Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Apr 22;10(1):6797.
doi: 10.1038/s41598-020-63669-2.

CRISPR-mediated gene targeting of CK1δ/ε leads to enhanced understanding of their role in endocytosis via phosphoregulation of GAPVD1

Affiliations

CRISPR-mediated gene targeting of CK1δ/ε leads to enhanced understanding of their role in endocytosis via phosphoregulation of GAPVD1

Rodrigo X Guillen et al. Sci Rep. .

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.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Intracellular localization of endogenous CK1δ-mNG and CK1ε-mNG. (A–C) Representative images of fixed HEK293 cells at indicated cell cycle stages producing CK1δ-mNG or CK1ε-mNG stained with (A) DAPI and anti-γ-tubulin, (B) DAPI and anti-γ-tubulin, or (C) DAPI and anti-MKLP1 antibodies. Scale bars, 10 μm. Insets correspond to centrosomes in A and B or the midbody in C. Scale bars, 0.5 μm. (D) Representative single z-sections of live-cell images of HEK293 CK1δ-mNG and CK1ε-mNG cells. Yellow arrows indicate examples of vesicle-like structures. Scale bars, 10 μm.
Figure 2
Figure 2
Analysis of CK1δ/ε interacting proteins. (A) Pie chart depicting the number of associated proteins with a gene-ontology (GO) annotation, that function in general cellular processes. Note that this chart includes 120/181 of the associated proteins. (B) Pie chart indicating the number of associated proteins with a GO annotation for localization to a specific cellular compartment. (C) The mean NSAF scores for the top ten proteins identified in four affinity purifications each of CK1δ and CK1ε. (D) NSAF values of the four identified proteins with a GO annotation for function in endocytosis.
Figure 3
Figure 3
CK1δ/ε interact with GAPVD1 throughout the cell cycle. (A,B) Immunoblots of the indicated proteins from whole cell lysates (WCL) or immunoprecipitates (IPs) of CK1δ (A) or CK1ε (B) from HeLa cell lysates. (C) Immunoblots of the indicated proteins from WCL (bottom panels) or IPs of CK1δ/ε from asynchronously (Asy) growing HeLa cells or HeLa cells synchronized in S-phase (S), metaphase (M), or G1-phase (G1). The black arrow and arrow-head indicate phosphorylated and dephosphorylated CK1δ/ε, respectively. pHH3, phosphohistone H3; a marker of mitotic cells. The red arrow and arrow-head indicate phosphorylated and dephosphorylated GAPVD1, respectively. (D) Yeast-two-hybrid showing direct interaction between GAPVD and CK1δ and CK1ε as indicated by growth on -His plates. (E) Bar graphs show β-galactosidase activity of the indicated bait and prey plasmids tested for growth in D (represented as ratio to empty bait and prey luminescence intensity). Each assay was performed in triplicate. ****p < 0.001 determined using a one-way ANOVA followed by Tukey’s posthoc test. ns, not significant. Error bars represent SEM. The entire images of immunoblots are presented in Supplementary Fig. 3. In some cases, membranes were cut for hybridization with multiple antibodies and then reassembled.
Figure 4
Figure 4
GAPVD1 is a substrate of CK1ε. (A) Cartoon representation of GAPVD1 protein domains. Green relates to the GAP domain, orange relates to the GEF domain. Red lines correspond to positions of serine or threonine residues that are mutated to alanine in the GAPVD1 38A mutant. (B) In vitro kinase assays of recombinant MBP-CK1ε WT and K38R (kinase-dead) detected by Coomassie brilliant blue (CBB) staining of SDS–PAGE gels, with GAPVD1 WT and 38 A as substrates. Phosphorylated GAPVD1 was detected by autoradiography (AutoRad). Black arrow indicates autophosphorylation of MBP-CK1ε. Red arrow indicates GAPVD1. (C) Immunoblots of WCLs and IPs of GAPVD1 from asynchronously (Asy) growing HeLa cells or HeLa cells synchronized in S-phase (S), metaphase (M), or G1-phase (G1). IPs were treated (+) or not (−) with lambda phosphatase (λPP) and blotted with the indicated antibodies. Red arrow and arrow-head indicate phosphorylated and dephosphorylated GAPVD1, respectively. pHH3, phosphohistone H3; a marker of mitotic cells. (D) Immunoblots of IPs from HeLa GAPVD1−/− cells transfected with vector only or vector expressing Flag3-V5-GAPVD1 or Flag3-V5-GAPVD1-38A. The entire images of immunoblots are presented in Supplementary Fig. 4. In some cases, membranes were cut for hybridization with multiple antibodies and then reassembled.
Figure 5
Figure 5
GAPVD1 phosphorylation promotes endocytosis. (A) Representative images of HeLa WT and GAPVD1−/− cells incubated with Tfn-594 in an endocytosis assay. Scale bar 10 μm. (B) Quantification of Alexa 594-coupled Tfn uptake in HeLa WT and GAPVD1−/− cells transfected with Flag3-V5 plasmids. The mean and SEM from 3 independent experiments (n ≥ 40 cells per experiment) are presented as a ratio to control. (C) Immunoblots of whole cell lysates from the experiments in (A,B). (D) Quantification of Alexa 488-coupled EGF uptake in HeLa WT and GAPVD1−/− cells transfected with Flag3-V5 plasmids. The mean and SEM from 3 independent experiments (n ≥ 40 cells per experiment) are presented as a ratio to control. In (B,D), ****p < 0.001, p values determined using a one-way ANOVA followed by Tukey’s posthoc test. ns, not significant. The entire images of immunoblots are presented in Supplementary Fig. 5. In some cases, membranes were cut for hybridization with multiple antibodies and then reassembled.

Similar articles

Cited by

References

    1. Knippschild U, et al. The casein kinase 1 family: participation in multiple cellular processes in eukaryotes. Cell Signal. 2005;17(6):675–689. doi: 10.1016/j.cellsig.2004.12.011. - DOI - PubMed
    1. Cheong JK, Virshup DM. Casein kinase 1: Complexity in the family. Int. J. Biochem. Cell Biol. 2011;43(4):465–469. doi: 10.1016/j.biocel.2010.12.004. - DOI - PubMed
    1. Cesaro L, Pinna LA. The generation of phosphoserine stretches in phosphoproteins: mechanism and significance. Mol. Biosyst. 2015;11(10):2666–2679. doi: 10.1039/C5MB00337G. - DOI - PubMed
    1. Vancura A, Sessler A, Leichus B, Kuret J. A prenylation motif is required for plasma membrane localization and biochemical function of casein kinase I in budding yeast. J. Biol. Chem. 1994;269(30):19271–19278. doi: 10.1016/S0021-9258(17)32163-4. - DOI - PubMed
    1. Wang PC, Vancura A, Mitcheson TG, Kuret J. Two genes in Saccharomyces cerevisiae encode a membrane-bound form of casein kinase-1. Mol. Biol. Cell. 1992;3(3):275–286. doi: 10.1091/mbc.3.3.275. - DOI - PMC - PubMed

Publication types

MeSH terms