Irreversible APC(Cdh1) Inactivation Underlies the Point of No Return for Cell-Cycle Entry
2016; 166 (1): 167-180
The Proliferation-Quiescence Decision Is Controlled by a Bifurcation in CDK2 Activity at Mitotic Exit
2013; 155 (2): 369-383
Proliferating cells must cross a point of no return before they replicate their DNA and divide. This commitment decision plays a fundamental role in cancer and degenerative diseases and has been proposed to be mediated by phosphorylation of retinoblastoma (Rb) protein. Here, we show that inactivation of the anaphase-promoting complex/cyclosome (APC(Cdh1)) has the necessary characteristics to be the point of no return for cell-cycle entry. Our study shows that APC(Cdh1) inactivation is a rapid, bistable switch initiated shortly before the start of DNA replication by cyclin E/Cdk2 and made irreversible by Emi1. Exposure to stress between Rb phosphorylation and APC(Cdh1) inactivation, but not after APC(Cdh1) inactivation, reverted cells to a mitogen-sensitive quiescent state, from which they can later re-enter the cell cycle. Thus, APC(Cdh1) inactivation is the commitment point when cells lose the ability to return to quiescence and decide to progress through the cell cycle.
View details for DOI 10.1016/j.cell.2016.05.077
View details for Web of Science ID 000380254400019
View details for PubMedID 27368103
Cell Cycle-dependent Phosphorylation and Ubiquitination of a G Protein alpha Subunit
JOURNAL OF BIOLOGICAL CHEMISTRY
2011; 286 (23): 20208-20216
Tissue 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.
View details for DOI 10.1016/j.cell.2013.08.062
View details for Web of Science ID 000325719800014
Selective Regulation of MAP Kinase Signaling by an Endomembrane Phosphatidylinositol 4-Kinase
JOURNAL OF BIOLOGICAL CHEMISTRY
2011; 286 (17): 14852-14860
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 G(1) phase. Here, we show that the G? 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 G(2)/M. Accordingly, phosphorylation of Gpa1 in G(2)/M phase leads to polyubiquitination in G(1) 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.
View details for DOI 10.1074/jbc.M111.239343
View details for Web of Science ID 000291267600008
View details for PubMedID 21521692
Systematic Analysis of Essential Genes Reveals Important Regulators of G Protein Signaling
2010; 38 (5): 746-757
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.
View details for DOI 10.1074/jbc.M110.195073
View details for Web of Science ID 000289788300015
View details for PubMedID 21388955
Regulators of G-protein Signaling accelerate GPCR signaling kinetics and govern sensitivity solely by accelerating GTPase activity
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2010; 107 (15): 7066-7071
The yeast pheromone pathway consists of a canonical heterotrimeric G protein and MAP kinase cascade. To identify additional 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 Skp, Cullin, F box (SCF) ubiquitin ligase complex. Further genetic and biochemical analysis reveals that SCF(Cdc4) acts together with the Cdc34 ubiquitin-conjugating enzyme at the level of the G protein; promotes degradation of the G protein alpha subunit, Gpa1, in vivo; and catalyzes Gpa1 ubiquitination in vitro. These 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.
View details for DOI 10.1016/j.molcel.2010.05.026
View details for Web of Science ID 000278950800014
View details for PubMedID 20542006
Selective role for RGS12 as a Ras/Raf/MEK scaffold in nerve growth factor-mediated differentiation
2007; 26 (8): 2029-2040
G-protein heterotrimers, composed of a guanine nucleotide-binding G alpha subunit and an obligate G betagamma dimer, regulate signal transduction pathways by cycling between GDP- and GTP-bound states. Signal deactivation is achieved by G alpha-mediated GTP hydrolysis (GTPase activity) which is enhanced by the GTPase-accelerating protein (GAP) activity of "regulator of G-protein signaling" (RGS) proteins. In a cellular context, RGS proteins have also been shown to speed up the onset of signaling, and to accelerate deactivation without changing amplitude or sensitivity of the signal. This latter paradoxical activity has been variably attributed to GAP/enzymatic or non-GAP/scaffolding functions of these proteins. Here, we validated and exploited a G alpha switch-region point mutation, known to engender increased GTPase activity, to mimic in cis the GAP function of RGS proteins. While the transition-state, GDP x AlF(4)(-)-bound conformation of the G202A mutant was found to be nearly identical to wild-type, G alpha(i1)(G202A) x GDP assumed a divergent conformation more closely resembling the GDP x AlF(4)(-)-bound state. When placed within Saccharomyces cerevisiae G alpha subunit Gpa1, the fast-hydrolysis mutation restored appropriate dose-response behaviors to pheromone signaling in the absence of RGS-mediated GAP activity. A bioluminescence resonance energy transfer (BRET) readout of heterotrimer activation with high temporal resolution revealed that fast intrinsic GTPase activity could recapitulate in cis the kinetic sharpening (increased onset and deactivation rates) and blunting of sensitivity also engendered by RGS protein action in trans. Thus G alpha-directed GAP activity, the first biochemical function ascribed to RGS proteins, is sufficient to explain the activation kinetics and agonist sensitivity observed from G-protein-coupled receptor (GPCR) signaling in a cellular context.
View details for DOI 10.1073/pnas.0912934107
View details for Web of Science ID 000276642100090
View details for PubMedID 20351284
Extracts from two marine sponges lower cyclin B1 levels, cause a G2/M cell cycle block and trigger apoptosis in SW-13 human adrenal carcinoma cells
2004; 43 (7): 841-846
Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by heterotrimeric G-protein alpha subunits and thus inhibit signaling by many G protein-coupled receptors. Several RGS proteins have a multidomain architecture that adds further complexity to their roles in cell signaling in addition to their GTPase-accelerating activity. RGS12 contains a tandem repeat of Ras-binding domains but, to date, the role of this protein in Ras-mediated signal transduction has not been reported. Here, we show that RGS12 associates with the nerve growth factor (NGF) receptor tyrosine kinase TrkA, activated H-Ras, B-Raf, and MEK2 and facilitates their coordinated signaling to prolonged ERK activation. RGS12 is required for NGF-mediated neurite outgrowth of PC12 cells, but not outgrowth stimulated by basic fibroblast growth factor. siRNA-mediated knockdown of RGS12 expression also inhibits NGF-induced axonal growth in dissociated cultures of primary dorsal root ganglia neurons. These data suggest that RGS12 may play a critical, and receptor-selective, role in coordinating Ras-dependent signals that are required for promoting and/or maintaining neuronal differentiation.
View details for DOI 10.1038/sj.emboj.7601659
View details for Web of Science ID 000245851500004
View details for PubMedID 17380122
Marine sponges have been shown to produce metabolites with cell growth- and endocrine-altering activities. We tested extracts from two species: the 'brown variable sponge' (Anthosigmella varians) and the 'West Indian bath sponge' (Spongia barbara), for effects on the cell cycle regulatory protein, cyclin B1; cell cycle growth-phase (sub-G1/apoptosis, G1, S, and G2/M); and cell survival in SW-13 human adrenal carcinoma cultures. Polyacrylamide gel electrophoresis studies indicated a 70-90% reduction in cyclin B1 levels by treatment with these agents. Microscopic examination of cultures with DAPI staining showed dense and fragmented DNA fluorescence, characteristic of apoptosis, in both sponge extract-treated cultures but not in controls. Flow cytometry analysis showed a 16-fold increase in the percentage of cells entering apoptosis (sub-G1 phase of cell cycle) by treatment with Anthosigmella varians extract (p <0.01) and a 10-fold increase using Spongia barbara extract (p <0.01) During this same time, the percentage of cells in G2/M was increased 1.6-fold by Anthosigmella varians extract (p <0.01) and 2.0-fold by Spongia barbara extract (p <0.01) Cell growth/survival studies also indicated a time-dependent decline in the percentage confluence of cell cultures exposed to Anthosigmella varians or Spongia barbara extracts. These experiments demonstrate that some species of marine sponges have metabolites which are capable of interfering with the mammalian cell cycle and with the survival of human adrenal carcinoma cells in culture.
View details for DOI 10.1016/j.toxicon.2004.03.017
View details for Web of Science ID 000221821700012
View details for PubMedID 15284019