Slp2-a inactivates ezrin by recruiting protein phosphatase 1 to the plasma membrane

• Slp2-a inactivates ezrin and regulates MDCK II cell size.
• Cytoplasmic signals of inactive ezrin are hardly detected in Slp2-a-KD cells.
• The Slp2-a linker domain interacts with protein phosphatase 1β.
• The Slp2-a−PP1β−Rap1GAP2 complex controls ezrin inactivation.

Synaptotagmin-like protein 2-a (Slp2-a) was originally described as a membrane trafficking protein that consists of a Slp homology domain (SHD), a linker domain, and tandem C2 domains (named the C2A domain and C2B domain). Slp2-a mediates docking of Rab27-bearing vesicles to the plasma membrane through simultaneous interaction with Rab27 and phospholipids in the plasma membrane. We have recently reported that Slp2-a regulates renal epithelial cell size through interaction with Rap1GAP2 via the C2B domain independently of Rab27 and demonstrated the presence of excess activation of ezrin, a membrane-cytoskeleton linker and signal transducer, in Slp2-a-knockdown Madin–Darby canine kidney II (MDCK II) cells. However, the precise mechanism of ezrin inactivation by Slp2-a in cell size control has remained largely unknown. In this study, we investigated the functional relationship between Slp2-a and ezrin in MDCK II cells. The results showed that activation of ezrin in control MDCK II cells either pharmacologically or by overexpression of a constitutively active ezrin mutant caused an increase in cell size, whereas inactivation of ezrin in Slp2-a-knockdown cells by a specific ezrin inhibitor restored them to their normal cell size. We also found that Slp2-a interacts via its previously uncharacterized linker domain with protein phosphatase 1β (PP1β), which inactivates ezrin, and that the interaction is required for the plasma membrane localization of PP1β. These results indicate that Slp2-a inactivates ezrin by recruiting PP1 to the plasma membrane.