Abstract
Human casein kinase 1 delta (CK1δ) and epsilon (CK1ε) 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 CK1δ and CK1ε during interphase and mitosis. CK1δ and CK1ε 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 CK1δ and/or CK1ε. 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 CK1δ/ε 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 CK1δ/ε for endocytosis.
Highlights
Human casein kinase 1 delta (CK1δ) and epsilon (CK1ε) are members of a conserved family of abundant, ubiquitously expressed serine/threonine kinases that regulate multiple cellular processes including circadian rhythm and endocytosis
We verified that all alleles in the selected clones had been modified to produce CK1δ-Venus multifunctional (VM), CK1ε-VM, CK1δ-mNG, or CK1ε-mNG by PCR amplifications of 1000 base-pair regions flanking the insert sites of VM or mNG (Supplementary Fig. 1B)
In addition to identifying known interacting partners involved in biological rhythms, such as Period protein 1 and 213,15,45,46, we identified additional interacting proteins involved in other biological processes that CK1δ/ε are involved in, such as endocytosis
Summary
Human casein kinase 1 delta (CK1δ) and epsilon (CK1ε) are members of a conserved family of abundant, ubiquitously expressed serine/threonine kinases that regulate multiple cellular processes including circadian rhythm and endocytosis. Though the four soluble CK1s are highly related, CK1δ and CK1ε (hereafter referred to as CK1δ/ε) have the greatest degree of sequence identity within their kinase domains, function in similar biological pathways, and share interacting partners and substrates[1,8,9,10,11,12,13,14]. An association between GAPVD1 and CK1δ/ε was identified previously, through affinity purifications and MS analysis[13,14], but the functional relevance of this interaction has not been previously reported
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