PMC

EPPK1/keratin filament association is induced by elevation of intracellular Ca²⁺ levels. (A) Live-cell imaging of shEpi/AK cells treated with 165 nM ionomycin. In standard cell culture conditions (upper panel), EPPK1 is not associated with keratin filaments but distributed diffusely in the cytoplasm. After ionomycin treatment (lower panel), EPPK1 translocated to keratin filaments. Plots on the right side of the panels show fluorescent signal intensities of EPPK1 (red) and HK13 (green) along the dotted white lines indicated in the images. Note the non-overlapping keratin and EPPK1 signal profiles in the absence of ionomycin (upper panel) and the almost perfect overlap of both after addition of ionomycin (lower panel). (B) Single frames from a time-lapse recording of shEpi-CaS cells overexpressing mScarlet-short EPPK1 and the Ca²⁺ sensor GCaMP6m-XC. Under standard culture conditions (left panel), EPPK1 (red) shows a diffuse non-filamentous localization pattern and the Ca²⁺ sensor (green) shows only a faint fluorescent signal, indicating low intracellular Ca²⁺ levels. A total of 30 s after the addition of 200 nM Tg (right panel), short EPPK1 shows a filamentous localization pattern (red) accompanied by an elevation of Ca²⁺ sensor fluorescence intensity (green), indicating a Ca²⁺-dependent EPPK1 translocation. Plots on the right side of the panels show fluorescent signal intensities of short EPPK1 (red) and the Ca²⁺ sensor (green) along the dotted white lines indicated in the images taken before (light colors) and after Tg treatment (dark colors). The signal profiles for EPPK1 changed from a uniform signal intensity throughout the cell before treatment to a signal profile with several high-intensity peaks after Tg treatment, typical for keratin filament association (compare with ). In the case of the Ca²⁺ sensor, the signal intensity profile increased after Tg addition, indicating elevated levels of intracellular Ca²⁺. Images correspond to . (C) The Ca²⁺ chelator BAPTA-AM inhibits EPPK1 re-localization. Live-cell imaging of shEpi/AK cells expressing mScarlet-short EPPK1 (red) and HK13-EGFP (green) cultivated in FluoroBrite DMEM medium with and without 20 µM BAPTA-AM (upper two and lower two panels, respectively) before and after treatment with 200 nM Tg. Before Tg treatment (first panel), EPPK1 showed a non-keratin-associated diffuse cellular localization, which changed to a filament-associated pattern after Tg addition (second panel). Plots on the right side of the panels show fluorescent signal intensities of EPPK1 (red) and HK13 (green) along the dotted white lines indicated in the images. The separated keratin and EPPK1 signal profiles (upper panel) almost perfectly overlap after the addition of Tg (lower panel) as shown before (see H). Cells grown in BAPTA-AM-containing medium showed a comparable diffuse EPPK1 localization pattern before (third panel) and after (fourth panel) the addition of Tg, indicating that chelation of Ca²⁺ inhibits Tg-induced re-localization of EPPK1. The plots next to the panels displaying the fluorescent signal intensities of short EPPK1 (red) and HK13 (green) corroborate this finding. Note the lower levels of EPPK1 signals when compared with those of HK13 due to stronger bleaching as a consequence of higher laser intensities used for imaging. (D) Intensity correlation quotient (ICQ) analysis shows inhibition of HK13/EPPK1 co-localization by BAPTA-AM. A comparison of the ICQ of EPPK1 and HK13 fluorescent signals from control cells and cells pretreated with 20 µM BAPTA-AM before and after Tg treatment is shown. ICQ values were calculated using the intensity correlation analysis plugin of ImageJ (see ). ICQ values vary from 0.5 (for perfect co-localization of signals; see right cell scheme) to −0.5 (for total mutual exclusion of signals; see left cell scheme). Cell schemes show keratin filaments in green and EPPK1 in red. Note that before Tg treatment, ICQ levels of signals from cells in BAPTA-AM containing medium are lower than those of cells cultivated in medium without BAPTA-AM, as all cytoplasmic Ca²⁺ is removed. Data represent at least three independent experiments. Statistical evaluation was performed using the student t-test in GraphPad Prism software; ns p > 0.05, **** p ≤ 0.0001. (E,F) EPPK1 associates with the insoluble keratin pool in a Ca²⁺-dependent manner. (E) Protein lysates from AK 13-1 cells obtained by using the buffers indicated (with and without EGTA) were subjected to a sedimentation assay followed by immunoblot analysis using antibodies against EPPK1 and K8. By comparison with the size of EPPK1 variants determined for HaCaT cells [] and the known size of mScarlet-short EPPK1 (see ), we estimated the sizes of the two endogenous EPPK1 variants expressed in tetraploid AK 13-1 cells to 672 kDa for a variant comprising 15 PRDs and 497 kDa for a variant with 12 PRDs, respectively. Supernatant (S) fractions represent soluble, keratin-unbound EPPK1, and pellet (P) fractions represent keratin filament-bound EPPK1. EPPK1 fractionation carried out with a lysis buffer without EGTA induced co-sedimentation with keratins in the pellet (P) fraction. Hardly any EPPK1 signals were observed in the supernatant (S) fraction, indicating that the entire amount of EPPK1 is associated with the insoluble keratin pool during cell lysis. EPPK1 fractionation carried out with a lysis buffer containing the Ca²⁺ chelator EGTA abolished co-sedimentation of EPPK1 with keratin filaments. The entire amount of EPPK1 can be found in the supernatant (S) fraction. (F) Protein lysates from EpiKI/AK cells, which were FACS-sorted for mScarlet expression, were prepared by using the buffers indicated (with and without EGTA) and subjected to a sedimentation assay followed by immunoblot analysis using antibodies against EPPK1, RFP, and K8. The sizes of the endogenous EPPK1-mScarlet variants are 700 and 525 kDa, and 672 and 497 kDa for the untagged endogenous EPPK1, respectively. Supernatant (S) fractions represent soluble, keratin-unbound untagged and mScarlet-tagged EPPK1, and pellet (P) fractions represent keratin filament-bound untagged and mScarlet-tagged EPPK1. A similar result as in (E) was obtained. Scale bar: 10 µm.

EPPK1-keratin association reduces the mean flow of keratins. Keratin mean flow is significantly reduced in EPPK1 WT cells expressing mScarlet-tagged endogenous EPPK1 (A) after Tg-induced binding of EPPK1 to keratins, but it is not slowed down in the two independent EPPK1^−/− cell lines, EpiKO/AK 2C4 and EpiKO/AK 3F2 (B). The phenotype of EPPK1^−/− cells could be rescued in the cell lines shEpi-KO/AK 2C4 and shEpi-KO/AK 3F2 by overexpression of short EPPK1 in an EPPK1^−/− background (C). Plots show the mean flow of keratin [µm/min] of 10 time points before Tg addition (EPPK1 not associated with keratins) and 10 time points after Tg addition (EPPK1 associated with keratins). CMove was used to calculate keratin flow as described in []. Data represent at least three independent experiments. Statistical evaluation was performed using the Wilcoxon matched-pairs signed-rank test in GraphPad Prism software; ns p > 0.05, **** p ≤ 0.0001. (D) Schematic depicting the newly discovered localization and function of EPPK1 in homeostatic and stressed cells.

EPPK1 is diffusely distributed in the cytoplasm of living A-431-derived cells and rapidly associates with keratin filaments during fixation with PFA and after stress induction. (A,B) Schematic representation of fluorescently tagged EPPK1 variants used for live-cell imaging in AK 13-1 cells. (A) Both endogenous EPPK1 alleles of AK 13-1 cells were tagged with an mScarlet-encoding gene fragment using CRISPR/Cas9 HDR at their carboxytermini. The corresponding fusion proteins differ in size due to copy number variations in their almost identical C-terminal PRDs (4 versus 7), encompassing either 12 or 15 PRDs. (B) An EPPK1 variant (short EPPK1) consisting of the first eight N-terminal PRDs and the last of the identical C-terminal PRDs was N-terminally tagged with mScarlet. (C,D) Live-cell imaging during PFA fixation of EPPK1-mScarlet (red) and HK13-EGFP (green) in EpiKI/AK cells (C) or shEpi/AK cells (D). The respective left panels show cells before fixation, the middle panels show cells after removal of medium and subsequent PBS wash, and the right panels show cells after 0.5% PFA addition. EpiKI/AK cells (E–G) and shEpi/AK cells (H–J) were treated with 200 nM Tg (E,H), 2.5 mM H₂O₂ (F,I)_, and 16 J/cm² UV-A light pulse (G,J). In standard cell culture conditions (upper panels E–J) EPPK1 localization was diffuse and not keratin-associated. Plots on the right side of the panels display fluorescent signal intensities (arbitrary units) of EPPK1 (red) and keratin (green) along the dashed lines (from left to right) indicated in the image panels. The mainly non-overlapping keratin and EPPK1 signal profiles plots indicate their lack of association. After stress induction (lower panels E–J), EPPK1 re-localizes to keratin filaments, demonstrating a stress-dependent association. The almost perfectly overlapping keratin and EPPK1 signal profiles in the corresponding plots indicate their co-localization after stress induction. Scale bar: 10 µm.

EPPK1 decreased in ESCC tissues and cells. (a) The expression level of EPPK1 in tumor tissues and normal tissues obtained from the TCGA+ GTEx database. (b) The expression level of EPPK1 in paired cancer and normal tissues obtained from the TCGA database. (c) The mRNA level of EPPK1 in ESCC tissues and para‐carcinoma tissues measured by qPCR (p < 0.05). (d) The mRNA level of EPPK1 in 14 pairs of ESCC tissues and para‐carcinoma tissues (p < 0.05). (e) The mRNA level of EPPK1 in ESCC and normal cells measured by qPCR (p < 0.05). Values represent means ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. P, patient