y CRISPR-mediated gene targeting of CK1 delta/epsilon leads to enhanced understanding of their role in endocytosis via phosphoregulation of GAPVD1

Human casein kinase 1 delta (CK1 delta) and epsilon (CK1 epsilon) are members of a conserved family of abundant, ubiquitously expressed serine/threonine kinases that regulate multiple cellular processes including circadian rhythm and endocytosis. Here, we have investigated the localization and interactomes of endogenously tagged CK1 delta and CK1 epsilon during interphase and mitosis. CK1 delta and CK1 epsilon localize to centrosomes throughout the cell cycle, and in interphase cells to the nucleus, and in both a diffuse and punctate pattern in the cytoplasm. Also, for the first time, they were detected at the midbody during cell division. Mass spectrometry analysis identified a total of 181 proteins co-purifying with a Venus multifunctional (VM)-tagged CK1 delta and/or CK1 epsilon. GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1), a protein required for efficient endocytosis, was consistently one of the most abundant interacting partners. We demonstrate that GAPVD1 is a substrate of CK1 delta/epsilon with up to 38 phosphorylated residues in vitro and in vivo. Wildtype and a phosphomimetic mutant of GAPVD1, but not a phospho-ablating mutant, were able to rescue defects in transferrin and EGF internalization caused by loss of endogenous GAPVD1. Our results indicate that GAPVD1 is an important interacting partner and substrate of CK1 delta/epsilon for endocytosis.