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

The endoplasmic reticulum (ER) interacts with virtually all other cellular organelles, and major players mediating these interactions are the ER-bound VAMP-associated protein (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 major sperm protein (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. Amyotrophic lateral sclerosis (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 predominantly 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.