Modification and movement: Phosphorylation and SUMOylation regulate endocytosis of GluK2-containing kainate receptors.

2012·0.62 MB·English

by  Wilkinson, Kevin A.

ARTICLEADDENDUM
Communicative&IntegrativeBiology5:2,223–226;March/April2012;G2012LandesBioscience
Modification and movement
Phosphorylation and SUMOylation regulate endocytosis of GluK2-containing
kainate receptors
Kevin A. Wilkinson,* Filip Konopacki and Jeremy M. Henley
MRCCentreforSynapticPlasticity;SchoolofBiochemistry;MedicalSciencesBuilding;UniversityofBristol;Bristol,UK
Kainate receptors (KARs) are tetra- confirmed as, SUMO substrates and
meric glutamate-gated ion chan- protein SUMOylation is strongly impli-
nels composed of combinations of the cated in a range of neurodegenerative
subunits GluK1-5. Depending on their disorders, suggesting a fundamental role
precise localization and subunit composi- for protein SUMOylation in regulating
tion,KARscanregulateneurotransmitter neuronal function2 and dysfunction.3,4
release, synaptic function and neuronal Ischemia and other forms of cell stress
excitability. Because of these diverse can enhance global protein SUMOyla-
roles,theregulatedandpreciselytargeted tion5-7buttheSUMOylationofindividual
© 2012 Landes Bioscience.
trafficking of KARs is of crucial impor- proteins is regulated in a substrate-specific
tance to neuronal function. We pre- manner. Therefore, a major unresolved
viously reported that the KAR subunit question for the majority of SUMO
Do not distribute.
GluK2 is post-translationally modified substrates is exactly how SUMOylation is
by attachment of Small Ubiquitin-like initiated and regulated. One way that this
Modifier 1 (SUMO-1) and that may be achieved is by the coordinated
SUMOylation is required for agonist- interplay between post-translational modi-
dependent endocytosis of GluK2. We fications at specific protein targets. For
recently extended these findings to example,SUMOylationcaninterferewith,
demonstrate that agonist activation or facilitate other lysine-based modifica-
leads to PKC-mediated phosphorylation tions such as ubiquitination or acetyla-
of GluK2 at serine 868, which directly tion, leading to complex cross-regulation
enhances GluK2 SUMOylation and, between these systems.7,8 Furthermore,
in turn, leads to endocytosis of the substratephosphorylationhasbeenreported
receptor. These new data demonstrate to either enhance or inhibit SUMOylation,
Keywords: posttranslational modification,
the importance of interplay between depending on the target protein.1
Protein kinase C, SUMOylation,
two post-translational modifications in As for most, if not all, other neuro-
kainate receptor, glutamate receptor,
orchestrating the temporal and spatial transmitter receptors, it is well-established
endocytosis
regulation of kainate receptor trafficking. that KAR function can be regulated by
Submitted: 12/22/11 phosphorylation.9-15 It is less clear, how-
ever, where and how direct phosphoryla-
Accepted: 12/22/11
The covalent attachment of a member tion of KAR subunits affects receptor
http://dx.doi.org/10.4161/cib.19195
of the SUMO family to lysine residues in trafficking, localization and function.
*Correspondenceto:KevinA.Wilkinson; target proteins occurs via an enzymatic We therefore investigated if GluK2 itself
Email:[email protected]
cascade analogous to, but distinct from, is phosphorylated following agonist-
that of the ubiquitin system. SUMOyla- stimulation and, if so, how this impacts
Addendumto:KonopackiFA,JaafariN,RoccaDL,
tion occurs via a three step pathway com- upon GluK2 SUMOylation.16 To do this
WilkinsonKA,ChamberlainS,RubinP,etal.
prising an E1, aheterodimer of SAE1 and we first confirmed that virally-expressed
Agonist-inducedPKCphosphorylationregulates
GluK2SUMOylationandkainatereceptor SAE2; a sole E2, Ubc9; and a growing GluK2 is robustly phosphorylated in
endocytosis.ProcNatlAcadSciUSA2011; number of identified E3 enzymes.1 neurons in response to kainate stimu-
108:19772–7;PMID:22089239;http://dx.doi.org/ Intriguingly, multiple synaptic proteins lation. This phosphorylation was reduced
10.1073/pnas.1111575108
have been proposed to be, or have been by the protein kinase C (PKC) inhibitor
www.landesbioscience.com Communicative&IntegrativeBiology 223
chelerythrine. In addition, in agreement phosphorylation of GluK2. Interestingly, other, as yet undefined, kinases also play a
with another recent study of GluK2 phos- some agonist-induced phosphorylation of role in regulating GluK2 trafficking. We
phorylation,17mutationofeitherserine846 GluK2 remained after incubation with next examined whether modulating PKC
or serine 868 also reduced kainate-evoked chelerythrine, raising the possibility that activity regulated GluK2 SUMOylation.
© 2012 Landes Bioscience.
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Figure1.SchematicillustratingtherolesofPKCphosphorylationandSUMOylationonthetraffickingofGluK2-containingKARs.Thetoppanelrepresents
themembranetopologyofGluK2,highlightingtheintracellularC-terminuswherePKCphosphorylationandSUMOylationoccur.Thesequenceofthis
regionisshownandthemodifiedaminoacidsindicated.AgonistactivationofGluK2leadstoPKC-mediatedphosphorylationatS868(aswellasS846,
notshown),whichdirectlyleadstoreceptorSUMOylationandendocytosis.Onceinternalized,receptorsaresubjecttopost-endocyticsortingeitherinto
recyclingpoolandexocytosedbacktotheneuronalplasmamembraneortheyaretargetedfordegradation.
224 Communicative&IntegrativeBiology Volume5Issue2
Consistent with SUMOylation being simulation of GluK2-containing KARs reportedtorecruittheSUMOE2enzyme
downstream of phosphorylation we found leads to activation of PKC and direct Ubc9, through the binding of a cognate
that activating PKC with phorbol ester phosphorylation of GluK2 at S846 and basic patch on Ubc9.18-20 While this
enhanced GluK2 SUMOylation. Taken S868. While the role of agonist-induced remains possible for GluK2, the PKC
together, these results indicate that PKC S846 phosphorylation remains to be fully phosphorylation site at S868 is relatively
phosphorylation is a requirement for elucidated, phosphorylation of S868 is distant from the SUMOylated lysine
agonist-induced SUMOylation of GluK2. required for the increased SUMOylation (K886) in comparison to other substrates
Importantly, we confirmed that this andconsequentendocytosisofthereceptor in which SUMOylation is increased
effectofPKCphosphorylationisdirectby (Fig.1). through phospho-mediated recruitment
demonstrating that in vitro phosphoryla- These findings shed new light on the of Ubc9. Thus, the direct mechanism of
tion of the C-terminus of GluK2 by regulatory mechanisms and complex the phosphorylation-mediated enhance-
PKC enhances its subsequent in vitro interactions involved in KAR trafficking. ment of GluK2 SUMOylation is another
SUMOylation.Phosphomimeticmutation This is important because the processes important question that awaits an answer.
of the PKC site S868 enhanced GluK2 that orchestrate KAR availability, local- While this study reaffirms the crucial role
SUMOylation in COS-7 cells whereas ization and function determine the res- of GluK2 SUMOylation in regulating
phosphomimetic mutation of S846 did ponsiveness and viability of neurons, and KAR endocytosis, perhaps the most press-
not, indicating that the facilitation of also profoundly influence neuronal net- ing outstanding question is how this
SUMOylation is site-specific. Thus, PKC work properties. More generally, our process works. SUMOylation may, for
phosphorylates both S846 and S868 but resultsconfirmandextendtheimportance example, recruit proteins of the endocytic
only phosphorylation of S868 acts to of the interplay between SUMOylation machinery,oracttodisrupttheanchoring
enhance receptor SUMOylation. Although and phosphorylation in dictating the of GluK2 at the neuronal membrane. We
S846has beenimplicatedintheregulation spatialandtemporalspecificityofsubstrate believe that future work aimed at directly
of basal GluK2 endocytosis17 it does not SUMOylation. addressingthesequestionswilldeepenour
affectagonist-inducedreceptorendocytosis.16 Of course, many intriguing questions understanding of both physiological and
© 2012 Landes Bioscience.
In reciprocal experiments, S868 was remain to be addressed. For example, pathophysiological KAR trafficking and
mutated to a non-phosphorylatable alanine kainate application causes activation of function, and could reveal potential for
residue. Following expression in neurons PKC but exactly how this occurs is eitherKARsortheSUMOylationpathway
Do not distribute.
wild-type GluK2 was SUMOylated in unclear. Further, while we have demon- as therapeutic targets in a number of
response to kainate, however this effect strated that PKC phosphorylation of disorders of the nervous system.
was lost for the S868A mutant. These GluK2 enhances SUMOylation in vitro,
results confirm that prior PKC phosphory- the underlying mechanism remains to Acknowledgments
lation at S868 is necessary for receptor be determined. The in vitro assay con- We are grateful to the MRC, BBSRC,
SUMOylation in response to kainate. tains only the E1 and E2 SUMOylation Wellcome Trust and ERC for financial
Further, in contrast to wild-type GluK2, enzymes, suggesting that phosphorylation support and to all of the authors on the
the S868 mutant did not undergo agonist- likely acts to recruit one (or both) of Proceedings of the National Academy of
induced endocytosis. Taken together, our these components to GluK2. Substrate Sciences of the United States of America
findings suggest a model whereby agonist phosphorylation has previously been paper for their invaluable contributions.
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