Phosphorylation of Protein Translation Initiation Factors as a Novel Component of Brassinosteroid Signal Transduction

North Carolina State University, Raleigh NC

Investigators

Abstract

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.