Activating and de-activating Ras

Key human members of Ras family KRAS, HRAS, NRAS Kick-start cell growth when activated Other signaling?

Some terms

Conserved proteins, genes, etc.: Appear in many organisms, presumably “conserved” by evolution

Allosteric site: a site (other than the catalytic site) that affects the rate of a catalytic reaction

Hydrolysis: cleavage of chemical bonds by the addition of water

GTPase: a protein that catalyzes hydrolysis of GTP to GDP

A Video: GTPases as Molecular Switches

Turning Ras On and Off Ras is normally tightly bound to GDP Ras is activated by binding to GTP instead of GDP Ras is a weak GTPase (i.e., it encourges hydrolysis of GTP

into GDP) A “guanine nucleotide exchange factor” helps Ras to

exchange GTP for GDP GEF is the generic term Sos1 is the specific GEF we will look at

A GTPase Activating Protein speeds up Ras GTPase activity GAP is the generic term RASA1 is the specific GAP we will look at

Turning Ras On and Off

Ras-GDP

Ras-GDP is the “off” state of Ras

Turning Ras On and Off

Ras-GDP

Ras-GTP

Ras-GTP is the “on” state of Ras

Turning Ras On and Off

Ras-GDP

Ras-GTP

The entire reversible reaction (????)

Turning Ras On and OffGDP

GTP

Ras-GDP

Ras-GTP

Ras-O

Ras can bind to either GDP or GTP (or not at all)

Turning Ras OnGDP

GTP

Ras-GDP

Ras-GTP

Ras-OGEF

A guanine nucleotide exchange factor (GEF) helps Ras exchange a GDP for a GTP

Turning Ras On and OffGDP

GTP

GAP

Ras-GDP

Ras-GTP

Ras-OGEF

A GTPase Activating Protein (GAP) helps Ras turn the GTP back into GDP

But wait: There’s More!!

GEF

Ras-GDP

Ras-GTP

GAP

How Fast Does Ras Turn On?

Sos1

Ras-GDP

Ras-GTP

Cdc25

REM

Guanine Nucleotide Exchange Rate

Ras-GDP

Sos1

Rate: kcat, Km

Part of Sos1

Ras-GDP

Cdc25

REM

Guanine Nucleotide Exchange Rate

Sos1

Rate: kcat, Km

Part of Sos1

Ras-GDP

GDP

Ras-O

Cdc25

REM

Guanine Nucleotide Exchange Rate

Ras-O

Ras-GTP

Sos1

GTP

Rate: kcat, Km

Part of Sos1

Cdc25

REM

Guanine Nucleotide Exchange Rate

Ras-GDP

Ras-O

Ras-GTP

Sos1

GDPGTP

Part of Sos1

Cdc25

REM

Guanine Nucleotide Exchange Rate

Ras-GDP

Ras-O

Ras-GTP

Sos1

GDPGTP

Rate: 5 times as fast

Ras-GDP

Part of Sos1

Cdc25

REM

Guanine Nucleotide Exchange Rate

Ras-GDP

Ras-O

Ras-GTP

Sos1

GDPGTP

Rate: 75 times as fast

Ras-GTP

Part of Sos1

Ras Deactivation (Hydrolysis)

Ras-GDP

Ras-GTP

RASA1

How Fast Does Ras Turn Off

Ras-GDP

Ras-GTP

Ras-GTP is a GTPase: It hydrolyzes GTP to GDP (slowly)

Ras Deactivation (Hydrolysis)

Ras-GDP

Ras-GTP

RASA1

RASA1 speeds up the hydrolysis by ???

Turning Ras On and Off: The Whole Story (… or is it?)

GDP

GTP

RASA1

Ras-GDP

Ras-GTP

Ras-O

Sos1

slow

fast

Super fast

Summary Ras is normally tightly bound to GDP Ras is activated by binding to GTP instead of GDP A “guanine nucleotide exchange factor” helps Ras to

exchange GTP for GDP GEF is the generic term Sos1 is the specific GEF we will look at Sos1 has an allosteric site that affects the reaction rate

A GTPase activating protein speeds up Ras GTPase activity (ie, hydrolyzing the GTP so it becomes GDP)

GAP is the generic term RASA1 is the specific GAP we will look at

YouTube Lectures on Ras

Wittinghofer Part 1 Wittinghofer Part 2