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Palmitoylation by DHHC3 is critical for the function, expression, and stability of integrin α6β4

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Abstract

The laminin-binding integrin α6β4 plays key roles in both normal epithelial and endothelial cells and during tumor cell progression, metastasis, and angiogenesis. Previous cysteine mutagenesis studies have suggested that palmitoylation of α6β4 protein supports a few integrin-dependent functions and molecular associations. Here we took another approach and obtained strikingly different results. We used overexpression and RNAi knockdown in multiple cell types to identify protein acyl transferase DHHC3 as the enzyme responsible for integrin β4 and α6 palmitoylation. Ablation of DHHC3 markedly diminished integrin-dependent cellular cable formation on Matrigel, integrin signaling through Src, and β4 phosphorylation on key diagnostic amino acids (S1356 and 1424). However, unexpectedly, and in sharp contrast to prior α6β4 mutagenesis results, knockdown of DHHC3 accelerated the degradation of α6β4, likely due to an increase in endosomal exposure to cathepsin D. When proteolytic degradation was inhibited (by Pepstatin A), rescued α6β4 accumulated intracellularly, but was unable to reach the cell surface. DHHC3 ablation effects were strongly selective for α6β4. Cell-surface levels of ~10 other proteins (including α3β1) were not diminished, and the appearance of hundreds of other palmitoylated proteins was not altered. Results obtained here demonstrate a new substrate for the DHHC3 enzyme and provide novel opportunities for modulating α6β4 expression, distribution, and function.

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References

  1. Rabinovitz I, Gipson IK, Mercurio AM (2001) Traction forces mediated by alpha6beta4 integrin: implications for basement membrane organization and tumor invasion. Mol Biol Cell 12:4030–4043

    CAS  PubMed  Google Scholar 

  2. de Pereda JM, Ortega E, Alonso-Garcia N, Gomez-Hernandez M, Sonnenberg A (2009) Advances and perspectives of the architecture of hemidesmosomes: lessons from structural biology. Cell Adh Migr 3:361–364

    Article  PubMed  Google Scholar 

  3. Georges-Labouesse EN, Messaddeq N, Yehia G, Cadalbert L, Dierich A, Le Meur M (1996) Absence of the alpha-6 integrin leads to epidermolysis bullosa and neonatal death in mice. Nat Genet 13:370–373

    Article  CAS  PubMed  Google Scholar 

  4. Pulkkinen L, Uitto J (1999) Mutation analysis and molecular genetics of epidermolysis bullosa. Matrix Biol 18:29–42

    Article  CAS  PubMed  Google Scholar 

  5. Kasirer-Friede A, Kahn ML, Shattil SJ (2007) Platelet integrins and immunoreceptors. Immunol Rev 218:247–264

    Article  CAS  PubMed  Google Scholar 

  6. Borland G, Cushley W (2004) Positioning the immune system: unexpected roles for alpha6-integrins. Immunology 111:381–383

    Article  CAS  PubMed  Google Scholar 

  7. Haworth O, Hardie DL, Burman A, Rainger GE, Eksteen B, Adams DH, Salmon M, Nash GB, Buckley CD (2008) A role for the integrin alpha6beta1 in the differential distribution of CD4 and CD8 T-cell subsets within the rheumatoid synovium. Rheumatology (Oxford) 47:1329–1334

    Article  CAS  Google Scholar 

  8. McMillan NA, Payne E, Frazer IH, Evander M (1999) Expression of the alpha6 integrin confers papillomavirus binding upon receptor-negative B-cells. Virology 261:271–279

    Article  CAS  PubMed  Google Scholar 

  9. Lipscomb EA, Mercurio AM (2005) Mobilization and activation of a signaling competent alpha6beta4integrin underlies its contribution to carcinoma progression. Cancer Metastasis Rev 24:413–423

    Article  CAS  PubMed  Google Scholar 

  10. Stipp CS (2010) Laminin-binding integrins and their tetraspanin partners as potential antimetastatic targets. Expert Rev Mol Med 12:e3

    Article  PubMed  Google Scholar 

  11. Nikolopoulos SN, Blaikie P, Yoshioka T, Guo W, Giancotti FG (2004) Integrin beta4 signaling promotes tumor angiogenesis. Cancer Cell 6:471–483

    Article  CAS  PubMed  Google Scholar 

  12. Yang XH, Flores LM, Li Q, Zhou P, Xu F, Krop IE, Hemler ME (2010) Disruption of laminin-integrin-CD151-focal adhesion kinase axis sensitizes breast cancer cells to ErbB2 antagonists. Cancer Res 70:2256–2263

    Article  CAS  PubMed  Google Scholar 

  13. M. Colombel, C. L. Eaton, F. Hamdy, E. Ricci, G. van der Pluijm, M. Cecchini, F. Mege-Lechevallier, P. Clezardin, G. Thalmann (2011) Increased expression of putative cancer stem cell markers in primary prostate cancer is associated with progression of bone metastases. Prostate

  14. Honeth G, Bendahl PO, Ringner M, Saal LH, Gruvberger-Saal SK, Lovgren K, Grabau D, Ferno M, Borg A, Hegardt C (2008) The CD44+/CD24− phenotype is enriched in basal-like breast tumors. Breast Cancer Res 10:R53

    Article  PubMed  Google Scholar 

  15. Berditchevski F (2001) Complexes of tetraspanins with integrins: more than meets the eye. J Cell Sci 114:4143–4151

    CAS  PubMed  Google Scholar 

  16. Sterk LM, Geuijen CA, van Den Berg JG, Claessen N, Weening JJ, Sonnenberg A (2002) Association of the tetraspanin CD151 with the laminin-binding integrins alpha3beta1, alpha6beta1, alpha6beta4 and alpha7beta1 in cells in culture and in vivo. J Cell Sci 115:1161–1173

    CAS  PubMed  Google Scholar 

  17. Hemler ME (2003) Tetraspanin proteins mediate cellular penetration, invasion and fusion events, and define a novel type of membrane microdomain. Ann Rev Cell Dev Biol 19:397–422

    Article  CAS  Google Scholar 

  18. Yang X, Kovalenko OV, Tang W, Claas C, Stipp CS, Hemler ME (2004) Palmitoylation supports assembly and function of integrin-tetraspanin complexes. J Cell Biol 167:1231–1240

    Article  CAS  PubMed  Google Scholar 

  19. Gagnoux-Palacios L, Dans M, Van’t Hof W, Mariotti A, Pepe A, Meneguzzi G, Resh MD, Giancotti FG (2003) Compartmentalization of integrin {alpha}6{beta}4 signaling in lipid rafts. J Cell Biol 162:1189–1196

    Article  CAS  PubMed  Google Scholar 

  20. Mitchell DA, Vasudevan A, Linder ME, Deschenes RJ (2006) Protein palmitoylation by a family of DHHC protein S-acyltransferases. J Lipid Res 47:1118–1127

    Article  CAS  PubMed  Google Scholar 

  21. Tsutsumi R, Fukata Y, Fukata M (2008) Discovery of protein-palmitoylating enzymes. Pflugers Arch 456:1199–1206

    Article  CAS  PubMed  Google Scholar 

  22. Ohno Y, Kihara A, Sano T, Igarashi Y (2006) Intracellular localization and tissue-specific distribution of human and yeast DHHC cysteine-rich domain-containing proteins. Biochim Biophys Acta 1761:474–483

    CAS  PubMed  Google Scholar 

  23. Fang C, Deng L, Keller CA, Fukata M, Fukata Y, Chen G, Luscher B (2006) GODZ-mediated palmitoylation of GABA(A) receptors is required for normal assembly and function of GABAergic inhibitory synapses. J Neurosci 26:12758–12768

    Article  CAS  PubMed  Google Scholar 

  24. Sharma C, Yang XH, Hemler ME (2008) DHHC2 affects palmitoylation and stability of tetraspanins CD9 and CD151. Mol Biol Cell 19:3415–3425

    Article  CAS  PubMed  Google Scholar 

  25. Abrami L, Kunz B, Iacovache I, van der Goot FG (2008) Palmitoylation and ubiquitination regulate exit of the Wnt signaling protein LRP6 from the endoplasmic reticulum. Proc Natl Acad Sci USA 105:5384–5389

    Article  CAS  PubMed  Google Scholar 

  26. Alvarez E, Girones N, Davis RJ (1990) Inhibition of the receptor-mediated endocytosis of diferric transferrin is associated with the covalent modification of the transferrin receptor with palmitic acid. J Biol Chem 265:16644–16655

    CAS  PubMed  Google Scholar 

  27. Percherancier Y, Planchenault T, Valenzuela-Fernandez A, Virelizier JL, Arenzana-Seisdedos F, Bachelerie F (2001) Palmitoylation-dependent control of degradation, life span, and membrane expression of the CCR5 receptor. J Biol Chem 276:31936–31944

    Article  CAS  PubMed  Google Scholar 

  28. Yanez-Mo M, Barreiro O, Gordon-Alonso M, Sala-Valdes M, Sanchez-Madrid F (2009) Tetraspanin-enriched microdomains: a functional unit in cell plasma membranes. Trends Cell Biol 19:434–446

    Article  CAS  PubMed  Google Scholar 

  29. Seehafer JG, Slupsky JR, Tang SC, Masellis-Smith A, Shaw AR (1990) Myristic acid is incorporated into the two acylatable domains of the functional glycoprotein CD9 in ester, but not in amide bonds. Biochim Biophys Acta 1039:218–226

    Article  CAS  PubMed  Google Scholar 

  30. Yang X, Claas C, Kraeft SK, Chen LB, Wang Z, Kreidberg JA, Hemler ME (2002) Palmitoylation of tetraspanin proteins: modulation of CD151 lateral interactions, subcellular distribution, and integrin-dependent cell morphology. Mol Biol Cell 13:767–781

    Article  CAS  PubMed  Google Scholar 

  31. Berditchevski F, Odintsova E, Sawada S, Gilbert E (2002) Expression of the palmitoylation-deficient CD151 weakens the association of alpha 3beta 1 integrin with the tetraspanin-enriched microdomains and affects integrin-dependent signalling. J Biol Chem 277:36991–37000

    Article  CAS  PubMed  Google Scholar 

  32. Charrin S, Manie S, Oualid M, Billard M, Boucheix C, Rubinstein E (2002) Differential stability of tetraspanin/tetraspanin interactions: role of palmitoylation. FEBS Lett 516:139–144

    Article  CAS  PubMed  Google Scholar 

  33. Keller CA, Yuan X, Panzanelli P, Martin ML, Alldred M, Sassoe-Pognetto M, Luscher B (2004) The gamma2 subunit of GABA(A) receptors is a substrate for palmitoylation by GODZ. J Neurosci 24:5881–5891

    Article  CAS  PubMed  Google Scholar 

  34. Tsutsumi R, Fukata Y, Noritake J, Iwanaga T, Perez F, Fukata M (2009) Identification of G protein alpha subunit-palmitoylating enzyme. Mol Cell Biol 29:435–447

    Article  CAS  PubMed  Google Scholar 

  35. Wang J, Xie Y, Wolff DW, Abel PW, Tu Y (2010) DHHC protein-dependent palmitoylation protects regulator of G-protein signaling 4 from proteasome degradation. FEBS Lett 584:4570–4574

    Article  CAS  PubMed  Google Scholar 

  36. Germain EC, Santos TM, Rabinovitz I (2009) Phosphorylation of a novel site on the {beta}4 integrin at the trailing edge of migrating cells promotes hemidesmosome disassembly. Mol Biol Cell 20:56–67

    Article  CAS  PubMed  Google Scholar 

  37. Kashyap T, Germain E, Roche M, Lyle S, Rabinovitz I (2011) Role of β4 integrin phosphorylation in human invasive squamous cell carcinoma: regulation of hemidesmosome stability modulates cell migration. Lab Invest 91:1414–1426

    Article  CAS  PubMed  Google Scholar 

  38. Fernandez-Hernando C, Fukata M, Bernatchez PN, Fukata Y, Lin MI, Bredt DS, Sessa WC (2006) Identification of Golgi-localized acyl transferases that palmitoylate and regulate endothelial nitric oxide synthase. J Cell Biol 174:369–377

    Article  CAS  PubMed  Google Scholar 

  39. Zhang XA, Kazarov AR, Yang X, Bontrager AL, Stipp CS, Hemler ME (2002) Function of the tetraspanin CD151-alpha6beta1 integrin complex during cellular morphogenesis. Mol Biol Cell 13:1–11

    Article  PubMed  Google Scholar 

  40. Wilhelmsen K, Litjens SH, Kuikman I, Margadant C, van Rheenen J, Sonnenberg A (2007) Serine phosphorylation of the integrin beta4 subunit is necessary for epidermal growth factor receptor induced hemidesmosome disruption. Mol Biol Cell 18:3512–3522

    Article  CAS  PubMed  Google Scholar 

  41. Greaves J, Salaun C, Fukata Y, Fukata M, Chamberlain LH (2008) Palmitoylation and membrane interactions of the neuroprotective chaperone cysteine-string protein. J Biol Chem 283:25014–25026

    Article  CAS  PubMed  Google Scholar 

  42. Greaves J, Chamberlain LH (2011) DHHC palmitoyl transferases: substrate interactions and (patho)physiology. Trends Biochem Sci 36:245–253

    Article  CAS  PubMed  Google Scholar 

  43. Vernon RB, Sage EH (1995) Between molecules and morphology. Extracellular matrix and creation of vascular form. Am J Pathol 147:873–883

    CAS  PubMed  Google Scholar 

  44. Davis GE, Camarillo CW (1995) Regulation of endothelial cell morphogenesis by integrins, mechanical forces, and matrix guidance pathways. Exp Cell Res 216:113–123

    Article  CAS  PubMed  Google Scholar 

  45. Kim TH, Kim HI, Soung YH, Shaw LA, Chung J (2009) Integrin (alpha6beta4) signals through Src to increase expression of S100A4, a metastasis-promoting factor: implications for cancer cell invasion. Mol Cancer Res 7:1605–1612

    Article  CAS  PubMed  Google Scholar 

  46. Takkunen M, Grenman R, Hukkanen M, Korhonen M, de Garcia HA, Virtanen I (2006) Snail-dependent and -independent epithelial–mesenchymal transition in oral squamous carcinoma cells. J Histochem Cytochem 54:1263–1275

    Article  CAS  PubMed  Google Scholar 

  47. Greaves J, Prescott GR, Fukata Y, Fukata M, Salaun C, Chamberlain LH (2009) The hydrophobic cysteine-rich domain of SNAP25 couples with downstream residues to mediate membrane interactions and recognition by DHHC palmitoyl transferases. Mol Biol Cell 20:1845–1854

    Article  CAS  PubMed  Google Scholar 

  48. Barrett AJ (1979) Cathepsin D: the lysosomal aspartic proteinase. Ciba Found Symp, pp 37–50

  49. Diment S, Leech MS, Stahl PD (1988) Cathepsin D is membrane-associated in macrophage endosomes. J Biol Chem 263:6901–6907

    CAS  PubMed  Google Scholar 

  50. van Weert AW, Dunn KW, Gueze HJ, Maxfield FR, Stoorvogel W (1995) Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump. J Cell Biol 130:821–834

    Article  PubMed  Google Scholar 

  51. Lafleur MA, Xu D, Hemler ME (2009) Tetraspanin proteins regulate membrane type-1 matrix metalloproteinase-dependent pericellular proteolysis. Mol Biol Cell 20:2030–2040

    Article  CAS  PubMed  Google Scholar 

  52. Linder ME, Deschenes RJ (2007) Palmitoylation: policing protein stability and traffic. Natl Rev Mol Cell Biol 8:74–84

    Article  CAS  Google Scholar 

  53. J. Korycka, A. Lach, E. Heger, D. M. Boguslawska, M. Wolny, M. Toporkiewicz, K. Augoff, J. Korzeniewski, A. F. Sikorski (2011) Human DHHC proteins: a spotlight on the hidden player of palmitoylation. Eur J Cell Biol

  54. Aicart-Ramos C, Valero RA, Rodriguez-Crespo I (2011) Protein palmitoylation and subcellular trafficking. Biochim Biophys Acta 1808:2981–2994

    Article  CAS  PubMed  Google Scholar 

  55. Lukk M, Kapushesky M, Nikkila J, Parkinson H, Goncalves A, Huber W, Ukkonen E, Brazma A (2010) A global map of human gene expression. Nat Biotechnol 28:322–324

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We thank Dr. Hong-Xing Wang for assistance with confocal microscopy and for providing a control vector. This work was supported by National Institutes of Health Grant GM38903 (to MEH).

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The authors declare no conflict of interest.

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Authors and Affiliations

  1. Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Rm D1430, 450 Brookline Ave, Boston, MA, 02215, USA

    Chandan Sharma & Martin E. Hemler

  2. Division of Cancer Biology and Angiogenesis, Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA

    Isaac Rabinovitz

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  1. Chandan Sharma
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  2. Isaac Rabinovitz
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Correspondence to Martin E. Hemler.

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Sharma, C., Rabinovitz, I. & Hemler, M.E. Palmitoylation by DHHC3 is critical for the function, expression, and stability of integrin α6β4. Cell. Mol. Life Sci. 69, 2233–2244 (2012). https://doi.org/10.1007/s00018-012-0924-6

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