CB201: Molecular Biology of the Cell

CB201: Molecular Biology of the CellLipids and SignalingMarch 31, 2014

Brendan D. ManningDept of Genetics & Complex DiseasesHarvard School of Public Health

The diverse role of lipids in signal transduction Acyl-glycerolsFatty AcidsSterolsIsoprenoids

IntracellularSignaling(2nd Messengers)GPCRLigandNuclearReceptors(ER, PPAR, etc)LipidRaftsSmall GProteinsDAGPIPsLeukotrienesProstaglandinsLPAhGHVitDEstrogenCholesterolFarnesyl

2ReceptorsTransducersAmplifiersMessengersSensors / effectorsCELLULAR RESPONSESSignalingPathwayStimuliSignals are amplified by the transducing pathwayHormoneReceptor (RTKs and GPCRs)AmplifierSecond MessengerEffectorSer/ThrKinaseTargetsSecond Messengers in SignalingMESSENGERSmetal ionslipidscyclic nucleotidesexamplesdissolved gasCa2+, Zn2+, Mn2+PIPs, DAGcAMP, cGMPNOSecond messengers interact with and induce alterations in the activity and/or cellular location of signaling proteins

Messengers must: Accumulate rapidly Accumulate locally Be mobile Be removed by active mechanisms

Ca2+ oscillations inhistamine-stimulated cells

Imaged with Fura-2: Ca2+-binding fluorescent dye

Calcium is rapidly mobilized to and fromintracellular stores upon stimulationCa2+ stimulates diverse cellular responsesin a time-dependent manner

7The concentration of Ca2+ in cells at rest is approximately 100 nM, but this increases to 500 nM or more following a stimulus that activates the Ca2+ ON reactions. When the stimulus is removed the Ca2+ OFF reactions return the concentration of Ca2+ back to its resting level. Ca2+ is a universal signal capable of activating many different cellular processes operating over a very wide time domain.cAMP PathwaysGPCRStimulatorySignalqGTP+ACCREB: cAMP Response element binding proteinPDE: PhosphodiesterasesPK: Phosphorylase kinaseCFTR: Cystic fibrosis transmembrane regulatorATPcAMPPKACREBLipasePDEsPKCFTRAmplifier(Adenylate Cyclase)2nd Messenger8Cyclic AMP is formed both by membrane-bound adenylyl cyclase and by the bicarbonate-sensitive soluble adenylyl cyclase. The former is regulated by both stimulatory agonists that act through the aS subunit or through inhibitory agonists that act through either the ai or the bg subunits. The increase in cyclic AMP then acts through three different effector systems. It acts through the exchange protein activated by cyclic AMP (EPAC), which functions to activate Rap1. It can open cyclic nucleotide-gated channels (CNGCs). The main action of cyclic AMP is to activate protein kinase A (PKA) to phosphorylate a large number of downstream targets. Some of these drive specific processes such as gene transcription through phosphorylation of cyclic AMP response element-binding protein (CREB), and activation of ion channels [e.g. AMPA receptors and cystic fibrosis transmembrane conductance regulator (CFTR)] and various enzymes that control metabolism [e.g. fructose 2,6-bisphosphate (F-2,6-P2) 2-phosphatase, lipase and phosphorylase kinase]. Other downstream targets are components of other signalling pathways such as the cyclic GMP phosphodiesterase (cGMP PDE), the phospholamban (PLN) that controls the sarco/endo-plasmic reticulum Ca2+-ATPase (SERCA), the ryanodine receptor (RYR) and the Ca2+ channels CaV1.1 and CaV1.2.

The Human KinomePKAPKCC1C2PHAkt/PKBcAMPDAGCa2+PIP3Many AGC family kinases areregulated by 2nd messengersLipids 101OOHHOHOHOFatty AcidGlycerolOOHOHOMonoacylglycerol(MAG)OOOHOPOOHOHLysophosphatidic Acid(LPA)LPA is the most abundantgrowth factor/mitogen in serum,and signals through GPCRs10There is more to GPCR signaling than just G subunits.G subunits also play fundamental roles because they couple to a distinct subset of effectors.G subunit effectors include: K+ channels, GRKs, adenylate cyclase, phospholipases (PLC, types 1, 2, 3), and phosphoinositide 3-kinases (PI 3-K, type ).As we will see later, different isoforms of both PLC and PI 3-K are also activated by and signal through receptor tyrosine kinases (RTKs).

OOHHOHOHOFatty AcidGlycerolOOHOODiacylglycerol(DAG)OOOOOTriacylglycerol(TAG)OODAGs and TAGs arestored in lipid dropletsLipids 10111There is more to GPCR signaling than just G subunits.G subunits also play fundamental roles because they couple to a distinct subset of effectors.G subunit effectors include: K+ channels, GRKs, adenylate cyclase, phospholipases (PLC, types 1, 2, 3), and phosphoinositide 3-kinases (PI 3-K, type ).As we will see later, different isoforms of both PLC and PI 3-K are also activated by and signal through receptor tyrosine kinases (RTKs).

Lipids 101OOOOPOOHOHPhosphatidic Acid(PA)OPA is a precursor formembrane phospholipidsPhosphatidylserinePhosphatidylethanolaminePhosphatidylcholinePhosphatidylinositolSerineEthanolamineCholineInositol12There is more to GPCR signaling than just G subunits.G subunits also play fundamental roles because they couple to a distinct subset of effectors.G subunit effectors include: K+ channels, GRKs, adenylate cyclase, phospholipases (PLC, types 1, 2, 3), and phosphoinositide 3-kinases (PI 3-K, type ).As we will see later, different isoforms of both PLC and PI 3-K are also activated by and signal through receptor tyrosine kinases (RTKs).

OOOOPOOHOHPhosphatidic Acid(PA)OPA is a precursor formembrane phospholipidsPhosphatidylserinePhosphatidylethanolaminePhosphatidylcholinePhosphatidylinositolSerineEthanolamineCholineInositolHOOH123456OHOHOHOHLipids 10113There is more to GPCR signaling than just G subunits.G subunits also play fundamental roles because they couple to a distinct subset of effectors.G subunit effectors include: K+ channels, GRKs, adenylate cyclase, phospholipases (PLC, types 1, 2, 3), and phosphoinositide 3-kinases (PI 3-K, type ).As we will see later, different isoforms of both PLC and PI 3-K are also activated by and signal through receptor tyrosine kinases (RTKs).

Phosphatidylinositol phosphates (PIPs) are of very low abundance but act as key second messengers in signalingOOOOPOOOHPhosphatidylinositol-4,5-bisphosphate(PtdIns-4,5-P2 or PIP2)OOH123456OPO32-OPO32-OHOHLipid % of totalPhophatidylserine..8.5%Phosphatidylinositol..1.0%Phosphatidylinositol-4,5-bisphosphate..0.05%14There is more to GPCR signaling than just G subunits.G subunits also play fundamental roles because they couple to a distinct subset of effectors.G subunit effectors include: K+ channels, GRKs, adenylate cyclase, phospholipases (PLC, types 1, 2, 3), and phosphoinositide 3-kinases (PI 3-K, type ).As we will see later, different isoforms of both PLC and PI 3-K are also activated by and signal through receptor tyrosine kinases (RTKs).

Phosphoinositide Kinases and PhosphatasesControl the localized production and removal of PIPsPtdInsPtdIns-3-PPtdIns-5-PPtdIns-4-PPtdIns-3,5-P2PtdIns-3,4-P2PtdIns-4,5-P2PtdIns-3,4,5-P3KinasePhosphatase15

PIP2 Controls Many Cellular ProcessesMultiple signalling functions of phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2)PtdIns4,5P2 has multiple signalling functions in the plasma membrane. It is the lipid precursor used for the generation of second messengers such as InsP3, DAG and PtdIns3,4,5P3. It functions in the regulation of the phospholipase D (PLD) signalling pathway. In addition, PtdIns4,5P2 can function as a messenger to control a whole variety of cellular systems including the cytoskeleton, phagocytosis, exocytosis, endocytosis and ion channels. All of these processes are sensitive to changes in the membrane level of this lipid.

16Phospholipase C hydrolyzes PIP2 to producetwo second messengers, DAG and IP3OOOOPOOOHPhosphatidylinositol-4,5-bisphosphate(PtdIns-4,5-P2 or PIP2)OOH123456OPO32-OPO32-OHOHOOOOHOHOPOOOHOH123456OPO32-OPO32-OHOHDiacylglycerol(DAG)Inositol-1,4,5-Trisphosphate(IP3)Phospholipase C(PLC)17There is more to GPCR signaling than just G subunits.G subunits also play fundamental roles because they couple to a distinct subset of effectors.G subunit effectors include: K+ channels, GRKs, adenylate cyclase, phospholipases (PLC, types 1, 2, 3), and phosphoinositide 3-kinases (PI 3-K, type ).As we will see later, different isoforms of both PLC and PI 3-K are also activated by and signal through receptor tyrosine kinases (RTKs).

GPCRStimulatory signalqGTP+PIP2DAGC1C2Phospholipase C hydrolyzes PIP2 to producethe two second messengers DAG and IP3PLCbIP318Mention phosphorylation of PKC in these pathways, to be covered in the RTK lecture with PI 3-K and Akt/PKB signaling. Also mention Calcium signaling, to be covered later in PLCgamma signaling by PDGFRasRTKPLCePIP2IP3DAGC1C2Phospholipase C hydrolyzes PIP2 to producethe two second messengers DAG and IP3Stimulatory signal19Mention phosphorylation of PKC in these pathways, to be covered in the RTK lecture with PI 3-K and Akt/PKB signaling. Also mention Calcium signaling, to be covered later in PLCgamma signaling by PDGFRTKPLCgPIP2IP3DAGC1C2Phospholipase C hydrolyzes PIP2 to producethe two second messengers DAG and IP3Stimulatory signal

PDGFR20Mention phosphorylation of PKC in these pathways, to be covered in the RTK lecture with PI 3-K and Akt/PKB signaling. Also mention Calcium signaling, to be covered later in PLCgamma signaling by PDGFPIP2DAGERCa2+Ca2+Ca2+-bindingEffectorsThe IP3 receptor on the ERis an IP3-gated Ca2+ channel Phospholipase C hydrolyzes PIP2 to producethe two second messengers DAG and IP3IP3RTKPLCgStimulatory signalPKCC1C2DAG binds to C1 domainson downstream effectors

Time (Min)Adapted from Newton J Lipid Res 200921Mention phosphorylation of PKC in these pathways, to be covered in the RTK lecture with PI 3-K and Akt/PKB signaling. Also mention Calcium signaling, to be covered later in PLCgamma signaling by PDGFPKCs, a Large Family of DAG Receptors

ConventionalNovelAtypicalSubstrates:Cell proliferationCell survivalCytoskeletal dynamicsMembrane trafficking

22Therefore, cannot use PMA/PdBu alone, and use combinatorial tools such as chemical inhibitors, siRNA, KO cells.

PIP2DAGCa2+PKC activates Erk signaling andfeedback inhibits PLC activityIP3PKCC1C2GPCRStimulatory signalqGTP+PLCbRaf

MEK

Erk

CellProliferation23Mention phosphorylation of PKC in these pathways, to be covered in the RTK lecture with PI 3-K and Akt/PKB signaling. Also mention Calcium signaling, to be covered later in PLCgamma signaling by PDGFPhosphatidylinositol phosphates (PIPs) are second messengersthat relay spatial cues to signaling pathwaysOOOOPOOOHPhosphatidylinositolOOH123456OHOHOHOH

Kutateladze et al., Nat Chem Biol 201024There is more to GPCR signaling than just G subunits.G subunits also play fundamental roles because they couple to a distinct subset of effectors.G subunit effectors include: K+ channels, GRKs, adenylate cyclase, phospholipases (PLC, types 1, 2, 3), and phosphoinositide 3-kinases (PI 3-K, type ).As we will see later, different isoforms of both PLC and PI 3-K are also activated by and signal through receptor tyrosine kinases (RTKs).

Many proteins contain domains that bind to specific PIPs, therebyrecruiting these proteins to specific membrane compartmentsOOOOPOOOHPhosphatidylinositolOOH123456OHOHOHOH

Signaling, Cytoskeletal, and Trafficking ProteinsKutateladze et al., Nat Chem Biol 201025There is more to GPCR signaling than just G subunits.G subunits also play fundamental roles because they couple to a distinct subset of effectors.G subunit effectors include: K+ channels, GRKs, adenylate cyclase, phospholipases (PLC, types 1, 2, 3), and phosphoinositide 3-kinases (PI 3-K, type ).As we will see later, different isoforms of both PLC and PI 3-K are also activated by and signal through receptor tyrosine kinases (RTKs).

OOOOPOOOHPhosphatidylinositolOOH123456OHOHOHOHPI3PFYVE Domain(Fab1,YOTB,Vac1,Eea1)PI3PPX Domain(Phox Homology)PI3,4,5P3 or PIP3PH Domain(Pleckstrin Homology)These PIP-binding domains contain conserved residues thatengage different combinations of phosphates on the inositol ringKutateladze et al., Nat Chem Biol 201026There is more to GPCR signaling than just G subunits.G subunits also play fundamental roles because they couple to a distinct subset of effectors.G subunit effectors include: K+ channels, GRKs, adenylate cyclase, phospholipases (PLC, types 1, 2, 3), and phosphoinositide 3-kinases (PI 3-K, type ).As we will see later, different isoforms of both PLC and PI 3-K are also activated by and signal through receptor tyrosine kinases (RTKs).

Phosphoinositide Kinases and PhosphatasesControl the localized production and removal of PIPsPtdInsPtdIns-3-PPtdIns-5-PPtdIns-4-PPtdIns-3,5-P2PtdIns-3,4-P2PtdIns-4,5-P2PtdIns-3,4,5-P3KinasePhosphatase273 classes of phosphoinositide 3-kinaseshave different substrate specificitiesPtdInsPtdIns-3-PPtdIns-5-PPtdIns-4-PPtdIns-3,5-P2PtdIns-3,4-P2PtdIns-4,5-P2PtdIns-3,4,5-P3KinasePhosphatasePI3K (class I)PI3K (class III)PI3K (class I or II)28Phosphoinositide 3-kinase produces PIP3in response to growth factors OOOOPOOOHPhosphatidylinositol-4,5-bisphosphate(PtdIns-4,5-P2 or PIP2)OOH123456OPO32-OPO32-OHOHPI3KOOOOPOOOHPhosphatidylinositol-3,4,5-triisphosphate(PtdIns-3,4,5-P2 or PIP3)OOH123456OPO32-OPO32-OHOPO32-Lipid % of totalPhophatidylserine..8.5%Phosphatidylinositol..1.0%Phosphatidylinositol-4,5-bisphosphate..0.05%Phosphatidylinositol-3,4,5-bisphosphate..0.0001%10-fold Increase upon stimulation29There is more to GPCR signaling than just G subunits.G subunits also play fundamental roles because they couple to a distinct subset of effectors.G subunit effectors include: K+ channels, GRKs, adenylate cyclase, phospholipases (PLC, types 1, 2, 3), and phosphoinositide 3-kinases (PI 3-K, type ).As we will see later, different isoforms of both PLC and PI 3-K are also activated by and signal through receptor tyrosine kinases (RTKs).

GPCRStimulatory signalsGTP+PIP2C1C2Class IB PI3K is a downstream effector of GPCRsPIP3p101p110gClass IB PI3K30Mention phosphorylation of PKC in these pathways, to be covered in the RTK lecture with PI 3-K and Akt/PKB signaling. Also mention Calcium signaling, to be covered later in PLCgamma signaling by PDGFPIP2C1C2PIP3p85Class 1A PI3K(p110 a / b / d)RasRTKStimulatory signal

Class IA PI3K is a downstream effector of RTKs and Ras

p110p85p110a31Mention phosphorylation of PKC in these pathways, to be covered in the RTK lecture with PI 3-K and Akt/PKB signaling. Also mention Calcium signaling, to be covered later in PLCgamma signaling by PDGFPIP2PIP3p85RTKStimulatory signalClass 1A PI3K(p110 a / b / d)

p110p85Class IA PI3K is a downstream effector of RTKs and RasThe SH2 domains of p85 bind to phospho-tyrosine residueson RTKs and scaffolding adaptors to bring PI3Kto its substrate PI4,5P2 at the plasma membranep110a32Mention phosphorylation of PKC in these pathways, to be covered in the RTK lecture with PI 3-K and Akt/PKB signaling. Also mention Calcium signaling, to be covered later in PLCgamma signaling by PDGFPHEffectorPIP2PIP3p85RTKStimulatory signalClass I PI3Ks generate the lipid second messenger PIP3, which recruits specific downstream effectors

PHGFPwortmanninEGFTime-lapse microscopy (10s intervals):60s stimulation with EGFFollowed by wortmannin treatment

Cullen et al., BJ (1998) 335; 139-146.PTENp110a33Mention phosphorylation of PKC in these pathways, to be covered in the RTK lecture with PI 3-K and Akt/PKB signaling. Also mention Calcium signaling, to be covered later in PLCgamma signaling by PDGFPHEffectorPIP2PIP3p85RTKStimulatory signalClass I PI3Ks generate the lipid second messenger PIP3, which recruits specific downstream effectors

PH >> PH domains found in 275 human proteinsKutateladze et al., Nat Chem Biol 2010>> 10-20% have strong preferencesfor specific PIP species(PI4,5P2 / PI3,4P2 / PI3,4,5P3)>> PH domains engage phosphateson the inositol head groupvia three variable loopsp110aPTEN34Mention phosphorylation of PKC in these pathways, to be covered in the RTK lecture with PI 3-K and Akt/PKB signaling. Also mention Calcium signaling, to be covered later in PLCgamma signaling by PDGFPHEffectorPIP2PIP3p85RTKStimulatory signalGrp1 An ARF GEF Vesicle trafficking

Vav2 A Rac GEF Cytoskeletal dynamics / Migration

Btk A Tyr Kinase PLC regulation (DAG/Ca2+)

Akt A Ser/Thr Kinase Many substratesClass I PI3Ks generate the lipid second messenger PIP3, which recruits specific downstream effectorsPIP3-binding Effectors of PI3Kp110aPTEN35Mention phosphorylation of PKC in these pathways, to be covered in the RTK lecture with PI 3-K and Akt/PKB signaling. Also mention Calcium signaling, to be covered later in PLCgamma signaling by PDGFPHAktPIP2PIP3p85RTKStimulatory signalAkt (aka protein kinase B or PKB) is a majordownstream effector of PI3K signalingp110aPHPDK1PIP3PIP3T308mTORC2S473PTEN

StochasticResponse(p-Akt)Debnath et al. Cell 2002

Rapid and timedependent

Dose dependent36Mention phosphorylation of PKC in these pathways, to be covered in the RTK lecture with PI 3-K and Akt/PKB signaling. Also mention Calcium signaling, to be covered later in PLCgamma signaling by PDGFPHAktPIP2PIP3p85RTKStimulatory signalAkt (aka protein kinase B or PKB) is a majordownstream effector of PI3K signalingp110aPHPDK1PIP3PIP3T308mTORC2S473CellSurvivalCellProliferationCellGrowthMetabolism(cell-tissue-system) PTEN37Mention phosphorylation of PKC in these pathways, to be covered in the RTK lecture with PI 3-K and Akt/PKB signaling. Also mention Calcium signaling, to be covered later in PLCgamma signaling by PDGFPHAktPIP2PIP3p85RTKPI3K-Akt is constitutively activated inover 50% of human cancersp110aPHPDK1PIP3PIP3T308mTORC2S473CellSurvivalCellProliferationCellGrowthMetabolism(cell-tissue-system) PTEN***XRas*38Mention phosphorylation of PKC in these pathways, to be covered in the RTK lecture with PI 3-K and Akt/PKB signaling. Also mention Calcium signaling, to be covered later in PLCgamma signaling by PDGF

PHAktPIP2PIP3p85RTKPIK3CA and PTEN are frequently alteredin human cancersp110aPHPDK1PIP3PIP3T308mTORC2S473PTEN*X

(data from ~69 tumor genome sequencing studies)UterineLungProstateBrainCervicalBreastHead & NeckColonOvarianBladderMelanomaKidneyRas39Mention phosphorylation of PKC in these pathways, to be covered in the RTK lecture with PI 3-K and Akt/PKB signaling. Also mention Calcium signaling, to be covered later in PLCgamma signaling by PDGFAkt has many downstream targets tocontrol diverse cellular processes

Manning and Cantley 2007 Cell 40Akt has many downstream targets tocontrol diverse cellular processes

R-x-R-x-x-S/T--x=any amino acid-=large hydrophobicAkt-mediated phosphorylationoften inhibits its substratesManning and Cantley 2007 Cell 41

Tran et al 2003 Science STKE FOXO proteins regulate transcription downstream of Akt

Greer and Brunet 2005 OncogeneVertebrateC. elegansAktAktS/T phosphorylation, like Tyr phosphorylation,creates protein-protein binding motifsYaffe and Elia 2001 Curr Opin Cell Biol

Sequence overlap between Akt substrate preferenceand phospho-binding motif of 14-3-3 proteins

R-x-R-x-x-S/T--xx-x-R-x-x-S/T-x-PPAkt substrate14-3-3 bindingReinhardt and Yaffe 2013 Nat Rev Mol Cell BiolR-P-R-S-C-T-W-PFOXO1-T24Akt Controls FOXO1 LocalizationPI3KAktFOXOPI3KAktFOXOTargetsXFOXOPPPAkt OFF - FOXO1 nuclearAkt ON - FOXO1 cytoplasmicTargets{14-3-3Apoptosis (Bim1, FasL)Cell Cycle Arrest (p21, p27)Cell SurvivalProliferation

Fat

eal

glucoseMusclePancreasLiverinsulininsulinInsulin control of systemic glucose homeostasisGlucoseProductionGlucoseUptakeIRSPIP2PIP3PIP3PAKTPI3KPIRPPInsulinInsulin signaling in muscleIRS-1PHPTBY608Y628Y891Y935PI3K bindingGLUT4GlucoseGlucoseUptakeGlycogenSynthesisProteinSynthesisGlucoseStorageMuscleMassIRSPIP2PIP3PIP3PAKTPI3KPIRPPGLUT4InsulinGlucoseGlucoseUptakeGlycogenSynthesisProteinSynthesisGSK3PAS160PPPTSC2PPPInsulin signaling in muscle

Glut4 is the major glucose transporter in muscle and adipose andrapidly translocates to the plasma membrane in response to insulin Watson and Pessin 2006 TiBS Glut4AKTAS160PPPRabRabGDPGTPAS160/TBC1D1is a Rab GAPGLUT4GLUT4Glut4VesicleIRSPIP2PIP3PIP3PAKTPI3KPIRPPGLUT4InsulinGlucoseGlucoseUptakeGlycogenSynthesisGSK3PAS160PPPGlycogenSynthasePPPPInsulin signaling in muscleProteinSynthesisTSC2PPPmTORC1LipidSynthesisIRSPIP2PIP3PIP3PAKTPI3KPIRPPInsulinGlucoseGluconeogenesisProteinSynthesisTSC2PPPInsulin signaling in livermTORC1LipidSynthesisFOXOPPPGlucoseReleaseG6PasePEPCK