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PXD033488-2

PXD033488 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleHuman RIPK3 C-lobe phosphorylation is essential for necroptotic signaling
DescriptionNecroptosis is a caspase-independent, pro-inflammatory mode of programmed cell death which relies on the activation of the terminal effector, MLKL, by the upstream protein kinase RIPK3. To mediate necroptosis, RIPK3 must stably interact with, and phosphorylate the pseudokinase domain of MLKL. While the precise molecular cue that prompts RIPK3 to activate MLKL is incompletely understood, it is known that RIPK3 is highly regulated by phosphorylation. Here, we sought to identify phosphorylation sites of RIPK3 and dissect their regulatory functions. Phosphoproteomics identified 21 phosphorylation sites in HT29 cells overexpressing human RIPK3. By comparing cells expressing wild-type and kinase-inactive D142N RIPK3, autophosphorylation sites and substrates of other cellular kinases were distinguished. Of these 21 phosphosites, extensive mutational analyses identified only pT224 and pS227 as crucial, synergistic sites for stable interaction with MLKL to promote necroptosis, while the recently reported activation loop phosphorylation at S164/T165 negatively regulate the kinase activity of RIPK3. Despite being able to phosphorylate MLKL to a similar or higher extent than the wild-type RIPK3, mutation of T224, S227, and RHIM in RIPK3 attenuated or compromised their abilities to mediate necroptosis. This finding highlights the stable recruitment of human MLKL by RIPK3 to the necrosome as an essential checkpoint in necroptosis signaling, which is independent from and precedes the phosphorylation of MLKL.
HostingRepositoryPRIDE
AnnounceDate2023-11-14
AnnouncementXMLSubmission_2023-11-14_08:56:05.943.xml
DigitalObjectIdentifier
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportUnsupported dataset by repository
PrimarySubmitterJarrod Sandow
SpeciesList scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationListphosphorylated residue
InstrumenttimsTOF Pro
Dataset History
RevisionDatetimeStatusChangeLog Entry
02022-04-28 00:25:42ID requested
12022-07-30 01:31:56announced
22023-11-14 08:56:06announced2023-11-14: Updated project metadata.
Publication List
Dataset with its publication pending
Keyword List
submitter keyword: TNF, programmed necrosis,Pseudokinase, protein kinase, RIPK1
Contact List
James Murphy
contact affiliationDivision Head, WEHI
contact emailjamesm@wehi.edu.au
lab head
Jarrod Sandow
contact affiliationWEHI
contact emailsandow@wehi.edu.au
dataset submitter
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Dataset FTP location
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