D. G., Dohlman, H. G., Buried
ionizable networks are an ancient hallmark of G protein-coupled
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
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,
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,
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
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
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,
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,
[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
[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
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
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
Proceedings of the National Academies of Sciences USA
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.
[See news articles by L. B. Ray (Science, 331:989 2011) and in Faculty
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
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
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
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,
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
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
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
Steve Cappell was a grad student and is now a Damon Runyan fellow with
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
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
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
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
Current Biology 18:211-5, 2008.
In this paper we show that Arr4 (now known as Get3) binds directly to
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
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
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
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
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)
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
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
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.
This paper was the first to map a protein ubiquitination site by mass
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
[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.
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.
This paper was the first to sequence a ligand-binding G
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).