Phosphorylation of the Amphipathic Helix Changes the Lipid Binding Capacities of PICK1

PICK1 (Protein Interacting with C-kinase 1) is a functionally important protein, which is distributed mainly in testis, pancreas and brain. It has been shown to play a central role in regulation of dense core vesicles from the golgi apparatus and trafficking of ionotropic glutamate receptors.PICK1 contains a N-terminal PDZ-domain, which we have earlier demonstrated to be important for interaction with a large number of proteins, including several important receptors and transporters. In addition, it has a BAR (Bin/Amphiphysin/Rvs) domain in the C-terminal end. BAR domains are generally believed to either recognize or induce curvatures of lipid membranes, but as we have demonstrated, proteins of the N-BAR family (incl. PICK1) binds lipids and recognizes membrane curvature (MC) through an associated amphipathic helix (AH) rather than through the BAR domain itself.Here we show that the lipid binding AH of PICK1 contains a phosphorylation-site, which, through PKC activation, is responsible for an altered cellular distribution of PICK1. To investigate whether the altered cellular distribution results directly from a change in the lipid binding capacities of the AH, we employ a Single Liposome Curvature Sensing (SLiC) assay. We use quantitivative fluorescence microscopy to evaluate the binding of the phospho-mimicking mutants to nanosized liposomes in terms of MC-sensing, lipid affinity and membrane deformation.Intriguingly, we find that this single phospho-mimicking mutation in the AH is sufficient to change the lipid binding capacities of the entire protein, likely causing the altered cellular distribution of the phosphorylated protein seen in the cells. As MC-sensing has been shown to be dependent on the AH of N-BAR proteins in general, we speculate that the finding may apply generally to phospho-regulation of N-BAR proteins.