Signal Transduction in
Schizosaccharomyces pombe
Benjamin Smith , Marcin Jurdzinski & Graham Ladds
The pheromone response in
Schizosaccharomyces pombe
GPCR Signalling in Sz. pombe
Working with the model
•Models like this are open to a range of techniques for mathematical analysis.
•Schizosaccharomyces pombe is a fission yeast and normally exists as 2 mating
types - M (Minus) and P (Plus).
•Upon conditions of nutrient starvation a mating response is induced [Davey,
1998].
•This mating response causes cells to secrete pheromones which bind to and
activate GPCRs (G-Protein Coupled Receptors) on the surface of cells of the
opposite mating type.
a
Pheromone
b
ACTIVATION
Mam2
Mam2
β γ
•Cells live and die by signalling. A large proportion of a cells energy is devoted to
processing information about its extracellular environment and translating this into
an adaptive response.
•The signalling network depicted in Figure 2 is a formal and more complete
representation of the one shown in Figure 1. It is a prototypical example of how a
cell may perform signal transduction and affect a response.
•Almost all of the components in this pathway are of considerable interest as they
operate in non-trivial ways and in so doing exert specific yet varied influence on
cell fate.
GDP
Plasma
Membrane
•The model will provide a clear framework within which all subsequent
experimental work can be analysed, interpreted and reviewed.
•Further work on this aspect of the project will look at robustness, stability
analysis, control analysis and quantifying information flux.
•The corollary of this is that understanding how these components operate within
the context of the whole system, has profound implications for understanding a
range of diseases as well as exposing potential avenues for treatment.
β γ
The Importance of Ras1
Efc25
Ste6
Gpa1
Ras1 GTP Ras1
Byr2
Scd1
Gpa1
•Initial analysis will involve making a library of in silico mutants, following similar
work by Kofahl and Klipp [Kofahl & Klipp, 2004] on S. cerevisiae. Numerical
simulations of signalling assays can be run on these in silico mutants, for
comparison with existing experimental results and in order to make predictions
against strains we can engineer.
P
Byr1
Cdc42
P
P
Spk1
Shk1
P
•As can be seen in Figures 1, 2 and 3, the Ras1 protein in
Sz. pombe plays a central rôle in the pheromone response
pathway. Figure 3 shows the effects of disrupting Ras1.
•Not only does it interact directly with the activated Gα
subunit but in doing so controls either cell growth
(morphology) or cell signalling (mating response) by
3. The effects of deleting Ras1
sending the signal down one of two downstream branches. Figure
on sexual response. (-) plates are in
•Efc25 → Morphology
Cytoplasm
P
•Ste6 → Mating Response
MORPHOLOGY
Ste11
Spk1
P
P
P
P
P
Nucleus
Ste11
sxa2
mam2
rgs1
ste6
ste11
•This suggests that competition between the two
proteins determine the extent of each response.
c
P
Mating Response Genes
Figure 2. A preliminary process diagram of the pheromone response pathway in Sz. pombe, constructed using
CellDesigner2.5. This model incorporates a number of working hypotheses which can now be tested.
•Gaining an holistic understanding of the the response is one of the central
objectives of this project.
•There are many clear, yet often counter-intuitive results on different components of
this pathway.
•Integrating as much of this biological data as possible will help to answer a
multitude of questions they give rise to.
Figure 1. A schematic representation of the pheromone response GPCR signalling pathway in Sz. pombe. a)
shows the preactivation complex where inactive Mam2 (Sz. pombe P-type GPCR) is bound to trimeric G-protein.
b) shows the how signalling proceeds, following activation of the GPCR by pheromone. Ras1 activates either of
two separate pathways, depending upon whether Ste6 or Efc25 interaction is involved. c) If signalling goes
through Ste6, a MAP kinase cascade is triggered resulting in phosphorylation of transcription factor Ste11 within
the nucleus.
•GPCR’s are a ubiquitous family of seven span transmembrane receptors that allow
cells to respond to a variety of extracellular signals [Ladds et al., 2003].
•GPCR activation results in the exchange of GDP for GTP on the Gα subunit (Gpa1) of
the G-protein which then leads to a signalling cascade as shown in Figure 1.
•Replacement of some of the response gene open reading frames with reporter genes
in modified strains of Sz. pombe allows us to investigate many aspects of GPCR
signalling.
normal Log phase growth, (+) plates
uare under starvation conditions.
[Papadaki et al., 2002]
•Using established techniques from dynamical systems theory, this process
diagram is being translated into a kinetic model of the form
r
dCi
= ∑η ij v j
dt
j =1
•Where Ci is the concentration of the ith species, ηij is the stoichiometric coefficient
representing the ith species and the jth reaction, and vj is the kinetic rate constant
of the jth reaction.
•The mechanism by which this happens is not fully
understood. The effect is shown in figure 4.
•To investigate this experimentally two gene
knockouts will be performed; one to remove Efc25
and the other to remove Ste6. We would expect to
see just mating response and just morphology
response, respectively.
Figure 4. Ste6 disruptants (ste6∆) and
Efc25 disruptants (efc25∆), transformed
with vectors conatining Ste6 (Ste6↑) and
Efc25 (Efc25↑) ORF’s. [Papadaki et al.,
2002]
•It has been shown [Hughes, 1995] that mutations in mammalian Ras proteins
are responsible for many forms of cancer. Investigating how it operates in its
capacity to control and direct signalling may open the way for important research
into controlling tumour formation by controlling Ras.
References:
DAVEY, J. 1998. Fusion of a fission yeast. Yeast 14: 1529-1566.
HUGHES, D. A. 1995. Control of signal transduction and morphogenesis by Ras. seminars in CELL
BIOLOGY 6: 89-94
LADDS, G.; DAVIS, K.; HILLHOUSE, E.W. and DAVEY, J. 2003 Modified yeast cells to investigate the
coupling of GPCRs to specific G proteins. Mol. Microbiol. 47: 781-792.
KOFAHL, B.; KLIPP, E. 2004. Modelling the dynamics of the yeast pheromone pathway, Yeast 21: 831850
PAPADAKI, P.; PIZON, V.; ONKEN, B.; CHANG, E. C.; 2002 Two Ras Pathways in Fission Yeast Are
Differentially Regulated byTwo Ras Guanine Nucleotide Exchange Factors. Mol. Cell Biol. 22(13): 45984606
The Author