Activation of a receptor. Assembly of the complex

Transcription

1 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. 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 1

2 The adaptor proteins recruit regulatory proteins Inactive Ras Ras Gef Active Ras Relay signal to cytoplasm adaptor protein The adaptor protein bound to the phosphorylated tyrposine kinase domain of the growth factor receptor recruits Ras Gef, a regulator of the small GTPase Ras Ras-Gef catalyzes the exchange of GDP and GTP GDP Ras-GEF GTP N N H O N NH NH 2 G = Guanine Ras-GDP Inactive Ras-GTP Active Ras-GAP promotes hydrolysis of GTP to P i GDP and Pi Ras-GAP Why do we need another protein (Ras-Gef ) to catalyze the exchange of GDP and GTP bound to Ras? 2

3 Timescales of cellular processes k off Ras-GDP k on Ras + GDP The lifetime of Ras-GDP is a measure of how long the complex survives. The equilibrium dissociation constant, K D, and the lifetime of a complex are correlated k off [Ras-GDP] = k on [Ras][GDP] k off / k on = [Ras][GDP]/[Ras-GDP] = K D k on = bimolecular rate constant = M -1 s- 1 k off = unimolecular rate constant = s -1 K D = M so you can estimate the lifetime if you know K D. Estimating lifetimes of biological complexes The rate of a reaction that depends on only one species displays a simple exponential decay [Reactants] Rate of Reaction t 1/2 = time (s) ln 2 k off 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 k off, the dissociation rate constant. K D = k off k on k off =K D x k on 3

4 Estimating lifetimes of biological complexes K D = [Ras][GDP]/[Ras-GDP]. These concentrations can be measured. k on can be estimated (or measured for greater accuracy) Typical on-rates: ~ 10 7 M -1 s M -1 s -1 for a small molecule colliding with a protein ~10 6 M -1 s M -1 s -1 for protein-protein interactions Typical biological processes occur in milliseconds to seconds K D x k on = M x 10 7 M -1 s -1 = k off = 10-4 s -1 t 1/2 = 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. 4

5 Small GTPases are used as switches and timers to regulate many processes in biology Traffic through secretory pathway Nuclear transport Signaling Translation Signal is transmitted from Ras to the cytoplasm Inactive Ras Active Ras adaptor protein Ras Gef Relay signal to cytoplasm Activated Ras relays the signal to a cascade of protein kinases 5

6 Activated Ras transmits the signal to the MAPK cascade Active Ras MAPKKK MAPKK MAPK Gene regulatory protein Signal (input) Amplification MAPKKK MAPKK MAPK Transcription factor (output) Transcription factor Exponential response (100 X 100 X 100 = 10^6) allows tremendous sensitivity and more... 6

7 Getting the signal to the nucleus and transcription Recognition 1 Signal transmission and processing 2 Getting into the nucleus and transcription 3 The end of the line (for the signal) MAPKKK MAPKK MAPK MAPK Nuclear Pore Phosphorylation and activation of MAPK triggers dimerization import into the nucleus MAPK phosphorylates transcription factors in the nucleus Transcription factor Activated Transcription factor 7

8 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) 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. 8