Cell signaling: responding to the outside world

1
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

2
Receiving the Signal G-protein Coupled Receptors
(GPCRs)

• GPCRs are an important and ubiquitous class of
  eukaryotic receptors (gt700 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

3
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

4
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
• Often the cellular response involves expression
  of previously inactive genes which requires
  effector proteins called transcriptional
  activators or 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 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

Changes in gene expression
Effector Protein
Cytoskeletal Rearrangement
Cell Cycle Arrest
5
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
  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
• 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 pheromone originated (in hopes of
  mating successfully), and expression of genes
  required for fusion to the opposite mating type

a cells, no pheromone
mating pheromone (alpha factor)
a cells, pheromone
6
Other MAPK Signaling Pathways in Yeast
Filamentation Pathway
HOG Pathway
Pheromone 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
• These pathways and the pheromone pathway share
  some components
• How do these pathways keep their prevent
  cross-talk and maintain signal specificity?

Nitrogen Starvation
Pheromone
7
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 signaling
  specificity are scaffolding and cross-pathway
  inhibition
• Scaffolds the pheromone pathway uses the
  scaffold Ste5, and the HOG pathway uses the
  scaffold/MAPKK Pbs2
• 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

Scaffolds in the pheromone and HOG pathways
Cross-pathway inhibition of the filamentation
pathway by the pheromone pathway Fus3
phosphorylates and triggers degrad-ation of
Tec1, the transcription factor required for
filimentation
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Engineering Cross-Talk Rewiring Yeast MAPK
Pathways

• If scaffolds promote specificity, it 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
• 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 theory
The construct
The result
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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. 7070354
• 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. 2991061