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Phospholipids, Phosphoinositols & Eicosanoids
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Phospholipids, Phosphoinositols & Eicosanoids
Common types ofPhospholipids:
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Phospholipids, Phosphoinositols & Eicosanoids
Second messenger – released through hydrolysis by phospholipases and/or – generated through the actions of lipid kinases
Phospholipases: Phosphatidylinositol-kinases:
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Phospholipids, Phosphoinositols & Eicosanoids
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:
– PLC is activated through GPCRs (Gq) => binding enhances its catalytic activity and in return the GTPase activity of Gq(similar to GAP function in ras signaling)
– PLC 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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Phospholipids, Phosphoinositols & Eicosanoids
Both phospholipases yield finally arachidonic acid (see below), in addition, PLC activity also produces DAG and IP3:
• DAG: remains membrane bound; diacylglycerol kinase phosphorylates DAG to generate phosphatidic 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 protein which binds to PLA2 and blocks its activity.
– Phorbol esters: strongest known tumor promotors; mimic DAG => bind PKC and activate it. Also potent activator of Ca2+ influx, MAPK pathway etc.
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Phospholipids, Phosphoinositols & Eicosanoids
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 the antiapoptotic 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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Arachidonic Acid Metabolism
• 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 synthesis catalyzed by PGH-synthase which has dual enzymatic activity:
cyclooxygenase (closes ring =>PGG2)
and peroxidase (=> 15-OH)
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Arachidonic Acid Metabolism
Biological functions of PGs:
• Vascular tone Relaxation: PGs E1, E2, F2 and I2
Constriction: PGs F2, TxA2
• Platelet aggregation Increase: PGs E1, TxA2
Decrease: PGs E2, I2
• Uterus tone Increase: PGs E1, E2, F1
• Bronchial muscle Constriction: PGFs
Relaxation: 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!!! => constricts bronchi, 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 intrinsic tyrosine 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, which abrogates all cellular responses => signaling depends on tyrosine phosphorylation
of 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 ligand
EGF-R, erbB2, erbB3, erbB4 (heregulin receptors)– Class II: heterotetrameric: 2 and 2 chains stabilized through S-S bonds: monomeric ligand
Insulin-R, IGF-1-R– Class III: Repeats of mmunoglobulin-like structure, dimeric ligand
FGF-R, NGF-R, PDGF-R, CSF-1-R, c-kit
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Growth Factor Receptors
Signaling through Adapter proteins:
• grb2: adapter with one SH2 domain which binds PY residue on RTK, and two SH3 domains which bind to
• Sos: “Son of Sevenless” (mutation in drosophila prevents 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 of ras.
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Growth Factor Receptors
Signaling through Adapter proteins:
• raf: ser/thr kinase of the MAPKKK/MEKK family (MEKK does normally NOT phosphorylate MEK, but rather MKK4/7 => Stress pathway); requires context of plasma membrane for activation (mixing GTP-ras and raf in a test tube fails to activate raf) => raf likely to be phosphorylated at the plasma membrane. Activated raf phosphorylates...
• 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 phosphorylate transcription factors such as fos and jun; also feedback loop to other signaling molecules
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Growth Factor Receptors
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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) = B Cell 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 IFN’s, but
each encoded by a distinct gene)
– genes segmented into 4 or 5 exons (exceptions are IFN and IFN: 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
• IFN R, IFN R
– Type III cytokine receptors = TNF receptor family: receptors contain 4 Cys rich regions in extracellular domain
• TNF R, TNF=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 (-family): IL-8, PF4, TG
• C-C subgroup (-family): RANTES, MCAF, MIP-1
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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:
TGF: EGF competitor, binds and signals through the EGF receptor,potent mitogen, but does not support anchorage-independent growth
overexpressed in epithelium of psoriasis patients (=> hyperproliferation of keratinocytes)
– TGF: acts through distinct receptor, it also is a potent mitogen, but it also does not support anchorage-independent growth (only combination does!)
TGF- receptors I and II:– external ligand binding domain and cytosolic serine/threonine kinase activity.
– betaglycan: proteoglycan required for TGF binding
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Ser/Thr-kinase Receptors
Signal Transduction:
SMADs:– 8 members - conserved MH1 and MH2 domain
– rapidly phosphorylated in response to TGF (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 TGFR are found in 90% of colon cancers
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NFB
– originally identified as a transcription factor binding an enhancer (B)in the -light chain immunoglobulin gene
– Activated by a variety of (proinflammatory) signals (IL-1, TNF, Phorbol esters...)
– Homo-or heterodimer composed of p50 and/or p65 subunits
– retained in its inactive form in the cytoplasm by the inhibitory protein IB
– dissociation of NFB from IB activates NFB’s DNA binding capabilities
– NFB/ IB association is regulated by serine phosphorylation of IB!
– Phosphorylated IB does not dissociate from NFB, rather is marked for degradation (NFB activation can be inhibited by protease inhibitors!)
– Phosphorylation of IB through IB-kinase (complex >600 kDa)
– IKK: two kinase subunits: IKK and IKK homo- or heterodimers IKK (NEMO): no kinase activity, required for complex assembly = regulatory subunit