Honkanen, R. | Search bioRxiv

bioRxiv2024

The role of liprin-α1 phosphorylation in its liquid-liquid phase separation: regulation by PPP2R5D/PP2A holoenzyme

Liprin-1 is a widely expressed scaffolding protein responsible for regulating cellular processes such as focal adhesion, cell motility, and synaptic transmission. Liprin-1 interacts with many proteins including ELKS, GIT1, liprin-{beta}, and LAR-family receptor tyrosine protein phosphatase. Through these protein-protein interactions, liprin-1 assembles large higher-order molecular complexes; however, the regulation of this complex assembly/disassembly is unknown. Liquid-liquid phase separation (LLPS) is a process that concentrates proteins within cellular nano-domains to facilitate efficient spatiotemporal signaling in response to signaling cascades. While there is no report that liprin-1 spontaneously undergoes LLPS, we found that GFP-liprin-1 expressed in HEK293 cells occasionally forms droplet-like condensates. MS-based interactomics identified Protein Phosphatase 2A (PP2A)/B56{delta} (PPP2R5D) trimers as specific interaction partners of liprin-1 through a canonical Short Linear Interaction Motif (SLiM) in its N-terminal dimerization domain. Mutation of this SLiM nearly abolished PP2A interaction, and resulted in significantly increased LLPS. GFP-liprin-1 showed significantly increased droplet formation in HEK293 cells devoid of B56{delta} (PPP2R5D knockout), suggesting that PPP2R5D/PP2A holoenzyme inhibits liprin-1 LLPS. Guided by reported liprin-1 Ser/Thr phosphorylation sites, we found liprin-1 phospho-mimetic mutant at serine 763 (S763E) is sufficient to drive its LLPS. Domain mapping studies of liprin-1 indicated that the intrinsically disordered region, the N-terminal dimerization domain, and the SAM domains are all necessary for liprin-1 LLPS. Finally, expression of p.E420K, a human PPP2R5D variant causing Houge-Janssens Syndrome type 1 (also known as Jordans Syndrome), significantly compromised suppression of liprin-1 LLPS. Our work identified B56{delta}-PP2A holoenzyme as an inhibitor of liprin-1 LLPS via regulation at multiple phosphorylation sites.

neuroscience↗

bioRxiv2023

Extended regulation interface coupled to the allosteric network and disease mutations in the PP2A-B56 delta holoenzyme

An increasing number of mutations associated with devastating human diseases are diagnosed by whole-genome/exon sequencing. Recurrent de novo missense mutations have been discovered in B56{delta} (encoded by PPP2R5D), a regulatory subunit of protein phosphatase 2A (PP2A), that cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Single-particle cryo-EM structures show that the PP2A-B56{delta} holoenzyme possesses closed latent and open active forms. In the closed form, the long, disordered arms of B56{delta} termini fold against each other and the holoenzyme core, establishing dual autoinhibition of the phosphatase active site and the substrate-binding protein groove. The resulting interface spans over 190 [A] and harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is close to an allosteric network responsive to activation phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations perturb the activation phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the wild variant.

biochemistry↗