Protein kinases that phosphorylate and regulate eIF2B

Interactions between proteins modulate essential cell functions ensuring that cells can grow and perform their tasks. Each protein is like a piece in a machine, each must be in the correct place and act at the correct time with all the other parts for the machine to function properly. But unlike most machines and their parts, proteins are continually renewed, move and can be modified to alter their function and/or location. Many proteins act together as parts of protein complexes with a common function. One group of proteins relevant to this proposal is required make (or synthesize) all new proteins in the cell. These are called protein synthesis factors. By increasing or reducing the activity of some of the protein synthesis factors cells can control exactly what new proteins are made at any one time. One common way to alter the activity of proteins is by the addition of phosphate groups to specific places on the surface of the protein. This is called phosphorylation and is performed by a class of proteins called protein kinases. Each kinase responds to specific signals that tell it when to act. We have found that one key protein synthesis factor called eIF2B is phosphorylated at multiple sites, and that this phosphorylation enhances the activity of eIF2B. We are proposing here a series of experiments designed to discover which of the protein kinases present in the cell is responsible for each phosphorylation and to discover how each modification alters eIF2B function in the cell. In yeast cells there are 122 protein kinases. Using modern technologies we will be able to assess the role each one plays in control of eIF2B simultaneously. eIF2B function is also inhibited by phosphorylation of a second protein synthesis factor called eIF2. eIF2 is known to be phosphorylated at a single site called ser51 in response to diverse cell stresses. We have already determined that three of the five parts (or subunits) of eIF2B are all needed to correctly detect whether or not eIF2 is phosphorylated at this key regulatory site. But how exactly do these three subunits of eIF2B act together to do this? In a second series of experiments we propose to use a combination of genetic and biochemistry tools to address this question. This work will provide important detailed molecular information on how the activities of these essential proteins are controlled by phosphorylation.

Understanding how interactions between protein complexes modulate essential cell functions is an important current research theme in biology. Eukaryotic translation initiation factor (eIF) 2B is a five subunit protein complex that is essential for protein synthesis and its control in all eukaryotic cells. eIF2B functions as a guanine-nucleotide exchange factor to activate its substrate (eIF2). Active eIF2 forms complex with initiator Met-tRNA and so eIF2B activity is required for each new round of translation initiation for all cellular mRNAs and almost all viral RNAs. By controlling eIF2B function the translation of many mRNAs can be modulated, although the number of translationally controlled genes remains unknown the best characterised examples are GCN4 and ATF4. There is increasing evidence that regulation of eIF2B activity can modulate the expression of a much wider proportion of genes than previously anticipated. eIF2B activity is regulated by phosphorylation. (1) We have found that yeast eIF2B is a phosphoprotein and that dephosphorylation of purified eIF2B preparations significantly impairs its activity in vitro. Using mass spectrometric techniques (LC-MS/MS), we have identified several phosphorylation sites within four eIF2B subunits. None of these sites have been previously characterised. We propose here to use a combination of modern high-throughput technologies (systems biology), molecular biology and yeast genetic tools to identify which of the approximately 120 yeast protein kinases are responsible for these events. We will then characterise their physiological significance. (2) eIF2B is also regulated by phosphorylation of its substrate eIF2. eIF2 kinases respond to diverse physiological stresses to inhibit eIF2B activity. Bizarrely, this response requires recognition of the phosphorylated ser51 residue of eIF2alpha by three homologous subunits of eIF2B. How 3 eIF2B subunits can all contribute to recognition of ser51 remains unknown. To address this we have initiated a suppressor screen and identified allele-specific interactions between one eIF2B subunit and eIF2alpha. We propose here to extend this suppressor screen to identify suppressor mutations for other eIF2B subunits and to undertake a comprehensive biochemical analysis of how the mutations alter the interactions between eIF2 and eIF2B. These studies will map interactions between these protein subunits to uncover how the regulatory subunits of eIF2B sense and respond to phosphorylation of eIF2.