Cell bio Exam 3 - Ras GTPase

• G-protein coupled receptors

• Enzyme linked receptors

• Ion channel receptors

EXAMPLES OF THE 3 TYPES OF SIGNAL TRANSDUCERS

• _______

• _____

• ______

• kinases

• tyrosine

• Ligand

• Phosphorylation

• dimerization and phosphorylation

• RTK

• Ras-GEF

• GEF

• Ras activation

• activates Ras

Ras GTPase PATHWAY - RECEPTOR TYROSINE KINASES

• Receptor are _____

  • phosphorylation occur on _____

• ______ (signaling molecule) induces dimerization of RTKs

• Cross phosphorylation occurs

• _______ sites are binding sites for other proteins

  • 10-20 different molecules

• Ligand induces _______

• Adaptor protein binds to _____

• _____ binds to adaptor protein

• localizes ____ to membrane

• Ras-GEF facilitates _____

• Close enough but not enough to have a meaningful interaction (penny vs dollar) 

• Ligand dimerizes (effective things can now happen!) 

• Cross phosphorylation (the two dimers phosphorylate each other!) 

• Now other proteins can bind there because it was phosphorylated (has different shape, so it can interact differently with proteins!) 

• Ras GEF gets closer to the membrane, which ______

• activates

• GTPase 

• GDP; GTP

• inactive

• GTPase

• GEFS

Ras GTPase PATHWAY - RAS GTPASE, GEFS AND GAPS

• Ras is a monomeric GTPase

• trimeric GTPase proteins are part of the GPCR pathway

• Most RTKs GTPases are monomeric

• Ras GEF gets closer to the membrane, which ____ Ras

• When Ras is activated, it's a _____

• Inactivate is ____, activated form is ____ 

• Ras-GEF makes GTP more active so there is more of it! 

• Ras-GAP causes more GTP hydrolysis, therefore it becomes GDP bound, which is the ____ state of Ras 

• Constitutively active Ras (always on, ignoring regulation, does not turn off!) 

• Ras has a natural hydrolyzation since its a _____… but ____ make it happen faster 

• GEFS

• GAPs

• constitutively active

Ras GTPase PATHWAY - RAS GTPASE, GEFS AND GAPS

• Ras activated by by many different factors

• General scheme

  • Ras has basal level of activity

  • Activate ____ to activate Ras

  • ____ and natural hydrolytic activity inactivate

• 30% of cancers contain mutant Ras that is ______

• Plants don’t have an equivalent family of GTPase

  • principles are the same

• MAP kinase (MAPK)

• MAP kinase kinase (MAPKK)

• MAP kinase kinase kinase (MAPKKK)

• Signal

• Feedback

• activity; transcriptional regulators

• Activity; transcriptional

Ras GTPase PATHWAY - MAP KINASE CASCADE

• ________ - phosphorylate effector molecules

• ________ - phosphorylate and activate MAP kinases

• _________ - phosphorylate and activate MAP kinase kinases

• ____ Amplification

• _____ regulation

• MAPK… at the end of the pathway that does the stuff 

• When Ras activates the MAPKKK, it phosphorylates MAPKK, which phosphorylates MAPK 

• It is regulated 

• MAPK can phosphorylate proteins that change ____ (turns on or off), they can also phosphorylate ________ (changes gene regulation, makes different proteins) 

• _____ is fast, _____ is slower 

• Tyrosine

• Remove

• MAPK

• endocytosis

• ligand

• GTP

• phosphatase

HOW DOES THE CELL TURN THIS OFF?

• _____ phosphotases

• ____ signal molecule

• Feedback regulation in ____ cascade

• Internalization of receptor and degradation by lysosome using _____

• Use competitive inhibitor to stop ____ 

• Hinder the creation of ____ (but this may have consequences on other things) 

• Add _____ (dephosphorylate a step) 

• culture

• ligand

• divide

HOW DO WE KNOW WHAT PROTEINS ARE IN THE PATHWAY AND WHAT ORDER THEY ARE IN?

• We _____ primary liver cells in a dish.

• When we add a _____ signaling occurs. The ligand we are adding is known to bind to an RTK

• Cell ____

• We have normal cells 

• Can grow it in a dish 

• Add in ligand, it gets activated and cell division happens 

• that means its involved in the pathway (if it was mutated and no longer gets a pathway!) 

• not involved in the pathway! 

QUESTION: WHAT PROTEINS ARE THE PATHWAY?

LOTS OF POTENTIAL PROTEINS MIGHT BE IN PATHWAY

TEST WITH CELL CULTURE - EXAMPLE

• Create a cell line with a mutant version of that protein, add ligand in and see what happens

• Nothing happens with X, no signal → _______

• Protein Y gets cell division → ______

• C and F 

RESULTS OF EXPERIMENT TO DETERMINE WHICH PROTEINS ARE IN THE PATHWAY

Which one of these is involved in this pathway for cell division?

• _____

read and comprehend

Test in cell culture- 

• RTK is the first thing, on the outside 

Options: 

• C → Ras → F (Yes… F)

• F → Ras → C (Yes… C) 

• Ras → C → F (Yes… C and F)

• Ras → F → C (Yes… F and C)

• C → F → Ras (Yes… Whatever is after this)

• F → C → Ras (Yes… whatever is after this) 

• Add in constitutively active Ras… knows there is signaling 

• When C protein is mutated, nothing happens

• When F protein is mutated, cell division still occurs 

• RTK (first before the rest of the pathway); ligand is added to get pathway going

• Adding constitutively active Ras (devil cat)... signals no matter if the ligand is present or not. Therefore, whatever is after Ras receives and sends a signal (yes for cell division!)

read and comprehend

If we mutate C- 

• C → Ras → F

• (RTK does not signal because ligand is not present. C does not receive a signal so it does not signal. Ras signals which means F signals) → gets cell division 

• F → Ras → C

• does not get cell division! 

• Ras → C → F

• Does not get cell division because C is broken! 

• Ras → F → C

• No cell division! Ras signals to F, F signals to C… but sense its broken there is no cell division

• C → F → Ras

• Ras creates cell division 

• F → C → Ras

• Has cell division 

read and comprehend

If we mutate F- 

• C → Ras → F

• No cell division

• F → Ras → C

• does get cell division! 

• Ras → C → F

• Does not get cell division because F is broken! 

• Ras → F → C

• No cell division! Ras signals to C, C signals to F… but sense its broken there is no cell division

• C → F → Ras

• Ras creates cell division 

• F → C → Ras

• Has cell division 

• ****Therefore, it is F → Ras → C because this is the one that lines up! 

read and comprehend

How would you stop this pathway- 

• Block the active receptor site of tyrosine kinase 

• Block any of them!! 

• If Ras is intuitively active, then block Y 

• Acetylcholine

CELLS CAN RESPOND DIFFERENTLY TO THE SAME SIGNAL

• _____ induces a different response in salivary cells and skeletal muscle cells

• same ligand can activate different kinds of Gproteins depending on the cell

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