Lecture #3 - Lipid Mediate Signal Transduction

Question 1

Major signlaing Lipids

Answer 1

Glycerolipids + Sphingolipids + Fatty Acids all play a major signlaing role

Metabolism of these lipids may lead to the generation of hydrophibic and/or hydrophilic signlaing molecules

Major enzymes involoved in lipid singlining includes lipases + kinases + synthases + phosphatases
- Regulation: post translation modifications + membrane binding (interfacial activation) + substrate availibility

Question 2

Hydrophilic Vs. Lipid mediated signaling

Answer 2

Hydrophilic signlaing process – Have Receptor/Adapter –> Receptor/adapter activats of enzyme that use water soluble substartes –> enzyme make a water soluble messenger –> water soluble messger goes to intracelular targets –> have effect

Hydrophobic Lipid mediated signaling –> Have a receptor/adapter –> Activate Lipid metabolizing enzyme –> Enzyme makes Lipid and/or water siluble metabolites OR modified lipids –> Lipid or water soluble metabolites go to intracellular target –> get effect
- Can have a hydrophobic agonist that goes into cell to affect target (Ex hormone)

Question 3

Major difference between lipid mediated signlaing and OTHER signlaing systems

Answer 3

Difference is that lipids are hydrophoblic (at least amphipathic)

Because lipids are hydrophobic –> means the generation of lipids + effects of lipids + metabolism of lipids must involoe a hydrophobic envirnment
- At some point in lipid signaling pathway there is a hydrophobic environment
- Exception - some substrates and metabolites in lipid signaling are hydrophilic (don’t need hydrophobic envirnment)

Question 4

Example physiologic process Lipids are involoved

Answer 4

Lipids mediate inflammatory responses

Ex. Arachidonic Acid is metabolized in 2 pathways by lipid metabolzing enzymes to produce prostaglandins and leukotrienes (inflamatory lipids)

Example - Asprin inhibits lipid metabolizing enzymes to inhibit the production of inflammatory lipids

Question 5

How did we know that Lipids are involoved in signaling

Answer 5

ALL showed lipids are involoved in signlaing
1. By finding they mediate physiologic processes (Ex. inflamatory response)
2. Finding lipid metabolsim can generate second messengers
3. Finding the PI cycle + its involovment in signaling cascade
4. Finding lipid diveristy between mebranes of different cells and membranes of a cell

Question 6

Key players in membrane lipids

Answer 6

MOSTLY phospholipids

1. Phosphatdic acid (PtDOH)
2. Phosphatidylethanolamine (PtdEh) –> Phosphatdic acid + ethanlonaline head group
3. Phosphatidylcholine (PtdCho)
4. Phosphatidylserine (PtdSer)
5. Phosphatidileinosital (PtdIns)
6. Sphingomyelin (Sphingolipids) –> sphingosine backbone AND a choline
   
   • Forms the major contistutive myelin in nuerons AND functions in signaling (sphingosine and Phosphorylated sphingosine are signlaing molecules)

Question 7

Lipid Diversity

Answer 7

Have Lipid diverity between membranes

Have a difference in the abundnece of the types of lipids between different kinds of cells
- Example - Proteins are 50% of membarne weight in most organsims but 75% in E.coli

Have a differences in the abudnence of the types of lipids in different membranes in one cell
- Example - Have a lot of choletral in the plasma membrane and less cgrostrol in the mitocondiral membranes

Question 8

Distrubution of membrane lipids

Answer 8

Membrane Lipids have preferential distrubutions in within membarnes (Composition of the outer leaflet is different from the composition of the inner leaflet)
- Inner leaflet has a concave curvature ; outer leaflet has a convex curvature –> affects how lipid head groups pack –> affects the distrubution of lipids

Outer leaflet –> has more choline contaning phospholipids and more cholestrol (Ex. phosphatidilecholine)

Inner leaflet –> Has more amino phospholipids (Ex. phosphatidylserine)

Neutral lipids (Diacyglycerol) can flip between the two leaflets in the membrane

Question 9

Signaling lipids

Answer 9

Function – modulate activities of proteins (including enzymes)

Proteins that signaling lipids modulate are involved in:
1. Cell Growth
2. Cell Agregation
3. Apoptosis
4. Inflamatory/immune response

Question 10

Types of lipid metabolsizing signaling enzymes

Answer 10

Signaling enzymes that metabolize lipids:
1. Phospholipases –> Phospholipases A2 (PLA2s) + Phospholipases C (PLC) + Phospholipases D (PLD)
2. Lipid Kinases –> Phosphoionsitide Kinases + Diacylglycerol Kinases + Sphingosine Kinases
- LIPID-OH –> Lipid-OPO (uses ATP to add Pi to make phosphorylated version of lipid)
3. Lipid Phosphatases –> Phospoinositide Phosphatase (PTEN-Phosphtase and Tensin Homolog) + Phosphatidic Acid Phosphatases
- Lipid-OPO –> Lipid-OH (removes Pi from phosphorlayed Lipid)

Other important enzymes in lipid metabolism = lipid oxidaes + desaturases + transferases

Question 11

Major signaling lipids and the enzymes involved in their metabolsim

Answer 11

Image - Shows the lipid metabolizing enzymes + the singling lipids that they make/act on

show enzymes use hydrophobic/hydrophilic/amphipathic substartes AND that lipid metabsolism can make amphipathic + hydrophobic+ hydrophilic products
- Some hydrophobic substrates and some of their products are membrane localized)

Question 12

Soluble Kinetics

Answer 12

Soluble - Enzymes and substrates are moving all around in 3D dimmensions of the solution –> eventually enzyme finds a substrate and makes a product
- Have some concentration of the substarte and a lower condetration of the enzume

Question 13

Interfacial Kinetics

Answer 13

Enzyme can be soluble OR always at the membrane –> Soluble Enzyme binds to membrane (by binding to substarte or different component) –> enzyme (bound to substarte) can do catalysis and make product
- Product can be soluble or can stay in the membrane

Shows the substrate is NOT moving about in 3D space (different from soluble kinetics) INSTEAD the substrate is only moving in the planar surface of the membrane

For interfacial kinetics - don’t care about the concentration of the soluble protein INSTEAD care about the percent of substarte in the membrane (‘mole percent’)
- How much of the membrane does the substrate take up

Question 14

Regulation of lipid metabolizing enzymes

Answer 14

Regulation of lipid metabolizing enzymes:
1. Substrate Availibility –> Enzyme is present (on or off the membrane) BUT only get enzymatic activity when the substrate is made and is near enzyme
2. Alteration of their intrinsic enzymatic activity –> Inactive enzyme bind to effector/be modified to be activated
3. Membrane localization (UNIQUE to lipid signaling)
- Includes interfacial activation –> Enzyme is slightly active or inactive when soluble (NOT bound to membarne) BUT becomes activated when it binds to the membrane (activity increases)

Mechanisms of regulation are NOT mutually exclusive

Question 15

Modes of Interfacial Enzymes

Answer 15

1. Scooting mode – Enzyme binds to and stays in the membrane and scoots around until it finds the substrate
   - Enzyme never leaves the surface of the membrane
   - Easier mode to evalute
2. Hopping Mode - Enzyme binds to the membrane and scoots around to find the substrate –> enzyme leaves the membrane and goes to a new membrane (hopping between membranes)
   
   • Could be agonist induced

Question 16

Phospholipase A2 (PLA2)

Answer 16

Reaction: Phospholipid –> Lysophospholipid (LysoPL) + Free fatty Acid

Phospholipids have a glycerol backbone with SN1, SN2, SN3 –> SN1 and SN2 have a Fatty Acid attatched

PLA2 hydrolyzes the Fatty Acid off of SN2 in the glycerol backbone of Phospholipid
- Free fatty Acid can be a sigling molecule itself OR can be metabolizd further to be a different signaling molecule

Question 17

PLA2 binding to Substrate

Answer 17

Substrate = phospholipid

Image - Shows PLA2 with the phospholipid substrate is PLA2 active site

Tryptrophan binds the enzyme to the phospholipid to anchors the enzume to the cell membrane –> PLA2 enzyme lifts the PL out of the membarne –> PLA2 cleaves the 2nd position of PL glycerol backbone –> relases a Free Fatty Acid –> PLA2 drops PL back down into the membrane –> PLA2 goes to a new substrate

Question 18

Major types of PLA2s

Answer 18

1. Cytosolic PLA2 (cPLA2)
   
   • Specific for Arachidonic Acid (Fatty acid that cPLA2 cleaves off of the phospholipid)
   • invloved in inflamatory responses
2. Small secreted PLA2s (sPLAs)
   
   • Not Acyl chain specific
3. Caclium independent PLAs (iPLA2)
4. Lipoprotein-Associated PLAs (Lp-PLA2)
5. PAF Achethlhydrolases (PAF-AH)

Question 19

What increases cPLA2 activity

Answer 19

Adding Agonists that increase Ca in the cytoplasm –> there is an increase in cPLA2 activity

How would you determine if cPLA2s membrane localization is regulated in an agonist and or/Ca depenedent manner?
Experiment Used GFP taged cPLA2 and Calcium ionopore:
1. Toxin (CryIC) –> stimulates arachidonic acid and Ca increase in a receptor dependent manner –> cPLA2 enzymes go to the membrane
2. Okadaic Acid –> stimulates Arachidonic Acid and Ca increase in a receptor indenpendent manner –> enzymes go to the membrane
3. Add nothing —> enzymes go to the membrane

SHOWS when agonist (arachnoid acid) AND Ca incudes tranlocarion of cPLA2 enzyme goes to the membarne –> Membrane localization is regulated by agonist (AA) and Ca

Question 20

Regulation of cPLA2

Answer 20

Regulation include Substrate availibility + Alteration of the instricnic activity of cPLA2 enzyme + interfacial Activation

Major regulatory mechanism: Ca binds to C2 domain of cPLA2 –> When Ca binds cPLA2 enzyme binds to the membarne –> When bound cPLA2 cleaves Arachnoid Acid off of the membrane phospholipid

Phospholipids in other parts of membrane help anchor PLA2 to the membrane

PLA2 is ALSO phosphorylated by MAPK (important at low agonist/Ca levels)

END - PLA2 is activated by binding to the membrane AND by a post translation modification (phosphorylation)

Question 21

Critical Micelle Concentration (CMC)

Answer 21

CMC = lipid concentration at which micelles form

Micelle = ball of lipids

Lipid concentration below CMC = lipid exists as monomers in solution
Lipid concentration at or above CMC = form lipid micelles

Question 22

How would you determine if PLA2 is regulated via interfacial activation

Answer 22

Uses the lipid CMC

Experiment to show that PLA2 is activated by Interfacial Activation (PLA2 Activity Vs. Concetration of Lipid Substrate)
- PLA2 is only actvated when substrate (lipid concentration) is at the CMC
- PLA2 has low activity at lower lipid concentrations –> THEN once hit CMC (lipids form micelles using substartes) the PLA2 enzymatic increases –> PLA2 is ACTIVATED by interfacial activation

No membranes then PLA2 has no mebrane to bind to in order to activate Vs. When can from membranes PLA2 can bind to membrane and be activated –> means activated by Interfacial Activation

Question 23

Platlet Activating factor

Answer 23

Platlet Activating factor –> lipid derived signaling molecules (activates platlets AND inducings Pro-inflamatory signlaing pathways)
- PAF-AH = type of PLA2 that degrades PAF

PAF (enzyme) – has a Critical Acetyl group at the 2nd position

PAF enzyme is activated at very low concetrations (good because don’t want to activate platlets easily)

Question 24

Eicosanoids

Answer 24

Eicosanoids (Ex. Arachinoic Acid = oxidized polyunsaturades 20 carbon Fatty Acid

Types of PLA2 Reaction – Phosphatidlcholine (PtdCho) (PL) –> 1-Lysophasphatidil choline (LysoPL) + Arachidonic Acid (FA)
- PLA2 = Generates Arachonoic Acid

Question 25

Arachidonic Acid

Answer 25

Arachidonic Acid is able to do many things AND Arachidonic Acid can be metabolzied Example - Arachidonic Acid is metabolized by Cyclooxygenase to make COX1 and COX2 - Arachidonic Acid can make prostaglandins + thromboxane --> involved in inflamatory response - Asprins and NSAIDS work by inhibiting the Cycloxygenase --> inhibits the production of downstream products that are involoved in inflamatory response (ex. prostgandins and thromboxane)

Question 26

Phospholipases C (PLC)

Answer 26

Involved in PI cycle PLC works at the membrane Function - PLC cleaves off the IP3 headgroup - Cleaves PIP2 --> makes Diacyglycerol (DAG) and IP3

Question 27

Discovery of PI Cycle

Answer 27

Add Acetylcholine (agonist) to cells --> increases synthesis of phosphatidylinositol (PtdIns/PIs) NOw know adding Acetylcholine (agonist stimulation) also: 1. Increases diacylglycerol (DAG) 2. Increases Ca 3. INcreases in Arachidonic Acid ALL these observations led to the PI Cycle hypothesis

Question 28

PI Cycle

Answer 28

Start - ptdINs (PI) --> Phosphorylate PtdINS to get PIP --> Phosphorylate PIP to get PIP2 Once have PIP2 - PtdIns Phospholipase C (PI-PLC) cleaves PIP2 --> makes Diacyglycerol (DAG) and IP3 - PI-PLC cleaves in receptor agonist way IP3 mobilizes the intracellular store of Ca to release Ca to the Cytosol DAG Can: 1. DAG can be acted on by lipase to makes Archndonic Acid (AA) because PI/PIP/PIP2 is rich in AA 2. DAG can activate Protein kinase C (PKC) 3. DAG can be phopshorylated by Diackglycerol Kinase (DGK) --> makes Phosphatidic acid --> Phosphatidic acid join with a Inosital to make PI END - Pathways affects proliferation + differentiation

Question 29

Affect of PI cycle of Ca

Answer 29

Reaction in PI Cycle = PI --> PIP --> PIP2 --> IP3 --> IP3 leads to increase in Ca2+ PI-PLC cleaves PIP2 --> IP3 - Pi-PLC can be activated through receptor activation

Question 30

How does IP3 increase Ca

Answer 30

PI-PLC - cleaves PIP2 to IP3 + DAG - IP3 = water soluble IP3 binds to IP3 receptor on ER and open the Ca2+ channel (part of the receptor) --> Ca goes form the ER to the cytoplasm - IP3 increase Ca Released Ca2+ binds to any things AND can bind to Calmodulin (Calmodulin can active proteins) - Ca2+ can activate enzymes/effectors in calmodulin dependent or independent way (binds to calcmodulin to activate things OR can activate things without calmodulin)

Question 31

How does Ca decrease after IP3 releases it

Answer 31

Ca binds to Callmodulin --> Calmodulin can activate Ca2+ pumps on the ER OR Ca pumps on the plasma membrane --> BOTH lowering Ca in cytoplasm The Ca WAS increased because IP3 bidning to IP3 receptor --> to lower Ca after release Ca can be pumped out of the cell OR can go back to the ER

Question 32

How was it determined that calcium increases occurs in response to agonists (IP3)?

Answer 32

Show increase in IP3 (agonostic) --> increase in Ca Use Fura-2 --> radiometric Ca indicator dye (color changes when Ca is there) - Increase in calcium --> increase in floruence at 340nm - Tested solutions with different concetration of agonist (IP3) --> found increase in flrounece (increase in Ca) when have higher concentration of the agonist

Question 33

How was it shown that the increases in Ca were from intracellula stores (came from Ca in the ER)?

Answer 33

Response is unaffected when extracellular calcium is removed - When have no extracellular Calcium still get the same response = means that the response is not due to extracelular calcium (instead due to intracellular stores) Video – Calcium rises in the middle of the cell and then spreads out - ALSO show that Calcium increases when the agonist is added (release of Ca correlates with the production of IP3)

Question 34

Major Classes of PI-PLCs

Answer 34

1. Beta --> Activated by G proteins 2. Gamma --> Activated by phosphorylation 3. Delta (don't know how it is activated)

Question 35

2nd activity of PI-PLC

Answer 35

PI-PLC have phosphodiestratse activity - Mechanism - phosphatidylinositol (PI) --> cyclic inosital phosphate (cIP) + DAG (fast step) ; cIP --> IP (slow step) Experiment to determine if PLC uses interfacial activation - Add PI-PLC substrate (cIP) to solution Conidtions: 1. No detergent --> Low PI-PLC activity 2. With detergent --> PI-PLC activity increases 3. Add diCPC --> high PI-PLC activity (hydrolyzed cIP to IP) - diCPC has a low CMC = easily forms memebranes (PLC is interfacially activated becaue activated when have membrane)

Question 36

How do you know if an enzyme uses interfacial activation

Answer 36

IF the enzymes is activated when have micelles then enzyme uses Interfacial activation Example - PLC is activated (hydrolyzes the cIP into IP) when micelles form --> uses interfcicial activation

Question 37

Protein Kinase C

Answer 37

PKC gets activated in PI cycle Phosphoyrlates Ser/Thr on proteins Activated by membrane association and SOME PKC are activated by calcium Isorforms: 1. Conventional PKCs ---> Activated by DAG/Phorbol esters (PMA) and calcium 2. Novel PKCs --> Activated by DAG and unsaturaed fatty Acids 3. Atipyical PKCs (PKD)

Question 38

How did they find the endogenous activator of PKC

Answer 38

Knew that PKC was activated by nonendogenous Phorbol esters (PMA) AND can be actiavted in agonist dependent manner but the endogenous activator was unknown FOUND - DAG is the endogenous activator - Found DAG activates PKC is the presence of phospholipids by lowing Km for Ca2+

Question 39

How did they find the endogenous activator of PKC - Experiment

Answer 39

HOW - Looked at the enzyme activity at different concentrations of CaCl2 (different concetrations of Ca) --> Found DAG activates PKC is the presence of phospholipids by lowing Km for Ca2+ Conditions: 1. Add Nuetral lipid (NL) --> nothing happens 2. Add diC18:1DAG (DAG)--> small bump 3. Add phospholipids --> more activity 4. add NL + PL --> more activity 5. Add DAG AND phospholipids --> HUGE increase in activity - When add DAG and PL the system is sensitive to Calcium (lowers Km for calcium) --> NOW PCK is actiavted in lower Ca

Question 40

DAG signaling

Answer 40

DAG = signaling molecule (lipid) Signaling activity: 1. DAG --> activates MUNC13 (Nuerotranismitter release) 2. DAG --> activates RasGEF (Increases RAS-GDP to RAS-GTP) 3. DAG --> activates RAC-GAP (increases Rac GTP --> Rac GDP) 4. DAG --> activates DGK (kinase) 5. DAG can activate PKC --> PKC can activate PKD 6. Affects cPKCs and nPKCs (kinases in signlaing pathways)

Question 41

Phospholipase D (PLD)

Answer 41

PLDs play major roles in physiological and pathophysiological processes PLD completes Hydrolytic or transphosphatidylation reactions Functions: 1. PLD catalyzes a transphosphorylation (‘transphosphorylation’ - exchange of a polar phospholipid head groups) 2. Catalyze a hydrolytic reaction using water 3. Use a small molecule weight primary alcohol in transphosphatidylation reactions

Question 42

PLD reactions

Answer 42

Hydroltic reaction (water is used) - Phospholipase D (PLD) cleaves Phosphatidyl choline - Water (nucelophile) hydrolases phosphate --> produces free choline and phosphatatic Acid Transphosphatidylation Reaction (Alchol used) - PLD cleaves phosphatatic acid - Small alcohol attacks phosphatatic acid --> produces free alchaol and Phosphatidyl ethanol (Pet) - THIS property can be used to determine whether or not PLD is activated

Question 43

Does PLD prefer to use water or alchol

Answer 43

PDL will preferentially use alcohol to do transphosphatidylation (uses the alcohol even in the presence of water) If there is alcohol then PLD will do Transphosphatidylation --> get PEt - PEt is metabolized slowley

Question 44

Given the fact that PLDs catalyze transphosphatidylation reaction how could you determine is PLD is actiavted in an agonist induced manner ; how do you know that the activity you are looking at is actually PLD (how do you know PLD is actually doing the reaction)

Answer 44

If you know there is a transphosphatidylation --> how do you know that PLD is doing the reaction (how do you know that PLD is activated by an agonist) Experiment - based PLD prefence for alchol --> add ethanol to a radiolabled cell --> look at the amount of PEt made to see if PDL is active (Y-Axis) - Control --> get small amount of PEt - Stimulate cells with agonist (ethanol) --> get lots of PEt - Since PEt is made = know PDL is active END - stimulate PLD with agonost = get more PEt (get more PDL activity) = shows that PLD is activated in agonist induced manner

Question 45

How do you test of an enzyme is activated in agonist dependent mannr

Answer 45

Add enzymes agonists --> See if get more of product when add the enzymes agnost IF get more product when add the enzymes agonist then the enzume is stimulated by the agonist

Question 46

Mammalian PLD isoforms

Answer 46

1. PLD1/PLD2 - Signaling roles (Catalytically active ) - PLD1 = activated by small G proteins (Rho/ARF) - PLD1 = business molecule in signaling cascades 2. PLD3/PLD4 --> Single stranded (RNA) exonucleases - Mediates formation of BMP in golgi + degrades gangliosides 3. PLD5 --> Catylyticlaly inactive 4. PLD6 (Mitocindrial) --> Single strand specific RNAse (used for piRNA that supress transposon expression) - NOT a lipase/phosphodiestares

Question 47

Why are PLD5/6 considered PLD enzymes

Answer 47

PLD5/6 are considered PLD enzymes because they are structurally similar to PLD enzymes BUT the catalytic domain of PLD5/6 is different from PLDs and their activities are different

Question 48

Phosphoinositide Kinase (TYPE OF lipid Kinase)

Answer 48

Lipid kinases work at the PM Example lipid Kinase - Phosphatidylinosital-3Kinase (PI3-K) PI3-K --> phosphorylates PIP2 --> makes PIP3 - PI3K phosphorylates the 3rd position of the inisitol ring in PIP2 (substrate) - PIP3 has been found in nuetrophils - PI3K = implicated in cell cycle control

Question 49

PI3K substrates

Answer 49

First though of PI3-K as a phosphoinositide kinase associated with polyoma middle T protein NOW know PI3-K prefers PtdInsP2 (PIP2) as a substarte (NOT polyoma middle T protein)

Question 50

Degredation of proteins that have been phosphorylated by PI3K

Answer 50

PI3-K phosphates PIP2 --> PI (phosphoinosine ; ex. PIP2) lipids that are phosphated at the D3 position of inisitol by PI3-K are NOT hydrolyzed (degraded) by PI-PLCs Example - PI3-K phosphorylates PIP2 to PIP3 on the 3rd position of the inisitol ring --> PIP3 can’t be hydrolyzed by PI-PLCs - Phospate at the 3rd position blocks PIP3 from entering the active site of PI-PLC

Question 51

Polyoma middle sized T antigen

Answer 51

Polyoma middle sized T antigen - membrane bound protein with tyrosine kinase containing activity - Polyoma T antigen can transfrom cells (cells have dysregulated growth) - PI3-K is associated with plyoma middle T protein

Question 52

How do you determine whether this activity (PI3-K activity and its associated with T antigen?) is asscoiated with polyoma induced transformation?

Answer 52

Activity = PI3-K regulating prolifertaion Experiment – Immunoprecipiate T antigen - Look at T antigen activity based on phosphorylated enolase ; Look at PI3-K activity by looking at PtdINs phosphorylation Results: - Mutant T antigen – When immunoprecipitate still have Tyrosine kinase activity BUT does not have PI3K activity AND does not transform cells - WT T antigen – has PI3K activity and can transform the cells FOUND - PI3-K associated with Polyma virus mediated transformation - Shows that PI3-K transforms cells (because mutant can do tyrosine kinase from T antigen BUT can’t transform cells so it is the PI3K not the T antigen that transforms the cells)

Question 53

PI3K activtaing enzymes

Answer 53

PI3K can activate Akt/PKB or PDK1 Akt/PKB and PDK1 = involoed in different processes (BOTH phosphoryate proteins in ATP dependent manner)

Question 54

Diacylglycerol Kinases

Answer 54

Diacylglycerol Kinases - phosphorylates Diacylglycerol (DAG) to Phosphatidic Acid (PtdOH) Reaction - DAG --> PtdOH DAG and PtdOH are BOTH second messengers

Question 55

Diacylglycerol Kinases affect on signling

Answer 55

DGK Reaction - DAG --> PtdOH DAG and PtdOH are BOTH second messengers - DGKs converts one second messenger to another - When DGK converts DAG to PtdOH it turns DAG factors off and turns PtdOH factors on SLIDE - Starred are things that are regulated by DGK (activated or inactive depending on DGK) - Regulated using DAG depletion (inactive DAG factors) OR PtdOH production (Actuvate PtDOH factors) - Example - mTOR + cPKC + nPCK

Question 56

Sphingosine Kinases

Answer 56

Reaction - Phosphorylates Sphingosine --> Sphingosine-Phosphate Reaction is Impacted in many pathways

Question 57

Spingolipid signlaing

Answer 57

Serine (precusor) and Palmitoyl-coA --> cermide (using SPT) - Cerimide = affects apoptosis Cerimide can go to: 1. Cermide --> Cerimiade-P (using CERK) 2. Cerimide is converted to form Spingomyelin 3. Cermide --> Spingosine --> Sphingosine-1-P - Get Sphingosine-1-P using SK - Sphingosine --> can cause growth arrest - Spingosine-1-P --> involoved in survival and proliferation

Question 58

PIP3 regulation

Answer 58

PIP3 = promote cell growth --> NEED a mechanism to remove PIP3 - NOTE - PI-PLC can't hydrolyze PIP3 Solution - PIP3 is removed using PTEN phosphatase or PTEN like phosphatase

Question 59

PTEN

Answer 59

PTEN is a phosphatase and Tensin homolog PTEN was first identified as a tumor suppressor Thought PTEN was a tyrosine phopshate phosphatase (protein phosphatase) BUT now know it is a lipid phosphatase - PTEN lipid phosphatase activity is critical for its tumor suppressor function PIP3 is the preferred substrate of PTEN (PTEN acts on PIP3)

Question 60

PTEN reaction

Answer 60

PTEN works at the membrane Reaction – PTEN removes a phosphate from PIP3 --> get PIP2 - PIP3 --> PIP2 (releases a Pi)

Question 61

How would you determine if an enzyme can hops or if it scoots (how do you show if PTEN hops or scoots)

Answer 61

Experiment – Add vesicles that contain the substrate and look at enzymatic acticity Chart – see the enzyme activity levels off (saturates) when only add vesciles that contain substrate - IF add more PTEN – get a increase bump in activity (seen by dark circle line) - IF add more vesicles with more substrate = get a huge increase in activity --> enzyme is hopping IF the enzyme was not hopping then nothing would happen when add the vesicles because the enzyme would be stuck in the first membrane

Question 62

How can you study PTEN phosphtase activity (how can you assay fro PTEN turning PIP3 to PIP2)

Answer 62

Experiment - Shows that PTEN dephospyrlates PIP3 Chart A - Shows PTEN releasing a phosphate (turning PIP3 --> PIP2) - Add substrate (PIP3) and show there is less PIP3 over time (more Pi released = more PIP2) Chart B --> Looks at PTEN turning PIP3 -> PIP2 then P13K turning PIP2 back to PIP3 - When add PIP2 with PI3K --> get PIP3 END - Shows that substate that was generated is what you thought it was (shows that PTEN does actually dephosphorylate PIP3 to get PIP2)

Question 63

Phosphatidate Phosphatase

Answer 63

Phosphatidate Phosphatase works at the membrane Example PtdOH phsphatase (aka PA phosphatase/PA phosphodrolase/PAH) Reaction - Phosphataic Acid --> Diaculglycerol (DAG) and Free phosphate

Question 64

Review

Answer 64

These should all make sense by end of review

Question 65

Steroid hormones

Answer 65

Don’t bind to a membrane receptor --> INSTEAD hormone goes through cell membrane --> bind to a soluble receptor in the cytoplasm --> receptor ligand complex then goes to the nucleus --> get transcriptional changes SHOWS NOT all lipid signaling needs to happen at the membrane surface HERE the agonist (hormone) is hydrophobic

Question 66

Other things that affect signlaing

Answer 66

1. Formation of rafts 2. Alterations in membrane architecture (curvature and pressure) - Pack lipid tighter or diffuse affects the pressure in the membrane --> Change in pressure can influence signaling molecules because proteins don't move as easily

Question 67

Fatty acid modification of proteins

Answer 67

Fatty acid modification of proteins is important Ex. Isoprenoids - modifying proteins (important in signaling) - In first part of cholestrol pathway isoproids are made --> isoproids modify oncogenes (Ex. RAS) Fun fact - People on Statins are less likely to get tumors because the RAS is not lipid modified (because Statins block cholestrol at the top of the pathway = don't get isoproids)