phospholipids, phosphoinositols & eicosanoids

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Phospholipids, Phosphoinositols & Eicosanoids

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Common types ofPhospholipids:

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Second messenger– released through hydrolysis by phospholipases and/or– generated through the actions of lipid kinases

Phospholipases: Phosphatidylinositol-kinases:

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Phospholipases:

• PLA2:– Cytoplasmic form (90 kDa) is regulated through nM Ca++ (Annexins) and

phosphorylation; AA specific => signaling function

– Secreted form (pancreas, 14 kDa) is also Ca++ dependent (mM range)=>digestive function

• PLC: coupled to a variety of (growth factor) receptors:

– PLCβ is activated through GPCRs (Gqα) => binding enhances its catalyticactivity and in return the GTPase activity of Gqα (similar to GAP function in rassignaling)

– PLCγ couples with its SH2 domains directly to growth factor receptors (EGFR,PDGFR) or the TCR, where it is activated through tyrosine phosphorylation

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Both phospholipases yield finally arachidonic acid (see below), in addition, PLC activity also producesDAG and IP3:

• DAG: remains membrane bound; diacylglycerol kinase phosphorylates DAG to generatephosphatidic acid which functions as a substrate for PLA2.Phosphatidyl-serine (PS), Ca++ and DAG activate PKC on the plasma membrane

• IP3: see Ca++ signaling!!

– Glucocorticoids: inhibit PLA2 by transcriptionally inducing Lipocortin, a proteinwhich binds to PLA2 and blocks its activity.

– Phorbol esters: strongest known tumor promotors; mimic DAG => bind PKC andactivate it. Also potent activator of Ca2+ influx, MAPK pathway etc.

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Lipid kinases:

• PI3-kinase:– binds to and becomes tyrosine phosphorylated in response to activation of

growth factor receptors or immune receptors– 85 kDa regulatory subunit (pY) and a 110 kDa catalytic subunit– regulatory subunit contains SH2 and SH3 domains– PIP3-phosphates can bind to the pleckstrin homology (PH) domain of Akt

=> Akt activation => phosphorylation of BAD, which dissociates from theantiapoptotic protein bcl-2 => inhibition of apoptosis

• Wortmannin: fungal metabolite, potent, irreversible inhibitor of PI3Kinase• Ly290004: synthetic compound, blocks ATP binding site of PI3Kinase

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Arachidonic Acid Metabolism

• Eicosanoids:collective name for derivatives of arachidonic acid (=5,8,11,14 - eicosatetraenic acid)

– AA is mainly generated through the action of PLA2 and DAG-lipase.– Rapidly metabolized by cyclooxygenase and lipoxygenase into

prostaglandins and leukotrienes:

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• Prostaglandins:– First observed in seminal fluid => name– Structure of cyclopentane ring defines letter– Double bonds in side chains account for number– Greek letter refers to the spatial position of the OH-group at C-9

Initial step in PG synthesiscatalyzed by PGH-synthasewhich has dual enzymaticactivity:

cyclooxygenase (closes ring =>PGG2)

andperoxidase (=> 15-OH)

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Biological functions of PGs:

• Vascular tone Relaxation: PGs E1, E2, F2α and I2Constriction: PGs F2α, TxA2

• Platelet aggregation Increase: PGs E1, TxA2

Decrease: PGs E2, I2• Uterus tone Increase: PGs E1, E2, F1α

• Bronchial muscle Constriction: PGFsRelaxation: PGEs

• Gastric secretion Inhibition: PGs E1, E2, I2• Temperature and pain Increase: PGEs

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Arachidonic Acid Metabolism• Leukotrienes:

– First found in leucocytes; contain 3 conjugated double bonds– Lipoxygenase generates Hydroperoxyeicosatetraenoic acid (HPETE)– LTC4, D4 and E4 mediate allergic reaction: Slow Reacting Substance of Anaphylaxis (SRS-A)

=> mediates anaphylactic shock 10,000 fold more potent than histamine!!! => constrictsbronchi, dilate blood vessels

– LTB4 is a very strong chemoattractant for macrophages

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Growth Factor Receptors

• Many growth factors (EGF, PDGF, IGF-1, CSF-1, ...) signal through receptors with intrinsictyrosine kinase activity

• Common features:– Large, glycosylated ligand binding domain– Single hydrophobic transmembrane domain– Activation occurs through ligand mediated oligomerization– Undergo ligand induced downregulation by internalization– Cytoplasmic tyrosine kinase domain:

• most highly conserved region• GlyXGlyXXGlyX(15-20)Lys

Lys is critical for ATP binding - mutation renders receptor kinase inactive, whichabrogates all cellular responses => signaling depends on tyrosine phosphorylationof receptor and cytoplasmic substrates

• Tyrosine kinase receptors also bind and activate cytoplasmic tyrosine kinases– Autophosphorylation sites:

• conserved in the C-term of each receptor class• autophosphorylation does not effect Km of receptor kinase activity• provide docking sites for SH2 domain containg signaling proteins

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Growth Factor Receptors• Three subclasses:

– Class I: two Cys-rich region in the EC, monomeric ligandEGF-R, erbB2, erbB3, erbB4 (heregulin receptors)

– Class II: heterotetrameric: 2 α and 2 β chains stabilized through S-S bonds: monomeric ligandInsulin-R, IGF-1-R

– Class III: Repeats of mmunoglobulin-like structure, dimeric ligandFGF-R, NGF-R, PDGF-R, CSF-1-R, c-kit

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Signaling through Adapter proteins:

• grb2: adapter with one SH2 domain which binds PYresidue on RTK, and two SH3 domains which bind to

• Sos: “Son of Sevenless” (mutation in drosophilaprevents development of the R7 photoreceptor cell).Functions as a GEF to facilitate GTP loading of

• ras: GTP binding protein, farnesylated; protooncogene,provides a docking site on the plasma membrane for raf.

• ras-GAP: negative regulator of groth factor signaling:promotes GTP hydrolysis through the GTPase activity ofras.

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Signaling through Adapter proteins:

• raf: ser/thr kinase of the MAPKKK/MEKK family (MEKKdoes normally NOT phosphorylate MEK, but ratherMKK4/7 => Stress pathway); requires context of plasmamembrane for activation (mixing GTP-ras and raf in atest tube fails to activate raf) => raf likely to bephosphorylated at the plasma membrane. Activated rafphosphorylates...

• MEK: Dual specificity kinase (in case of Stress pathway:SEK) phosphorylates ERKs or MAPKs on tyr and thr ->

• ERKs: migrate to the nucleus where they phosphorylatetranscription factors such as fos and jun; also feedbackloop to other signaling molecules

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Cytokine Receptors

• “Classical” hormones:– produced by cells organized into endocrine organs,– often referred to as “endocrine signal molecules”– target cells usually distant from the site of synthesis– hormone carried by blood stream from producing gland to target cells– signal through receptors coupled to G-proteins (e.g. epinephrine receptor), ion-channels– (e.g. acetylcholine receptor) or receptors with intrinsic enzymatic activity

• Cytokines:– single producing and effector cell– only affect target cells in close proximity (autocrine or paracrine)– almost exclusively involved in regulating immunological processes– sometimes subdivided into different groups based on their origin (lymphokines, monokines,

interleukines)– often carry several (old) names based on their multiple biological functions: e.g. Lymphocyte

Activating Factor (LAF) = Mitogenic Protein (MP) = T Cell Replacing Factor III (TRF-III) = BCell Activating Factor (BAF) = B cell Differentiation Factor (BDF) = INTERLEUKIN 1

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Cytokine Receptors

• Basic characteristics:– only one copy of encoding gene per haploid cell (~20 different IFNα’s, but each

encoded by a distinct gene)– genes segmented into 4 or 5 exons (exceptions are IFNα and IFNβ: no introns)– mature protein usually between 8 and 25 kDa– barely any structural resemblances– often N-glycosylated– often form oligomers– some carry signal sequence in precursor– expression is tightly regulated on a transcriptional level– generally pleiotropic– usually highly species specific (up and down)

• Multiple (old) classifications:– Based on origin (Lymphokines, Monokines..)– Based on action (inhibitory, stimulating, antiviral, chemotactic...)– Based on composition of receptor (single-chain vs. multi-chain)

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Cytokine Receptors

• Current nomenclature based on structure of receptors:– Type I cytokine receptors = hematopoietin receptor family:

receptors contain W-S-X-W-S motif in the C-terminus• IL-2 R, IL-3 R, IL-4 R, IL-5 R, IL-6 R (has also Ig-like domain), IL-7 R, IL-9 R, IL-11 R

IL-13 R, IL-15 R, GM-CSF R, EPO R, G-CSF R (has also Ig-like domain)

– Type II cytokine receptors = Interferon receptor family:receptors contain IRH1 (200aa extracellular) and IRH2 (50 aa cytoplasmic) domain• IFNα/β R, IFNγ R

– Type III cytokine receptors = TNF receptor family:receptors contain 4 Cys rich regions in extracellular domain• TNFα R, TNFβ=LT R, NGF R (trk), fas, CD40

– Type IV cytokine receptors = Immunoglobulin family:receptors contain an Ig like repeat in the extracellular domain• IL-1 R, M-CSF R (c-fms), SCF R = steel factor R (kit) (tyrosine kinase activity), (IL-6 R) (has

also W-S-X-W-S motif), (G-CSF R)

– (Chemokine receptors):• C-X-C subgroup (α-family): IL-8, PF4, βTG• C-C subgroup (β-family): RANTES, MCAF, MIP-1β

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Cytokine Receptors

Signal Transduction:Receptors lack intrinsic catalytic activity but associate w/ cytosolic enzymes

STAT: Signal transducer and activator of transcription– contain SH2 domains– become tyrosine phosphorylated after stimulation– 6 family members– homo or heterodimerize– translocate to nucleus and bind enhancers

JAK: Janus kinase– large cytoplasmic tyrosine kinases (130-140 kDa)– NO SH2 or SH3 domains– kinase-like domain

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Ser/Thr-kinase Receptors

Transforming Growth Factors = TGFs:– Murine sarcoma virus infected cells can not bind EGF => cells produce a growth factor that

competes for EGF binding (=sarcoma growth factor, SGF).– SGF promoted anchorage independent growth (reversible) => first evidence that transformed

cells produce their own growth factor!– SGF was found to consist of two subunits:

TGFα: EGF competitor, binds and signals through the EGF receptor,potent mitogen, but does not support anchorage-independent growthoverexpressed in epithelium of psoriasis patients (=> hyperproliferation of keratinocytes)

– TGFβ: acts through distinct receptor, it also is a potent mitogen, but it also does not support anchorage-independent growth (only combination does!)

TGFβ- receptors I and II:– external ligand binding domain and cytosolic serine/threonine kinase activity.– betaglycan: proteoglycan required for TGFβ binding

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Ser/Thr-kinase Receptors

Signal Transduction:

SMADs:– 8 members - conserved MH1 and MH2 domain– rapidly phosphorylated in response to TGFβ (2,3) or

BMP (1,5,8)– Smads function in heteromeric complexes– “common” Smad = Smad-4 (Smad-4 required for all

Smad signaling)– translocate to the nucleus– phosphorylated by the receptor itself– phosphorylation motif: SSXS– Smad1 potentially also activated by MAPK– Mutations of Smads and /or TGFβR are found in 90%

of colon cancers

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NFκB

– originally identified as a transcription factor binding anenhancer (κB)in the κ-light chain immunoglobulin gene

– Activated by a variety of (proinflammatory) signals(IL-1, TNF, Phorbol esters...)

– Homo-or heterodimer composed of p50 and/or p65subunits

– retained in its inactive form in the cytoplasm by theinhibitory protein IκB

– dissociation of NFκB from IκB activates NFκB’s DNAbinding capabilities

– NFκB/ IκB association is regulated by serinephosphorylation of IκB!

– Phosphorylated IκB does not dissociate from NFκB,rather is marked for degradation (NFκB activation canbe inhibited by protease inhibitors!)

– Phosphorylation of IκB through IκB-kinase (complex>600 kDa)

– IKK: two kinase subunits: IKKα and IKKβ, homo- orheterodimers IKKγ (NEMO): no kinase activity,required for complex assembly = regulatory subunit

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