Activation of a receptor ligand 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.