bimm118 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:

– 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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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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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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• 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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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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• 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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• 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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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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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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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

bimm118 phospholipids, phosphoinositols & eicosanoids

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