PXD033488-2

PXD033488 is an original dataset announced via ProteomeXchange.

Title	Human RIPK3 C-lobe phosphorylation is essential for necroptotic signaling
Description	Necroptosis 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.
HostingRepository	PRIDE
AnnounceDate	2023-11-14
AnnouncementXML	Submission_2023-11-14_08:56:05.943.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Jarrod Sandow
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue
Instrument	timsTOF Pro

Revision	Datetime	Status	ChangeLog Entry
0	2022-04-28 00:25:42	ID requested
1	2022-07-30 01:31:56	announced
⏵ 2	2023-11-14 08:56:06	announced	2023-11-14: Updated project metadata.

Dataset with its publication pending

submitter keyword: TNF, programmed necrosis,Pseudokinase, protein kinase, RIPK1

James Murphy
contact affiliation	Division Head, WEHI
contact email	jamesm@wehi.edu.au
lab head
Jarrod Sandow
contact affiliation	WEHI
contact email	sandow@wehi.edu.au
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD033488
     1. Label: PRIDE project
     2. Name: Human RIPK3 C-lobe phosphorylation is essential for necroptotic signaling