Transcript Phospholipid signaling

Cellular mechanisms of signaling evolve
from available components
H2O2
Hydrogen peroxide
Phospholipids constitute a
readily available reservoir
that regulates many
intracellular events
PtdIns in yeast
Phospholipase C activation initiates the IP3 pathway
Cellular mechanisms of
signaling evolve from
available components
Phospholipids act as a readily
available reservoir that regulate
many intracellular events
Pathways of phosphoinositide synthesis and degradation.
The phosphatidylinositol (PI) includes inositol
1-phosphate bound via its phosphate group to
1-stearoyl,2-arachidoyl diacylglycerol,
prevalent in mammal cells favouring exposure
of the inositol ring and its interaction with
PIBMs.The 7 known PIs in eukaryotes are
PI4P,PI5P,PI3P,PI45P2, PI35P2, PI34P2 and
PI345P3. Each PI is indicated according to
colour codes. Blue and arrows indicate routes
of PI phosphorylation and dephosphorylation,
respectively. Unlike phosphoinositides, the
soluble inositol phosphates (IPs) can be
phosphorylated in all of the six
positions,giving rise to more than 60
soluble species.This is because other IP-specific
enzymes are present in the cell as well as the
kinases/phosphatases acting on the
phosphoinositides and IPs.The PIs can be
hydrolysed by PLC to generate inositol 1,4,5trisphosphate and diacylglycerol from PI45P2;
by PLA2 to LPIs; PLA/lysophospholipases
(LPLA1) to form the GPIs;and by PLD to form
phosphatidic acid. PLC acts preferentially on
PI45P2, whereas the other phospholipases
may act on the different PIs (for simplicity in
the figure,all the phospholipases are shown
acting only on PI45P2).
Proposed functions of PX-domain proteins. a, Recruitment
of the NADPH oxidase complex. Upon neutrophil activation,
PI3K-I converts PI(4,5)P2 into PI(3,4,5)P3. The cytosolic
subunits of the NADPH oxidase complex (p40, p47, p67) are
recruited to developing phagosome at the plasma membrane
by binding of the PX domain of p47phox to PI(3,4)P2 (green),
generated upon dephosphorylation of PI(3,4,5)P3 by the 5phosphatase SHIP-1. PI(3,4)P2 is then dephosphorylated by a
PI(3,4)P2 4-phosphatase to generate PI(3)P (red), which
binds to the PX domain of p40phox. The correct assembly of
p40phox, p47phox and p67phox with the membrane-bound
(cytb558) components of the complex results in a functional
Phox complex that produces O2b, Membrane trafficking. PI3K-II is recruited to the plasma
membrane through binding of its PX domain to PI(4,5)P2 and
may promote the formation of clathrin-coated vesicles. Snx3
binds to PI(3)P (red) in the sorting early endosome and
augments transport of transferrin (Tf) from the sorting to
the recycling endosome. The yeast SNARE Vam7p is
recruited by PI(3)P on multivesicular bodies (MVBs) and
vacuoles to complex with other SNAREs and thereby
promote vacuolar membrane docking and fusion.
Simplified overview of the main synthetic pathways involved in the formation of
polyphosphoinositides in higher plant cells
O
Phosphatidic Acid serves as the precursor from which
many of these second messenger lipids are derived →
CH
O
OH
2
HC
P OH
OC R2
O
CH2 OC R1
O
The two kinases, phosphatidylinositol 3-kinase (PtdIns 3K) and PtdIns(3)phosphate [PtdIns(3)P]
5-kinase (Fab1) are shown. Routes of synthesis that are established are shown by unbroken
arrows, whereas steps that still need confirmation or are less well defined in vivo are indicated
by broken arrows. Abbreviations: PtdIns(4)P, PtdIns(4)phosphate; PtdIns(5)P,
PtdIns(5)phosphate; PtdIns(4,5)P2, PtdIns(4,5)bisphosphate; PtdIns(3,4)P2,
PtdIns(3,4)bisphosphate; PtdIns(3,5)P2, PtdIns(3,5)bisphosphate
Lipid substrates and messengers produced by
phospholipids- and/or galactolipid-hydrolyzing enzymes,
and their downstream physiological effects
Note that the substrate lipids can be located on the plasma membrane or other membranes,
depending on the nature of a specific enzyme and its intracellular location
Phosphoinositides involved in classical phagocytosis
PtdIns(4,5)P2 accumulates in pseudopods during extension of the phagocytic cup. As the phagosome
seals, PtdIns(4,5)P2 disappears. This could be explained in part by its catabolism by phospholipases
but also by its conversion into PtdIns(3,4,5)P3, and indeed, PtdIns(3,4,5)P3 appearance coincides
with PtdIns(4,5)P2 clearance. PtdIns(3,4,5)P3 accumulates transiently in the phagocytic cup and is
required for its closure. Once the phagosome is formed, PtdIns(3)P is produced on its surface and
recruits proteins that control phagosome fusion and maturation. Other phosphoinositide species are
present in the trans-Golgi complex (PtdIns(4)P) or in the nucleus (PtdIns(5)P), leading to the proposal
that membrane identity can be mediated by compartmentalization of specific phosphoinositides
Phospholipid signaling under salt stress, drought,
cold, or ABA. Osmotic stress, cold, and ABA
activate several types of phospholipases that
cleave phospholipids to generate lipid
messengers (e.g., PA, DAG, and IP3), which
regulate stress tolerance partly through
modulation of gene expression. FRY1 (a 1phosphatase) and 5-phosphatase-mediated IP3
degradation attenuates the stress gene regulation
by helping to control cellular IP3 levels.
PLD and PA in response to H2O2
PLD , is activated in response to H2O2 and the resulting PA functions in
amplification of H2O2 -promoting PCD
Stress stimulates production of H2O2 that activates PLD associated with the plasma membrane.
Potential activators: Ca2+ and oleic acid. This increases PLD affinity to its substrates, stimulating lipid
hydrolysis and PA production. PA may bind to target proteins, such as Raf-like MAPKK, that contain a
PA binding moti, leading to the activation of MAPK cascades. PA may also function by modulating
membrane trafficking and remodeling. These interactions modulate the cell's ability to respond to
oxidative stress and decrease cell death. Dashed lines - hypothetical interactions.
PLD & PA
• Knockout of PLD renders Arabidopsis plants more sensitive
to the reactive oxygen species H2O2 and to stresses
• H2O2 activates PLD , and PLD -derived PA functions to
decrease the promotion of cell death by H2O2. These results
suggest that both PLD and its product PA play a positive role
in signaling stress responses
• PLD and its derivative PA provide a link between
phospholipid signaling and H2O2-promoted cell death. PLD
and PA positively regulate plant cell survival and stress
responses.
The role of PLD in vesicular trafficking & signal transduction
A) PLD catalytic activity. In the first step of the reaction (left panel), PLD removes the head group of
a structural phospholipid, such as PC, forming covalent bond with the resulting phosphatidyl moiety,
the PLD-PA intermediate (middle panel). In the second step (right panel), PLD transfers the
phosphatidyl moiety to a nucleophile. Under physiological conditions, this is water, representing the
hydrolysis of PC to generate PA. Primary alcohols, such as 1-butanol, can also be used as
acceptors, resulting in the formation of PBut, a reaction that is used to measure PLD activity in vivo
and in vitro (3, 8, 32). (B) Cytokinesis in plant cells. (C) Model of PLD binding to microtubules and
membranes. PLD binds vesicular and plasma membranes through its covalent PLD-PA intermediate
(Fig. 1A, middle panel). (D) PLD's contribution in PA signaling. A summary of factors activating PLD
in plants and the role of PA in signaling
Phospholipid signalling pathways that are involved
in plant defence responses.
PLA2 generates lyso-phospholipids (LPL) and FFAs that stimulate the plasma membrane H+ATPase, and free fatty acids can be metabolised via octadecanoid pathway to JA. PLC hydrolyses
PIP2 into IP3 and DAG. IP3 diffuses into the cytosol, where it could release Ca2+ from intracellular
stores, or is metabolised further to IP6. DAG remains in the membrane to be phosphorylated by
DGK to PA. Activation of PLD generates PA directly by hydrolysing structural phospholipids such as
PC. PA can activate MAPK, CDPK, ion channels, and NADPH oxidase, all of which are involved in typical defencerelated responses. PA signalling is attenuated by its conversion to DGPP by PA kinase. All lipids or their
derivatives that are involved in signalling are shown in red. Solid arrows indicate metabolic
conversion; dashed arrows indicate activation (directly or indirectly) of downstream targets.
PI metabolism in Arabidopsis
The different steps in the synthesis of PIs and the lipid kinases catalyzing the different reactions
are indicated. PtdIns(3,4,5)P3 is present in animal cells but has not been detected in plant
tissues, so far. In animal cells, PtdIns(3,4)P2 can be generated from PtdIns4P by a PtdIns 3kinase or by an as-yet-unidentified PIPkin from PtdIns3P. Plant cells do not contain any homolog
of the heterodimeric inositol lipid 3-kinases that are able to phosphorylate PtdIns4P to
PtdIns(3,4)P2 and PtdIns(4,5)P2 to PtdIns(3,4,5)P3. PtdIns(4,5)P2 can be synthesized by type I
and type II PIPkins from PtdIns4P and PtdIns5P, respectively. On the basis of sequence
comparison, plants cells do not possess type II PIPkins. PtdIns5P is present in plants, but an
enzyme capable of producing it has not been identified.
.
PLD is involved in O2 - production in Arabidopsis
PLD suppression decreases Phosphatidic acid (PA) production
PA levels increase during various stress conditions.
PA-stimulated production
of superoxide in PLD deficient and wt leaves
Plant Physiology, 2004, Vol. 134, pp. 129
Plant Physiol. 126 (2001) 1449-1
PA specifically induces leaf cell death in
Arabidopsis
A)
WT plants were infiltrated with PA or PC and photographed 24 h after treatment with
the lipids. Arrows indicate the area of liposome infiltration.
B)
Leaves of WT plants were floated on phospholipid liposomes
C)
Trypan blue staining was used to visualize dying cells in areas of turgor loss in PAtreated leaves. Leaves of WT plants were detached, floated on PA (left), or PC (right)
suspensions for 2 h, and stained with Trypan blue
Phospholipase C Activity
These phospholipases are involved
in second messenger generation
from membrane phosphoinositides
Phosphatidylinositol –4,5-bisphosphate (PIP2)
O
O
PLA2
Diacylglycerol (DAG)
O
O
Phospholipase C (PLC)
hydrolysis PIP2 to yield two
second messengers
PLC
O-
O
O-
P
O
O
O
P OO
5
PLD
N.B. Different phospholipid
specificities (releases different PIs)
1
O
OH
H
Inositol-1,4,5-triphosphate
(IP3)
4
HO
O
O P OO-
The receptor for inositol 1,4,5-triphosphate (IP 3 )is located on the tonoplast and ER
membranes
Conformational changes in this receptor transduce subsequent signaling.
Certain ion channel receptors,including the IP3 receptor,are composed of
four subunits. Each subunit contains four membrane-spanning domains (not
shown). When IP3 binds to the receptor,conformational changes result in
movement of two of the subunits.The distribution of positive and negative
charges stabilizes the open conformation of the channel and allows the entry
of Ca2+ into the cytoplasm.
Domain structures of PLDa, PLDb, and PLDg in Arabidopsis
XX in the PLD C2 marks the loss of two acidic residues potentially
involved in Ca2+ binding; XX in the PPI-binding motifs marks the loss of
the number of basic residues potentially required for PPI binding.
Direct and derived products of PLD activation
LysoPA and free fatty acid (FA) can be formed from PA by nonspecific acyl hydrolase or by
PLA. PA is dephosphorylated to DAG by PA phosphatase. CDP-DAG is the precursor for the
synthesis of PS, PI, and PG. XOH, Primary alcohol used for transphosphatidylation; Ptd,
phosphatidyl; NAE, N-acylethanolamine.
PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; ARF, ADP-ribosylation factor; LPA,
lysophosphatidic acid; PLA, phospholipase A; PA, phosphatidic acid; DGK, diacylglycerol
kinase; PAP, PA phosphohydrolase; PIP5K, phosphatidylinositol 4-phosphate 5-kinase; MAPK,
mitogen-activated protein kinase; MEK, MAP kinase kinase; ERK, extracellular signal-regulated
kinase; SPHK, sphingosine kinase; Edg, endothelial differentiation gene
Classes of Phospholipase C
Four main isoforms (+variants)
b
900-1315
Animals only
G protein activated
Two main isoforms
g 1220-1285
Animals only
Tyrosine kinase activated
Four mammalian isoforms + four splice variants
d
600-870
All non-animal PLCs are in this class
Ca2+ activated?
The Structure of PLCg
Phosphotyrosines
Pleckstrin homology –
phosphoinositide binding
Catalytic X and
Y domains
P
C3 – part of catalytic
domain? Phospholipid
interaction?
P
P
SH2 – phosphotyrosine
binding
EF hand-like
Ca2+ binding?
SH3 – interaction with
cytoskeleton?
Activation of PLCg by EGFR complex
EGF binds to receptor
EGF
PLCg hydrolyses PIP2
to yield IP3 and DAG
PLCg
P
PIP2
DAG
IP3
P
PLCg phosphorylation may release it
from interaction with inhibitor
PLCg phosphorylated
Inactive PLCg
Receptor
phosphorylated
PIP2-derived Second Messengers
Inositol-1,4,5-triphosphate (IP3)
O-
O
O-
P
Diacyleglycerol (DAG)
O
O
P
O-
O
O
O
O
5
1
OH
OH
O
4
O
HO
O
O
P
O-
O-
Hydrophilic
Hydrophobic
Binds to receptor on ER
Remains in plasmalemma
IP3 Receptor is Ca2+ channel
Activates Protein Kinase C (PKC)
AMPLIFICATION – many IP3/DAG per bound ligand
Summary
Phosphatidylinositol-specific PLC hydrolyses membrane PIP2
PLCg has domains that allow binding to phosphotyrosine (SH2)
PLCg associates with activated receptor tyrosine kinases
PLCg is activated by tyrosine phosphorylation
IP3 – soluble, induces Ca2+ release
DAG – hydrophobic, activates protein kinase C
Loewen, et al (2004). Phospholipid Metabolism Regulated by a Transcription Factor
Sensing Phosphatidic Acid. Science 304, 1644-1647.
Inositol-induced alteration in phospholipid synthesis.
Phosphatidylinositol 3’-Kinase (PI3K) Activity
O-
O
O-
P
O
O-
OH
O
1
O
O
O
4
HO
PI4K
1
O-
OH
O
5
OH
OH
P
P
4
HO
1
PI5K
O-
P
O
Headgroup of PIP2
O
O
5
OH
OH
O
5
OH
OH
4
HO
O
O
OH
P
O
O-
P
O
O-
O-
O-
OO
O
O
O
O-
P
O
OH
O
1
O-
O
4
OH
OH
1
4
O
O-
PI5Ptase
O
O
P
O-
O
O-
P
O-
O-
P
O
O
5
OH
OH
1
4
O
OH
O-
P
O
5
O
P
O-
OH
O
5
OH
OH
P
O
O
OO
P
O
O-
O-
PI3K phosphorylates inositol on the 3 position
PTEN dephosphorylates inositol on 3 position
P
O-
O-
Class I PI3K
p85 binding
ras binding
Catalytic
p110a, b , d kinases
SH2
p85a
SH3
p110-binding
p85b
adapters
p55a, g
Proline-Rich
p50a
CLASS IA
p110g kinase
p101 adapter
CLASS IB
Class I PI3K Regulation
p110d autophosphorylation
inhibits PI3K activity
regulation by p21ras
Subunit interaction
Proline-rich repeats bind SH3
domains of e.g. src, fyn or lck
p110a phosphorylation of p85a
(S608) inhibits PI3K activity
SH2 bind pY-X-X-M
SH2 also binds PI(3,4,5)P3 this
binding competes with pY binding
Inter-SH binds PI(4)P and PI(4,5)P2
Protein Kinase C (PKC)
Three Classes
All forms require phosphatidylserine
(PS) for activity
Classical cPKC
Novel nPKC
Atypical aPKC
a, b1, b2 and g
d, e,  and 
z, i and l
Activated by DAG and Ca2+
Activated by DAG but do
not require Ca2+
Do not require DAG or Ca2+
cPKC have two zinc finger domains
C1 – binds PS and Ca2+
C2 – binds DAG
PKC Substrates
PKC phosphorylation sites
– release from membrane
P P P
MARCKS
Myristoylation site – membrane
association
Calmodulin (CAM) binding
Associated with
Proliferation
• Decreased MARCKS- F actin association
• Actin polymerisation
• Decrease CAM-dependent mlc phosphorylation
MARCKS Protein
MARCKS Phosphorylation
VEGF
Effects mediated by PKC
Proliferation - insulin
MAP Kinase Pathway – ras independent
Differentiation – wnt pathway
+Apoptosis – UV-B, neutrophils (PKCd activated by caspase 3)
-Apoptosis – suppresses Fas-induced PCD (PKCa?)
Cell Polarity – atypical PKC and interacting protein
Feedback Inhibition of IP3/Ca2+
Receptor Downregulation - e.g. EGF
Inhibition of PLCg - -ve feedback
STAT inhibition – PKCd blocks STAT DNA association
Summary
Three classes of PKC
All require phosphatidylserine for activity
Pre-activation of PKC requires PDK-1 phosphorylation
Activation completed by DAG (except aPKC class)
MARCKS – major substrate for PKC
MARCKS role in proliferation and cell morphology
PKCs many roles in proliferation,differentiation and death
Summary
PI3 kinases phosphorylate phosphoinositides at position 3
PTEN dephosphorylates phosphoinositides at position 3
p110 contains catalytic activity
p85 responsible for recruiting enzyme to RTK
PI-3,4,5-P3 recruits PDK1, PDK2 and Akt
PI-3,4,5-P3 recruits other proteins and regulates cytoskeleton and transport
Ligand-activated
RTK
PI3K and Akt Activation
P110 phosphorylates PIP2
PDK1 phosphorylates Akt
T308 (activation loop)
PI(3,4,5)P3
PI(4,5)P2
pS124
T308
p110
S473
pT450
PDK2 phosphorylates Akt
S473
pS124
pT308
pS473
pT450
p85
PDK1
p85 binds to
activated RTK
PDK2
Pleckstrin homology domain
S124
T308
Fully-activated Akt
Pre-activation of Akt
T450 phosphorylation
S473
Akt
T450
Kinase Domain
Akt, PDK1 and PDK2 all bind PIP3
(plekstrin homology domains)
Other PI-3,4,5-P3 Functions
Regulation of Vesicle Transport either…
• Binding to FYVE domain proteins
• Regulating small GTP-binding protein Arf
Rearrangement of actin cytoskeleton (rac)
Recruitment of Tyrosine kinases – PH domains in Btk
Enhancement of PLCg – direct interaction