ZDHHC7-mediated S-palmitoylation of Scribble regulates cell polarity

Abstract

Scribble (SCRIB) is a tumor-suppressor protein, playing critical roles in establishing and maintaining epithelial cell polarity. SCRIB is frequently amplified in human cancers but does not localize properly to cell-cell junctions, suggesting that mislocalization of SCRIB disrupts its tumor-suppressive activities. Using chemical reporters, here we showed that SCRIB localization was regulated by S-palmitoylation at conserved cysteine residues. Palmitoylation-deficient mutants of SCRIB were mislocalized, leading to disruption of cell polarity and loss of their tumor-suppressive activities to oncogenic YAP, MAPK and PI3K/AKT pathways. We further found that ZDHHC7 was the major palmitoyl acyltransferase regulating SCRIB. Knockout of ZDHHC7 led to SCRIB mislocalization and YAP activation, and disruption of SCRIB's suppressive activities in HRas(V12)-induced cell invasion. In summary, we demonstrated that ZDHHC7-mediated SCRIB palmitoylation is critical for SCRIB membrane targeting, cell polarity and tumor suppression, providing new mechanistic insights of how dynamic protein palmitoylation regulates cell polarity and tumorigenesis.

Figure 1. SCRIB is S-palmitoylated at evolutionarily conserved N-terminal cysteine residues

(a) Biochemical validation of SCRIB palmitoylation by metabolic labeling using chemical reporters of palmitoylation and streptavidin blot. (b) Treatment with hydroxylamine significantly decreased SCRIB palmitoylation. (c) Streptavidin pull-down and western blotting confirmed that endogenous SCRIB is palmitoylated in HEK293A cells. (d) Mutation of cysteine 4 and/or 10 abolished palmitoylation of SCRIB. (e) Pulse-chase analysis showed dynamic S-palmitoylation of SCRIB. All blots are representatives of at least three independent experiments. See Supplementary Figure 33 for full images of the blots in a–e.

Figure 2. Palmitoylation of SCRIB regulates its membrane localization and epithelial cell polarity

(a) SCRIB (C4/10S or P305L) mutant disrupted its membrane localization in MDCK cells. Cells were stained with anti-Flag antibody (red), anti-ZO1 antibody (green), and nuclei (DAPI, blue). The yellow line indicates the position of the Z stack. (b) The percentage of cells with mislocalized SCRIB in MDCK cells were quantified. (c) SCRIB (C4/10S or P305L) mutant were mislocalized in MCF10A acini at day 8 of 3-D culture. Cells were stained with anti-Flag antibody (red) and DAPI (blue). (d) The percentage of acini with mislocalized SCRIB in MCF10A cells were quantified. Expression levels of SCRIB were measured by the fluorescence intensity of SCRIB (in a–d). (e–f) SCRIB (C4/10S or P305L) mutant disrupted the acinar morphogenesis of MCF10A cells at 3-D culture at day 14. Acini were stained with anti-GM130 antibody (red) and DAPI (blue) (e), or anti-α6-integrin antibody (red) and DAPI (blue) (f). (g) The percentage of MCF10A acini with filled lumen was quantified. SCRIB (C4/10S or P305L) mutant showed increased luminal filling in MCF10A acini. Scale bars represent 20 μm in all images. All data are represented as mean ± SEM, n=3. P values were determined using two-tailed t-tests. *, P<0.05, **, P<0.005, N.S., not significant.

Figure 3. Palmitoylation of SCRIB is required to suppress YAP, MAPK and PI3K/AKT pathways

(a) Palmitoylation-deficient mutant of SCRIB (C4/10S or P305L) lost its suppression to YAP reporter (8xGTIIC-Luc) activity. EGFP signal was detected to show the similar SCRIB expression level in different cell lines. (b) Palmitoylation-deficient SCRIB mutant reduced YAP S127 phosphorylation in HEK293A cells. At 36 hours post transfection, cells were harvested for western blot analysis. (c) Palmitoylation-deficient mutant of SCRIB (C4/10S or P305L) lost the suppressive activities of YAP target gene expression (CTGF and Cyr61) in MCF10A cells. Relative mRNA levels were normalized to vector control. (d) Palmitoylation-deficient mutant of SCRIB (C4/10S or P305L) did not suppress YAP nuclear localization in MDCK cells. MDCK stable cells were stained with anti-Flag antibody (Flag-SCRIB, red), anti-YAP antibody (green) and nuclear DAPI (blue). The yellow line indicates the position of the Z stack. Scale bars represent 20 μm. (e) Quantification of the percentage of YAP nuclear localization in MDCK cells. (f) Palmitoylation-deficient SCRIB mutant (C4/10S or P305L) is not sufficient to suppress EGF-stimulated MAPK and PI3K/AKT pathways activation. All blots are representatives of at least three independent experiments. All data are represented as mean ± SEM, n=3. P values were determined by two-tailed t-test. *, P<0.05.***, P<0.001. See Supplementary Figure 34 for full images of the blots in b and f.

Figure 4. ZDHHC7 is a major palmitoyl acyltransferase regulating SCRIB palmitoylation

(a) Overexpression of ZDHHC7 significantly increased SCRIB palmitoylation levels. HEK293A cells were co-transfected with Flag-SCRIB construct and HA-ZDHHC constructs or empty vector. (b) Flag-SCRIB physically interacted with HA-ZDHHC7 in co-immunoprecipitation assays when co-transfected into cells. (c) Endogenous SCRIB interacted with HA-ZDHHC7 in co-immunoprecipitation assays. (d) The catalytic inactive mutant of ZDHHC7 (C160S) failed to induce SCRIB palmitoylation. (e) The N-terminal LRR domains of SCRIB are required and sufficient for ZDHHC7-SCRIB interaction. (f) Expression of siRNA-resistant construct of mouse WT HA-ZDHHC7 rather than the catalytically inactive mutant (C160S) restored SCRIB palmitoylation. All blots are representatives of at least three independent experiments. See Supplementary Figure 35 for full images of the blots.

Figure 5. ZDHHC7-mediated palmitoylation regulates SCRIB localization and YAP translocation

(a) Knockout of ZDHHC7 in MCF10A cells led to SCRIB mislocalization. Cells were stained with anti-SCRIB antibody (red), anti-ZDHHC7 antibody (green) and DAPI (blue). (b) Quantification of the percentage of cells with mislocalized SCRIB in ZDHHC7 knockout cells. (c) Expression of WT ZDHHC7, but not the C160S mutant partially restored SCRIB membrane localization in ZDHHC7 KO cells. Cells were stained with anti-SCRIB antibody (red), anti-ZDHHC7 antibody (green) and DAPI (blue). (d) Quantification of the percentage of cells with mislocalized SCRIB in different ZDHHC7 knockout cells. (e) Knockout of ZDHHC7 in MCF10A cells led to increased YAP nuclear localization. Cells were stained with anti-SCRIB antibody (red), anti-YAP antibody (green) and DAPI (blue). (f) Quantification of the percentage of YAP nuclear localization in ZDHHC7 knockout cells. In all images, the yellow line indicates the position of the Z stack (XY or XZ). Scale bars represent 20 μm. Data are represented as mean ± SEM, n=3. P values were determined by two-tailed t-test. *, P<0.05, **, P<0.01.

Figure 6. Palmitoylation of SCRIB is required to suppress HRas^V12-induced cell invasion

(a) Representative images of different MCF10A stable cell lines cultured in Matrigel/collagen mixture for 6 days. Scale bars represent 50 μm. (b) Quantification of the percentage of acini with invasive protrusions in each cell line. At least 100 acini were analyzed for each cell line in three independent experiments. (c) Representative images of control or ZDHHC7-knockout cells cultured in Matrigel/collagen mixture for 6 days. High magnification (upper, 20×) view and low magnification (lower, 10×) view are shown, respectively. Scale bars represent 50 μm, and 100 μm, respectively. (d) Quantification of the percentage of acini with invasive protrusions in vector control and ZDHHC7-knockout MCF10A cells. At least 100 acini were analyzed for each cell line in three independent experiments. All data are represented as mean ± SEM, n = 6. P values were determined by two-tailed t-test, compared with the vector control cells. *, P< 0.05; ***, P< 0.001.