Phosphorylation-dependent interaction between VAP proteins and INF2 influences ER morphology

Abstract

Summary The endoplasmic reticulum (ER) interacts with virtually all other cellular organelles, and major players mediating these interactions are the ER-bound VAP proteins VAPA and VAPB. Here we show that VAP proteins interact with the actin polymerization factor INF2 in a phosphorylation-dependent manner. Similar to many other VAP-interacting proteins (the ‘VAPome’), an FFAT motif in INF2’s C-terminus interacts with the MSP domain of VAPs. Phosphorylation of a serine within the FFAT (S1100 in mouse, S1077 in human INF2) is necessary for high-affinity interaction both in cells and with purified proteins. The position of this phosphoserine, at position -1 of the FFAT consensus, is novel to the VAPome. Biochemical assays show that the phospho-FFAT binds both VAPA and VAPB, but not the related VAP family protein MOSPD2. Increased cytoplasmic calcium stimulates both INF2 phosphorylation and the INF2/VAP interaction. ALS-associated mutations in the MSP disrupt the INF2/VAP interaction. Both major INF2 isoforms interact with VAPs: the INF2-CAAX isoform, which is constitutively ER-bound; and the INF2-nonCAAX isoform, which is predominately cytosolic. For INF2-nonCAAX, VAP proteins cause ER recruitment in a phosphorylation-dependent manner. Disruption of INF2/VAP binding does not affect INF2-mediated actin polymerization, but has a clear effect on ER morphology, causing the tubule/sheet balance to shift toward sheets. Cross-species evaluation suggests that only INF2 from placental mammals possesses an FFAT. These results suggest that interaction between VAP proteins and the actin polymerization factor INF2 plays a role in mediating ER morphology.