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 HRasV12-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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: SCRIB is S-palmitoylated at evolutionarily conserved N-terminal cysteine residues.
Figure 2: Palmitoylation of SCRIB regulates its membrane localization and epithelial cell polarity.
Figure 3: Palmitoylation of SCRIB is required to suppress YAP, MAPK and PI3K/AKT pathways.
Figure 4: ZDHHC7 is a major palmitoyl acyltransferase regulating SCRIB palmitoylation.
Figure 5: ZDHHC7-mediated palmitoylation regulates SCRIB localization and YAP translocation.
Figure 6: Palmitoylation of SCRIB is required to suppress HRasV12-induced cell invasion.

References

  1. 1

    Martin-Belmonte, F. & Perez-Moreno, M. Epithelial cell polarity, stem cells and cancer. Nat. Rev. Cancer 12, 23–38 (2012).

    CAS  Article  Google Scholar 

  2. 2

    Bilder, D. & Perrimon, N. Localization of apical epithelial determinants by the basolateral PDZ protein Scribble. Nature 403, 676–680 (2000).

    CAS  Article  Google Scholar 

  3. 3

    Bilder, D., Li, M. & Perrimon, N. Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors. Science 289, 113–116 (2000).

    CAS  Article  Google Scholar 

  4. 4

    Elsum, I.A., Martin, C. & Humbert, P.O. Scribble regulates an EMT polarity pathway through modulation of MAPK-ERK signaling to mediate junction formation. J. Cell Sci. 126, 3990–3999 (2013).

    CAS  Article  Google Scholar 

  5. 5

    Zhan, L. et al. Deregulation of scribble promotes mammary tumorigenesis and reveals a role for cell polarity in carcinoma. Cell 135, 865–878 (2008).

    CAS  Article  Google Scholar 

  6. 6

    Pearson, H.B. et al. SCRIB expression is deregulated in human prostate cancer, and its deficiency in mice promotes prostate neoplasia. J. Clin. Invest. 121, 4257–4267 (2011).

    CAS  Article  Google Scholar 

  7. 7

    Feigin, M.E. et al. Mislocalization of the cell polarity protein scribble promotes mammary tumorigenesis and is associated with basal breast cancer. Cancer Res. 74, 3180–3194 (2014).

    CAS  Article  Google Scholar 

  8. 8

    Dow, L.E. et al. Loss of human Scribble cooperates with H-Ras to promote cell invasion through deregulation of MAPK signalling. Oncogene 27, 5988–6001 (2008).

    CAS  Article  Google Scholar 

  9. 9

    Godde, N.J. et al. Scribble modulates the MAPK/Fra1 pathway to disrupt luminal and ductal integrity and suppress tumour formation in the mammary gland. PLoS Genet. 10, e1004323 (2014).

    Article  Google Scholar 

  10. 10

    Cordenonsi, M. et al. The Hippo transducer TAZ confers cancer stem cell-related traits on breast cancer cells. Cell 147, 759–772 (2011).

    CAS  Article  Google Scholar 

  11. 11

    Mohseni, M. et al. A genetic screen identifies an LKB1-MARK signalling axis controlling the Hippo-YAP pathway. Nat. Cell Biol. 16, 108–117 (2014).

    CAS  Article  Google Scholar 

  12. 12

    Santoni, M.J., Pontarotti, P., Birnbaum, D. & Borg, J.P. The LAP family: a phylogenetic point of view. Trends Genet. 18, 494–497 (2002).

    CAS  Article  Google Scholar 

  13. 13

    Zeitler, J., Hsu, C.P., Dionne, H. & Bilder, D. Domains controlling cell polarity and proliferation in the Drosophila tumor suppressor Scribble. J. Cell Biol. 167, 1137–1146 (2004).

    CAS  Article  Google Scholar 

  14. 14

    Navarro, C. et al. Junctional recruitment of mammalian Scribble relies on E-cadherin engagement. Oncogene 24, 4330–4339 (2005).

    CAS  Article  Google Scholar 

  15. 15

    Audebert, S. et al. Mammalian Scribble forms a tight complex with the betaPIX exchange factor. Curr. Biol. 14, 987–995 (2004).

    CAS  Article  Google Scholar 

  16. 16

    Linder, M.E. & Deschenes, R.J. Palmitoylation: policing protein stability and traffic. Nat. Rev. Mol. Cell Biol. 8, 74–84 (2007).

    CAS  Article  Google Scholar 

  17. 17

    Hannoush, R.N. & Sun, J. The chemical toolbox for monitoring protein fatty acylation and prenylation. Nat. Chem. Biol. 6, 498–506 (2010).

    CAS  Article  Google Scholar 

  18. 18

    Hang, H.C. & Linder, M.E. Exploring protein lipidation with chemical biology. Chem. Rev. 111, 6341–6358 (2011).

    CAS  Article  Google Scholar 

  19. 19

    Zheng, B. et al. 2-Bromopalmitate analogues as activity-based probes to explore palmitoyl acyltransferases. J. Am. Chem. Soc. 135, 7082–7085 (2013).

    CAS  Article  Google Scholar 

  20. 20

    Martin, B.R., Wang, C., Adibekian, A., Tully, S.E. & Cravatt, B.F. Global profiling of dynamic protein palmitoylation. Nat. Methods 9, 84–89 (2012).

    CAS  Article  Google Scholar 

  21. 21

    Kang, R. et al. Neural palmitoyl-proteomics reveals dynamic synaptic palmitoylation. Nature 456, 904–909 (2008).

    CAS  Article  Google Scholar 

  22. 22

    Wei, X., Song, H. & Semenkovich, C.F. Insulin-regulated protein palmitoylation impacts endothelial cell function. Arterioscler. Thromb. Vasc. Biol. 34, 346–354 (2014).

    CAS  Article  Google Scholar 

  23. 23

    Martin, B.R. & Cravatt, B.F. Large-scale profiling of protein palmitoylation in mammalian cells. Nat. Methods 6, 135–138 (2009).

    CAS  Article  Google Scholar 

  24. 24

    Li, Y., Martin, B.R., Cravatt, B.F. & Hofmann, S.L. DHHC5 protein palmitoylates flotillin-2 and is rapidly degraded on induction of neuronal differentiation in cultured cells. J. Biol. Chem. 287, 523–530 (2012).

    CAS  Article  Google Scholar 

  25. 25

    Yang, W., Di Vizio, D., Kirchner, M., Steen, H. & Freeman, M.R. Proteome scale characterization of human S-acylated proteins in lipid raft-enriched and non-raft membranes. Mol. Cell. Proteomics 9, 54–70 (2010).

    CAS  Article  Google Scholar 

  26. 26

    Zhang, M.M., Tsou, L.K., Charron, G., Raghavan, A.S. & Hang, H.C. Tandem fluorescence imaging of dynamic S-acylation and protein turnover. Proc. Natl. Acad. Sci. USA 107, 8627–8632 (2010).

    CAS  Article  Google Scholar 

  27. 27

    O'Brien, L.E., Zegers, M.M. & Mostov, K.E. Building epithelial architecture: insights from three-dimensional culture models. Nat. Rev. Mol. Cell Biol. 3, 531–537 (2002).

    CAS  Article  Google Scholar 

  28. 28

    Elsum, I.A. & Humbert, P.O. Localization, not important in all tumor-suppressing properties: a lesson learnt from scribble. Cells Tissues Organs 198, 1–11 (2013).

    CAS  Article  Google Scholar 

  29. 29

    Johnson, R. & Halder, G. The two faces of Hippo: targeting the Hippo pathway for regenerative medicine and cancer treatment. Nat. Rev. Drug Discov. 13, 63–79 (2014).

    CAS  Article  Google Scholar 

  30. 30

    Moroishi, T., Hansen, C.G. & Guan, K.L. The emerging roles of YAP and TAZ in cancer. Nat. Rev. Cancer 15, 73–79 (2015).

    CAS  Article  Google Scholar 

  31. 31

    Yang, C.C. et al. Differential regulation of the Hippo pathway by adherens junctions and apical-basal cell polarity modules. Proc. Natl. Acad. Sci. USA 112, 1785–1790 (2015).

    CAS  Article  Google Scholar 

  32. 32

    Li, X., Yang, H., Liu, J., Schmidt, M.D. & Gao, T. Scribble-mediated membrane targeting of PHLPP1 is required for its negative regulation of Akt. EMBO Rep. 12, 818–824 (2011).

    CAS  Article  Google Scholar 

  33. 33

    Legouis, R. et al. Basolateral targeting by leucine-rich repeat domains in epithelial cells. EMBO Rep. 4, 1096–1102 (2003).

    CAS  Article  Google Scholar 

  34. 34

    Pedram, A., Razandi, M., Deschenes, R.J. & Levin, E.R. DHHC-7 and -21 are palmitoylacyltransferases for sex steroid receptors. Mol. Biol. Cell 23, 188–199 (2012).

    CAS  Article  Google Scholar 

  35. 35

    Vaira, V. et al. Aberrant overexpression of the cell polarity module scribble in human cancer. Am. J. Pathol. 178, 2478–2483 (2011).

    CAS  Article  Google Scholar 

  36. 36

    Hungermann, D. et al. Influence of whole arm loss of chromosome 16q on gene expression patterns in oestrogen receptor-positive, invasive breast cancer. J. Pathol. 224, 517–528 (2011).

    CAS  Article  Google Scholar 

  37. 37

    Harvey, K.F., Zhang, X. & Thomas, D.M. The Hippo pathway and human cancer. Nat. Rev. Cancer 13, 246–257 (2013).

    CAS  Article  Google Scholar 

  38. 38

    Chan, P. et al. Autopalmitoylation of TEAD proteins regulates transcriptional output of the Hippo pathway. Nat. Chem. Biol. 12, 282–289 (2016).

    CAS  Article  Google Scholar 

  39. 39

    Rossin, A. et al. Fas palmitoylation by the palmitoyl acyltransferase DHHC7 regulates Fas stability. Cell Death Differ. 22, 643–653 (2015).

    CAS  Article  Google Scholar 

  40. 40

    Fukata, Y. & Fukata, M. Protein palmitoylation in neuronal development and synaptic plasticity. Nat. Rev. Neurosci. 11, 161–175 (2010).

    CAS  Article  Google Scholar 

  41. 41

    Rocks, O. et al. The palmitoylation machinery is a spatially organizing system for peripheral membrane proteins. Cell 141, 458–471 (2010).

    CAS  Article  Google Scholar 

  42. 42

    Wan, J., Roth, A.F., Bailey, A.O. & Davis, N.G. Palmitoylated proteins: purification and identification. Nat. Protoc. 2, 1573–1584 (2007).

    CAS  Article  Google Scholar 

  43. 43

    Duncan, J.A. & Gilman, A.G. A cytoplasmic acyl-protein thioesterase that removes palmitate from G protein alpha subunits and p21(RAS). J. Biol. Chem. 273, 15830–15837 (1998).

    CAS  Article  Google Scholar 

  44. 44

    Rocks, O. et al. An acylation cycle regulates localization and activity of palmitoylated Ras isoforms. Science 307, 1746–1752 (2005).

    CAS  Article  Google Scholar 

  45. 45

    Dekker, F.J. et al. Small-molecule inhibition of APT1 affects Ras localization and signaling. Nat. Chem. Biol. 6, 449–456 (2010).

    CAS  Article  Google Scholar 

  46. 46

    Debnath, J., Muthuswamy, S.K. & Brugge, J.S. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30, 256–268 (2003).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by Stewart Rahr–MRA (Melanoma Research Alliance) Young Investigator Award, Research Scholar Award from American Cancer Society (124929-RSG-13-291-01-TBE), and grants from National Institutes of Health (R01CA181537 and R01DK107651-01) to X.W. We appreciate W.-L. Yan for his generous philanthropic donation to Massachusetts General Hospital to support Y.S.B.'s training and research. We thank M. Fukata (National Institute for Physiological Sciences, Japan) for the expression vectors of DHHC proteins, the Confocal Imaging Core at Cutaneous Biology Research Center of Massachusetts General Hospital, and the Shared Instrumentation Grant that covered the purchase of the microscope (1S10RR027673-01), and the Taplin Mass Spec Core at Harvard Medical School for proteomic studies.

Author information

Affiliations

Authors

Contributions

X.W. conceived the concept and supervised the project. B.C. and X.W. designed the experiments. B.Z. and G.K.J. synthesized the probes, and B.Z. identified Scribble from proteomic studies. B.C. performed biochemistry and cell biology experiments with the help from M.D., G.K.J., J.F., and Y.S.B. B.C. and X.W. wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Xu Wu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results and Supplementary Figures 1–49. (PDF 32493 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Chen, B., Zheng, B., DeRan, M. et al. ZDHHC7-mediated S-palmitoylation of Scribble regulates cell polarity. Nat Chem Biol 12, 686–693 (2016). https://doi.org/10.1038/nchembio.2119

Download citation

Further reading

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing