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

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

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