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. 2021 Feb 19;220(4):e202010048. doi: 10.1083/jcb.202010048

Figure 4.

Figure 4.

MAPK14 phosphorylates SNX27 at Ser51 in vitro and in vivo. (A) Schematic representation of human SNX27, indicating the different domains (top). Pull-down assay to assess interaction between MAPK14 and different domains of SNX27. HEK293T cells were cotransfected with FLAG-tagged MAPK14 and GST-tagged SNX27 FL, PDZ, PX, or FERM domain for 24 h. Control cells (vector) were transfected with empty vector. One tenth of the cell lysate was prepared as input, and the rest was used for pull-down with GST resin, followed by immunoblotting using antibodies against GST-tagged SNX27 and FLAG-tagged MAPK14. Representative images from one of three independent experiments are shown. (B) MAPK14 phosphorylates SNX27 at Ser51 in vitro. HEK293T cells were transfected to FLAG-tagged MAPK14 WT, inactive MAPK14 (MAPK14-AF), or active MAPK14 (MAPK14-ac), with or without FLAG-tagged active MKK6 (MKK6E), as indicated. MAPK14 was immunoprecipitated with anti-FLAG beads and mixed with purified GFP-tagged SNX27 and ATP. The assay was performed at 37°C and analyzed using the anti-phospho-SNX27-Ser51 antibody. WB, western blotting. Representative of three independent iterations. (C) MAPK14 phosphorylates SNX27 at Ser51 in vivo. HEK293T cells were cotransfected with FLAG-tagged SNX27 and various versions of FLAG-tagged MAPK14 as in B. Kinase and substrate proteins were immunoprecipitated with anti-FLAG beads, followed by immunoblotting using anti-FLAG and phospho-SNX27-Ser51 antibodies. Representative of three independent iterations. (D) In vivo MAPK14 kinase assay in the presence of kinase inhibitor, B202190. HEK293T cells were cotransfected with GST-tagged SNX27 and FLAG-tagged MAPK14-AF, MAPK14-ac, or MAPK14-ac with or without SB202190 (10 µM for 2 h). One tenth of the cell lysate was prepared as input, and the rest was used for pull-down with GST-affinity resin, followed by immunoblotting for FLAG-MAPK14, GST-SNX27, and p-SNX27-Ser51. Representative of three independent iterations. (E) ATG7 deficiency decreases the phosphorylation of p38. WT and ATG7 KO MEF cells were starved in the presence or absence of p38 inhibitor (SB203580, 10 µM for 2 h), followed by immunoblotting using antibodies against total p38, p-p38, LC3B, and GAPDH. Representative of two independent iterations. The numbers under the bands represent the represent the p-p38/t-p38 ratio in each group. Band intensities were quantified with ImageJ and normalized to the MEF-WT control group (first lane). (F) Confocal imaging for EEA1 (green) and SNX27 (red) immunofluorescence staining of HeLa cells. Cells were transfected with FLAG-tagged MAPK14-ac for 24 h and treated with 10 µM SB203580 or 10 µM SB202190 for 2 h. Representative images from two independent experiments are shown. Scale bars: 10 µm. Arrows indicate SNX27 puncta. Statistical analysis was performed based on the numbers and average sizes of the SNX27 puncta in each of the cells. Results for individual cells are plotted, along with the mean and SD for each group (n = 59 cells for DMSO, n = 58 for SB203580, and n = 51 for SB202190); P values were calculated using one-way ANOVA by Tukey’s HSD test or t test. ****, P < 0.0001; *, P < 0.05. HSD, honestly significant difference.