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82 Cards in this Set
- Front
- Back
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PIP2
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PI-4,5-P2
1-5% of total phospholipid (~5mM in plane of mmb) global concentrations don't change much Compartmentalization important highly charged (binds a lot) PH domains mediate lipid binding PLC-->IP3, DAG |
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DAG
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Diacylglycerol
Activates PKC |
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IP3
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Releases Ca from ER
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PLC
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Phospholipase C
6 families, 13 isoforms |
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PLC delta
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3 subtypes
regulatory mechanism unclear role unclear |
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PLC beta
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4 subtypes
Can be activated by either a(q) or bg subunits of GPCR's |
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PLC gamma
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Activated by growth factor receptors (RTKs)
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PKC epsilon
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Can interact with Ras (as well as many others)
Physiological role unclear |
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PI3K
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Phosphoinosotide 3-Kinase
Forms PI-3,4,5-P3 |
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Experimental evidence for PIP3 as second messenger
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levels increase upon receptor stimulation
Wortmannin inhibits formation (not formed passively) PIP3 can activate signaling cascades PIP3 can bind and activate downstream targets |
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Regulation of PIP3
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Receptor and non-receptor TK's (EDGF, PDGF, NGF, etc.)
Some GPCR's |
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Class I-A PI3K
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regulated by TK's
p110, p85 heterodimer p110--catalytic p85--regulatory, has SH2,3 domains Both subunits phosphorylated upon receptor activation |
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SH domains
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Src homology domains
Present on p85 subunit of class IB PI3K |
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Class I-B PI3K
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G-protein regulated form (bg subunit)
-PTX-sensitive p110, p101 heterodimer p110(gamma)-catalytic domain p101-regulatory domain |
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PH domains
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Pleckstrin homology domains
~100 aa sequence Structural homology Rel. low sequence homology-->diversity! |
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TGF B involved in: (4 things)
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1-Growth inhibition/cell cycle ctrl
2-Epithelial-mesenchymal transition 3-Fibrosis 4-in vivo stuff (KO) |
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Myc
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Downregulated by TGF-B
-->leads to cell cycle arrest Smad3/E2F4/p107 inhibit myc transcription |
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P15(ink4b)
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induced by TGF-B
Inhibits cdk's TGF-B disinhibits myc control of P15 |
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P21(cip1)
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Induced by TGF-B via FoxO binding site
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PI3K in TGF-B signaling
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Induce FoxO nuclear export via Akt
w/out FoxO, no induction of P21 |
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Cdk4/6
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TGF-B phosphorylates Tyr residue
Inhibitory Result:decreased kinase activity |
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cdc25a
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downregulated by TGF-B (just like myc)
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P27
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Binds and activates D/K4
Released from D/K4 by TGF-B THEN binds and inhibits E/K2 |
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Cycin E / cdk2
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Activates H1 (histone 1) kinase
inhibited by TGF-B |
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Mechanisms of TGF-B cell cycle control (5 ways)
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1-inhibit myc
2-induction of P15 (via dis-inhibition of myc) 3-induction of P21 (same as P15) 4-Removal of P27 from D/K4 complex by P15/21 AND downregulation of cdc25a 5-P27 inhibition of E/K2 complex |
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TGF-B target genes
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Induction:
P15 P21 Downregulation: myc cdc25a |
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TGF-B1 KO mice phenotypes
(6) |
1-early inflammation, wasting death at 3-4 wks
2-more lymphocytes, neutrophils 3-more MHC molecules 4-more adhesiveness of leukocytes 5-more prolif. in spleen, lymph nodes 6-more IL-2 |
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Multistep skin chemical carcinogenesis
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Apply DMBA (mutates Ras)
10-15 wks: small benign papilloma 25-30wks: malignant squamous later: EMT to form spindle cell carcinoma |
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TGF-B, skin carcinogenesis
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Suppresses benign papillomas
Enhances malignancy! Triggers EMT |
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Fibrosis and TGF-B
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TGF-B overexpression (via adenovirus) induces fibrosis in lung
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How was TGF-B pathway elucidated?
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1-Yeast 2 hybrid to find prot-prot interactions
2-co-IP to confirm this w/ WT proteins 3-RNAi to see effects +/- proteins |
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Rsmads
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1,5,8
2,3 Mad (drosophila) Sma (c elegans) |
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Co smads
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Smad 4
Medea (drosophila) sma 4 (c elegans) |
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Antagonistic smads
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6,7
Dad (drosophila) |
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Chordin
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Xenopus
Binds and sequesters BMP4 (inhibiting signaling) Proteolysed by Xolloid |
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TGF-B and dorsal-ventral patterning
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Dorsal: Chordin (frog), Dpp (fly)
Ventral: BMP4 (frog), Sog (fly) |
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TGF-B type II receptor
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Constitutively phosphorylated
Recruits and phosphorylates TGF-B IR when ligand-bound |
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Smad domains
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MH1---Linker---MH2
MH1=DNA binding MH2=Prot-prot interations |
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TGF-B:
drosophila vs man |
frog/fly: determines dorsal/ventral patterning
mammals: controls cell cycle |
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Strategy to determine phosphoinositide-domain interactions
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1-overexpress domain (ie PH)
2-Tag domain w/ GST 3-add radiolabeled PIP 4-purify w/ GST tag 5-see how much radioactivity |
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Why should Phosphoinosotides bind PH domains?
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1-Localization at Plasma mmb
ie IP3 and PLC compete for access to substrate (PIP2) 2-Allosteric regulation: changing prot. conformation ie: PIP3 changes conformation of Akt (PI3K pathway) |
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GFP-base PI biosensors
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Responsive to changes in [PI] resulting in different ability of PH domains to localize to mmb
ie: PH from mmb to cytosol with PLC activation (increased IP3 competes for PH domain of PIP3 and others) |
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Other non-PH-related roles of PIP2
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1. Regulation of cytoskeleton
2. Regulation of ion channels 3. Neutrophil chemotaxis (PI3K/Akt) |
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PTEN
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Tumor suppressor
Dephosphorylates PIP3 |
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Evidence for PIP2 regulation of ion channels
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Patch clamp expt's
1-RUN DOWN 2-ATP eliminated run down |
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Run down
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When a channel is excised w/ patch clamp, I decreases
Can be restored by addition of ATP The result of PIP2 hydrolysis-->PIP2 holds channel open |
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M1-AChR
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Muscarinic
Gq alpha subunit activates PLC to hydrolize PIP2, forms DAG, IP3 |
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DAG
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Diacylglycerol
Result of PIP2 hydrolysis Activates PKC |
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IP3
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Inositol triphosphate
Result of PIP2 hydrolysis Activates ER Ca channel |
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Katp
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Transiently inhibited by ATP, then activates
PIP2 can activate this channel directly Explanation: ATP is used to make PIP2 (from PIP). Thus the delay in channel activation |
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Disease-causing mutations in channels
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MANY are in residues that bind PIP2
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Water-soluble PIP2 analogs
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diC8-PIP2
AASt-PIP2 Showed that PIP2-protein interactions ARE specific. Analogs could activate channels like normal |
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Channels Regulated by PIP2
(4 types) |
K: Kir, Kcnq, A-type
Na: ENaC Ca: N, P-type Cl-CFTR (Cystic fibrosis) |
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PIP2/Channels take home message
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-PIP2 necessary for activity of many ion channels
-Dynamic regulation of PIP2 modulates channel function -Disease-linked mutations can change channel-PIP2 interactions |
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PIP2 and caspase
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1-inhibits caspase activity
Proven by IP experiments showing PIP2,3 bind caspase 2-Overexpression of PI5K (PI4,5P2) rescues apoptosis |
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PIP2 and DNA damage
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H2O2, UV cause PIP2 dephosphorylation
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Discovery of NF-kB
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Some NF binds k3 in differentiated B cells
Binding is POST-translational (CHX no effect) |
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How were P50, P105 discovered to be the same?
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1-TAG myc-p105 and p105-HA
2-transfect plasmids 3-S-35 label 4-IP (anti myc or HA) 5-autoradiography (only newly synthesized prots radiolabeled) Results: 5' myc tag shows p50 band. 3' HA tag NO band. So processing is 3'->5' |
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Evidence for IkB
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EMSA
-No DNA binding w/out treatment -Nuclear fraction binds w/ treatment -w. DOC (slight denaturant), cytosol of untreated fraction binds DNA -w. DOC + Treatment, nuclear fraction binds *SOMETHING in cytosol prevents DNA binding, and this is released upon activation |
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Ankyrin repeats
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Responsible for IkB prot-prot interactions
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Ways IkB keeps NFkB in cytoplasm
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1-masks nuclear localization signal
2-IkBa has nuclear export signal 3-prevention of DNA binding |
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Regulation of IkB
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Phosphorylated and degraded upon NF-kB activation
Induced by NF-kB TRANSIENT |
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IkB super repressor
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Ser 32,36 mutated, no longer can be phosphorylated. NEVER degraded
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Evidence for IKKa
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Activity induced by TNF,IL-1
Showed IKKa binds IkBa by co-IP Mutate K44A, no more IkBa binding IKKa accelerated IkBa degradation |
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Dom neg IKKa,b
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Prevents NF-kB nuclear translocation (by inhibiting IkB phosphorylation and degradation)
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NEMO
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NF-kB Essential Modulator
aka IKKg Complexes w/ IKKa,b NO DNA binding in NEMO-KO |
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IKKb
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CRUCIAL to NF-kB activation
-/-, +/- By: 1-IP-->Kinase assay (add, then blot for phosphorylated substrate) 2-EMSA (NFkB-DNA) |
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IKKa
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NOT crucial to canonical NF-kB activation
Thus, IKKb-IKKb homodimer is enough (also need IKKg/NEMO) BUT, KEY to alternate pathway (BAFF,CD40,LTbR) |
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Induction of IKK's
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Phosphorylated in response to TNF
Two key Ser residues |
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IP-Kinase assay
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1-Pull down kinase of interest
2-Add P-32 ATP 3-Add substrate 4-Electrophoresis 5-autoradiography to see if/where P-32 went |
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IKK kinases
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NIK
TAK1 MEKK3 |
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NIK
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An IKK Kinase
Not responsive to cytokines (as seen in NIK-/-) BUT, NIK-/- abnormal in responst to LTbR stimulation *conclusion: IKK-kinases are specific to cytokine NIK req'd for P100-->P52 processing |
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P100-->P52 processing
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In alternate NF-kB pathway
NIK phosphorylation of IKKa is required for this |
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Canonical Pathway
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TNF-->IKKb/a/g(NEMO)-->P105/RelA-->P50/RelA-->DNA
MANY ways of activating this pathway on many levels! |
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Alternate Pathway
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BAFF-->NIK-->IKKa/a-->P100/RelB-->P52/RelB--DNA
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NFkB and cancer
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ALL cancers
NF-kB involved in: 1-proliferation 2-Inflammation 3-Anti-apoptosis 4-Invasion, angiogenesis |
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Possible mechanism of NF-kB cancer
P50 overexpression |
Constitutive activity, not enough IkB to sequester
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Possible mechanism of NF-kB cancer
Translocation of Bcl-3 |
Contains transactivation domain, can translocate otherwise inactive complexes to nucleus
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Possible mechanism of NF-kB cancer
NF-kB gene alteration |
Would result in constitutive processing of P100 (alt pathway)
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Possible mechanism of NF-kB cancer
Deletion/mutation of TRAF2/3 |
TRAF's are Ub ligases, but not for degradation but as scaffolds. Loss off scaffolding
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Possible mechanism of NF-kB cancer
Overexpression of NIK |
Would result in increased phosphorylation of IKKa, thus increased degradation of IkB
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Possible mechanism of NF-kB cancer
CD40 overexpression |
Increased signaling on alternate pathway
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