Transcript Document

Cell signaling: responding to the outside world
•Cells interact with their environment by
interpreting extracellular signals via
proteins that span their plasma
membrane called receptors
•Receptors are comprised of
extracellular and intracellular domains
•The extracellular domain relays
information about the outside world to
the intracellular domain
•The intracellular domain then interacts
with other intracellular signaling
proteins
•These intracellular signaling proteins
further relay the message to one or
more effector proteins
•Effector proteins mediate the
appropriate response
Receiving the Signal: G-protein Coupled Receptors (GPCRs)
•GPCRs are an important and ubiquitous class of
eukaryotic receptors (>700 in humans)
•The extracellular domain connects to the intracellular
domain through 7 transmembrane spans
•The intracellular domain is coupled to a heterotrimeric Gprotein
•The heterotrimeric g-protein is composed of 3 subunits:
G, G, and G
•When the G subunit is bound to GDP it is “OFF”; when it
is bound to GTP it is “ON”
•When the extracellular domain binds
to the signal molecule, it causes a
conformational change relayed
through the transmembrane spans to
the intracellular domain
•The conformational change relayed
to the intracellular domain causes the
G subunit to release GDP and bind
to GTP thereby activating both the G
and G/G subunits
Transmitting the Signal: Protein Kinases
•Activated receptors frequently transmit
signals through through intracellular
signaling proteins called kinases
•Protein kinases are enzymes that add a
phosphate group from ATP onto a
substrate protein; this reaction is called
phosphorylation
•Phosphorylation frequently serves to
activate the substrate of the kinase, but
can also target the substrate for
degradation
•Kinases are often themselves activated
by other kinases via phosphorylation and
can organize into phosphorylation
cascades
•One important class of phosphorylation
cascade is called a mitogen activated
protein kinase (MAPK) cascade
P
Kinase 1
P
Kinase 2
P
Kinase 3
Phosphorylation Cascade
Responding to the Signal: Effector Proteins
•The final step in cell signaling is activation of the
effector proteins
•The effector proteins carry out the cellular response
to the signal
Changes in gene expression
•Often the cellular response involves expression of
previously inactive genes which requires effector
proteins called transcriptional activators or
transcription factors
Effector Protein
•Transcription factors are proteins that bind to
specific DNA sequences called promoters that are
upstream of the genes that are turned on
•Promoters that are upstream of genes that are only
activated during specific cellular responses are
called response elements
•Effector proteins can also directly act on proteins
that regulate cell shape to induce changes in
morphology by rearranging the cytoskeleton
•Other types of effector proteins directly regulate cell
growth by arresting the cell cycle or altering cellular
metabolism
Cytoskeletal Rearrangement
Effector protein
Cell Cycle Arrest
A model signaling pathway: The Yeast Pheromone Pathway
•There are two mating types (“sexes”) of yeast, a
and  (in the lab we generally study the a mating
type)
•They can mate by responding to an extracellular
signal, called a pheromone (13 amino acid peptide),
released by one mating type and received by the
other
a cells, no pheromone
•The  mating type pheromone, alpha factor, binds
to a GPCR on the surface of an a cell to initiate
signaling
•The GPCR undergoes a conformational change
that is transmitted to the G-protein whose G
subunit releases GDP and binds to GTP
+ mating pheromone
(alpha factor)
•The GTP-bound G subunit then dissociates from
the G/G subunit which in turn initiates a MAPK
phosphorylation cascade where a MAP kinase
kinase kinase (MAPKKK) activates a MAP kinase
kinase (MAPKK) which activates a MAP kinase
(MAPK)
•The activated MAPK then activates several effector
proteins: a transcription factor and a cell-cycle
inhibitor
a cells, + pheromone
•The net results are cell cycle arrest, cytoskeletal
rearrangements to “grow” toward where the
pheromone originated (in hopes of mating
successfully), and expression of genes required for
fusion to the opposite mating type
Other MAPK Signaling Pathways in Yeast
Pheromone Pathway
Filamentation Pathway
HOG Pathway
•In addition to the pheromone pathway,
yeast have several other pathways that
use the MAPK architecture to transmit
signals
Pheromone
Nitrogen Starvation
•Two other commonly studies MAPK
pathways in yeast are the High Osmolarity
Glycerol pathway (HOG pathway), which
responds when there is high salt in the
environment, and the filamentation
pathway which responds to lack of
nitrogen in the environment
•These pathways and the pheromone
pathway share some components
•How do these pathways keep their
prevent cross-talk and maintain signal
specificity?
Achieving Specificity in Signaling in Yeast MAPK Pathways
Ste11
Ste11
Ste7
Fus3
Pbs2
•Two mechanisms yeast employ to achieve
signaling specificity are scaffolding and
cross-pathway inhibition
Ste5
•With so many components in common,
how do yeast cells keep their signals
straight?
Hog1
•Scaffolds: the pheromone pathway uses
the scaffold Ste5, and the HOG pathway
uses the scaffold/MAPKK Pbs2
Scaffolds in the pheromone and HOG pathways
•Scaffolds promote signaling efficiency by
localizing all the proteins in one cascade
close together
•Scaffolds promote signaling specificity by
preventing upstream activators (e.g.
MAPKKK or MAPK) from interacting with
inappropriate downstream proteins (e.g. the
wrong MAPK or effector)
•Cross-pathway inhibition promotes
signaling specificity by having the activated
pathway make sure the other pathway
stays off by actively inhibiting it
Cross-pathway inhibition of the filamentation pathway by the
pheromone pathway: Fus3 phosphorylates and triggers degradation of Tec1, the transcription factor required for filimentation
Engineering Cross-Talk: Rewiring Yeast MAPK Pathways
•If scaffolds promote specificity, it
should be possible to rewire
pathways by engineering
scaffolds with new connections
The theory:
•Goal: link the pheromone
pathway to the HOG pathway, so
when you add pheromone, you
induce the salt survival response
•Step 1: Fuse the pheromone
scaffold, Ste5, to the HOG
pathway scaffold, Pbs2
•Step 2: Remove the binding site
in the Ste5 for the pheromone
MAPKK (Ste7)
•Step 3: Remove the connection
between the Pbs2 and the
upstream salt response proteins
•Now pheromone induces the
HOG response
The construct:
The result:
References
•Alberts et al. Molecular Biology of the Cell, Chapter 15
•Dohlman, H. and Thorner, J. Regulation of G-Protein
initiated signal transduction in yeast: paradigms and principles.
Annu. Rev. Biochem. 2001. 70:703–54
•Bao et al. Pheromone-dependent destruction of the Tec1
transcription factor is required for MAP kinase signaling
specficity in yeast. Cell. 2004. 119: 991
•Schwartz and Madhani. Principles of MAP kinase signaling
specificity in Saccharomyces cerevisiae. Annu. Rev. Genet.
2004. 38: 725
•Park, Zarrinpar and Lim. Rewiring MAP kinase pathways
using alternative scaffold assembly mechanisms. Science
2003. 299:1061