Phosphatidylinositol Lect 25-28 Flashcards
what kind of receptor can activate phospholipase C? what “version” of phospholipase C gets activated by each receptor?
G-protein-coupled receptor activates PLC-beta
Receptor tyrosine kinase activates PLC-gamma
what are the downstream effects of PLC activation?
Kinase activation: CaM-kinase or PKC
name cell responses in the liver, pancreas, smooth muscle, and blood platelets in which g-protein coupled receptors activate the inositol phospholipid signaling pathway
- liver: glycogen breakdown (signaling molecule = vasopressin)
- pancreas: amylase secretion (acetylcholine)
- smooth muscle: contraction (acetylcholine)
- blood platelets: aggregation (thrombin)
what charge does PI (phosphatidylinositol) have?
negative charge
in what membranes is PI found?
in all eukaryorit membranes
describe the structure of PI?
phosphatidic acid backbone, linked to an inositol molecule via the phosphate head group
where is PI synthesized?
mainly in the ER
what catalyzes PI synthesis?
phosphatidylinositol synthase
how does phosphatidylinositol synthase work?
it used CDP-diacylglycerol and L-myo-inositol as substrates to produce PI and cytidine monophosphate (CMP)
how does PI gets transported to other cellular membranes?
via vesicular transport or transport proteins
the breakdown of what inositol phospholipid is the most critical? why?
PI(4,5)P2 (compared to PI and PI(4)P) because it generates 2 intracellular mediators
what inositol lipid is the less abundant?
PI(4,5)P2 (less than 10% of total inositol lipids
whats another words for inositol phospholipids?
phosphoinositides
what positions of the inositol ring are usually not phosphorylated? why?
hydroxyl groups at position 2 and 6;
due to steric hindrance
in which leaflet are phosphtodylinositols typically found?
mostly in the outer leaflets. They comprise only 0.5-1% of the total lipids of the inner leaflet (unlikely to have a role).
what was the first demonstration that PI phosphorylation is linked to cellular signaling?
Stimulation of amylase secretion by acetylcholine substitute in pigeon pancreatic slices increased 32P incorporation into phospholipids 7 times to what is observed in control tissues
what are PLCs?
a group of enzymes that cleave PIP2 just before the phosphate group to generate IP3 and diacylglycerol (DAG)
what are the main domains of PLC found in the 6 PLC families? what is their function?
- PH = pleckstrin homology, binds phosphoinositides
- EF = EF-hand motif, binds Ca2+
- C2 = Ca2+-dependent phospholipid binding domain
what domains are only found in the PLC-epsilon family?
RasGEF
RA1/2 = Ras association domain
what domains are only found in PLC-gamma (y)?
2 SH2 and 1 SH3 domain (Src homology domain)
what is the first step in the chain of events leading to PIP2 breakdown?
activation of PLC-B (via the dissociation of trimeric Gq alpha subunit following the activation of G-protein coupled receptor)
what else can activation PLC-B (other than Gq alpha subunit)?
the beta-gamma complex of another G-protein
what happens once PLC-B is activated?
hydrolization of PI(4,5)P2 gives two intracellular messenger molecules: IP3 (inositol 1,4,5-triphosphate) and diacylglycerol
what does IP3 do?
binds to and opens IP3-gated Ca2+-release channels in the ER membrane which releases Ca2+ from the ER
what does diacylglycerol (DAG) do?
remains in the PM. With phosphatidylserine and Ca2+, it helps activate PKC
remember: what activated PLC-B vs PLC-y?
- G-protein coupled receptor activates PLC-B
- receptor tyrosine kinase activates PLC-y
phosphatidate phosphatase causes DAG to become what?
phosphatidic acid
what kind of domains does protein kinase C have?
regulatory and catalytic domain
name PKC’s regulatory domains
- C1: DAG and phorbol esters binding domain (zinc finger)
- C2: Ca2+ binding domain. also binds phospholipid cofactors via Ca2+ and PIP2 for localization at PM
- Pseudosubstrate site: autoinhibitory domain that binds the substrate site in the absence of cofactors and activators
on what side of PKC are regulatory domains found?
N terminus (amino terminus)
name PKC’s catalytic domains
C3: binds ATP
C4: binds substrate (it is the active site)
what is V3 region in PKC?
V3 is the hinge region; highly sensitive to proteolytic cleavage by cellular processes
what kind of kinases are PKCs?
serine/threonine kinases
what differs among PKCs groups?
their conserved domains, regulatory motifs, and substrate binding sites.
BUT mostly C1 and C2 alterations. (C3 and C4 stay the same in all categories)
how many PKCs are in the PKC family?
9 grouped in 3 categories
what are the 3 categories of PKCs?
conventional (a, B, y), novel, and atypical
what do the conventional PKCs bind to?
DAG and calcium (gets activated by them)
what domain is non-functional in novel PKC?
C2 domain
what can novel PKCs bind to?
DAG only, not calcium
what increases novel PKC’s binding affinity to DAG x2 compared to conventional?
the tryptophan (W)
what domains are non-functional in atypical PKC?
C1 and C2 domain
how do atypical PKCs work withour a C1 or C2 domain?
they have PB1 domain that mediates binding to protein scaffolds (serves similar function as DAG binding to C1 domains to relieve autoinhibition by pseudosubstrate)
what are atypical PKCs mostly regulated by?
phosphatidylserine
the action of what compound do phorbol esters mimic? how?
mimick the action of DAG by binding to C1 domain of PKC
mouse-skin models were instrumental in defining what?
the multi-stage nature of carcinogenesis
what did they find after exposing mice to a cancer initiator and to promoters?
cancer developped if promoter was given after the initiator and at a short enough interval.
what cancer promoter did they use in the mice experiment?
phorbol-esters (PMA and TPA)
what gives phorbol ester its potency as a tumour promoter?
the hydrophobicity of the acyl chains
remember what is the receptor for phorbol ester?
PKC (C1 domain)
what domain of PKC retains it in an inactive state?
pseudosubstrate domain (its auto-inhibitory)
in what “part” of PKC is the pseudosubstrate domain located?
in the regulatory domains
how does the pseudosubstrate domain inhibit PKC?
it sits in the active site to block the acess. Unlike PKC substrate, it can not be phosphorylated because of its alanine residue
what residue usually found in PKC substrate is replaced by alanine in PKC’s pseudosubstrate domain?
serine or threonine is replaced by alanine (nonphosphorylatable)
where is the active site located in PKC?
in the catalytic domain
name the 4 main mechanisms of PKC regulation
- maturation
- activation
- subcellular localization
- inactivation
what controls PKC’s maturation?
phosphorylation
what controls PKC’s activation?
DAG, calcium, and/or phosphatidyl serine
what controls PKC’s subcellular localization?
protein-protein interactions
what controls PKC’s inactivation?
proteolysis and degradation
what is PKC’s maturation?
bringing PKC away from the membrane and into a closed state via phosphorylation
out of the 4 mechanisms of PKC regulation, which one is constitutive?
maturation, the others are agonist evoked
in what state is PKC synthesized?
open, degradation-sensitive configuration
what region of PKC’s regulatory modules is membrane targetting? how?
C2: its a basic patch that binds PIP2
explain in details PKC maturation
- PKC is open
- C2 targets it to the PM
- PKC binds molecular chaperones via PXXP motif in C-term
- PDK-1 phosphorylates threonine on the activation loop
- this triggers mTORC2 to phosphorylate 2x PKC hydrophobic motifs
- fully phosphorylated PKC goes into closed conformation
which PKCs require phosphorylation by mTORC2?
PKCepsilon (novel PKCs) (others don’t need it but can still be phosphorylated by mTORC2)
what PKCs do NOT get phosphorylated at the hydrophobic motif? why?
atypical PKCs: because serine is substituted by glutamic acid (E)
what is the difference between a mature and an active PKC?
- mature PKC is inactive (closed conformation; C2 domain blocks the kinase domain)
- active PKC is in an open conformation
describe PKC’s activation in details
- calcium is released from the ER and and binds C2 domain, displacing it from kinase domain
- this recruits PKC to the PM again
- C2 binds PIP2
- C1b binds DAG, which expels the pseudosubstrate from the substrate binding cavity
what is different about C2 domain in novel PKCs? what does this change about their location?
it does not sense calcium or PM;
novel PKCs mostly localize the DAG-rich golgi membranes
how are ATYPICAL PKCs activated compared to conventional and novel?
they don’t have a C2 domain; they have a PB1 domain that binds protein scaffolds and tether the pseudosubstrate out of the substrate-binding cavity
PKC membrane translocation is a hallmark of PKC; what reflects the activation and deactivation of PKC activity
intracellular calcium rise reflects the activation level;
DAG level at the PM decreases with decrease in PKC activity.
you can say that what molecule sustains PKC activity?
DAG
PKC activity is proportional to what else?
its localization at the PM
what is the general function of PKC-interaction proteins C-KIPS?
Confer specificity to individual PKC isoforms by regulating their activity and sub-cellular localization.
name the 4 types of C-KIPs and their function
- upstream activators: direct PKC to PLC and DAG
- intracelllular compartments: target various PKC isoform to the compartment
- substrates: bring together PKC isoforms and their substrates for efficient phosphorylation
- signaling molecules: for interaction with signaling molecules
give an example of a C-KIPs (PKC-interacting protein) and its function relating to PKC?
RACKs: scaffolding proteins that mediate PKC sub-cellular localization and function
what is PDX domain?
protein-interaction domain in C-KIPs in scaffolding proteins that has a highly conserved carboxylate-binding loop
what are LIM domains?
protein-interaction domain in C-KIPs located at defined intervals
what are WD40 repeats?
protein-interaction domain in C-KIPs;
contains 4 to 16 rpeating regions with dipeptides
what is PH domain?
protein-interaction domain in C-KIPs;
about 100 amino acids, adopts a 7-stranded B sandwich structure with a C-terminal alpha helix;
binds polyphosphoinositides and proteins
what mediates the interactions between PKC and PKC binding proteins?
protein interacting domains
explain the downregulation of PKC in details
- dephosphorylation at the hydrophobic motid by PHLPP: destabilizes the kinase domain
- dephosphorylation at the turn and at the activation loop by PP2A phosphatases
- ubiquitination -> proteosomal degradation
they hypothesize that the tumor promoting effects of phorbol esters could be due to what?
chronic loss of PKC
name biological processes regulated by PKC
mitogenesis, cell proliferation and differentiation, apoptosis, cell survival, cell adhesion
(distinct and sometimes opposing)
Differential signaling regulation by discrete PKC isozymes is strongly dependent on what?
on the stimuli and cell type
give an example of a PKC downstream signaling pathway
phosphorylates MAPK -> transcription -> regulate cell proliferation and differentiation
2/3 of cancers associated mutations in pkCs result in what modification of the protein? what does this suggests?
loss of function of PKC; suggests its role as a tumor suppressor
specifically what does PKCepsilon mostly promote?
cell survival
specifically what does PKCdelta mostly promote?
apoptosis
name another random downstream effect of PKC (not related to cell sruvival)
actin skeleton remodelling:
how doe sPKC remodel actin cytoskeleton (2 ways)
- PKC phosphorylates adducin -> reduces F-actin capping and assembly with spectrin
- PKC phosphorylates fascin -> reduces F-actin bundling which is important for protrusion formation
how is the expression of PKC isoforms in different cancers?
sometimes increased, sometimes decreased
what does PI3K (Phosphoinositide 3-kinase) do?
phosphorylates inositol lipids at the 3rd position of the inositol ring to generate 3-phosphoinositides
what modifications does PI3K bring to the following inositol phospholipids?
PI
PI4P
PI4,5P
PI -> PI3P
PI4P -> PI(3,4)P2
PI4,5P -> PI(3,4,5)P3
what are PTEN and SHIP?
phosphatases
at what position does SHIP remove the phosphate?
position 5
at what position does PTEN remove the phosphate?
position 3
name the different classes of PI3K and how they each get activated
class I, class II = activated by cell surface receptors
class III = activation is cell surface receptor-independent
briefly describe the structures (domains) of each class of PI3Ks
- Class I PI3Ks: catalytic subunit (p110) + regulatory subunit (of different sizes)
- Class II and III lack a regulatory subunit
what lipids do Class I PI3K preferentially phosphorylate?
PI4,5P to PI(3,4,5)P3
how many isoforms of catalytic subunits does each class of PI3K have?
class I: 4
II: 3 isoforms
III: 1 isoform
name 2 functions of class II PI3K
endocytosis,\ and vesicular trafficking
name 2 functions of class III PI3K
autophagy and vesicular trafficking
what domain do PI3Ks and PKCs have in common?
C2 domain (ca2+ domain)
what is the PI3Kalpha domain?
PI3K accessory domain also known as the helical domain
how does class IA vs class IB get activated?
class IA gets activated by receptor tyrosine kinases;
class IB gets activated by G-protein-linked receptors
what lipid is the preffered substrate for the class I PI3Ks?
PIP2
Class I PI3 Kinases Function as what?
heterodimers
what is called the catalytic subunit of Class I PI3K? which isoforms are for class 1a vs 1b?
p110 binding:
Class 1A = p110a, p110b, p110d
Class 1B = p110y
how many regulatory isoforms do class I PI3Ks have (a and b)? name them
- class 1A has 5 regulatory isoforms;
P85a, P85b, p55a, P55b, p50 - class 1B has p101/p84 or p101/p87
we can say that the expression of p110 catalytic domain amongst Class 1 PI3Ks is what?
ubiquitous
what activates class IA PI3K?
RTKs
where are the catalytic and regulatory subunits of PI3Ks when the cell is unstimulated?
in the cytoplasm
what are the 3 ways how a Class 1A PI3K can get activated?
- p85 SH2 domain directly interacts with pYXXM (phosphorylated tyrosine motif) on RTK
- p85 SH2 domain interacts wiht RTK via an adaptor protein that has pYXXM motif
- p85 SH2 domain interacts with RTK viaRAS/Grb2/Gab2 complex that has pYXXM motif
what kind of domain is p85 SH2 part of in class 1A PI3Ks?
PI3K regulatory domain
what delivers class 1A PI3K to PIP2 on the PM?
p110 domain
class 1A PI3K going to PIP2 on the membrane triggers what?
generation of PIP3
PCR activation of class 1B PI3Ks leads to the formation of what?
GBy dimers (Gbeta gamma)
how is p110y (catalytic domain of class 1B PI3Ks) recruited to the membrane?
via the p101/p84 regulatory subunit that has a GBy binding domain
what happens when class 1B PI3K is recruited to the membrane?
PIP2 converted to PIP3
what are the 3 steps for the initiation of the signaling cascade of PI3K?
- binding of growth factors to their receptors -> activates receptors
- reeceptor activation recruit PI3K to the inner PM via protein-protein interactions
- PI3K phosphorylates inositol lipids at the 3rd position
what methods have been used to study the downstream effects of PI3K activity?
genetic studies
PI3K inhibitors
PI3K overexpression
these studies have implicated PI3K in what cellular processes?
- regulation of cell proliferation and survival
- transformation
- metabolism
- cytoskeletal reorganization
- membrane trafficking
How do these membrane imbedded phospholipids (PI3P)act as intracellular second messengers?
recruit a broad variety of proteins to the membrane by interacting with 3’phosphoinositide binding modules in these proteins
give 2 examples of proteins that are recruited to the membrane by phosphorylated lipids? name what lipid exactly attracts them
- FYVE domain: binds PI3P
- Pleckstrin homology domain (PH): binds PI(3,4)P2 and/or PI(3,4,5)P3
what is FYVE domain?
a cysteine-rich zinc finger-like MOTIF that coordinates two zinc atoms and characteristically has basic amino acids surrounding the third cysteine (binds PI3P)
FYVE domain is found in what proteins?
in the zinc finger domains of Fab1, YOTB, Vac1, and EEA1
what is the PH domain?
Structurally conserved modules of ~100 amino acids that can adopt a seven-stranded β sandwich structure with a C-terminal α helix that bind inositol lipids and their head groups
basically, PH domain is important to recognize what?
PI3K action
like what happens to PH domain-containing proteins after PI3P production?
translocation to the plasma membrane
give 2 examples of PH domain-containing proteins that are recruited to PI(3,4)P2 and PI(3,,4,5)P3?
PDK-1 and Akt
what is Akt?
protein serine/threonine kinases: a cellular homolog of the transforming oncogene of ADT8 retrovirus in rodent T cell lymphomas
where / what is PDK1?
phosphoinoside-dependent protein kinase-1: constitutively
active and present at the plasma membrane in nonstimulated cells.
Its activity is upregulated by PI3K-generated phospholipids.
basically, what does PIP3 provide for PDK1 and Akt?
docking sites
where is Akt initially when inactive?
cytoplasm
what happens to Akt when it is recruited to the membrane?
it undergoes phosphorylation at Thr and Ser
more specifically, Akt is recruited to the PM via what domain interactions?
PIP3/PH domain interactions
what mediates Akt’s phosphorylation?
PDK1 (at Thr/T) and TORC2 (at Ser/S)
what happens after Akt gets phosphorylation at Thr vs Ser?
1) Thr phosphorylation by PDK1 = conformational change = loss of membrane binding affinity of the PH domain.
2) Ser phosphorylation by TORC2 = on c-term HM (hydrophobic motif) = stabilizes the conformational change that exposes the kinase domain
what happens to Akt’s localization when it gets activated?
it releases from the membrane and phosphorylates downstream substrates in other subcellular locations
what happens to the PH domain of Akt when it is active?
it is blocked from membrane association by direct interaction with the catalytic domain or other proteins
What is Akt’s function in the cell? therefore what disease is it linked to?
promote cell survival by phosphorylation: linked with certain cancers
what happens if you overexress Akt?
blocks apoptosis
What happens when there is expression of dominant-negative mutants of Akt?
blocks the ability of growth factors to promote survival
flies defective in Akt show ectopic what?
apoptosis during embryogenesis
name 3 substrates of Akt
BAD, IKK, FOXO
the phosphorylation of what genes by Akt promote cell survival?
tumor suppressor, cycle-dependent kinase inhibitors, telomerase, p27, p21, IKK (NFKB)
the phosphorylation of BAD by Akt leads to what? how?
inhibition of apoptosis; phosphorylation inactivates Bad
without phosphorylation, what does BAD do? what family is it part of?
it’s a pro-apoptotic factor part of thr Bcl-2 family
more specifically how does Akt block BAD action?
it phosphorylates BAD at ser136, creating a binding site for the adaptor protein 14-3-3. Binding to 14-3-3 blocks the heterodimerization of BAD with BCL-2 and Bcl-XL.
kinda confusing: what type of activity do Bcl-2 and Bcl-XL have?
opposite of BAD: they are anti-apoptotic
how does Bcl-Xl and Bcl-2 interaction with BAD affect their activity?
The binding of BAD to Bcl-2 or Bcl-XL inhibits the survival activity of the anti-apoptotic proteins Bcl-2 and Bcl-XL.
Therefore when they can’t bind to BAD because it is phosphorylated, they have cell survival effects.
what is the first step in the activation of NFkB by Akt?
Phosphorylation of IKB-kinase (IKK) by Akt
once phosphorylated by Akt, what does IKK do?
it phosphorylates IKB
what happens to IKB once it gets phosphorylated by IKK? what does this trigger?
IKB gets degraded; this unmasks the nuclear localization signal of NFkB (because IKB is like stuck to NFkB)
what happens when the NFkB’s localization signal is unmasked?
NFkB translocates to the nucleus to mediate the transcription of anti-apoptotic genes
give examples of anti-apoptotic genes whose transcription is regulated by NFkB?
BclXL, Bcl2, cIAPs
NFkB signalling cascade involving IKK, IKB and Bcl-XL/2/IAPS is one of the mechanism by which NFkN regulate what?
apoptosis
what is FOXO?
transcription factors that increase the transcription of p21 and p27
how does Akt phosphorylation affects FOXO?
Akt phosphorylates serine residues, allowing 14-3-3 proteins to bind FOXO proteins
what does the binding of 14-3-3 to FOXO TFs trigger?
1) accelerates FOXO nuclear export
2) Blocks nuclear import
3) Blocks FOXO DNA binding activity
in the 2 cases we saw (BAD and FOXO), what happens when 14-3-3 adaptor protein binds to the proteins?
it inhibit their apoptotic activity aka promotes cell survival
what type of PI3K mutations are involved in cancer?
mutations in the catalytic region (p100a / PIL3CA gene)
more specifically in what part of the catalytic domain are the PI3K mutations linked to cancer found?
- helical domain beyween p110a and p85 regulatory domain
- kinase dmain
PI3K mutations in the catalytic domain that lead to cancer have what effect on PI3K?
cause p85-independent constitutive activation of the PI3K pathway
the mutations causing constiturive activation of PI3K pathway have what downstream effecT?
constituvely activate the Akt pathway
what is PIK3CA?
catalytic domain of PI3K
they found that the loss of PIK3CA expression decreased the tumors of what kind of cancer?
decreases breast tumor proliferation and survival
dox-inducible expression of PIK3CA mutant (H1047R) showed what?
- expression in the mammary epithelial
- increased mammary tumor formation
what is studied at a cancer treatment?
PI3K pharmacological inhibitors
unexpected: what other molecules could inhibit PI3K pathway?
inositol phosphatases!
what is PTEN? what deos it do?
lipid phosphatase that removes the phosphate on 3rd position of inositol group, initally added by PI3K
what is SHIP? what does it do?
also lipid phosphatase, but that dephosphorylates phosphatidyl inositides at the 5’ position
so what does PTEN generate?
PIP2 from PIP3
what domain does PTEN have that is present in proteins that interact with PKC?
PDZ-binding domain
name all the PTEN domains?
- phosphatase domain: catalytic domains that interacts with substrate (contains short active site)
- C2 domain: membrane binding domain
- C-term region: PEST sequence
- PDZ-binding domain: protein binding domain
what kind of PTEN mutation is found in 50-80% of carcinomas?
deletion or mutation of PTEN (almost as frequent as p53), including loss of heterozygosity, and missense mutations
the missense mutations in PTEN affect what dunction?
- affects phosphatase domain that abrogate lipid phosphatase function
- affects C terminus that reduce protein stability
what was found when PTEN was overexpressed?
PTEN overexpression lowers the basal levels of phosphoinositides with phosphates at the 3 position
what did they find in PTEN-/- mice?
elevated levels of phosphoinositides with a phosphate at the 3 position and they die during embryogenesis as a result of failure in developmental apoptosis, not from cancer
PTEN -/- mouse embryonic fibroblasts are resistant to what?
apoptotic stimuli
(PTEN -/-??) heterozygotes show increased what?
tumor incidence
what are 2 things downregulated by PTEN?
PI3K-dependent Akt activity and BAD phosphorylation
how is PTEN protected from degradation?
via the C-terminal that interacts with the C2 domain via phosphate groupe interactions
the phosphates on PTEN c-terminal interact with what part of the C2 domain?
the + charged residues
more precisely how does the c-term-C2 domain interaction protect the PTEN from proteosomal degradation?
blocking the binding site for NEDD4 E3 ubiquitin ligase which, which would otherwise target PTEN for proteasomal degradation
name the 4 mechanisms by which PTEN gets targeted for degradation
1) De-phosphorylation of the C-terminal fragment.
2) C-terminal interacting proteins
3) Mutations and/or truncations of the C-terminal fragment
4) PTEN membrane localization (the positively charged C2 domain interacts with anionic phospholipids).
remember what kind of protein is PTEN?
inositol phosphatase and tumor suppressor
remember: mutations in the catalytic subunit of PI3K constituvely activate what?
the Akt pathway and increase tumor formation and survival
