Signalling Lecturenotesb-1

inactive, monomeric active, dimeric • When activated by growth factor binding, the growth factor receptor tyrosine kinase phosphorylates the neighboring receptor.

As we learned, when growth factor binds to the extracellular domain of the growth factor receptor, it causes the receptor to dimerize, which in turn activates the intracellular kinase domain. The kinase domain of one monomer of growth factor receptor phosphorylates tyrosine side chains in the neighboring monomer, and the phosphorylated tyrosines have different structural properties than their unphosphorylated counterparts. You will see on the next slide that the phosphorylated groups on the intracellular domain of growth factor receptor are “recognized” by other proteins.

1 Assembly of the complex

adaptor proteins

• The phosphorylated receptor recruits other signaling proteins • The phosphorylated amino acids on the receptor are recognized and bound by proteins called adaptor proteins

The phosphorylated receptor recruits other signaling proteins, called adaptor proteins, which bind to the phosphorylated amino acids on the receptor. Different adaptor proteins can recognize phosphorylated tyrosines on different proteins (or on different parts of the same protein) because they also contact amino acids adjacent to the phosphorylated tyrosine. That is, they bind only to particular phosphopeptide sequences. Adaptor proteins are so-called because, as we will see, they bind to other proteins, which transmit the information about the altered state of growth factor receptor (the signal) into the cytoplasm by changing the activity of still other proteins. (Think of adaptors for electronic equipment.)

2 The adaptor proteins recruit regulatory proteins

Inactive Ras Active Ras Relay signal to cytoplasm

Ras Gef

adaptor protein The adaptor protein bound to the phosphorylated tyrosine kinase domain of the growth factor receptor recruits Ras Gef, a regulator of the small GTPase Ras

As we just discussed, the adaptor proteins that bind to the phosphorylated receptor recruit other proteins. In the case of growth factor receptor, one adaptor protein recruits a protein called Ras-GEF. Ras-GEF activates a very important signaling protein called Ras. Ras is modified with a lipid group that inserts into the membrane so that Ras is anchored to the cytoplasmic surface of the membrane. Activated Ras then relays the signal to the cytoplasm. On the next slides you will learn how Ras-GEF activates Ras.

3 Ras-GEF O GDP GTP •Ras-Gef catalyzes N NH the exchange of N N NH GDP and GTP H 2 G = Guanine

Ras-GDP Ras-GTP Inactive Active

Ras-GAP promotes hydrolysis of GTP to Pi GDP and Pi Ras-GAP Why do we need another protein (Ras-Gef ) to catalyze the exchange of GDP and GTP bound to Ras?

Ras is a member of a large family of proteins called small GTPases. These proteins can exist in two forms: an inactive form in which Ras is bound to the nucleotide GDP and an active form in which Ras is bound to GTP. In its active form, Ras binds to kinases and activates them. GTPases are enzymes that can hydrolyze GTP to form GDP and inorganic phosphate. Ras, like many small GTPases, is a poor enzyme, meaning that it hydrolyzes GTP very slowly. A protein called Ras-GAP, for Ras-GTPase Activating Protein, regulates Ras activity by binding to the Ras-GTP complex and accelerating hydrolysis of GTP. Ras-GAP thus turns off the activity of Ras-GTP by converting it to inactive Ras- GDP. GAPs play important roles in many signaling pathways by turning off small GTPases. Another important regulator of Ras is Ras-GEF, which is a Guanine Nucleotide Exchange factor. Ras-GEF binds to Ras- GDP and promotes release of the nucleotide. Since the cellular concentration of GTP is higher than that of GDP, Ras-GEF promotes exchange of GTP for GDP, thereby activating Ras. Ras plays a role in many signaling pathways inside eukaryotic cells. A large fraction of human cancers contain mutations in Ras that cause it to be locked in the GTP-bound, active state. These tumor cells therefore activate signaling pathways even in the absence of growth factor signals, eliminating the normal controls that keep cell growth and proliferation in check.

4 Timescales of cellular processes

off k Ras-GDP Ras + GDP k on The lifetime of Ras-GDP is a measure of how long the complex

survives. The equilibrium dissociation constant, KD, and the lifetime of a complex are correlated…

koff[Ras-GDP] = kon[Ras][GDP]

koff/ kon = [Ras][GDP]/[Ras-GDP] = KD

-1 1 kon= bimolecular rate constant = M s- -1 koff= unimolecular rate constant = s KD = M

…so you can estimate the lifetime if you know KD.

We learned that Ras bound to GDP is in its inactive state. In order to attain its active state, Ras must release the GDP and bind GTP. Ras-GEF is required for the exchange of GDP for GTP because Ras binds GDP very tightly -- so tightly, in fact, that it can’t let go of GDP by itself on a timescale that would be compatible with a biological process. To understand this statement, you need to know something about the timescales of biological processes and how to think about them. First, you should know that there is a correlation (inverse) between the equilibrium dissociation constant (KD) for a binding event and the lifetime of the complex. That is, the more tightly something binds (the lower the KD), the longer it stays bound. Second, you should know that you can come up with a ballpark estimate of the lifetime of a complex if you know the equilibrium dissociation constant for a binding event. We will learn how to estimate the lifetime of the Ras-GDP complex in the absence of GEF, but before we do that, you also need to know that biological processes must take place on a timescale that is relatively short. Some cells divide quickly. You learned that your body is producing enormous numbers of cells while you sit in this class. Those cells are carrying out lots of biochemical reactions. Those reactions must occur on timescales of milliseconds to seconds for the biology to work.

5 Timescales of cellular processes

off k Ras-GDP Ras + GDP k on The lifetime of Ras-GDP is a measure of how long the complex

survives. The equilibrium dissociation constant, KD, and the lifetime of a complex are correlated…

koff[Ras-GDP] = kon[Ras][GDP]

koff/ kon = [Ras][GDP]/[Ras-GDP] = KD

-1 1 kon= bimolecular rate constant = M s- -1 koff= unimolecular rate constant = s KD = M

…so you can estimate the lifetime if you know KD.

Now that you know that, you should also know that Ras-GDP has a KD of about 10-11 M. On this slide, we show the equilibrium equation for dissociation of Ras-GDP to Ras and GDP. At equilibrium, the dissociation rate constant (koff) times the molar concentration of Ras-GDP is equal to the association rate constant (kon) times the molar concentrations of Ras and GDP. Rearranging this equation shows that koff/kon is equal to the ratio of the product of the molar concentrations of Ras and GDP to Ras-GDP. These ratios (of rate constants or of concentrations of chemical species) are equal to the equilibrium dissociation constant, KD, for the reaction shown. Although in this course we have not focused much on units, you should pay attention to the units in this equation because they help you to understand the physical processes. The on-rate constant (kon) is a bimolecular rate constant with units of inverse time X inverse concentration. The concentration term reflects the fact that two species must collide in solution for a binding event to occur. The time dependence reflects the fact that the collisional frequency for two molecules depends on their size and shape (as well as the viscosity and temperature of the medium). Large molecules move more slowly than small molecules and so they collide more slowly. Thus, on rate constants for big molecules tend to be smaller than on rate constants for small molecules. The time dependence in the on-rate constant also reflects the fact that the molecules not only need to collide, but they need to collide and then fit together in a productive way. Sometimes they need to undergo conformational changes to allow a good fit. On-rate constants are smaller when reorganization is required for productive binding. The off-rate constant is a unimolecular rate constant with units of inverse time. It is not concentration dependent because it is a measure of the dissociation of a single species to two species. The time dependence of the off-rate reflects how hard it is to break the favorable interactions that keep the two molecules stuck together.

As you already know, the equilibrium dissociation constant has units of molar. 6 Estimating lifetimes of biological complexes

The rate of a

]

s t

reaction that n a t Rate of Reaction ln 2 depends on only c t =

a 1/2 e

one species R k off [ displays a simple exponential decay time (s)

t 1/2 = the time that it takes for 1/2 the complex to dissociate. It is a measure of the lifetime of the complex and can be determined from koff, the dissociation rate constant.

k off KD = koff=KD x kon k on

We said on the previous slide that if you know the equilibrium dissociation constant, or KD, for a complex, you can estimate the lifetime of the complex. Dissociation of a small molecule from a macromolecule as a function of time can be represented by a single exponential decay. A typical plot for such a process is shown on this slide. We use half-life (t1/2), the time it takes for half the complex to dissociate, to quantify the lifetime of a complex. T1/2 is given by the simple relationship shown above. This equation tells us that we can calculate the half life of any complex if we know the off rate constant for dissociation of the complex.

That is pretty simple, but begs the question: How can we get the off- rate constant? One way is to measure it directly, and there are various experimental techniques to do so, but we won’t talk about them. Instead, let’s focus on the relationship between the equilibrium constant for dissociation and the off-rate constant for the reaction. You learned on the previous slide that the equilibrium constant for dissociation of a complex (KD) is given by the ratio koff/kon. Therefore, the off rate constant for dissociation is simply the product of the equilibrium constant (M) for dissociation of the complex times the on-rate constant(M-1s-1) for formation of the complex. Equilibrium constants are usually easier to measure than rate constants for association or for dissociation because all you have to do is measure the concentrations of the free and bound species. 7 Estimating lifetimes of biological complexes

KD = [Ras][GDP]/[Ras-GDP].

These concentrations can be measured.

kon can be estimated (or measured for greater accuracy)

Typical on-rates: ~ 107 M-1s-1 --108 M-1s-1 for a small molecule colliding with a protein ~106 M-1s-1 --107 M-1s-1 for protein-protein interactions

As we said, the equilibrium constant for dissociation of a complex can be obtained by measuring the concentrations of the free and bound species, and there are many straightforward techniques for doing so. The on-rate constant for the formation of a complex can be measured as well, but with more difﬁculty. Even without measuring the on-rate, however, it is possible estimate the lifetime of a complex by making some assumptions. These assumptions will give an order of magnitude estimate of the lifetime. The assumptions will not be valid for all cases, but they are reasonable for many cases. One assumption is that the on-rate constant for binding of two molecules simply reﬂects how fast they can collide in the right orientation in a particular medium (in this case, aqueous solution around physiological temperatures). The fastest rate at which two molecules can collide in solution is given by the diffusion limit, a measure of how fast the molecules can move in the particular medium. For small molecules, the diffusion limit is close to 109M-1 s-1. Large molecules move more slowly and so they collide more slowly . On-rates are typically somewhat slower than the diffusion limit because some reorientation must take place. Having said all that, a reasonable estimate for an on-rate constant for a small molecule binding to a protein is between 107-108M-1 s-1. For two proteins binding, it is a little slower.

8 Typical biological processes occur in milliseconds to seconds

-11 7 -1 -1 -4 -1 KD x kon = 10 M x 10 M s = koff = 10 s

t1/2 = 6900 seconds!

It would take > 100 min for Ras to release GDP without help. This timescale is not compatible with biology.

Ras-GEF regulates the activity of Ras by facilitating nucleotide exchange.

The equilibrium dissociation constant for Ras-GDP binding has been measured and is around 10-11, as we said previously. Using an estimated on-rate constant of 107 M-1 s-1 gives an off-rate constant of 10-4 s-1. That would mean that the half life for the complex is 6900 seconds, or more than 100 minutes. We said earlier that biological reactions must occur on timescales of milliseconds to seconds for biology to work. You can immediately see that a half life of 100 minutes is not compatible with life.

In a cell, the lifetime of the Ras-GDP complex can be much shorter if Ras- GEF has been recruited by an adaptor protein so that it is localized near the membrane-associated Ras-GDP complex and able to facilitate nucleotide exchange. Thus, tight binding interactions in biology allow for complex regulatory mechanisms involving other proteins.

Kds in biology range between about 10-4 M and 10-11 M (some lie outside that range, but not many). You can calculate the range of half-lives that biological complexes display using the relationships and assumptions we have described. Complexes with very long half-lives must be regulated in some way so that dissociation can occur on a reasonable time scale. Complexes with short half lives are characteristic of many enzyme-substrate interactions involved in primary metabolic pathways where the cellular concentrations of substrates are relatively high (micromolar).

9 Small GTPases are used as switches and timers to regulate many processes in biology

. Trafﬁc through secretory pathway . Nuclear transport . Signaling . Translation

Small GTPases are used as switches and timers to regulate many biological processes, including trafficking though the secretory pathway, nuclear transport, signaling, and translation.

10 Signal is transmitted from Ras to the cytoplasm

Inactive Ras Active Ras

Relay signal to cytoplasm Ras adaptor Gef protein

• Activated Ras relays the signal to a cascade of protein kinases

Now that we have learned about the role of GTPases in general, we will continue with our story about Ras. When Ras is activated (binds GTP) it interacts with other proteins that have kinase activity and starts a kinase cascade. Thus, it relays the signal -- that growth factor is bound to growth factor receptor -- to a set of protein kinases in the cytoplasm.

11 Activated Ras transmits the signal to the MAPK cascade

MAPKKK

Active Ras

MAPKK

MAPK

Gene regulatory protein

Activated GTP-bound Ras, which is bound to the cytoplasmic membrane, activates a kinase cascade. The kinases in the cascade are called the MAP kinases (for Mitogen Activated Protein Kinases; a mitogen is an agent that induces mitosis). In the inactive form, the kinases in the MAP kinase cascade are unphosphorylated. Activated Ras binds to a membrane-associated kinase called MAPKKK (for mitogen-activated protein kinase kinase kinase), which turns on its kinase activity. MAPKKK then phosphorylates MAPKK, activating it so that it phosphorylates MAPK, which then phosphorylates one or more gene regulatory proteins that influence which genes are transcribed (and thus, which proteins are expressed). Thus, the signal has been communicated from the outside of the cell to the nucleus and changes in protein expression have occurred as a result.

12 Ampliﬁcation Signal (input) MAPKKK

MAPKK

MAPK

Transcription factor Transcription factor (output)

Exponential response (100 X 100 X 100 = 10^6) allows tremendous sensitivity and more . . .

The MAPK cascade plays an important role in amplifying the signal. Each activated MAPKKK can activate several MAPKKs, which can activate several MAPKs, which in turn phosphorylate multiple copies of transcription factor substrate. If we consider a simple example of how this might work when each kinase can phosphorylate and activate 100 molecules of the downstream target, activation of single molecule of MAPKKK will lead to phosphorylation of 10^6 molecules of transcription factor. This amplification of the signal from 1 to 10^6 greatly increases the sensitivity of the signaling pathway, allowing it to be sensitive to the presence of even a very small number of bound receptors. This is in part how growth factor signaling systems are able to detect the presence of growth factor present in the blood at very low concentrations.

In addition, having a cascade of kinases as part of a signaling pathway, allows for integration of multiple signals from many different extracellular inputs.

13 Getting the signal to the nucleus and transcription Recognition 1

Signal transmission 2 and processing

Getting into the nucleus 3 and transcription

You might be wondering how a signalling cascade that starts at the cell membrane leads to changes in the phosphorylation state of proteins that are in the nucleus. The next slide illustrates how this can occur.

14 The end of the line (for the signal)

MAPKKK • Phosphorylation and activation of MAPKK MAPK triggers dimerization MAPK import into the nucleus MAPK • MAPK

Nuclear Pore phosphorylates transcription factors in the nucleus

Transcription factor Activated Transcription factor

MAPKKK is membrane-associated, as you learned, and it phosphorylates a soluble, cytoplasmic kinase, MAPKK. MAPKK, now in its active state, then phosphorylates MAPK. Phosphorylation and activation of MAPK lead to dimerization of MAPK. Dimerized MAPK is somehow recognized for import into the nucleus. (Proteins that move into the nucleus interact with nuclear membrane proteins that form a kind of import channel called the nuclear pore.) Once inside the nucleus, MAPK can activate various transcription factors. In other signalling pathways, the gene regulatory proteins are cytoplasmic in their inactive states, but when they get phosphorylated, they move into the nucleus.

15 The phosphorylated transcription factor . . .

• Binds DNA • Interacts with other proteins that are required for transcription in eukaryotic cells

Transcription factors regulated by MAPK affect the regulation of genes involved in: • cell growth (increases protein production) • cell division (cell cycle control)

Phosphorylation regulates the activity of transcription factors, allowing them to bind DNA and interact with other proteins required for transcription in eukaryotic cells. The transcription factors phosphorylated by MAPK promote expression of genes important for the response to growth factors. Many of these genes are involved in controlling cell growth and cell cycle, and cell survival. When this signals controlling growth and division are misregulated, terrible consequences follow.

16 Take home messages

• All cells have mechanisms to sense and respond to the environment. • Many signals are transduced by phosphorylation cascades, with ATP as the phosphodonor. • ATP is thermodynamically labile but kinetically stable, and enzymes called kinases are required to effect phosphotransfer. • Phosphorylation changes the physical properties and behavior of proteins. • Phosphorylation cascades allow for signal ampliﬁcation. • Small GTPases act as switches or timers for biological processes. • Equilibrium dissociation constants are inversely correlated with lifetimes of complexes. • There are many ways to regulate gene expression (i.e., which genes are on and which are off). • Jacob and Monod discovered gene regulation.