Jennifer Benanti received her B.S. from the University of California, San Diego in 1996, and her Ph.D. from the University of Washington and the Fred Hutchinson Cancer Research Center in 2003. She did her postdoctoral work at the University of California, San Francisco from 2004-2010, where she was supported by a Damon Runyon Cancer Research Foundation Fellowship and a Pathway to Independence Award from the NIH. She joined the Program in Gene Function and Expression at the University of Massachusetts Medical School in spring 2010.
Regulation of Cell Growth and Division
Misregulation of cell division is the underlying cause of a number of human diseases, including cancer. Our lab is interested in understanding the molecular mechanisms that control how cells grow and divide. We study how protein degradation by the ubiquitin proteasome system controls both the cell cycle and metabolic transitions. Identification of ubiquitin ligase targets
Proteins are marked for degradation by the covalent attachment of ubiquitin chains, which target proteins to the proteasome for destruction (see Figure 1). Ubiquitin ligases recognize specific protein substrates and catalyze the final step of the ubiquitin conjugation pathway. This is the most highly regulated step of the pathway, consistent with the fact that there are approximately 1000 distinct ubiquitin ligases in human cells. Despite their critical roles in cellular biology, substrates have only been identified for a small fraction of these ligases.
Figure 1. The ubiquitin proteasome system. Proteins are marked for degradation in the proteasome by the addition of chains of ubiquitin molecules to a lysine side chain of a target protein. The conjugation of ubiquitin to a substrate requires the action of three enzymes. First, the energy of ATP is used to generate a thioester bond between ubiquitin (Ub) and a ubiquitin activating enzyme (E1). This Ub molecule is then transferred from the E1 to a ubiquitin conjugating enzyme (E2). Finally, a ubiquitin ligase (E3) directs transfer of the ubiquitin to a specific protein substrate. Subsequent ubiquitin molecules are added to a lysine side chain in the first Ub molecule, and when the chain reaches at least 4 ubiquitin molecules in length, the protein is shuttled to the proteasome and degraded.
We are using budding yeast as a model organism to identify new targets of ubiquitin ligases. To this end, we have screened the entire yeast proteome using a collection of yeast strains in which each open reading frame is tagged with GFP, in order to identify all targets of individual ubiquitin ligases. Using quantitative, high-throughput microscopy we identified GFP-tagged proteins that increase in abundance when a specific ubiquitin ligase gene is deleted (see Figure 2). This approach has led to the identification of new targets of two ubiquitin ligases that have central roles in controlling cell cycle progression, the Skp1-Cullin-F-box (SCF) complex and the Anaphase-Promoting Complex (APC). We are currently examining the biological significance of several of these new targets.
Figure 2. Identification of APC To identify proteins that increase in levels when the APC activator Cdh1 is deleted, wild type (RFP negative) and Cdh1 targets. cdh1Δ::RFP (RFP positive) cells, each expressing the same GFP-tagged protein, were mixed together and imaged in 96-well plates. Average GFP intensity per cell was calcuated, and levels were compared between wild type and cdh1Δ populations. Shown here are the APC Cdh1 targets Spo12 and Cik1, both of which are brighter green in cells that are RFP positive ( cdh1Δ). Cell cycle control by ubiquitin ligases
Although the SCF and APC have well established roles in regulating the cell cycle, deletion of several other ubiquitin ligase genes cause cell cycle defects. To gain a better understanding of how other ligases may contribute to cell cycle regulation, we recently identified 75 cell cycle proteins that are degraded rapidly, and have screened all ubiquitin ligases in budding yeast (~50) to identify which ubiquitin ligase targets each of these proteins for degradation. Ongoing projects focus on elucidating the mechanism by which these proteins are targeted to ubiquitin ligases, constructing non-degradable versions of these proteins, to assess the importance of their turnover for cell cycle progression, and identifying the functions of uncharacterized ubiquitin ligases.
Regulation of metabolic switches
Cell growth is tightly coupled to the cell cycle so that cells only divide when the necessary resources are available. We are interested in the role that ubiquitin-mediated degradation plays in how cells adapt to nutrient availability and growth conditions. One ubiquitin ligase that controls nutrient sensing in yeast is SCF
Grr1. Previously, we found that SCF Grr1 has several targets that function to activate or deactivate the glycoloysis pathway, depending on the availability of extracellular glucose. Surprisingly, each of these targets is ubiquitinated by SCF Grr1 following phosphorylation by a different kinase. We are working to identify the signalling pathways that lead to destruction of these glycolytic regulators.
Hyperactivation of glycolysis is also a feature of many cancer cells, and mammailan homologues of glycolytic SCF
Grr1 targets are upregulated in a number of cancer types. We are also investigating whether glycolytic regulators are regulated by the ubiquitin proteasome pathway in mammalian cells, and attempting to identify ubiquitin ligases that regulate this pathway.