Unformatted text preview: Signaling via protein modifications
1. Enzyme-coupled receptors (and protein phosphorylation)
2. Use of proteolysis in signaling 1 1 Signaling via protein modifications
1. Enzyme-coupled receptors (and protein phosphorylation)
There are six main classes of enzyme-coupled receptors
i)
ii)
iii)
iv)
v)
vi) Receptor tyrosine kinases RTKs
Tyrosine-kinase-associated receptors
Receptor serine/threonine kinases
Histidine-kinase-associated receptors
Receptor guanylyl cyclases
Receptorlike tyrosine phosphatases 2 2 Receptor tyrosine kinases RTK ~60 encoded in the human genome
Some bind secreted proteins and others bind cell surface proteins tyrosine kinase domain
T M proteins 3 3 Receptor tyrosine kinases mediate essential functions cell growth and proliferation
(related to cancer) 4 4 one phosphorylate another Signaling molecules induce the transautophosphorylation
of receptor tyrosine kinases
Ligand binding dimerizes the receptor
Their kinase domains are brought together and phosphorylate each other
dimerize bring 2 RTK together kinase domains are together== activated Phosphorylation of the kinase domains enhances their activity
Phosphorylation of other regions creates docking sites to assemble a signaling complex
5 5 If a receptor tyrosine kinases with a defective kinase domain was
expressed together with the normal receptor what would happen? A) The cell would respond
normally to the signal
B) The cell would have no
response to the signal
C) The cell would have a
reduced response to the
signal
depends on how much the mutant form is expressed.
If there are a lot of mutant receptors, the cell will
have
no response 6 6 Phosphorylated receptor tyrosine kinases recruit proteins
transduce the signal into the cytoplasm
which mediate downstream signaling 7 7 The recruited proteins contain domains (SH2 or PTB domains)
that bind to phosphotyrosine and neighbouring sequences Many molecules in the cell have
phosphotyrosine. This binding site
adds specificity. 3D structure of an SH2 domain The basic functionality of an SH2 domain
8 8 Examples of proteins recruited to phosphorylated PDGF receptor The proteins often contain additional domains
(e.g. SH3 domains which bind proline-rich regions)
They can function as adaptors to recruit proteins without SH2 domains9 9 Genetic studies in the Drosophila eye identified core components
of the receptor tyrosine kinase signaling pathway The Drosophila compound eye consists of ~800 ommatidia little circles that collect light The ommatidia are composed of 8 photoreceptor cells
and 12 support cells
They arise from a simple epithelial sheet through the
sequential differentiation of the photoreceptor cells
The R7 photoreceptor cell is needed to detect UV light
A screen was performed to identify mutants that failed
to specify R7 > based on their
10
insensitivity to UV light 10 Genetic studies in the Drosophila eye identified core components
of the receptor tyrosine kinase signaling pathway The first mutant identified was called Sevenless (Sev)
the normal Sevenless protein was shown to be a receptor tyrosine
kinase expressed in R7 cells Sevenless protein = RTK in R7 cell 11 11 Genetic studies in the Drosophila eye identified core components
of the receptor tyrosine kinase signaling pathway The second mutant identified was called Bride-of-sevenless (Boss)
the normal Bride-of-sevenless protein was shown to be the
ligand for Sevenless expressed on R8 cells 12 12 Genetic studies in the Drosophila eye identified core components
of the receptor tyrosine kinase signaling pathway Drk and Son-of-sevenless (Sos) were identified in subsequent screens
Drk links Sevenless to Son-of-sevenless
Son-of-sevenless is a GEF for Ras 13 Drk SH3 domain that binds to proline rich region of Sos (Ras GEF) 13 Genetic studies in the Drosophila eye identified core components
of the receptor tyrosine kinase signaling pathway This basic mechanism is used in different contexts in all animals
What is Ras? 14 14 molecular
switch 15 15 Ras is a molecular switch downstream of receptor tyrosine kinases
•monomeric GTPase
•attached to the cytoplasmic face of the plasma membrane by a lipid anchor
•activated by Ras-GEFs and inactivated by Ras-GAPs
•its activity leads to cell proliferation or differentiation
•30% of human tumours have hyperactive mutant forms of Ras 16 16 Ras activates a mitogen-activated protein kinase module
(MAP kinase module) to change protein activity and gene expression Downstream of Ras is a
cascade of kinases
(MAP kinase module). Mitogen is a secreted protein that
binds to a receptor and induces cell
proliferation.
Mitogen binds to Tyrosine Kinase
and start signalling.
Ras activates MAP kinase module. 17 17 At least 5 parallel MAP kinase modules can operate in a mammalian cells
(there are at least 12 MAPKs, 7 MAPKKs, and 7 MAPKKKs)
How is non-specific cross-talk controlled?
Why wouldn't MAPKKK phosphorylate all MAPKK in the cell? In yeast, scaffolds bind specific MAP kinase modules, insulating them from other
modules and increasing response specificity 18 18 Other pathways downstream of RTKs and overlap with GPCR pathways DAG 19 19 Other pathways downstream of RTKs and overlap with GPCR pathways 20 20 Signaling via protein modifications
1. Enzyme-coupled receptors (and protein phosphorylation)
There are six main classes of enzyme-coupled receptors
i)
ii)
iii)
iv)
v)
vi) Receptor tyrosine kinases
Tyrosine-kinase-associated receptors
Receptor serine/threonine kinases
Histidine-kinase-associated receptors
Receptor guanylyl cyclases
Receptorlike tyrosine phosphatases Tyrosine-kinase-associated receptors interact
non-covalently with a cytoplasmic tyrosine kinase…
The receptor itself does not have a kinase domain.
It pulls a TK and interact with it non-convalently. …but their mechanisms of action are similar to receptor tyrosine kinases 21 21 Signaling via protein modifications
1. Enzyme-coupled receptors (and protein phosphorylation)
There are six main classes of enzyme-coupled receptors
i)
ii)
iii)
iv)
v)
vi) Receptor tyrosine kinases
Tyrosine-kinase-associated receptors
Receptor serine/threonine kinases
Histidine-kinase-associated receptors
Receptor guanylyl cyclases
Receptorlike tyrosine phosphatases 22 22 Receptor serine/threonine kinases
-the largest class of cell surface receptors in plants (also function in animals)
~6 major families in plants
the largest is the leucine-rich repeat receptor kinase family
(175 members in Arabidopsis [e.g. the Clavatal1/Clavatal2 complex])
Clv1/Clv2 signaling suppresses
the development of
stems, leaves and flowers It's suppressing the differentiation of stem cells
into flowers, leaves… Gene regulation can remove the P
when these cells left
the meristem and
need to differentiate. Clv3 travels to cells with Clv2 and Clv1.
Clv3 binds to Clv2 and Clv1.
Clv2 and Clv1 phosphorylate themselves and sends signal
onwards (Rho-family GTPase)
23
Rho family GTPase is a switch. It will block genes that will form stem,
leaves and flowers. 23 Histidine-kinase-associated receptors
-activate a “two-component” signaling pathway
-used by bacteria, yeast and plants, but not animals
e.g. regulation of bacterial chemotaxis
flagella rotate counterclockwise, they will propel the
bacteria forward. attractant flagella rotate clockwise. repellent CheA is the histidine kinase CheZ can remove the Pi and cell can
move forward (counterclockwise) It phosphorylates itself and then
transfers the phosphate to an
aspartic acid on CheY
24 24 Signaling via protein modifications
1. Enzyme coupled receptors and protein phosphorylation
2. Use of proteolysis in signaling
Notch signaling
Hedgehog signaling
Wingless signaling
TNF / NF B signaling 25 25 Lateral inhibition by Notch signaling 26 26 The force of endocytosis pulls away Notch segment,
When Notch binds to Delta, endocytosis happens. Delta binding
leads to Notch 1st cleavage is
on
Golgi
cleavage… noncovalent
interaction a fragment of Notch enters
the nucleus. …and movement
of Notch fragment
to the nucleus
27 27 Organizer function
in vertebrate limb development Source of the morphogen sonic hedgehog (Shh)
Shh spreads from this source
The Shh gradient controls
the formation of distinct digits
POSTERIOR 28 28 Without hedgehog signaling
the transcriptional activator
Ci is sequestered in the cytoplasm
by a microtubule-associated complex
Plus, this complex promotes the
proteolysis of Ci to create a
transcriptional repressor Hedgehog is always off when
29
there is no Hedgehog signalling. 29 Patched sequesters Smoothened. At the same time, the Smoothened protein
is sequestered in intracellular vesicles
by Patched
30 30 Hedgehog binds to patch Smoothened translocates onto the plasma membrane. The binding of Hedgehog to Patched
allows Smoothened to transfer to the plasma membrane
where it releases Ci from its inhibitory complex Smoothened+Pi inhibits Ci
proteolysis and Ci can be
released and activate
transcription 31 31 Signaling via protein modifications
1. Enzyme coupled receptors and protein phosphorylation
2. Use of proteolysis in signaling
Notch signaling
Hedgehog signaling
Wingless signaling
TNF / NF B signaling The release of a transcriptional activator from an inhibitory complex
is also at the core of Wingless and TNF / NF B signaling 32 32 How can we understand all of the signaling occurring in a cell? PNAS 104:12890 e.g a network controlling human pancreatic cancer
33 33 How can we understand all of the signaling occurring in a cell? 34 34 How can we understand all of the signaling occurring in a cell? 35 35 How can we understand all of the signaling occurring in a cell? Mapping Brain Circuits: The Connectome
http://www.sfn.org/index.aspx?pagename=brainBriefings_09_mapping 36 36 Lecture 9 reading
921-931
956-957
941-943 (not figure 15-70)
946-947
950-951 37 37 ...
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- Fall '11
- Harris
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