Research in my laboratory is aimed at understanding how cellular behavior
is dictated by external signals. Currently, our studies stress three general
topics: What is the role of subcellular compartmentalization in signal transduction?
How do cells judge the location of extracellular cues and mount a directional
response? How are different signaling pathways with shared components
kept insulated from each other?
To investigate these issues, we use the simple eukaryotic cells of bakers
yeast, Saccharomyces cerevisiae, as a model for more complex cells.
(see Figure 1
The role of subcellular localization in signal transduction
has been highlighted by our efforts to understand how the heterotrimeric
G protein Gbg complex activates
its downstream MAP kinase cascade. Here, our observations suggested
that this activation involves the recruitment of a kinase cascade “scaffold”
protein, Ste5, from the cell interior to the periphery. Furthermore,
our work suggests that the factor waiting for the MAP kinase cascade to arrive
at the membrane is the PAK-family kinase Ste20, and that the function of
Ste20 depends on another membrane-resident protein, the GTPase Cdc42.
A second project is focused on how cells polarize toward localized
signals. This behavior, exhibited by many types of cells, implies communication
between the molecules that sense the signal and those that govern cell shape
and polarity. Our work suggests that, in addition to its role in activating
the MAP kinase cascade, the yeast heterotrimeric G protein has a separate
function in orienting cell polarity along gradients of pheromone chemoattractants.
This appears to involve the formation of multiprotein complexes between G
bg and polarity-control proteins (see Butty et al, 1998). In
addition, our work suggests that Gbg
must be regulated in a spatially-asymmetric manner by the receptor and G
a subunit in order for to properly guide cell polarization along external
gradients. (see Figure 2
We are also interested in the fidelity of signaling molecules,
and have recently begun a project investigating the “insulation” of different
signal transduction pathways. In yeast, as in human cells, multiple
independent signaling pathways operate to generate different responses to
different stimuli. Remarkably, individual protein molecules sometimes
function in multiple pathways, raising the question of how these pathways
are kept insulated from each other to avoid “crosstalk” between them.
We have been able to show that association with pathway-specific binding
partners causes signaling molecules to become sequestered into discrete complexes
that are dedicated to single pathways. To do this, we developed a
method for forcing signaling molecules to associate with a subset of their
possible partners. We anticipate that this approach will be of wide
interest, as it allows one to create custom signaling molecules that are
targeted toward chosen pathways. (see Figure 3
Figure 1. Mating reaction (left) and pheromone
response pathway (right) of Saccharomyces cerevisiae. Mating
pheromones (a factor and a
factor) secreted by each cell stimulate the partner cell to arrest cell
division and alter cell polarity. Many proteins have been identified as signaling
intermediates in pheromone response (right). Our efforts have been
directed at understanding the function of the G
bg dimer, the “scaffold” protein Ste5, the PAK family kinase Ste20,
and the control of polarity by the receptor/G-protein system.
Figure 2. (left) Pheromone-stimulated
membrane recruitment of the kinase cascade scaffold protein, Ste5.
This recruitment is dependent on the G
bg dimer, but not on downstream signaling events, and is thought to
allow the Ste5-associated kinase cascade to become activated by the membrane-bound
PAK kinase, Ste20 (see Pryciak and Huntress, 1998). (right) Cartoon
depicting yeast cells being exposed to a gradient of pheromone released from
a micropipette. Wild-type cells produce projections that grow in a
directional fashion toward the source of pheromone (Segall, 1993).
Chemotropism mutants are unable to orient their projections along the pheromone
gradient. Defects in chemotropism result when the G
bg-Far1-Cdc24 complex is disrupted by mutation in Far1 (Valtz et al,
1995; Dorer et al, 1995; Butty et al, 1998), Cdc24 (Nern and Arkowitz, 1998),
or Gbg (Pryciak et al, in preparation).
Figure 3. Distinct pathways share a common component.
The kinase Ste11 functions in at least three separate signaling pathways:
mating, filamentous growth, and the high osmolarity glycerol (HOG) response.
Though sharing a common kinase, these pathways are insulated from each other,
as each stimulus activates only a single pathway. Pathway-specific
scaffold proteins such as Ste5 and Pbs2 are thought to provide this insulation.
In addition to binding kinases, these scaffolds bind pathway-specific activators
at the membrane, possibly coupling each stimulus to a discrete set of kinases.
Membrane recruitment of Ste5 by Gbg
is implicated in the activation mechanism of the mating pathway (Pryciak
and Huntress, 1998); a related role for interaction of Pbs2 with the transmembrane
protein Sho1 has been recently proposed (Raitt et al, 2000). Note that
Ste20 can function in all three pathways as the activator of Ste11; the suggestion
that stimuli bring select substrates to Ste20 (Pryciak and Huntress, 1998)
may explain how Ste20 avoids cross-talk. Whether a scaffold exists
for the filamentation pathway is unknown.
Lamson, R.E., Winters, M.J., and Pryciak, P.M. (2002).
Cdc42 regulation of kinase activity and signaling by the yeast PAK Ste20.
Mol. Cell. Biol. 22: 2939-2951.
Harris, K., Lamson, R.E., Nelson, B., Hughes, T.R., Marton,
M.J., Roberts, C.J., Boone, C., and Pryciak, P.M. (2001). Role
of scaffolds in MAP kinase pathway specificity revealed by custom design
of pathway-dedicated signaling proteins. Curr. Biol. 11:
Moskow, J.J., Gladfelter, A.S., Lamson, R.E., Pryciak, P.M.,
and Lew, D.J. (2000). Role of Cdc42p in pheromone-stimulated signal transduction
in Saccharomyces cerevisiae. Mol. Cell. Biol. 20: 7559-7571.
Pryciak, P.M. and Huntress, F.A. (1998). Membrane recruitment
of the kinase cascade scaffold protein Ste5 by the G
bg complex underlies activation of the yeast pheromone response pathway.
Genes Dev. 12: 2684-2697.
Butty, A.-C., Pryciak, P.M., Huang, L.S., Herskowitz, I. and
Peter, M. (1998). The role of Far1p in linking the heterotrimeric G protein
to polarity establishment proteins during yeast mating. Science 282:
Pryciak, P.M. and Hartwell, L.H. (1996). AKR1 encodes
a candidate effector of the Gbg
complex in the Saccharomyces cerevisiae pheromone response pathyway
and contributes to control of both cell shape and signal transduction.
Mol. Cell. Biol. 16: 2614-2626.
Dorer, R., Pryciak, P.M. and Hartwell, L.H. (1995). Saccharomyces
cerevisiae cells execute a default pathway to select a mate in the absence
of pheromone gradients. J. Cell Biol. 131: 845-861.
Project #1: How the heterotrimeric G protein
bg complex (Gbg) activates
a downstream MAP kinase cascade. We know that G
bg stimulates the translocation of a kinase cascade "scaffold" protein,
Ste5, to the plasma membrane. This project would address whether factors
in addition to Gbg can contribute
to recruitment or retention of Ste5 at the plasma membrane; and if so, identifying
these factors by a genetic approach.
Project #2: How cells polarize toward localized signals. In addition
to activating the MAP kinase cascade, the yeast G
bg complex has a separate function in orienting cell polarity along
gradients of chemoattractant pheromones, by complexing with cytoskeletal
proteins. This project would address how the receptor/G-protein system organizes
a focus of morphogenesis to a discrete region of the cell periphery.
Project #3: The function of a novel transmembrane G
bg-binding protein, called Akr1. We suspect Akr1 helps traffic the
Gbg complex to and away from
the plasma membrane. This project would use fluorescence microscopy to determine
the subcellular location of Akr1, which is likely to be Golgi or endosomal
membranes. A related project would test the role of endocytosis in regulating
Gbg signaling or desensitization.
B.S., University of California, Los Angeles, 1983.
Ph.D., University of California, San Francisco 1992.
Postdoctoral Fellow, University of Washington, Seattle, 1992-1996.