So we now continue with the second talk and the second talk will be given by Professor Edmond Fischer.
He received the Nobel Prize in physiology or medicine in 1992.
And the quotation was for the reversible protein phosphorylation as a biological regulatory mechanism.
You are from the University of Washington, in Seattle, USA and you illustrate a general principle.
Many governments and awarding institutions have asked how to distribute money to get Nobel Prize in good research.
And the only common feature perhaps is that your Nobel Prize laureates are very mobile.
And in the official book from the Nobel Foundation, Doctor Fischer is listed as China, Switzerland and USA.
So you are mobile.
So your talk now is protein cross talk in cell signalling.
Please.
(Applause).
Thank you, Hans.
I am also delighted to be here and have this opportunity to speak to all of you.
What I propose to do today is to tell a few generalities about cell signalling.
That is the very complex series of reactions that will induce a cell to grow,
to proliferate and eventually to die in response to a multitude of internal or external signals.
And Tim just gave you a good example of the complexity of those reactions.
To give you another example, you all know about stem cells and the enormous potential
they have of curing a variety of diseases because of their ability to develop and differentiate
and to various tissues and organs, they can be coaxed to become a heart cell that beats,
a pancreatic cell that will produce insulin, a brain cell that might one day help us to cure Parkinson’s or Alzheimer’s disease
or a nerve cell that might repair a spinal cord injury.
But we don’t know all the commands that will tell those cells to go where they are supposed to go,
to become what they are supposed to become.
Though we begin to understand today the intricate number of signals and precisely time signals
coming in part from other cells, the addition or removal of hormones and growth factors and neuromediators,
second messengers, cytokines and other unknown factors.
We don’t know what those signals are, though we begin to understand how they are received by the cells
and the mechanism by which they are regulated.
And this is where cell signalling comes into the picture.
Now, I will limit my remarks only to regulation by tyrosine phosphorylation, starting from growth factor receptors,
because those are some of the main ways by which external signals are received, and they are all tyrosine kinases.
This is a slide borrowed from Yossi Schlessinger, it shows a family, about 20 sub family of growth factor receptors,
at last count there are 59 of them.
And they all have the same architecture, a single trans-membrane segment which separates the tyrosine kinase domain.
Inside this is the business end of those molecules
and outside a variety of classical structural motifs assist the enriched region.
Fibronectin type III repeats, immunoglobulin-like loops, EGF, the epidermal growth factor-like,
cadherin-like, factor 8-like proline glycine rich.
A number of classical structural motifs that will impart to those structures, the selectivity to recognise their specific ligands.
Here are the receptors for the epidermal growth factor, insulin or IGF1, platelet-derived growth factor and so forth.
All those receptors respond to circulating ligands, though for instance FGF has to bind to a glycoprotein such as heparin.
And the large family of F receptor here recognise membrane-bound ephrins.
More than a dozen are known, that interact with about 10 different ephrin molecules,
these can act both as ligand and as receptor which means that they can induce reverse signalling.
The F receptors are mainly, if not exclusively found in the nervous system,
where they promote communication during and after neurogenesis by promoting cell migration,
axon path finding, angiogenesis, topographic mapping and so forth.
The main function is to guide the developing axon towards its proper targets, sometimes over very long distances.
And path finding during neurogenesis, the nerve growth cone must explore its immediate environment to make the proper choice,
to go where it’s supposed to go and sometimes over very long distances.
And in certain cases, like in the optic tectum of the chicken brain,
these receptors are arranged according to a gradient of concentration from back to forth and top to bottom,
forming a sort of a grid, positional grid that will guide the axon to where it is supposed to go.
We begin to understand pretty well now how signal is transduced down from those receptors.
In the resting state they are inactive, they are separated one from the other and monomeric.
Upon binding of a ligand, they will dimerise and immediately undergo transphosphorylation.
And then they will be active by virtue of the fact that the tyrosine phosphate that has been generated
will bind to target proteins, to link proteins because these have SH2 domain, which stands for Src Homology 2 domain.
Those domains have a high affinity for tyrosyl phosphate groups, not anyone but within a specific sequence.
And then they will also recruit some other link or protein,
as a matter of fact Grb2 is known to be linked always to the next one, Sos, this is an enzyme,
it has one nucleotide exchange activity and it will exchange GDP for GTP in the small G protein called RAS.
So you see this type of communication, and then of course that will trigger a whole bunch of reaction,
the map kinase pathway, gene activation and so forth.
You see that this type of communication will allow a receptor
to interact almost directly with a protein at a distance and without this system it would have difficulties to do that.
This type of communication is practically mechanical.
You have proteins interlocking with other proteins or binding to various compartments of the cell,
as if you bind together structures, tinkertoy structures or snapping together elements of a Lego block.
And this type of communication guarantees that only the right protein will be drawn in the signalling pathway
and guarantees the selectivity and fidelity of the signalling process.
And today we know many dozens of those binding modules.
I mentioned already the SH2 module, the SH3 that was also involved, recognises proline rich regions,
the WW module also recognises proline rich blobs but of a different structure.
The PH module, which stands for pleckstrin homology module, recognises an ionic head groups at the membrane, PIP2, PIP3.
And contrary to the others that promote protein-protein interaction, its function is to recruit the proteins to the membrane.
And I’ll come back to that in a little while.
The PTP for phosphotyrosine binding domain also recognises tyrosine phosphates but of a completely different type of structure.
SH2 recognises the downstream sequences, whereas the PTP recognises upstream sequences from the tyrosine.
And it has a very strong affinity for this arginine-proline-x-tyrosine to such an extent
that it will bind to these even when the tyrosine is not phosphorylated.
The PDZ domain is a ubiquitous protein-protein interaction domain whose main function is to aggregate,
to coordinate ion channels and receptors and link them to the cytoskeleton.
And it recognises hydrophobic C-termini, C-termini having leucine, valine, isoleucine
and also sharp beta hairpins that mimic hydrophobic C-terminal and so forth.
And one after another of those binding modules of those which have been all determined by homologous sequence analysis.
Now, let me give you a few examples as to how those modules function.
The SH2 domain has been found now in about 100 proteins and very often links adaptor proteins to receptors, as I just mentioned.
But it has in addition a very important other function.
In that several proteins, this is the case, for instance, of all the Src kinases,
several proteins have both an SH2 domain and a tyrosine phosphorylated at the C-terminus.
And therefore, in the resting state the SH2 domain binds to this tyrosyl phosphate, which serves as a negative determine,
and shields the catalytic site.
Or the SH2 domain will bind to an inhibitory region, even when it is not phosphorylated,
this is the case of the abl kinase, the Src kinases,
this is the case of several kinases that function in B and T cell signalling, SAP70, the sick kinases
and then tyrosine phosphatases, the ship 1, 2 and 3, which have two SH2 domains.
Activation occurs when this tyrosine phosphate is dephosphorylated by tyrosine phosphatase
and then it opens up the molecule or when the SH2 domain binds to another protein which has a higher affinity for it.
And therefore the SH2 switch its allegiances from the enzyme to this other protein,
to the receptor, for instance, and activate the kinase.
So you see that the SH2 can be seen as a conformational switch, internal conformation switch,
just like a phosphate group, addition or removal can serve as an external molecular switch to activate or inhibit the enzymes.
The PH domain, as I told you, will recruit proteins to the membrane.
It serves the same function as a myristyl or as a palmitoyl group.
And this is the case for instance of the insulin receptor substrate 1, 2, 3, 4.
Those molecules usually have a lot of tyrosyl residues
that can become phosphorylated after activation of a growth factor receptor.
So, for instance, when you activate the insulin receptor, it will recruit its substrate to the membrane because of the PH module.
The PTB module has a very high affinity for a sequence on the receptor,
it will slap this molecule against the receptor and then the insulin receptor
will begin to phosphorylate many of those tyrosyl residues.
So you see it serves as a sort of an annex molecule to amplify the signal.
The importance of the PH domain can well be seen in the Bruton kinase.
Bruton kinase plays a very important role in B-cell-signalling
where a single mutation of an arginine to a cysteine in the PH domain will lead to growth functional abnormalities
resulting in agammaglobulinemia.
The PDZ domain, as I told you, has the main capacity of promoting protein-protein interaction
and to appear in multiple copies within its members.
And here, it’s a slide borrowed from a review by Harris and Lim, which shows a few examples of this large family of proteins
which are called MAGUK and this stands for membrane associated proteins with guanylate kinase-like domain.
And the MAGI proteins, or MAGUK with an inverted configuration, and that’s the guanylate kinase domain,
is towards the N-terminus, rather than being at the C-terminus.
And then the SH3, the proline binding domain is replaced by 2 WW domains, which also bind to proline residue.
And then you find many other modules of serine-threonine kinases, LIM domains, DEP domain, RBD domains,
which allow those proteins to bind, interact with a vast array of target proteins.
And these have obviously been added in the course of evolution to allow multi cellular higher organisms
to deal with the complexity of their signalling pathways.
And here are a few example of these.
You have PSD, the post synaptic density protein 95 with 3 PDZs.
You have InaD which serves as a scaffolding protein for the phototransducing system of Drosophila, it has 5 PDZ’s,
its human homolog has 8 PDZs in a string and attaches, has a high affinity for other elements of the cell.
Here are multiple PDZ proteins, MAP1 has 13 PDZs in a string.
And let me give you one or two examples of how those MAGUK, how those proteins function.
As I already said, they have no catalytic activity,
their only function is to participate in the organisation of signalling assemblies.
The first two PDZ domains, the PSD9-5 will bind to subunits of points of the NMDA glutamate receptor
and dimerise the receptor and activate it.
And the close proximity of those 2 PDZs will allow for synergistic binding to the, let’s say hydrophobic tail of the receptor.
The second PDZ domain will bind to the neural NO synthase and you heard about that yesterday from Ferid Murad,
what he called NOS1 and, as he told you, this NO synthase is a calcium calmodulin depending enzyme.
And the close proximity of the synthase to the channel
will allow for very efficient production of nitric oxide following calcium entry in the cell.
And the third PDZ domain will bind to CRIP, it’s a protein which participates in the microtubule system
and then the guanylate kinase-like domain will bind to its associated protein.
So with all these domains you can have the formation of a large complex around the receptor,
which will participate in signalling, in binding of the system to the cytoplasmic system of the cell.
And also participate in the transport, trafficking of the receptor.
InaD, as I told you, has 5 PDZ domains and its main function is to organise all the elements of the phototransducing cascade.
Activation of the Rhodopsin by a photon will activate the receptor and activate the PLC beta,
which is linked to the G-protein complex of the receptor.
An activation of PLC will produce diacylglycerol and IP3, which in turn will activate the ion channel
and causes calcium entry and cell depolarisation.
The inactivation process is again a calcium-dependent adaptation system,
which involves an I-specific protein kinase, C calmodulin, arrestin and other enzymes.
And here again the formation of this very large supermolecular complex will allow or result
in a huge amplification of the system according to which a single photon will activate
more than 100 calcium channels within I think 20 millisecond time scale.
The complexity of signalling is well demonstrated by this oncogenic protein p95 Vav.
And with all its modules, it looks like one of those Swiss army knives that can do anything.
P95 Vav is an enzyme, it has guanine nucleotide exchange activity, catalytic activity,
and it is activated upon receptor activation and also it is phosphorylated by the BCR abl protein,
that Tim spoke a moment ago, this oncogenic protein involved in chronic myelogenous leukaemia.
It has a very hydrophobic N-terminus, which must serve a repressing function,
because if you delete the N-terminal of it then Vav becomes oncogenic.
Now, I showed you this Vav molecule with all its module structural elements
to illustrate an important point we have learned about the structure of proteins
is that all of them in fact have this molecular multimodular molecular type of structure.
They are mosaics made up of a number of structural motifs, they are sort of necklaces of different beads
whose selection and whose disposition in the 3-dimensional structure of the molecule will determine its function.
With a number of modular structures like that, those proteins can bind simultaneously to several proteins.
And therefore they can stand at the intersection, if you want, of several signalling pathway.
They can serve the role of those traffic turnarounds that you saw in Lindau,
where a car arrives and then can go in different directions.
The complication for the cell is to decide which car should go in which direction and at which particular time.
Because any receptor can bring about a number of physiological processes, it can be involved in metabolic regulation,
gene expression, immune response, cell differential, oncogenesis and so forth.
So how can a cell decide in which direction the signal should go?
Obviously no one receptor can do all those things, it’s only when several receptors act in concert,
generating a combinational type of signals that selectivity can be introduced and that one response will be chosen over the other.
And, as you can well imagine, any mutation in those receptors will result in physiological aberration,
pathological conditions of different severities, diabetes, dwarfism, etc.
But probably the most dramatic of those mutations are those that result in oncogenesis.
And indeed we know today that about 70% of all forms of cancer involve tyrosine phosphorylation and tyrosine kinases,
which underscores the huge importance of tyrosine phosphorylation in determining cell behaviour.
Now, in most cases oncogenesis can result either from an overexpression of the normal non-mutated receptor,
or to mutations that will freeze, that will block the kinase in the active conformation
in such a way that it can no longer be controlled.
And we would say that it would be like if it were constitutively active.
And then the cell behaves like a car that goes wild, because its accelerator is stuck to the floor.
With so much evidence that oncogenicity is effected by mutations in tyrosine kinases,
it’s obvious that people became interested about the tyrosine phosphatases
with the idea that perhaps it might block or even reverse oncogenicity.
And we thought so, too.
And about 20 years ago we decided to go after the tyrosine phosphatases
and within a couple of years a very bright postdoctoral fellow by the name of Nick Tonks,
he’s now at Cold Spring Harbour, succeeded in isolating the first tyrosine phosphatase from human placenta.
It was extremely active but the big surprise came
when two other colleagues in the department determined the amino acid sequence of the tyrosine phosphatase.
By the way they determined better than 90% of the sequence on 0.2 mg of protein, which I thought was pretty spectacular.
Surprise because it showed no homology whatsoever with any of the other phosphatase, serine threonine phosphatase.
But a search of the database told us that this tyrosine phosphatase was homologous to a surface antigen
already wellknown by the immunologists, which they had called the leukocyte common antigen or CD45.
And the leukocyte CD45 is a large family of high molecular weight trans-membrane proteins
that are found in all hematopoietic cells, except mature erythrocytes, which are a-nucleated.
It’s heavily O and N glycosylated.
It has a single trans-membrane segment and then the intracellular moiety has two internally homologous regions
of about 30 KDA each.
And it is each of these regions that had sequence similarities with the phosphatase
which had been named PTP 1B 40% sequence, identity with one domain, 33% sequence identity with the other.
And CD45 was known already to participate in B- and T- cell signalling in cyclotoxicity and cell proliferation.
I won’t have time to discuss how it functions but we know today that, as you probably know,
the immune response depends on a lot of Src-type kinases.
And as I had told you before, those enzymes are repressed by interaction of the SH2 domain with the phosphates.
Well, it is known that CD45 is one of those tyrosine phosphatase, particularly dephosphorylate P56, LCK,
activate the system and then triggers a whole cascade of tyrosine phosphorylation.
So what you see here is that you need a tyrosine phosphatase to activate a tyrosine kinase.
And this is why when you express that enzyme and we have other tyrosine phosphatases in animals,
injected with an oncogene, it increases oncogenecity.
So you can’t use that.
In any case, since then a whole bunch of trans-membrane tyrosine phosphatase have been cloned and characterised
and they all have a structure very similar to that of CD45.
They have a single trans-membrane segment, catalytic domain inside and then a variety of different structures on the outside.
But the enormous surprise, at least for me, was that all those structural motifs are those of cell adhesion molecules.
The tyrosine phosphatase trans-membrane are cell adhesion molecules,
which means that, contrary to growth factor receptors that respond to circulating growth factors,
no circulating growth factors have been found for the tyrosine phosphatase.
Which implies of course that they either are involved in cell-cell interaction
or regulated by cell-cell or cell-matrix interaction.
With the exciting possibility that they might be directly implicated in contact inhibition,
which plays such an important role in oncogenicity.
And I’m sure that many of you know that when you grow a cell in a Petri dish, it would need,
a normal cell would need a solid support to grow, it will need the addition of growth factors
and it will grow until those cells reach confluency.
The moment cells come into contact into one another, they stopped grow - this is what we call contact inhibition –
and only transform cells continue to grow one on top of the other and this is how they can form tumours.
And this is one of the hall marks of transformation,
it’s a cell which no longer abides by the constraints under which a normal cell, don’t need a solid support anymore,
it can grow on soft agar.
It doesn’t need growth factors anymore, it produces its own and it’s no longer contact inhibited.
And so there is the exciting possibility that trans-membrane tyrosine phosphatase can exist as anti carcinogenic agents.
A sort of tumour suppressors by promoting contact inhibition, but this has not been proven yet.
Now, from what Tim told you a moment ago, obviously cancer is much more complicated than that.
And we know well today that it results from a series of genetic changes, mutations that occur over a period of many years,
clonal mutation that have been selected, because presumably they provide a proliferative advantage to the cell.
And of course in turn those mutations will result in a number of oncogenic events
that cumulatively will contribute to the advanced stage of the disease.
But we do know many of these events, we do know many of the pathways leading to tumour regenesis,
tumour progression and mutagenesis and other reactions that undergo when you have cancer.
And I personally think that sooner or later, and probably sooner than later,
we will be able to put much of this information together and bring many forms of cancer under control.
But what I can’t tell you is when this will occur, ok.
Well, this is the area that I have been involved in for about 150 years and thank you for your attention.
(Applause).