Cell Signaling IV: Receptor Tyrosine Kinase Signaling

Tyrosine Kinase-Mediated Signaling

I finally want to consider a kind of signal transduction mechanism in which the receptor protein in the plasma membrane of the cell is in effect its own second messenger. Before I do that, I want to briefly to discuss a particular kind of signaling molecules that we’ll be coming back to throughout the rest of the course. These signaling molecules are mostly small proteins that are important during development--that is, during the formation of an organism and in circumstances where particular cell types in adult organisms have to grow or differentiate. As examples, the formation of almost every organ in an animal or plant requires the division and differentiation of a large but relatively fixed number of cells. Formation of the heart or the kidney or a leaf or a wing must be tightly controlled so that they are big enough to function, but not so big as to be problematic. A heart that’s too big for the chest or one of a pair of wings that’s large than the other would be trouble. In adult organisms, cells must be able to respond to a variety of environmental and internal stimuli. For example, plants make flowers during certain seasons, so the cells that form these flowers must be activated to divide and differentiate to form the tissue in a mature plant. In animals, tissues must divide to repair wounds, to replace lost cells--such as skin or other epithelium, or in the immune system--T and B lymphocytes can be induced to divide and to differentiate when they encounter molecules that are foreign to the organism, called antigens. The point here is that cell division and differentiation not only occurs during formation of an organism, but continues throughout its lifetime.
We don’t know everything about these processes, but it’s becoming clearer that many of them are controlled by signals that are generated by the environment or by some cells that act on other cells. In plants an important signal for flower development is day length; in the immune system the presence of the antigen induces some cells, called helper T cells, to secrete proteins that induce other B or T lymphocytes to differentiate. These substances that carry signals between cells are called, as a class, growth factors or cytokines. "Growth" because they cause cells to grow (i.e., divide); but they also usually induce differentiation as well. "Factors" comes from the Latin word meaning "to do" or "to make" and this implies only that they do something related to growth. This neutral term was adopted when nobody knew anything about the chemical nature of these substances, and even though it’s clear that almost all of them are proteins or peptides, they aren’t called growth proteins for historical reasons. Since most of the growth factors induce cells to divide, in most cases, they are also sometimes called mitogens--which means generators of mitosis.
Now it stands to reason that if binding of a growth factor--or mitogen--to a receptor on the surface of the cell causes the cell to begin to divide--to undergo mitosis--then somehow the nucleus must get the clue that the growth factor is present at the cell surface. Why do I say that? Because for a cell to divide it must replicate its DNA, and the DNA is in the nucleus. So somehow a signal must reach the nucleus that says, among other things, "copy all your DNA and get ready to divide." And that’s the problem that has confronted people who are interested in how this stuff all works---what’s the nature of the signal that reaches the nucleus?
This general subject is of interest for a variety of reasons. Not only does it inform us about cell signaling, which we’ve been discussing, it informs us about the processes that determine whether a cell sits quiescently without dividing--as neurons do for all their lives--or whether they divide constantly, as epithelial cells do--or whether they divide at some times and not at others, like lymphocytes. In short it tells us about the processes that regulate the cell division cycle. This is again of interest in its own right, but people are also very interested in it for another reason--a medical reason which is--what? Cancer--cancer is uncontrolled cell division. That is, if cells are no longer subject to the normal controls that regulate the rate at which they divide, they can divide continuously, rapidly, and uncontrollably, and this unregulated growth is what we call cancer. So if we understood how mitogens regulate cell growth, if we knew what internal processes transmit the mitogenic signal from the cell surface to the nucleus, if we knew how that nuclear signal determined whether a cell would divide or not, we’d probably have a good idea about what kinds of things could go wrong to cause cells to become cancerous. And that’s why, for the rest of this course, we’re going to be concerned mostly with cell division and cancer, because they really go to the very heart of what regulates the growth, development, and function of organisms.
So that’s the brief introduction about why people are interested in this subject and why you might want to pay attention to the confusing business about MAP kinases and so forth. In fact a great deal about these signaling pathways has been learned because people who were interested in cancer identified genes that were defective or mutated in cancer cells, and then later realized that the proteins encoded by these genes normally function in the cell’s internal signaling pathways, and that what goes wrong in cancer is often that the cell thinks it is being continuously given a signal to divide even though it is not. That is, the signaling pathway for cell division gets permanently stuck in the "on" position, so that the "off" mechanisms that usually reverse the effects of a signal don’t work. Similar mechanisms are also important in the development of organisms, and some mutations that cause developmental abnormalities--"birth defects"--also affect signaling pathways.
Let me first summarize what seem to be the general principles that have emerged from these studies and then talk about one signaling pathway that seems to be a very important one, though by no means the only one, that mediates the effects of growth factors.

One principle is that the receptors for growth factors themselves, or sometimes a tightly associated protein, become covalently modified as a result of the binding of the growth factor. The covalent modification is, of course, phosphorylation, but this occurs mostly on tyrosine residues in the protein, rather than the "classical" serine or threonine residues. (Tyrosine phosphorylation was first discovered in the early 1980s by a man named Tony Hunter). This phosphorylated protein can then interact with a group of proteins that recognize and bind to phosphotyrosine, and this activates these binding proteins, which sets in motion a cascade of events, mostly carried out by kinases--both tyrosine kinases and serine/threonine kinases--that participate in a cascade of activation. Eventually what gets modified in these pathways are proteins that act in the nucleus to affect the rate of transcription or DNA replication that occurs there. That is, these nuclear regulatory factors, are activated by being phosphorylated by some kinase that in turn was activated by a series of reactions that was initiated by the binding of the growth factor to the cell surface. The activation of the nuclear regulatory factors apparently can be accomplished in several ways; that is, there’s not just one way to induce a cell to divide, but different signals can converge on the same end product. Activated nuclear regulatory factors can then kick a cell into high gear and promote division.
I spent all this time giving you some background because I think it’s easy to get lost in the trees here and not see the forest, and because this is laying the groundwork not only for the end of signaling transduction, but the beginning of cell growth and regulation.
The book does a pretty good job of providing you with lots of details about tyrosine kinase mediated pathways, but it’s pretty easy to get overwhelmed by detail. I want to consider one such pathway at some depth, because it seems to be the best studied and because other systems are likely to work in similar ways. This is signal transduction mediated by a monomeric GTP binding protein called Ras which was initially discovered as a mutated protein in a line of cancerous cells, and subsequently shown to be an oncogene, a gene that can cause cancer if it is mutated. (How was it shown to be an oncogene?--by placing the mutant ras into normal cells and "transforming" them into cancer-like cells that grew uncontrollably.) Ras subsequently turned out to be required for normal signaling in many cellular pathways. The mutant form of ras, then, is one that cannot be turned off, so it gives a constant signal to the cell.
But let’s back up and look a the early events in signaling through this pathway. The signal begins when a protein growth factor, like epidermal growth factor, platelet derived growth factor, or insulin binds to its receptor. These receptors are different from the ones we’ve been discussing because they mostly do not interact with and modify G proteins to activate a pathway. Rather they modify themselves. These receptors are either monomeric proteins that dimerize when they bind the ligand, such as PDGF or EGF receptors, or multimeric proteins, such as the insulin receptor. In all the cases, the binding of the ligand causes two identical cytosolic domains of the receptor to come close to each other and activates an enzymatic activity on these domains. The enzymatic activity is a tyrosine kinase, and it autophosphorylates--that is, each of the cytosolic domains attaches phosphate groups to tyrosine residues on the domain of the other subunit. Thus as a result of ligand binding, the receptor alters its own properties by adding phosphate groups to tyrosine. (In the case of insulin, the protein phosphorylated is both the receptor itself and another protein called the insulin receptor substrate-1 or IRS-1). (See Fig. 20-23 in Lodish et al.)
Now these newly phosphorlyated proteins can bind to phosphotyrosine binding proteins in the cell. In all cases studied so far, there seem to be common structures in these PB proteins that allow them to bind phosphotyrosine. Some are called simply phosphotyrosine-binding (PTB) domains, while others are called the SH2 domains (because they are homologous to the structure of Src, a well known oncogene. So it was figured if Src was an oncogene, structurally similar proteins might also be).). A domain, you will recall, is a kind of self-contained functional part of a protein that operates more or less independently from the rest of the protein; it's a three dimensional tertiary structure of the protein. For example, the steroid receptor proteins all contain three similar, i.e., "homologous" domains--one to bind the steroid hormone, one that binds the steroid receptor element in DNA, and one that binds RNA polymerase and other transcription factors). Proteins that contain these phosphotyrosine-binding domains appear to interact well with phosphotyrosine-containing proteins. Some of these phosphotyrosine-binding proteins also contain a second src-homology domain, called SH3, that allows them to bind with another protein that can catalyze the swapping of GTP and GDP. That is they serve as molecular bridges between the receptor and the swapping protein (called a guanine nucleotide releasing protein (GNRP), or guanine nucleotide exchange factor (GEF)).
In many mammalian systems--EGF, PDGF, and Insulin, for instance--the bridge protein is called GRB-2 (growth factor receptor binding protein #2) and the GEF is called Sos (son of sevenless, because it is nearly identical to a drosophila protein called son of sevenless for reasons explained in the book; it’s important in the development of fly eyes). So what apparently happens is that the growth factor receptor or IRS-1 gets phosphorylated. (Again refer to Fig. 20-23.) The GRB-2 now binds to it. The Sos binds in turn to the GRB-2 and this whole complex can catalyze an exchange reaction in which Ras-GDP becomes Ras-GTP. Now, things get interesting. Ras exists in an active and inactive form, and like many other small GTP-binding proteins, it is active when bound to GTP, but inactive with GDP. It is itself a GTPase, so it can turn itself off, but only when another protein, a GTPase-activating protein (GAP), binds to Ras. So Ras is activated when Sos causes exchange of GTP for GDP, and inactivated when GAP induces hydrolysis of GTP to GDP. (See Fig. 20-22).
Before I go on, let’s take a breather. Early on people discovered that when cells were treated with mitogens like insulin, EGF, PDGF, etc., they acquired a new protein kinase activity, which was named the mitogen-activated protein kinase (originally microtubule-associated protein kinase), abbreviated MAP Kinase. (This kinase is also sometimes called the Extracellular Receptor-Activated Protein Kinase or ERK). Soon it was found that the MAP kinase was activated at a pretty late step in the signaling pathway, but before nuclear regulatory factors were altered. Thus there were some steps that occurred earlier in the signaling pathway (so-calledupstream events) and some steps that occurred later in the pathway as a result of the activity of the MAP kinase (naturally called downstream events). MAP kinase is normally inactive, but when the cell is stimulated by a mitogen, MAP kinase gets activated by being phosphorylated, on both a threonine and a tyrosine residue, by an enzyme called MAP kinase kinase by some people and MEK by others. (MEK is another acronym for MAP and ERK Kinase). So MEK, a MAP kinase kinase, is upstream of MAP kinase. However, MEK is also inactive mostly unless it is phosphorylated by another serine-threonine kinase, called Raf, which is farther upstream of MAPK. By now you are probably asking: Is there any meeting ground, or is the pathway infinitely long? Well fortunately, there is a meeting point between the early and late parts of the pathway and it’s the point at which Ras is activated by Sos, because Ras in turn activates Raf (which stands for Ras-associated factor).
So it looks as if the extracellular signal activates the receptor that gets phosphorylated that catalyzes the exchange of GTP for GDP with activates Ras. (Fig. 20-28). (Remember Rab in the vesicle transport pathway and Ran in nuclear transport--these small GTP-binding proteins similar in structure to Ras). When Ras-GTP is bound to the membrane, it can bind to Raf and activate Raf to become a kinase. This may be the only role of Ras, because if you make a genetically engineered form of Raf that binds itself to membranes, then Ras is unnecessary; i.e., the pathway is active in cells that lack Ras. Now, the active Raf can phosphorylate MEK, the active MEK can in turn activate MAP kinase, and MAP kinase is really the business end of the pathway because it activates a whole slew of other proteins by phosphorylation. Most of these are thought to act in the nucleus and to alter the transcription of genes (back to first overhead)--some were already identified as products of oncogenes such as myc and jun/fos. These are then the downstream effectors. It’s not entirely clear what they do, though it’s clear that they turn on the activity of a bunch of different genes--that is, they activate the genes to increase the rate at which they make proteins.
So one effect of activating the growth factor, tyrosine-kinase receptor pathway is to turn on transcription of particular genes in the nucleus. Another thing that the MAP kinase can do in the insulin dependent pathway is to turn on the translation of inactive mRNA. (Show overhead). Here it appears that a protein called PHAS-1 normally binds in cells to some proteins called initiation factors in the cells and prevents them from working. MAP kinase can phosphorylate PHAS-1 and cause it to dissociate from these initiation factors, allowing them to bind to mRNA and attach the mRNA to ribosomes where it can be translated into proteins. This means that the Ras pathway not only activates gene transcription but can turn on protein synthesis as well.
One of the potential virtues of having such a complex pathway as this one is that there is the potential for many "branch points", places where different signalling pathways in the cell can converge or diverge. One such branch point is in the activation of Raf by Ras. It turns out that signals that increase the level of cAMP in fat cells, fibroblasts and smooth muscle, such as epinephrine--adrenaline--inhibit the activation of the MAP kinase. Several groups, including Phil Stork’s of OHSU, have recently reported that the reason for this inhibition is that PKA, which is activated by cAMP, can phosphorylate and inhibit Raf, which interrupts the growth factor receptor mediated pathway. (This may have clinical significance because if an overactive Ras pathway causes cancer, then something that slows it down could be very beneficial). Recent experiments show that a different signaling pathway--the Jak/STAT pathway can also act on, and be acted on by?, the Ras pathway, thereby modulating it.
Finally, of course all this stuff is reversible. In particular the phosphotyrosines get rapidly dephosphorylated by phosphatases, proteins called GTPase activating proteins (GAPs) bind to Ras and cause it to split GTP to GDP, turning itself off. (Again recall Rab). The reason Ras is an oncogene is that a mutation in Ras that destroys its ability to hydrolyze GTP locks it into the on position, so it continues to stimulate all the downstream molecules. You might guess that any downstream molecule that could be similarly locked into the active state would also cause cancer. And all the other phosphorylated proteins can also get dephosphorylated by phosphatases, so that if, say, EGF production is turned off, then the target cells cease dividing.
Well, this is an area of intense interest because of its relationship to cancer, and so even though 15 years ago, little was known about what insulin did--its receptor wasn’t even identified--now we know much more, though still obviously not everything. However, the major details of the pathway from cell surface to nucleus seem to be worked out in this system, though there are other systems--such as the so-called Janus kinases--that use a different mechanism. Look for lots of new information in this area in the months and years to come.