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Cell Signaling - PowerPoint Pres

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					               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 G-
                      protein

                      •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               P
degradation
                                                     Kinase 1
•Kinases are often themselves activated
by other kinases via phosphorylation and                        P
can organize into phosphorylation                    Kinase 2
cascades
                                                                P
•One important class of phosphorylation
cascade is called a mitogen activated                Kinase 3
protein kinase (MAPK) cascade
                                             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                                        Effector Protein
transcription factors

•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                           Cytoskeletal Rearrangement
activated during specific cellular responses are
called response elements
                                                           Effector protein
•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
                                                                                   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

                                                   •The  mating type pheromone, alpha factor, binds
                           a cells, no pheromone
                                                   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
      + mating pheromone                           subunit releases GDP and binds to GTP
         (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

                                                   •The net results are cell cycle arrest, cytoskeletal
                                                   rearrangements to “grow” toward where the
                           a cells, + pheromone    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

                                                                       •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
         Pheromone                           Nitrogen Starvation

                                                                       •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

•With so many components in common,
how do yeast cells keep their signals
straight?

•Two mechanisms yeast employ to achieve                  Ste11                                   Ste11




                                                                                                            Pbs2
                                                Ste5
signaling specificity are scaffolding and                Ste7
cross-pathway inhibition
                                                         Fus3                                    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             Cross-pathway inhibition of the filamentation pathway by the
                                                pheromone pathway: Fus3 phosphorylates and triggers degrad-
stays off by actively inhibiting it
                                                ation of Tec1, the transcription factor required for filimentation
Engineering Cross-Talk: Rewiring Yeast MAPK Pathways
•If scaffolds promote specificity, it    The theory:
should be possible to rewire
pathways by engineering
scaffolds with new connections

•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                  The construct:   The result:

•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
                    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

				
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