Principles of cell signaling - UT Southwestern

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activated protein kinase (MAPK) cascade, including
a MAP3K (Ste11p), a MAP2K (Ste7p)
and a MAPK (Fus3p). (The MAPK cascade will
be discussed in 14.32 MAPKs are central to many
signaling pathways). The function of Ste5p is partially
retained even if the positions of its binding
sites for the kinases are shuffled in the linear
sequence of the protein, indicating that a major
role is to bring the enzymes into proximity, rather
than to precisely orient them. Ste5p also binds
to the subunits of the heterotrimeric G protein
that mediates the actions of mating
pheromones, linking the membrane signal to
the intracellular transducers. Yeast that lack
Ste5p cannot mate, demonstrating that Ste5p is
required for this biological function (but not all
functions) carried out by the pathway.
In addition to facilitating signaling in their
own pathways, scaffolds can enhance signaling
specificity by limiting interactions with other
signaling proteins. Scaffolds thus insulate components
of a signaling pathway both from activation
by inappropriate signals and from
producing incorrect outputs. For example, the
mating and osmosensing pathways in yeast
share several components, including the MAP3K
Ste11p, but each pathway maintains specificity
because it employs different scaffolds that restrict
signal transmission.
In contrast, the presence of excess scaffold
can inhibit signaling because the individual signaling
components will more frequently bind
to distinct scaffold proteins rather than forming
a functional complex. Such dilution among scaffolds
causes separation rather than concentration
of the components, preventing their
productive interaction.
14.9
Independent, modular
domains specify proteinprotein
interactions
Key concepts
• Protein interactions may be mediated by small,
conserved domains.
• Modular interaction domains are essential for
signal transmission.
• Adaptors consist exclusively of binding domains or
motifs.
Modular protein interaction domains or motifs
occur in many signaling proteins and confer the
ability to bind structural motifs in other molecules,
including proteins, lipids, and nucleic
acids. Some of these domains are listed in FIGURE
14.9. In contrast to scaffolds, which bind specific
proteins with considerable selectivity, modular
interaction domains generally recognize
not a single molecule but a group of targets that
share related structural features.
Modular interaction domains important for
signal transduction were first discovered in the
protein tyrosine kinase proto-oncogene Src,
which contains a protein tyrosine kinase domain
and two domains named Src homology
(SH) 2 and 3 domains. The modular SH2 and
SH3 domains were originally identified by comparison
of Src to two other tyrosine kinases, Fps
and Abl. One or both of these domains appear
in numerous proteins and both are critically involved
in protein-protein interactions.
SH3 domains, which consist of approximately
50 residues, bind to specific short proline-rich
sequences. Many cytoskeletal proteins
and proteins found in focal adhesion complexes
contain SH3 domains and proline rich sequences,
suggesting that this targeting motif
may send proteins with these domains to these
sites of action within cells. In contrast to phosphotyrosine-SH2
binding, the proline-rich binding
sites for SH3 domains are present in resting
and activated cells. However, SH3-proline interactions
may be negatively regulated by phosphorylation
within the proline-rich motif.
SH2 domains, which consist of approximately
100 residues, bind to Tyr phosphorylated
proteins, such as cytoplasmic tyrosine
kinases and receptor tyrosine kinases. Thus, Tyr
phosphorylation regulates the appearance of
SH2 binding sites and, thereby, regulates a set
of protein-protein interactions in a stimulusdependent
manner.
A clever strategy was used to identify the
binding specificity of SH2 domains. An isolated
recombinant SH2 domain was incubated with
cell lysates and then recovered from the lysates
using a purification tag. The proteins associated
with the SH2 domain were some of the same
proteins that were recognized by antiphosphotyrosine
antibodies. By this and other methods,
it was discovered that SH2 domains recognize
sequences surrounding Tyr phosphorylation
sites and require phosphorylation of the included
Tyr for high affinity binding.
Information on specific amino acid sequences
that recognize and bind to modular
binding domains is being accumulated as these
individual interactions are identified. In addition,
screening programs using cDNA and/or
peptide libraries to assess binding capabilities
600 CHAPTER 14 Principles of cell signaling