November 8, 20135:39 PM
Non covalent interactions. We saw how small molecules interacted
with proteins. Small molecules , they interacted through non covalent
interactions. Changed the structure of the protein through downstream
Today we center on two covalent modifications
1. Phosphorylation - forming covalent binds
2. Proteolysis - breaking covalent bonds
There are 6 main classes.
BIO230 Page 1 Here we have receptor tyrosine kinases.
- Transmembrane proteins which are receptors for signals.
- With tyrosine kinases on the cytoplasmic face
- Two main classes. 60 encoded in human genome
- some bind to secreted proteins. Proteins in the extracellular face
diffusing around. Shown with the blue box.
- Others bind cell surface proteins. They bind to different ligands
on the EC surface.
- There is a large variation if you look across. We can hypothesize
that They will be binding to different ligands in extracellular
- Look at everything they have in common. They all have a
transmembrane domain. Can send signal from extracellular
space to intracellular space into the cytoplasm.
- If we look at the cytoplasmic tails of their proteins they all have a
common domain, tyrosine kinase domain.
- Different signals in the outside but feeding into the tyrosine
kinase signal from the inside.
There are whole number of these receptors.
Table shows some.
All of the details in the table is not critical
The theme as we look across the information
The name of the signaling protein and various growth factors.
In response to the signaling there are stimulations of:
Stimulate from self-growth
Stimulation of proliferation
So this is a common theme across the table signaling which promotes
cell proliferation and cell growth.
This is critical for development. For cells to build body. Critical for
cancer as well. Misregulation of these pathways leads to cell
overgrowth leading to cancer.
BIO230 Page 2 Here is the basic mechanisms of any of the tyrosine receptors are
We have an extracellular ligand, and this is bivalent. Has two binding
sites. Because it has two binding sites it dimerizes the receptor.
This brings the kinase domains together and they phosphorylate each
other.. This is called transautophosphorylation. Signal molecule in the
outside is inducing the trans auto phosphorylation.
Trans means across , one of the pair is phosphorylating the other one.
And vice versa.
Auto because same receptor. Two copies of the same receptor comes
together and phosphorylate each other.
Phosphorylation of one another increases their activity and then there
will be phosphorylation of other targets along the cytoplasmic tail.
There will be docking sites for assembling signal complexes.
Inactive RTKs is not completely correct, because there is low activity
there. When the ligands binds to them they have very low activity on
their own, but when the ligand brings them together to their kinase
target they pair and phosphorylate each other.
Step 1: initial phosphorylation which involves low level of activity. First
step increases the activity of the kinases domain. Which goes from low
to high level of activity. With that high activity you get the
phosphorylation of the additional sites to build the signaling complex.
What's the organization of this complex and mechanism of signaling?
Imagine a Scenario where you have a mutant tyrosine kinase receptor
with a mutation in the kinase domain. one has the potential to
phosphorylate, the other doesn’t. so the signal molecule comes in and
What would happen? Will this create a signal?
A, B or C?
A. There would be an inactive complex. We would think NO. but I
think this is the answer based on his explanation. Ask from me.
B. No response?
C. Reduced response
We have to think about signaling through copulation of molecules not
just as a single pair, a cell does not have two copies of the receptor has
only two copies on the surface that binds to the ligand and sends he
signal in. there are 1000s of 100s of copies of the receptor on the
surface of the cell.
We should think about how it's actually functioning in the cell we have Imagine when there are like two copies of the cell and one is mutated
and the other one is normal it would from a normal complex. But when
to think that there are large populations of molecules that are
conducting this activity. Not only as a single signal conductor. there is a thousands of copies it really depends on the ratio of how
much of the mutant protein do you have vs. how much of the normal
protein you would have.
When you think about how it's actually functioning in the cell. Then
that is what matters.
BIO230 Page 3 If we do move inwards from the activated receptor you can see that
when there is extra phosphorylation sites on the dimer it creates
docking sites for other proteins. Downstream component sof the
signaling pathway. This would create a relay of signals.
One of the key domains that would recruits proteins to these
receptors is an SH2 domain.
SH2 domain or a PTB domain. And the key thing about these
domains is that they have a binding site for phosphorylated
In the previous slide it’s a phosphate specifically on tyrosine residue.
Tyrosine kinase domain it will specifically add phosphate domains on
the tyrosine residue and you have protein domains that have a
binding site for the phosphorylated tyrosine so it only binds tyrosine
when its phosphorylated and doesn’t bind when it's not. So its
specifically gets recruited to this complex.
They often have binding sites next to it as well which gives them
Phospho tyrosine is relatively simple residues binding sites next door
that binds to a specific sequence of AA residues that can provide
some extra specificity for the recruitment.
Crystal structure of this. Important thing to remember is that
these domains are like the knobs on top of Lego bricks so one
protein can have multiple domains and they can come together in
BIO230 Page 4 We can here that the PDGF receptor is phosphorylation at multiple
residues. These are phosphotyrosines. They recruit specific proteins
with SH2 domains which is shown here in red. And they binds to these
specific phosphotyrosines because it’s a slight different surroundings aa
So this SH2 binds to there…etc.
There's slight differences in the surrounding AA residues.
In addition to contain the SH2 domains the proteins can also contain
other domains too.
G protein coupled receptors acts through phospholipase C which
cleaves the pep 2 and creates DAG and IP3. Q is: this occurred
downstream of receptor tyrosine kinase signaling. It can. This protein
has a phospholipase domain( another domain the protein) and the
particular phospholipase domain has an SH3 domain, so it will response
to phosphotyrosines signaling because it gets recruited to that
receptor. But phospholipase C gets activated by GPCR and instead have
a binding site for G protein. Rather than a binding site for
phoshotyrosine. So this is a way of linking up different proteins to
different signaling pathways. Additionally the domains present in the
domain recruit adapters to recruit proteins that don’t have SH2
domains. So SH3 domain binds to polyrpoline, so that can recruit
addiotnal proteins to the local complex.
Eg: on how this pathway was found.
This pathway was first identified in genetic streams of drosophila. Led
to the development if drosophila eye. Its eye is a compound eye.
There are 800 ommatidia covering the surface of the eye. Each of them
are composed with 8 photoreceptor cells and 12 support cells. You
have 800*8 are the photoreceptors crossing the entire surface of the
drosophila eye. So the screen was based on the development of the 8
photoreceptor cells from a single epithelial sheet during development.
The fact that there is a sequential differentiation of the photoreceptor
cells. That is shown at the top where you Start by forming R8 cell.( we
are going from 1-8 photoreceptor cells), it induce the formation of R5
and R2, R4 and R3 and R6 and R1 and then R8 signals to R7.
This signaling to R7 , the screen revealed, was based on RTK signaling.
We are talking about the Developmental pathway which needs R8 to
tell to make R7.
Special thing about R7 is that its specifically needed to detect UV light.
Though as soon as R7 differentiate in response to the signal it expresses
the proteins the fly needs to detect UV light.
This is a nice way to set up a screen to know if the fly had set up the R7
cell or not.
If it hasn’t formed the R7 cell it won't respond to UV, it has, then it has
Based on intensity to UV light
BIO230 Page 5 Screen was formed to identify flies that failed to develop R7 which
meant there was a failure in the signaling pathway.
They exposed the cells to UV and watched if there was a response or
When through the screen and one of the first mutants that was
identified was seven less. In Drosophila the names of the genes
expressed the mutant phenotype.
It was called sevenless because it didn’t have receptor cell number 7.
The gene that was mutated , was found as the gene that encoded the
sevenless proteins which is a RTK. It was shown to the a receptor
tyrosine kinase which was specifically expressed in R7 cells.
Here is the RTk wihch was mutated in the first line and its expressed in
the R7 and not in R8.
R8 is on top. R7 is in bottom. In order to activate R 7 you need the protein sevenless. Logic is if you mutate sevenless
you will be sevenless. If you mutate sevenless you will be missing R7. you need sevenless to get R7. R8 is
communicating with sevenless.
Subsequent mutation found was called bride of sevenless. BOSS.
BOSS was shown to be the ligand for sevenless, and it was expressed on
R8 cells. They Cloned the gene ,made antibodies for it and saw where it
was expressed on R8, and observed that it acted as a ligand for the
The ligand receptor pair, through the same screen was able to identify
additional component downstream.
You also need mutations boss or bride of sevenless to activate
sevenless. You also need
BIO230 Page 6 They were able to identify additional components downstream.
Drk and Sos was identified.
Drk is one of the adaptor proteins. You can imagine now that the
activated RTK phosphorylate itself, so now it has the phsophotyrosine
right there. SH2 domain of Drk binds to that. And because it has SH3
domains on it , it can recruit proteins identified as Sos.
So Sos has a polyprolin region which binds to the Sh3 regions which
were brought to the complex and then Sos is a GEF, and exchange
factor for the small G protein Ras.
Here we have GEF which turns on Ras and it then drives downstream
signals. This was identified in the drosophila. This has a major
implication in cancer development and growth in all animals.
You also need Drk, and son of sevenless (Sos) , Drk is downstream if receptor kinase. Downstream if receptor kinase
activate Sos. Sos is a GEF for Ras. So Ras in bound to GDP and its inactivated. So Sos helps to remove it and then activate
Sevenless is a receptor…kinase. Drk is activated in the same scenario. SH2 domain binding to …same scenario. Make sure
you know the domains. This is a really good review slide. They have shown you what happens in the R7.
You see RAS everywhere.
What is Ras?
BIO230 Page 7 Ras is the founding member of the Ras superfamily of G proteins.
We have talked about the Rab protein and Rho proteins. All of these
proteins are switches that can be switched on and off to control the
pathway of the cell.
Ras is for growth regulation. For the growth of cells. For the division of
cells to growth of cells.
RAS is a huge superfamily, RAS is a protein. Then there is all these other
proteins in the RAS super protein. RAS is particularly interesting
because 30% human cancers are RAS.
If a molecular switch downstream of RTK.
It's in close proximity to RTK and can be brought in to proteins.
They have Ras that cannot be turned on. Drives the proliferation of
They have a form of Ras that cannot be turned off.
RAS is a monomeric GTPase.
BIO230 Page 8 What does Ras signal through? Ras activates a mitogen activated
protein kinase module. MAP kinase module. When you say mitogen,
that is the name for a secreted molecule that promotes cell growth é
Mitogen is secreted, in the extracellular space, bound to the receptor
tyrosine kinase and activates Ras.
MAP KKK This pathway is given the name mitogen activated protein kinase
because it acting downstream of the mitogen. If you look at the base of
the pathway, then this is a serine threonine kinase. So it phosphorylate
serines and threonines now. And at the base of the pathway it is
MAPKK phosphorylating a whole range of proteins that is promoting cell
growth and division, changes in cell behavior. Gene expression.
Changes in other machinery that changes cell division and growth.
MAP K That was the last map kinase. This is a relay of signals. So the molecule
upstream is called MAPkk, this is another serine threonin kianse which
phosphorylate MAPk. Then we have another MAPKk, we have MAPKKK
at the top.
So we have three kinases which phosphorylates one another in a relay.
So Ras is linked upto MAPKKK and then MAPK is at the bottom that
phosphrylates the target.
This is an exmaple of relays. It is the same basic signal phosphorylation
event which activates the downstream signal.
Ras will activate a map kinase kinase kinase. This is going to take ATP and phosphorylate kinase kinase. Once MAP
kinase kinase take ATP it's going to phosphorylate kinase. And then activate protein activity, changes in gene
expression etc. MAP KKK can be called a MAC?
Here we have one of these relays and the key thing is there is known 5
different map kinases cascades can function at the same time in a
mammalian. There are at least 12 map kinases and 7 mapkk and
7mapkkk in the genome.
When we look at this we can ask how is nonspecific cross talk
5 different relays, why don’t they criss cross their wires?
We can look at yeast and get a really good example of how this works.
We are talking about a different receptor at the top in both cases, and
we can think of them as TRK as well. Think only about the M