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The phosphatase activity of the PPP2R5D-PP2A holoenzyme modulates liquid-liquid phase separation of the scaffold protein liprin-α1

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Rita Derua

LAB HEAD: Veerle Janssens

PROVIDER: PXD063716 | Pride | 2025-06-23

REPOSITORIES: Pride

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			Action	DRS
	202112_26_52AMFileSize_731607744ByteF040172.mzid	Mzid
	202112_26_52AMFileSize_731607744ByteF040172.mzid_202112_26_52AMFileSize_731607744ByteF040172.MGF	Mzid
	20216_16_29PMFileSize_734036072ByteF040170.mzid	Mzid
	20216_16_29PMFileSize_734036072ByteF040170.mzid_20216_16_29PMFileSize_734036072ByteF040170.MGF	Mzid
	20216_37_13AMFileSize_729458888ByteF040174.mzid	Mzid

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Project description:Background: Acute respiratory distress syndrome (ARDS) is characterized by refractory hypoxemia caused by accumulation of pulmonary fluid, which is related to inflammatory cell infiltration, impaired tight junction of pulmonary epithelium and impaired Na, K-ATPase function, especially Na, K-ATPase α1 subunit. Up until now, the pathogenic mechanism at the level of protein during lipopolysaccharide- (LPS-) induced ARDS remains unclear. Methods: Using an unbiased, discovery and quantitative proteomic approach, we discovered the differentially expressed proteins binding to Na, K-ATPase α1 between LPS-A549 cells and Control-A549 cells. These Na, K-ATPase α1 interacting proteins were screened by co-immunoprecipitation (Co-IP) technology. Among them, some of the differentially expressed proteins with significant performance were identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The protein interaction network was constructed by the related Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Several differentially expressed proteins were validated by Western blot. Results: Of identified 1598 proteins, 89 were differentially expressed proteins between LPS-A549 cells and Control-A549 cells. Intriguingly, protein-protein interaction network showed that there were 244 significantly enriched co-expression among 60 proteins in the group control-A549. while the group LPS-A549 showed 43 significant enriched interactions among 29 proteins. The related GO and KEGG analysis found evident phenomena of ubiquitination and deubiquitination, as well as the pathways related to autophagy. Among proteins with rich abundance, there were several intriguing ones, including the deubiquitinase (OTUB1), the tight junction protein zonula occludens-1 (ZO-1), the scaffold protein in CUL4B-RING ubiquitin ligase (CRL4B) complexes (CUL4B) and the autophagy-related protein sequestosome-1 (SQSTM1). Conclusions: In conclusion, our proteomic approach revealed targets related to the occurrence and development of ARDS, being the first study to investigate significant differences in Na, K-ATPase α1 interacting proteins between LPS-induced ARDS cell model and control-A549 cell. These proteins may help the clinical diagnosis and facilitate the personalized treatment of ARDS.

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The phosphatase activity of the PPP2R5D-PP2A holoenzyme modulates liquid-liquid phase separation of the scaffold protein liprin-α1

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