Investigating the cellular functions of casein kinase 1 δ and ε through identification of interacting partners and substrates
Human casein kinase 1 delta (CK1δ) and epsilon (CK1ε) are members of a conserved family of ubiquitously expressed serine/threonine kinases that regulate multiple cellular processes including endocytosis, circadian rhythm, and ribosome maturation. We sought to investigate the molecular mechanisms by which CK1δ/ε modulate these processes by determining their interacting partners and substrates. As observed previously in fixed cells with indirect immunofluorescent microscopy, CK1δ and CK1ε endogenously fused with a fluorescent tag localize to the centrosome, nucleus and cytoplasm and were newly detected at the midbody. Multifunctional affinity purification (MAP)-tagged CK1δ and CK1ε were used as bait to purify and identify associated proteins by mass spectrometry from asynchronous and mitotic cell populations. Mitotic substrates of CK1δ and CK1ε were identified using quantitative mass spectrometry. GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1), a protein required for efficient transferrin internalization, was demonstrated to be a substrate of CK1δ/ε in vitro and in vivo, with 38 phosphorylation sites located in GAPVD1’s unstructured region. A GAPVD1 mutant unable to be phosphorylated by CK1δ/ε is unable to rescue defects in transferrin internalization caused by loss of endogenous GAPVD1 function, indicating GAPVD1 is a key interacting partner and substrate of CK1δ/ε in endocytosis. Multiple new mitotic proteins were identified as putative CK1δ/ε substrates, including CK1δ itself. Further, several mitotic defects were associated with inhibition of CK1δ/ε. The results found in this dissertation provide several new avenues of research into the mitotic functions of CK1δ/ε.