Serine 77 in the PDZ Domain of PICK1 Is a Protein Kinase Cα Phosphorylation Site Regulated by Lipid Membrane Binding

PICK1 (protein interacting with C kinase 1) contains an N-terminal protein binding PDZ domain and a C-terminal lipid binding BAR domain. PICK1 plays a key role in several physiological processes, including synaptic plasticity. However, little is known about the cellular mechanisms governing the activity of PICK1 itself. Here we show that PICK1 is a substrate in vitro both for PKC alpha (protein kinase C alpha), as previously shown, and for CaMKII alpha (Ca2+-calmodulin-dependent protein kinase II alpha). By mutation of predicted phosphorylation sites, we identify Ser77 in the PDZ domain as a major phosphorylation site for PKC alpha. Mutation of Ser77 reduced the level of PKC alpha-mediated phosphorylation similar to 50%, whereas no reduction was observed upon mutation of seven other predicted sites. Addition of lipid vesicles increased the level of phosphorylation of Ser77 10-fold, indicating that lipid binding is critical for optimal phosphorylation. Binding of PKC alpha to the PICK1 PDZ domain was not required for phosphorylation, but a PDZ domain peptide ligand reduced the overall level of phosphorylation similar to 30%. The phosphomimic S77D reduced the extent of cytosolic clustering of eYFP-PICK1 in COS7 cells and thereby conceivably its lipid binding and/or polymerization capacity. We propose that PICK1 is phosphorylated at Ser77 by PKC alpha preferentially when bound to membrane vesicles and that this phosphorylation in turn modulates its cellular distribution.