, vector construction, DNA sequencing, and protein kinase biochemistry was provided to these individuals as part of our NSF funded project. Several of these individuals have moved on to permanent scientific positions in both academia and industry.
