protein mod and cell regulation 2 Flashcards
what are the 4 phospholipid heads and there relative charges
phosphatidylserine negative
phosphatidylinositol negative
phosphatidylcholine neutral
phosphatidylethanolamine neutral
what can happen to theses head groups if phosphorylated
become signalling molecules
the internal monolayer of the plasma membrane is -ve because
of anionic phospholipids eg PS
external monolayer is normally neutral because
it is composed of zwitterionic phospholipids
PC, PE and sphingomyelin
distribution of the 2 leaflets is maintained by
flipases
why is phospholipid asymmetry important
primary event in apoptosis is PS exposure - facilitates blood clotting
anionic surface
why is phospholipid asymmetry important
bacteria
they have anionic membranes
different from ours
we produced selective cationic antibacterial proteins taht affects them and not us
types of phospholipases
PLA1
PLA2- will relase arachidonic acid
PLC releases DAG and IP3
PLD releases phosphatidic acid
key enzymes when discussing inflammation is
cytosolic PLA2
cytosolic PLA2 is specific for
arachidonic acid at position 2 (sn-2)
CPLA2 is a
cytosolic enzyme but translocates to membranes in the presence of (micro)M ca2+
cPLA2 has 2 domains which are
N terminal C2 domain
ca2+ dependent phospholipid binding domain
c terminal domain containing the active site
the cPLA2 enzyme is also a substrate for
activates the enzyme in vitro
MAP kinase
which phosphorylates at ser - 505
the cPLA2 enzyme in cell culture is activated by
pro-inflammatory cytokines
eg interleukin 1
tumour necrosis factor and mitogens
MAP kinase is also stimulated by
A23187 - ca ionophore
which increases cells [ca]
ser-505 needs to be phosphorylated because
it goes into the membrane allowing the active site to open
the entire activity of the Ca2+ binding is in
the C2 domain
effect of binding Ca2+ to C2 domain is
it causes confomational change in the 3 Ca2+ binding loops
they become more hydrophobic
2 of the loops that are now hydrphobic can
penetrate the bilayer providing a stable membrane protein interaction
prefer to bind to neutral membranes
is phosphorylation important for activity
normal gene + s505A mutant
serine mutated to alanine
confirmed translocation with A23187 but failed to comoplete stimulation of arachidonic acid release
what is believed that phosphorylation does
produces correct alignment of 2 domains on the membrane surface to produce maximal activity
site of cPLA2 translocation
treated cells with A23187 translocate due to
and how do we know
increased [ca]
fixed cells and treat with rabbit ab to cPLA2
with fluorescent goat ab for rabbit igG
where is it translocated
to the perinuclear membrane
no translocation in control as they were not made Ca2+ permeable
COX is found in the same location within the cell
cox is the next enzyme in the formation of prostaglandin
c2 domains target specific membranes which are
PC rich membranes - neutral - perinuclear membrane
plasma membrane is rich in PS
PKC also has a C2 domain but when ca binds it targets anionic PL found in the plasma membrane
how is specific translocation achieved
A site specific docking or adaptor protein is required
enzyme colocalizes with COX-2
