Systematic analysis of essential genes reveals new regulators of G protein signaling

Heterotrimeric G proteins are molecular switches that respond to a wide range of stimuli including light, neurotransmitters, small molecules and peptides. Due to their role in a variety of physiological responses, it is no surprise that over 50% of drugs modulate G protein signaling pathways. While many drugs function at the level of the G protein-coupled receptor, downstream signaling components are increasingly being investigated as drug targets. Therefore, discovery of new components and regulators will help identify new ways to exploit G protein-coupled signaling pathways for therapeutic utility. Previous attempts to systematically identify new components of G protein pathways have focused on genome-wide knockout screens including gene-deletion mutants. However, these methods are inherently limited because they exclude the essential genes. In this thesis, we present studies to identify new signaling components by systematically analyzing 870 essential genes using repressible-promoter strains. Specifically, we show that the SCFCdc4 E3 ubiquitin ligase complex regulates G protein turnover and catalyzes ubiquitination of the G protein [alpha] subunit, Gpa1. Also, we demonstrate that Pik1, a phosphatidylinositol (PtdIns) 4-kinase, regulates the mitogen-activated protein kinase (MAPK) cascade and helps maintain signaling fidelity. These findings reveal the essential-genome as an untapped resource for identifying new components and regulators of signal transduction pathways. Furthermore, work on this thesis has expanded our understanding of G protein signaling networks and could lead to future opportunities for drug discovery

Systematic Analysis of Essential Genes Reveals Important Regulators of G Protein Signaling

The yeast pheromone pathway consists of a canonical heterotrimeric G protein and MAP kinase cascade. To identify new signaling components we systematically evaluated 870 essential genes using a library of repressible-promoter strains. Quantitative transcription-reporter and MAPK activity assays were used to identify strains that exhibit altered pheromone sensitivity. Of the 92 newly identified essential genes required for proper G protein signaling, those involved with protein degradation were most highly-represented. Included in this group are members of the SCF (Skp-Cullin-F-Box) ubiquitin ligase complex. Further genetic and biochemical analysis reveals that SCFCdc4 acts together with the Cdc34 ubiquitin conjugating enzyme at the level of the G protein, promotes degradation of the G protein α subunit, Gpa1, in vivo and catalyzes Gpa1 ubiquitination in vitro. These new insights to the G protein signaling network reveal the essential-genome as an untapped resource for identifying new components and regulators of signal transduction pathways

Nuclear pore protein NUP210 depletion suppresses metastasis through heterochromatin-mediated disruption of tumor cell mechanical response.

Mechanical signals from the extracellular microenvironment have been implicated in tumor and metastatic progression. Here, we identify nucleoporin NUP210 as a metastasis susceptibility gene for human estrogen receptor positive (ER+) breast cancer and a cellular mechanosensor. Nup210 depletion suppresses lung metastasis in mouse models of breast cancer. Mechanistically, NUP210 interacts with LINC complex protein SUN2 which connects the nucleus to the cytoskeleton. In addition, the NUP210/SUN2 complex interacts with chromatin via the short isoform of BRD4 and histone H3.1/H3.2 at the nuclear periphery. In Nup210 knockout cells, mechanosensitive genes accumulate H3K27me3 heterochromatin modification, mediated by the polycomb repressive complex 2 and differentially reposition within the nucleus. Transcriptional repression in Nup210 knockout cells results in defective mechanotransduction and focal adhesion necessary for their metastatic capacity. Our study provides an important role of nuclear pore protein in cellular mechanosensation and metastasis

EMI1 switches from being a substrate to an inhibitor of APC/CCDH1 to start the cell cycle.

Mammalian cells integrate mitogen and stress signalling before the end of G1 phase to determine whether or not they enter the cell cycle1-4. Before cells can replicate their DNA in S phase, they have to activate cyclin-dependent kinases (CDKs), induce an E2F transcription program and inactivate the anaphase-promoting complex (APC/CCDH1, also known as the cyclosome), which is an E3 ubiquitin ligase that contains the co-activator CDH1 (also known as FZR, encoded by FZR1). It was recently shown that stress can return cells to quiescence after CDK2 activation and E2F induction but not after inactivation of APC/CCDH1, which suggests that APC/CCDH1 inactivation is the point of no return for cell-cycle entry 3 . Rapid inactivation of APC/CCDH1 requires early mitotic inhibitor 1 (EMI1)3,5, but the molecular mechanism that controls this cell-cycle commitment step is unknown. Here we show using human cell models that cell-cycle commitment is mediated by an EMI1-APC/CCDH1 dual-negative feedback switch, in which EMI1 is both a substrate and an inhibitor of APC/CCDH1. The inactivation switch triggers a transition between a state with low EMI1 levels and high APC/CCDH1 activity during G1 and a state with high EMI1 levels and low APC/CCDH1 activity during S and G2. Cell-based analysis, in vitro reconstitution and modelling data show that the underlying dual-negative feedback is bistable and represents a robust irreversible switch. Our study suggests that mammalian cells commit to the cell cycle by increasing CDK2 activity and EMI1 mRNA expression to trigger a one-way APC/CCDH1 inactivation switch that is mediated by EMI1 transitioning from acting as a substrate of APC/CCDH1 to being an inhibitor of APC/CCDH1

The Proliferation-Quiescence Decision Is Controlled by a Bifurcation in CDK2 Activity at Mitotic Exit

SummaryTissue homeostasis in metazoans is regulated by transitions of cells between quiescence and proliferation. The hallmark of proliferating populations is progression through the cell cycle, which is driven by cyclin-dependent kinase (CDK) activity. Here, we introduce a live-cell sensor for CDK2 activity and unexpectedly found that proliferating cells bifurcate into two populations as they exit mitosis. Many cells immediately commit to the next cell cycle by building up CDK2 activity from an intermediate level, while other cells lack CDK2 activity and enter a transient state of quiescence. This bifurcation is directly controlled by the CDK inhibitor p21 and is regulated by mitogens during a restriction window at the end of the previous cell cycle. Thus, cells decide at the end of mitosis to either start the next cell cycle by immediately building up CDK2 activity or to enter a transient G0-like state by suppressing CDK2 activity

Cell Cycle-dependent Phosphorylation and Ubiquitination of a G Protein α Subunit*

A diverse array of external stimuli, including most hormones and neurotransmitters, bind to cell surface receptors that activate G proteins. Mating pheromones in yeast Saccharomyces cerevisiae activate G protein-coupled receptors and initiate events leading to cell cycle arrest in G1 phase. Here, we show that the Gα subunit (Gpa1) is phosphorylated and ubiquitinated in response to changes in the cell cycle. We systematically screened 109 gene deletion strains representing the non-essential yeast kinome and identified a single kinase gene, ELM1, as necessary and sufficient for Gpa1 phosphorylation. Elm1 is expressed in a cell cycle-dependent manner, primarily at S and G2/M. Accordingly, phosphorylation of Gpa1 in G2/M phase leads to polyubiquitination in G1 phase. These findings demonstrate that Gpa1 is dynamically regulated. More broadly, they reveal how G proteins can simultaneously regulate, and become regulated by, progression through the cell cycle

Selective Regulation of MAP Kinase Signaling by an Endomembrane Phosphatidylinositol 4-Kinase*

Multiple MAP kinase pathways share components yet initiate distinct biological processes. Signaling fidelity can be maintained by scaffold proteins and restriction of signaling complexes to discreet subcellular locations. For example, the yeast MAP kinase scaffold Ste5 binds to phospholipids produced at the plasma membrane and promotes selective MAP kinase activation. Here we show that Pik1, a phosphatidylinositol 4-kinase that localizes primarily to the Golgi, also regulates MAP kinase specificity but does so independently of Ste5. Pik1 is required for full activation of the MAP kinases Fus3 and Hog1 and represses activation of Kss1. Further, we show by genetic epistasis analysis that Pik1 likely regulates Ste11 and Ste50, components shared by all three MAP kinase pathways, through their interaction with the scaffold protein Opy2. These findings reveal a new regulator of signaling specificity functioning at endomembranes rather than at the plasma membrane