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Isom, D. G., Dohlman, H. G., Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation.
Proceedings of the National Academies of Science U S A. 112:5702-7, 2015.

  • This is a structural bioinformatics analysis of G protein coupled receptors. It identifies an electrostatic network that constitutes a “signature” of receptor activation, and shows that the same network is conserved in archael 7-transmembrane light-driven proton pumps.

English, J. G., Shellhammer, J. P., Malahe, M., McCarter, P. C., Elston, T. C. and Dohlman, H. G., MAPK feedback encodes a switch and timer for tunable stress adaptation in yeast. Science Signaling 8:ra5, 2015.

  • The MAPK Hog1 has long served as a model for cellular stress adaptation systems. This paper demonstrated that Hog1 has features of both a switch and a rheostat. Whereas Hog1 is activated as a switch, it phosphorylates protein substrates in a graded manner. Through computational modeling, single cell measurements, mutational analysis, and quantification of protein phosphorylation we were able to reconcile these seemingly incongruous behaviors. In particular, we showed that Hog1 converts input strength to signal duration, that the duration of activation is graded, and that this behavior serves to engage a graded gene induction program that is optimized to meet the needs of individual cells. Finally we showed that feedback by Hog1 is essential for the switch-to-rheostat conversion.

Kelley, J. B., Dixit, G., Sheetz, J. B., Venkatapurapu, S. P., Elston, T. C. and Dohlman, H. G., RGS proteins and septins cooperate to promote chemotropism by regulating polar cap mobility. Current Biology 25:1-11, 2015.

Dixit, G., Kelley, J. B., Houser, J. R., Elston, T. C., and Dohlman, H. G., Cellular noise suppression by the regulator of G protein signaling Sst2.
Molecular Cell 55:85-96, 2014.

  • Sst2 is the founding member of the Regulator of G protein Signaling (RGS) family. We had shown earlier that Sst2 promotes G protein GTPase activity and thereby dampens the activity of downstream effectors (32). In addition, Sst2 is one of a subset of RGS proteins that binds to the receptor and dampens signaling by a distinct mechanism (33). Using microfluidics and single-cell analysis (Fig. 4), Dixit et al. (26) showed that Sst2 suppresses cell-to-cell variability (noise) in transcription and morphogenesis. Kelley et al. (1) showed that Sst2 is required for the organization of septin structures, which dictate the position of the polar cap. Using point mutants that selectively uncouple Sst2 from either the receptor or the G protein, we determined that noise suppression and proper morphogenesis are specifically attributable to the GTPase-activating function of Sst2, and are independent of interactions with the receptor. Without this GTPase-activating function, the septin collar and polar cap are disorganized and the cells can no longer track a pheromone gradient. Finally, these findings revealed that proper assembly of septins is essential for proper gradient tracking. Thus by selectively uncoupling Sst2 from its two known binding partners, in each case by a single amino acid substitution, we could compare strains that produce equivalent signal outputs but widely different cell growth and noise characteristics. Collectively, these findings revealed how feedback inhibition contributes to gradient tracking and noise suppression.

Isom, D. G., Sridharan, V., Baker, R., Clement, S. T., Smalley, D. M., and Dohlman, H. G., Protons as second messenger regulators of G protein signaling.
Molecular Cell 51:531-538, 2013.

Clement, S. T., Dixit, G., and Dohlman, H. G., Regulation of yeast G protein signaling by the kinases that activate the AMPK homolog Snf1.
Science Signaling 6:ra78, 2013.

  • [See persepectives by M. Schmidt (Science Signaling, 6:pe28, 2013) and S. Sprang (Molecular Cell, 51:405-406, 2013)]
  • G proteins are well known to transmit signals from hormone and neurotransmitter receptors. The Isom paper shows that G proteins have an additional role as direct sensors of intracellular pH, which decreases substantially during glucose limitation. The Clement paper shows that the G protein is phosphorylated in response to glucose limitaiton, and does so via the same kinases and phosphatase that act on the glucose-sensing AMPK, Snf1. These findings reveal a new way for cells to fine tune receptor signals, depending on nutrient availability.
  • Sarah Clement is now working at PAREXEL, a nearby CRO.

Baker, R., Lewis, S. M., Wilkerson, E. M., Sasaki, A. T., Cantley, L. C., Kuhlman, B., *Dohlman, H. G., and *Campbell, S. L., Site-Specific Monoubiquitination Activates Ras by Impeding GTPase Activating Protein Function.
Nature Structural & Molecular Biology 20:46-52, 2012. *corresponding authors

  • [See news article by K. H. Wrighton (Nature Reviews Molecular Cell Biology, 14:66-7 2013) and in Faculty of 1000]
  • This paper demonstrates that monoubiquitination activates Ras by impeding the normal function of GTPase activating proteins. These findings suggest an entirely new mode of Ras activation, in which Ras signaling can occur in the absence of an extracellular stimulus or gene mutation, through a post-translational modification (ubiquitination).
  • Rachael Baker has defended her thesis and is now Assistant Professor of Chemistry and Biochemistry at Calvin College in Michigan.

Lien, E., Nagiec, M. J., and Dohlman, H. G., Proper protein glycosylation promotes mitogen-activated protein kinase signal fidelity.
Biochemistry 52:115-24, 2013.

  • This paper describes a large-scale screen for mutations that alter MAPK signal specificity. Analysis of the nonessential gene deletion collection revealed two mutants that allow inappropriate activation of the MAPK Kss1 under conditions that normally stimulate the MAPK Hog1. Both genes identified, MNN10 and MNN11, encode α-1,6-mannosyltransferases. Substitution of a single glycosylation site in the signaling mucin Msb2 likewise resulted in inappropriate activation of Kss1, revealing Msb2 as a likely target of regulation by Mnn10 and Mnn11.
  • Evan Lien was an undergraduate and is now a grad student with Alex Toker at Harvard Medical School.
  • Michal Nagiec was a grad student and is now a postdoc with John Blenis at Harvard Medical School.

Jones, J. C., Jones, A. M., Temple, B. R. S., and Dohlman, H. G., Differences in intradomain and interdomain motion confer distinct activation properties to structurally similar Gα proteins.
Proceedings of the National Academies of Sciences USA 109:7275-9. 2012.

Jones, J. C., Duffy, J. W., Machius, M., Temple, B. R. S., *Dohlman, H. G., and Jones, A. M., The crystal structure of a self-activating G protein α-subunit reveals its distinct mechanism of signal initiation.
Science Signaling 4:ra8, 2011. *corresponding author

  • [See news articles by L. B. Ray (Science, 331:989 2011) and in Faculty of 1000]
  • These two papers describe our analysis of a self-activating G protein in Arabidopsis, AtGPA1. Whereas most G proteins are activated by cell surface receptors, AtGPA1 is permanently activated and has no known receptor binding partner. In the Science Signaling paper we solved the AtGPA1 crystal structure. That work revealed that the helical domain of AtGPA1 is distinct from that of its animal counterparts. Other investigators later showed that a receptor/G protein complex triggers a dramatic rearrangement of the helical domain away from the ras-like domain, one that would allow GTP binding and signal initiation. In our PNAS paper we used molecular dynamics simulations to compare the animal and plant Gα proteins. That analysis revealed unexpected differences in inter- and intra-domain motion in Gα; follow up experimental analysis using chimeric proteins established that a small subdomain – the αA helix, within the helical domain – is almost entirely responsible for activation of AtGPA1. These results were particularly surprising given that the αA helix is quite distant from regions involved in binding to receptors, effectors and guanine nucleotides. More broadly these findings highlight the utility of distinct model systems (plants, animals, fungi) as of integrated structural and computational approaches for understanding G protein function.
  • Jan Jones was a postdoc and is now a scientist at AgBiome in Research Triangle Park, NC.

Nagiec, M. J., and Dohlman, H. G., Checkpoints in a Yeast Differentiation Pathway Coordinate Signaling During Hyperosmotic Stress.
PLoS Genetics, 8(1): e1002437, 2012.

  • All cells must prioritize responses when confronted with competing signals. However the molecular mechanisms that govern signal prioritization are poorly understood. This article investigated signal coordination by the pheromone mating (differentiation) pathway and the high osmolarity glycerol (stress) pathway. These pathways respond to competing stimuli despite sharing pathway components. By monitoring both short-term and long-term outputs and by using multiple cellular and biochemical measures of activity, we made the unexpected observation that yeast cells delay cell mating in the presence of an osmotic stress. To understand the mechanism of delayed differentiation, we used synthetic pathway activators together with genetic and molecular approaches to dissect the signaling network. We found that the stress-responsive MAP kinase Hog1 phosphorylates two different substrates that together serve to transiently limit activation of the differentiation MAP kinase Fus3. One substrate acts upstream and is required for Fus3 activation, while the other lies downstream and is required for induction of Fus3 expression. These findings revealed that pathway cross-inhibition is not a single process, but rather a network of events that work together to postpone cell differentiation until the cell adapts to stress conditions.
  • Michal Nagiec was a grad student and is now a postdoc with John Blenis at Harvard Medical School.

Cappell, S. D., Baker, R., Skowyra, D., and Dohlman, H. G., Systematic analysis of essential genes reveals important regulators of G protein signaling.
Molecular Cell 38:746-57, 2010.

  • [See news articles by L. B. Ray (Science Signal. 3:ec191, 2010) and in Faculty of 1000]
  • The pheromone response in yeast is arguably the best characterized of any signaling pathway. In this paper we considered whether there might be additional signaling components that are also essential for cell viability and had therefore eluded detection. Indeed the signaling function of essential genes had not previously been studied in any systematic manner. Using a powerful new resource for analysis of the “essential genome” we identified new components and regulators of the G protein signaling apparatus, and characterized several of these in detail. Our analysis revealed an important role for SCF- and ubiquitin-mediated proteolysis in G protein regulation. More generally it revealed considerable overlap among genes required for cell viability and signal transduction.
  • We subsequently screened the protein kinome and found that a cell-cycle regulated kinase, Elm1, phosphorylates Gpa1 and triggers its polyubiquitination by SCF (Torres M., et al. JBC 286:20208-16, 2011).
  • Steve Cappell was a grad student and is now a Damon Runyan fellow with Tobias Meyer at Stanford University.
  • Matt Torres is now Assistant Professor of Biology at Georgia Tech.

Hao, N., Behar, M., Parnell, S. C., Torres, M. P., Borchers, C. H., Elston, T. C., and Dohlman, H. G., A systems-biology analysis of feedback inhibition in the Sho1 osmotic stress-response pathway.
Current Biology 17:659-67, 2007.

Hao, N., Nayak, S., Behar, M., Shanks, R. H., Nagiec, M. J., Errede, B., Hasty, J., Elston, T. C., and Dohlman, H. G., Regulation of cell signaling dynamics by the protein kinase-scaffold Ste5.
Molecular Cell 30:649-56, 2008.

  • [See news article in Faculty of 1000]
  • In the Current Biology paper we developed mathematical models of the yeast osmotic stress-response pathway. These models predicted the existence of a desensitization event early in the pathway that requires Hog1 MAP kinase. We then demonstrated that Hog1 phosphorylates the plasma membrane osmosensor Sho1, mapped the phosphorylation site by mass spectrometry, and demonstrated that Sho1 exists normally as a homo-oligomer. Further, we showed that feedback phosphorylation leads to diminished oligomerization of Sho1, diminished activation of Hog1, and diminished growth in high salt conditions. Based on these findings we proposed that feedback regulation allows the cells to respond to a wide range of signal inputs, in addition to providing an effective means of signal amplification.
  • In the Molecular Cell paper we developed mathematical models of the yeast pheromone-response pathway. Yeast, which are otherwise non-motile, will expand in the direction of a weak pheromone stimulus and thus towards a distant mating partner. We constructed a microfluidic growth chamber capable of exposing cells to a precisely-controlled pheromone gradient, and showed that the kinase scaffold Ste5 is needed to discriminate between pheromone doses appropriate for chemotropic growth versus doses that trigger growth arrest, and does so by altering the time- and dose-dependent behavior of the MAP kinase Fus3.
  • Given the burgeoning interest in single-cell analysis and in computational modeling of signaling networks and pathways, the approach is likely to guide future efforts to understand temporal- and spatial-control mechanisms in animal cells.
  • Nan Hao was a grad student and later a postdoc with Erin O’Shea at HHMI/Harvard College. He is now Assistant Professor Biological Sciences at UC San Diego.

Lee, M. J., and Dohlman, H. G., Coactivation of G protein signaling by cell-surface receptors and an intracellular exchange-factor.
Current Biology 18:211-5, 2008.

  • In this paper we show that Arr4 (now known as Get3) binds directly to the G protein and promotes G protein activation (exchange of GDP for GTP), in the manner of cell surface receptors. In contrast to receptors however, Arr4 is expressed in the cytoplasm rather than at the cell surface. These findings reveal that receptor-initiated signals outside the cell are sustained by non-receptor exchange-factors expressed inside the cell.
  • Mike Lee was a grad student and later a postdoc with Mike Yaffe at MIT. He is now Assistant Professor of Systems Biology at UMass Medical School.

Slessareva, J. E., Routt S. M., Temple, B., Bankaitis, V. A., and Dohlman, H. G., Activation of the phosphatidylinositol 3-kinase Vps34 by a G protein α subunit at the endosome.
Cell 126:191-203, 2006.

  • [See perspectives by M. Koelle (Cell 126:25-7, 2006), by L. Bardwell (Curr. Biol., 2006), in Science STKE (tw234, 2006) and in Faculty of 1000]
  • In this paper we showed that the G protein α subunit Gpa1 signals via the phosphatidylinositol 3-kinase components Vps34 and Vps15. In contrast to previously-identified G protein effectors, Vps34 and Vps15 are located at endosomes rather than at the plasma membrane. We found that the activated (GTP-bound) form of Gpa1 binds selectively to the catalytic subunit Vps34 and promotes increased phosphatidylinositol 3-phosphate production. In contrast, unactivated (GDP-bound) Gpa1 binds to Vps15, in the manner of known G protein β subunits. We proposed a mechanism of G protein-effector interaction, analogous to that used for GIRK channels, in which the Gα (Gpa1) and Gβ-like subunits (Vps15) bind simultaneously to the effector (Vps34) during the cycle of signal activation and inactivation.
  • We subsequently solved the X-ray crystal structure of the Vps15 WD domain, which revealed a seven-bladed propeller resembling that of typical Gβ subunits. The WD domain is sufficient to bind Gpa1 as well as to Atg14, a potential Gγ protein that binds directly to Vps15 (Heenan E., et al. Biochemistry 48:6390-401, 2009). Thus Vps15 and Atg14 appear to function as an atypical, endomembrane-associated Gβγ pair.
  • Janna Slessareva is now a scientist in Research Triangle Park, NC. Erin Heenan is in law school.

Older Papers of Note

Guo, M., Aston, C., Burchett, S. A., Dyke, C., Fields, S., Rajarao, S. J. R., Uetz, P, Wang, Y., Young, K., and Dohlman, H. G., The yeast G protein α subunit Gpa1 transmits a signal through an RNA-binding effector protein Scp160.
Molecular Cell 12:517-24, 2003.

  • This paper was the first to establish a positive signaling function for the G protein α subunit in yeast, Gpa1. The effector in this case, the RNA-binding protein Scp160, had not previously been recognized to act in the pheromone response pathway. More generally, RNA-binding proteins had not previously been identified as G protein effectors.
  • It has since been shown that Gpa1 promotes Scp160-mediated mRNA trafficking to the tip of the mating projection, chemotropism and completion of the mating program (Gelin-Licht R., et al. Cell Reports 1:483-94, 2012).

Marotti, L., Newitt, R., Wang, Y., Aebersold, R., and Dohlman, H. G., Direct identification of a G protein ubiquitination site by mass spectrometry.
Biochemistry 41:5067-74, 2002.

  • This paper was the first to map a protein ubiquitination site by mass spectrometry.

Dohlman, H. G., Apaniesk, D., Chen, Y., Song, J., and Nusskern, D., Inhibition of G-protein signaling by dominant gain-of-function mutations in Sst2p, a pheromone desensitization factor in Saccharomyces cerevisiae.
Molecular and Cell Biology 15:3635-43, 1995.

  • [See news articles by W. Roush (Science 271:1056-8, 1996) and R. Iyengar (Science 275:42-3, 1997)].
  • This paper was the first to show that Sst2 regulates the G protein. Sst2 is the founding member of the RGS protein family, the GTPase activating proteins for heterotrimeric G proteins.

Dixon, R. A. F., Kobilka, B. K., Strader, D. J., Benovic, J. L., Dohlman, H. G., Frielle, T., Bolanowski, M. A., Bennett, C. D., Rands, E., Diehl, R. E., Mumford, R. A., Slater, E. E., Sigal, I. S., Caron, M. G., Lefkowitz, R. J. and Strader, C. D., Cloning of the gene and cDNA for mammalian β-adrenergic receptor and homology with rhodopsin.
Nature 321:75-9, 1986.

  • [See:]
  • This paper was the first to sequence a ligand-binding G protein-coupled receptor.
  • Follow up studies revealed the existence of a seven-span topology, sites of phosphorylation in the third intracellular loop and C-tail, as well as sites of glycosylation and disulfide bond formation in the outer loops (Dohlman H., et al. JBC 262:14282-8, 1987; Dohlman H., et al. Biochemistry 27:1813-7, 1988).