THE IMPORTANCE OF S-PALMITOYLATION IN CELL SIGNALLING … · THE IMPORTANCE OF S-PALMITOYLATION IN...
Transcript of THE IMPORTANCE OF S-PALMITOYLATION IN CELL SIGNALLING … · THE IMPORTANCE OF S-PALMITOYLATION IN...
THE IMPORTANCE OF S-PALMITOYLATION IN CELL SIGNALLING
INTRODUCTION Post-translational fatty-acid acylation of some receptors and signalling molecules is a
transient modification that alters the interaction of these signalling proteins with cell
membranes. It provokes the relocation of specific individual proteins to the plasma
membrane, controls their residence time in the plasma membrane, and their activity
often by regulating the macromolecular complexes they can form. The most
common lipid modification is S-palmitoylation1, which is the addition of palmitic acid
to the sulphur atom in the side chain of a cysteine residue, through a thioester bond.
Figure 1. A simple animation to show the thioester bond
between palmitic acid and a sulphur atom in
the side chain of a cysteine residue
What is S-palmitoylation? S-palmitoylation is a reversible post-translational protein modification process, whereby palmitic acid forms a thioester bond with the sulphur atom of the cysteine residue of a protein. How is this relevant in cell signaling? S-palmitoylation is involved in efficient trafficking and anchoring of multiple proteins to the plasma membrane amongst other targets. These include GPCRs, G-proteins, eNOS and multiple kinases. S-palmitoylation acts as a ‘switch’ to regulate receptor expression, intracellular protein activity and retrograde signals in neurons. What if this applies to my research? The CAPTUREome™ S-Palmitoylated Protein Kit can be used to determine whether this fatty acid modification is occurring in your experimental systems.
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Summary
KEY POINTS
u Regulate receptor expression on the plasma membrane
u Regulate the activity of intracellular signalling proteins
u Regulate protein kinase-dependent actin dynamics
u Relay long-distance retrograde signals in neurons
S-palmitoylation acts as a switch to…
1. Regulate receptor expression on the plasma membrane The modification of receptors by S-palmitoylation in the Endoplasmic Reticulum (ER)
and Golgi apparatus (GA) initiates the trafficking of receptors to the plasma
membrane. Removal of these palmitate groups at the plasma membrane often
leads to internalisation of the protein3. This cycle of S-palmitoylation-
depalmitoylation of certain receptors causes a switch from high-level to low-level
cell signalling.
One good example of this is the S-palmitoylation of G-protein coupled receptors
(GPCRs), a process thought to be mediated by zinc finger-DHHC type proteins. These
include ZDHHC-5 that has been shown to S-palmitoylate somatostatin receptor
subtype 54. Research shows S-palmitoylation of GPCRs leads to efficient trafficking
of the receptors to the plasma membrane. When S-palmitoylation of the δ opioid
receptor was inhibited, cell surface expression of the GPCR fell by 61.4%; suggesting
S-palmitoylation contributes to, but is not absolutely necessary for, the targeting of
GPCRs to the membrane and/or trafficking vesicles5. 2
A unique feature of S-palmitoylation is the reversibility of the modification, causing it
to act as a switch that regulates cell signalling by altering membrane localisation,
subcellular trafficking and the binding capacity of different proteins2. Extensive
research has elucidated the regulatory roles of S-palmitoylation throughout the body,
with a focus on transmembrane proteins and signalling molecules.
Additionally, data from the chemokine receptor CCR56 and the A1 adenosine
receptor6 suggest many GPCRs that do not undergo S-palmitoylation, are
degraded. Some hypothesise this may be due to a misfolding of the protein due to
free cysteine sulphydryl group(s)3. This leads to a fall in concentration of
membrane-bound GPCRs, and a decrease in cell signalling.
Another feature of S-palmitoylation is its ability to mediate the level of GPCR
phosphorylation by PKA, and therefore internalisation of the receptor. A non-S-
palmitoylated β2-adrenergic receptor mutant showed phosphorylation levels ~4-
times that of wild type (WT), with the authors suggesting depalmitoylation of the
receptor exposes additional phosphorylation sites8. Increased levels of
phosphorylation may lead to internalisation and/or desensitisation of GPCRs,
resulting in a reduced level of cell signalling3.
2. Regulate the activity of intracellular signalling proteins Evidence suggests S-palmitoylation regulates the activity of some signalling proteins,
for example endothelial nitric oxide synthase (eNOS). eNos is dually acylated, first
with the co-translational addition of a myristoyl group at the N-terminal (glycine-2),
followed by the post-translational addition of two palmitate groups at cysteines 15
and 269. Five DHHC domain proteins have been associated with eNOS S-
palmitoylation: DHHC-2, 3, 7, 8, and 21; significantly DHHC-21 co-localises with eNOS
in vascular endothelial cells (the principal site of eNOS activity)10.
Not only does S-palmitoylation regulate GPCRs, but it
also exhibits effects on the G-proteins themselves. All
Gα subunits, excluding transducin, have been shown to
be S-palmitoylated at the Golgi apparatus. This fatty
acid modification, in combination with myristoylation
and βγ association, is necessary for the targeting and
binding of the α-subunit to the plasma membrane3.
Research suggests S-palmitoylation also increases the
affinity of the α-subunit for the βγ-complex, which in
turn regulates the activity of the α-subunit and the
intracellular signalling pathway3.
What about G-proteins?
?
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Firstly, co-translational myristoylation and S-palmitoylation of eNOS at the Golgi
apparatus causes the enzyme to be trafficked to caveolae11,12, a similar mechanism
to that of aforementioned GPCRs. A non-S-palmitoylated eNOS mutant exhibited
deficient localisation at the plasma membrane, with a significant increase in
concentration at the Golgi apparatus and cytoplasm11. Additionally, each acylation
process caused a 10-fold increase in trafficking of eNOS to cavolae, suggesting both
modifications are essential for optimal membrane targeting12.
Furthermore, myristoylation followed by S-palmitoylation increases the affinity of
eNOS for the caveolae membrane13. Myristoylation results in hydrophic interactions
between eNOS and the lipid membrane, however these interactions were found to
be readily reversible14 and consequently would need stabilising, potentially by the
two palmitate groups. This supposition is supported by data that indicate the loss of
two palmitate groups causes a 2-fold decrease in membrane-bound eNOS, with an
additional loss of the myristoyl group removing any remaining eNOS-membrane
associations13.
Finally, S-palmitoylation contributes to the subcellular trafficking and membrane
localisation of eNOS, which results in regulation of the enzyme via caveolin-1 (Cav1)
and calmodulin (CaM). eNOS activity, that mediates the conversion of L-arginine to
NO and L-citrulline10, was undetectable in plasma membrane fractions that did not
contain caveolae. Furthermore, eNOS activity was 9-fold greater in caveolae in
relation to the whole plasma membrane15. This is due to signalling proteins (Cav1,
CaM), which contribute to the acute regulation of eNOS, also residing within
caveolae. These include Cav1, which causes a direct, reversible inhibition of eNOS,
due to the disruption of electron flow that prevents the production of NO. Inhibition
is relieved in the presence of excess CaM, causing electron transport to resume and
subsequently NO synthesis11. If eNOS does not undergo S-palmitoylation, it will not
be trafficked and anchored to the cavealae membrane, and therefore cannot be
regulated.
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Figure 2. A simplified schematic
showing the regulation
of dually acylated eNOS
by Cav1 and CaM
3. Regulates protein kinase-dependent actin dynamics There are over 500 protein kinases in the human genome and to date 20 have been
reported to be S-palmitoylated16. This includes LIM kinase-1 (LIMK1), an actin-
binding kinase that inactivates cofilin and subsequently promotes actin
polymerisation. S-palmitoylation occurs near the N-terminus of LIMK1 at cysteines
7 and 8 and contributes to the regulation of LIMK1-dependent cytoskeletal
dynamics in dendritic spines17.
S-palmitoylation is necessary for the targeting of LIMK1 to dendritic spines, shown
by a significant reduction in localisation of a non-S-palmitoylated LIMK1 mutant17.
S-palmitoylation also contributes to actin turnover in spines and activity-dependent
spine enlargement. LIMK1 knock down in hippocampal neurons led to a reduction in
actin turnover and activity-dependent spine enlargement. These phenotypes could
be rescued by WT LIMK1, but not a non-S-palmitoylated mutant17.
In addition, S-palmitoylation is essential for LIMK1 activation, via its upstream
kinase: p21-activated kinase-3 (PAK3), in vivo. When tested in vitro mutation of
cysteines 7 and 8 had no significant effect on the activation or activity of LIMK1.
However, when tested within neurons, the loss of palmitoyl-LIMK1 led to a 10-fold
decrease in phosphorylation by PAK317. This may be because the non-S-
palmitoylated LIMK1 mutant cannot co-localise with PAK3 on the dendritic spine
membrane.
Abnormal spine morphology is associated with various cognitive disabilities, such as
Autism Spectrum Disorder or schiozphrenia. These data indicate that S-
palmitoylation of LIMK1 is involved in the regulation of neuronal actin dynamics and
therefore impairments in S-palmitoylation may lead to various neurological
disorders.
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Figure 3. A simplified schematic to show LIMK1
localisation to the plasma membrane by
S - p a l m i t o y l a t i o n . H e r e i t i s
phosphorylated by PAK, and in turn
phosphorylates coffilin. Activated cofilin
then acts to reassemble actin filaments.
4. Relay long-distance retrograde signals in neurons Dual leucine-zipper kinase (DLK) is another kinase that is S-palmitoylated, leading to
the facilitation of the enzyme’s retrograde signalling. One role for DLK is to mediate
signalling from the axon to the soma in response to peripheral nerve injury, which is
essential for both axon degeneration and regeneration17. Activation of DLK in
peripheral axons sequentially activates the c-Jun N-terminal Kinase (JNK)18. This
results in the phosphorylation of the transcription factor c-Jun (p-c-Jun). However,
DLK does not alter basal physiological JNK activity19 and therefore a mechanism by
which DLK propagates these retrograde signals was largely unknown…until now!
Holland et al. (2016) concluded that S-palmitoylation exerts three distinct effects on
DLK-dependent signalling: enzyme location, macromolecular complex formation
and enzyme activity20.
Firstly, S-palmitoylation targets DLK to axonal trafficking vesicles, providing an
explanation as to how the soluble enzyme propagates long-range signals. Using
fluorescent tagging, DLK was shown to accumulate in the axonal puncta in cultured
sensory neurons, whilst a non-S-palmitoylated mutant form of DLK was distributed
throughout the axon20. Therefore, at a cellular level S-palmitoylation contributes to
the localisation of the enzyme, by targeting palmitoyl-DLK to axonal trafficking
vesicles, resulting in a retrograde signal.
Secondly, S-palmitoylation is essential for oligomerisation with MAP2K4, MAP2K7
and JNK-interacting Protein-3 (JIP3). Mutating the DLK S-palmitoylation site led to a
disruption of DLK association with JIP3, MAP2K4 and MAP2K7, but did not alter DLK-
DLK homodimerisation20. Preceding evidence suggests JIP3 is involved in dynein-
based retrograde transport of JNK and lysosomes21. Together these data suggest S-
palmitoylation facilitates the formation of multiprotein complexes that bind to
microtubules, aiding retrograde transport.
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Figure 4. A simplified model to show
axonal retograde signaling by
p a l m i t o y l a t e d D L K - J N K
pathway kinases
Thirdly, S-palmitoylation may regulate DLK activity. Non-S-palmitoylated mutant
DLK showed significantly reduced phosphorylation of JNK3 and downstream
MAP2Ks20. These data indicate that S-palmitoylation contributes to DLK activity,
possibly by preventing an auto-inhibitory interaction of the acidic N-terminal
domain with the basic kinase domain20.
Looking towards the future, these findings point towards an exciting therapeutic
target in palmitoyl-DLK. Molecules that inhibit S-palmitoylation could be used to
reduce DLK-mediated neurodegeneration and conversely, molecules that inhibit
depalmitoylation enzymes could be a target for DLK-mediated neuronal
regeneration20. Additionally, further research addressing which other MAP3Ks
contain S-palmitoylation sites and consequently the role of S-palmitoylation on
these kinases may be of interest.
Author: Eleanor Eisenstadt
References:
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