PXD063716-1

PXD063716 is an original dataset announced via ProteomeXchange.

Title	The phosphatase activity of the PPP2R5D-PP2A holoenzyme modulates liquid-liquid phase separation of the scaffold protein liprin-α1
Description	Liprin-α1 is a widely expressed scaffolding protein that regulates cellular processes such as cell motility and synaptic transmission through assembly of localized higher-order molecular complexes. The dynamic regulation of these complexes remains poorly understood. Liquid-liquid phase separation (LLPS) is a process that concentrates proteins into cellular nanodomains, facilitating efficient spatiotemporal signaling. Whether liprin-α1 undergoes regulated LLPS remains unclear. MS-based interactomics identified PPP2R5D, the regulatory B56δ subunit of PP2A, as a liprin-α1 interaction partner through a canonical short linear motif (SLiM) in its N-terminal dimerization domain. Mutation of SLiM4 nearly abolished liprin-α1 interaction with PP2A holoenzyme and resulted in a significant increase in GFP-liprin-α1 LLPS in HEK293 cells. Consistently, GFP-liprin-α1 exhibited increased droplet formation in PPP2R5D knockout HEK293 cells. Phospho-analysis of liprin-α1 SLiM4 mutant via MS revealed increased phosphorylation of multiple Ser/Thr sites, including S763, as validated by a novel phospho-specific antibody. A liprin-α1 S763E phospho-mimetic mutant appeared sufficient to drive LLPS. Expression of the PPP2R5D missense variant E420K, recurrently found in Houge-Janssens Syndrome Type 1 compromised suppression of liprin-α1 LLPS, correlating with increased liprin-α1 S763 phosphorylation. Mechanistically, a liprin-α1 E942A mutant unable to bind liprin-β1 underwent increased LLPS, despite preserved PPP2R5D holoenzyme binding. Furthermore, liprin-α1/β1 heterodimerization significantly decreased under conditions where liprin-α1 LLPS was promoted, i.e. upon SLiM4 or S763E mutation in wild type cells, or in PPP2R5D knockout and PPP2R5D E420K knock-in cells. Our findings identify both liprin-β1 and PPP2R5D-PP2A as potent inhibitors of liprin-α1 LLPS, with PP2A contributing to liprin-α1/β1 heterodimerization via phosphorylation of at least liprin-α1 S763.
HostingRepository	PRIDE
AnnounceDate	2025-06-23
AnnouncementXML	Submission_2025-06-23_13:10:49.519.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD063716
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Rita Derua
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2025-05-07 02:44:14	ID requested
⏵ 1	2025-06-23 13:10:49	announced

10.6019/PXD063716;

10.1016/J.JBC.2025.110349;

submitter keyword: protein phosphatase 2A (PP2A)

fluorescence recovery after photobleaching (FRAP)

mass spectrometry (MS)

phosphorylation

liprin-α1

liprin-β1

intrinsically disordered protein domain

phase separation

Houge-Janssens syndrome (HJS)

Veerle Janssens
contact affiliation	Department of Cellular and Molecular Medicine, KU Leuven, Belgium
contact email	veerle.janssens@kuleuven.be
lab head
Rita Derua
contact affiliation	KU Leuven
contact email	rita.derua@kuleuven.be
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD063716
     1. Label: PRIDE project
     2. Name: The phosphatase activity of the PPP2R5D-PP2A holoenzyme modulates liquid-liquid phase separation of the scaffold protein liprin-α1