A strategy to identify protein-N-myristoylation-dependent phosphorylation reactions of cellular proteins by using Phos-tag SDS-PAGE

Autoři: Emiko Kinoshita-Kikuta aff001;  Ayane Tanikawa aff002;  Takuro Hosokawa aff002;  Aya Kiwado aff002;  Koko Moriya aff002;  Eiji Kinoshita aff001;  Tohru Koike aff001;  Toshihiko Utsumi aff002
Působiště autorů: Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan aff001;  Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi, Japan aff002;  Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan aff003
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225510


To establish a strategy for identifying protein-N-myristoylation-dependent phosphorylation of cellular proteins, Phos-tag SDS-PAGE was performed on wild-type (WT) and nonmyristoylated mutant (G2A-mutant) FMNL2 and FMNL3, phosphorylated N-myristoylated model proteins expressed in HEK293 cells. The difference in the banding pattern in Phos-tag SDS-PAGE between the WT and G2A-mutant FMNL2 indicated the presence of N-myristoylation-dependent phosphorylation sites in FMNL2. Phos-tag SDS-PAGE of FMNL2 mutants in which the putative phosphorylation sites listed in PhosphoSitePlus (an online database of phosphorylation sites) were changed to Ala revealed that Ser-171 and Ser-1072 are N-myristoylation-dependent phosphorylation sites in FMNL2. Similar experiments with FMNL3 demonstrated that N-myristoylation-dependent phosphorylation occurs at a single Ser residue at position 174, which is a Ser residue conserved between FMNL2 and FMNL3, corresponding to Ser-171 in FMNL2. The facts that phosphorylation of Ser-1072 in FMNL2 has been shown to play a critical role in integrin β1 internalization mediated by FMNL2 and that Ser-171 in FMNL2 and Ser-174 in FMNL3 are novel putative phosphorylation sites conserved between FMNL2 and FMNL3 indicate that the strategy used in this study is a useful tool for identifying and characterizing physiologically important phosphorylation reactions occurring on N-myristoylated proteins.

Klíčová slova:

Cell membranes – Membrane proteins – Phosphorylation – Protein kinases – Protein sequencing – Sequence motif analysis – Polyacrylamides – Metabolic labeling


1. Udenwobele DI, Su RC, Good SV, Ball TB, Varma Shrivastav S, Shrivastav A. Myristoylation: An important protein modification in the immune response. Front Immunol. 2017;8:751. doi: 10.3389/fimmu.2017.00751 28713376

2. Peng T, Thinon E, Hang HC. Proteomic analysis of fatty-acylated proteins. Curr Opin Chem Biol. 2016;30:77–86. doi: 10.1016/j.cbpa.2015.11.008 26656971

3. Giglione C, Fieulaine S, Meinnel T. N-terminal protein modifications: Bringing back into play the ribosome. Biochimie. 2015;114:134–146. doi: 10.1016/j.biochi.2014.11.008 25450248

4. Resh MD. Trafficking and signaling by fatty-acylated and prenylated proteins. Nat Chem Biol. 2006;2:584–590. doi: 10.1038/nchembio834 17051234

5. Farazi TA, Waksman G, Gordon JI. The biology and enzymology of protein N-myristoylation. J Biol Chem. 2001;276:39501–39504. doi: 10.1074/jbc.R100042200 11527981

6. Resh MD. Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim Biophys Acta. 1999;1451:1–16. doi: 10.1016/s0167-4889(99)00075-0 10446384

7. Zha J, Weiler S, Oh K-J, Wei MC, Korsmeyer SJ. Posttranslational N-myristoylation of BID as a molecular switch for targeting mitochondria and apoptosis. Science. 2000;290:1761–1765. doi: 10.1126/science.290.5497.1761 11099414

8. Utsumi T, Sakurai N, Nakano K, Ishisaka R. C-Terminal 15 kDa fragment of cytoskeletal actin is posttranslationally N-myristoylated upon caspase-mediated cleavage and targeted to mitochondria. FEBS Lett. 2003;539:37–44. doi: 10.1016/s0014-5793(03)00180-7 12650923

9. Sakurai N, Utsumi T. Posttranslational N-myristoylation is required for the anti-apoptotic activity of human tGelsolin, the C-terminal caspase cleavage product of human gelsolin. J Biol Chem. 2006;281:14288–14295. doi: 10.1074/jbc.M510338200 16556605

10. Martin DD, Beauchamp E, Berthiaume LG. Post-translational myristoylation: Fat matters in cellular life and death. Biochimie. 2011;93:18–31. doi: 10.1016/j.biochi.2010.10.018 21056615

11. Dyda F, Klein DC, Hickman AB. GCN5-related N-acetyltransferases: A structural overview. Annu Rev Biophys Biomol Struct. 2000;29:81–103. doi: 10.1146/annurev.biophys.29.1.81 10940244

12. Tillo SE, Xiong WH, Takahashi M, Miao S, Andrade AL, Fortin DA, et al. Liberated PKA catalytic subunits associate with the membrane via myristoylation to preferentially phosphorylate membrane substrates. Cell Rep. 2017;19:617–629. doi: 10.1016/j.celrep.2017.03.070 28423323

13. Arbuzova A, Schmitz AA, Vergeres G. Cross-talk unfolded: MARCKS proteins. Biochem J. 2002;362:1–12. doi: 10.1042/0264-6021:3620001 11829734

14. Fong LWR, Yang DC, Chen C-H. Myristoylated alanine-rich C kinase substrate (MARCKS): A multirole signaling protein in cancers. Cancer Metastasis Rev. 2017;36:737–747. doi: 10.1007/s10555-017-9709-6 29039083

15. Moriya K, Yamamoto T, Takamitsu E, Matsunaga Y, Kimoto M, Fukushige D, et al. Protein N-myristoylation is required for cellular morphological changes induced by two formin family proteins, FMNL2 and FMNL3. Biosci Biotechnol Biochem. 2012;76:1201–1209. doi: 10.1271/bbb.120069 22790947

16. Wang Y, Arjonen A, Pouwels J, Ta H, Pausch P, Bange G, et al. Formin-like 2 promotes β1-integrin trafficking and invasive motility downstream of PKCα. Dev Cell. 2015;34:475–483. doi: 10.1016/j.devcel.2015.06.015 26256210

17. Kühn S, Erdmann C, Kage F, Block J, Schwenkmezger L, Steffen A, et al. The structure of FMNL2-Cdc42 yields insights into the mechanism of lamellipodia and filopodia formation. Nat Commun. 2015;6:7088. doi: 10.1038/ncomms8088 25963737

18. Saito S, Hamamoto S, Moriya K, Matsuura A, Sato Y, Muto J, et al. N-Myristoylation and S-acylation are common modifications of Ca2+-regulated Arabidopsis kinases and are required for activation of the SLAC1 anion channel. New Phytol. 2018;218:1504–1521. doi: 10.1111/nph.15053 29498046

19. Kinoshita E, Kinoshita-Kikuta E, Kubota Y, Takekawa M, Koike T. A Phos-tag SDS-PAGE method that effectively uses phosphoproteomic data for profiling the phosphorylation dynamics of MEK1. Proteomics. 2016;16:1825–1836. doi: 10.1002/pmic.201500494 27169363

20. Takamitsu E, Fukunaga K, Iio Y, Utsumi T. Cell-free identification of novel N-myristoylated proteins from complementary DNA resources using bioorthogonal myristic acid analogues. Anal Biochem. 2014;464:83–93. doi: 10.1016/j.ab.2014.07.006 25043870

21. Utsumi T, Matsuzaki K, Kiwado A, Tanikawa A, Kikkawa Y, Hosokawa T, et al. Identification and characterization of protein N-myristoylation occurring on four human mitochondrial proteins, SAMM50, TOMM40, MIC19, and MIC25. PLoS One. 2018;13:e0206355. doi: 10.1371/journal.pone.0206355 30427857

22. Moriya K, Nagatoshi K, Noriyasu Y, Okamura T, Takamitsu E, Suzuki T, et al. Protein N-myristoylation plays a critical role in the endoplasmic reticulum morphological change induced by overexpression of protein Lunapark, an integral membrane protein of the endoplasmic reticulum. PLoS One. 2013;8:e78235. doi: 10.1371/journal.pone.0078235 24223779

23. Kinoshita E, Kinoshita-Kikuta E. Improved Phos-tag SDS-PAGE under neutral pH conditions for advanced protein phosphorylation profiling. Proteomics. 2011;11:319–323. doi: 10.1002/pmic.201000472 21204258

24. Kinoshita-Kikuta E, Kinoshita E, Matsuda A, Koike T. Tips on improving the efficiency of electrotransfer of target proteins from Phos-tag SDS-PAGE gel. Proteomics. 2014;14: 2437–2442. doi: 10.1002/pmic.201400380 25266391

25. Kinoshita E, Kinoshita-Kikuta E, Takiyama K, Koike T. Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Mol Cell Proteomics. 2006;5:749–757. doi: 10.1074/mcp.T500024-MCP200 16340016

26. Kinoshita E, Kinoshita-Kikuta E, Matsubara M, Yamada S, et al. Separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate-affinity SDS-PAGE. Proteomics. 2008;8:2994–3003. doi: 10.1002/pmic.200800243 18615432

27. Kinoshita E, Kinoshita-Kikuta E, Koike T. Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE. Nat Protoc. 2009;4:1513–1521. doi: 10.1038/nprot.2009.154 19798084

28. Campellone KG, Welch MD. A nucleator arms race: Cellular control of actin assembly. Nat Rev Mol Cell Biol. 2010;11:237–251. doi: 10.1038/nrm2867 20237478

29. Schonichen A, Geyer M. Fifteen formins for an actin filament: A molecular view on the regulation of human formins. Biochim Biophys Acta. 2010;1803:152–163. doi: 10.1016/j.bbamcr.2010.01.014 20102729

30. Goode BL, Eck MJ. Mechanism and function of formins in the control of actin assembly. Annu Rev Biochem. 2007;76:593–627. doi: 10.1146/annurev.biochem.75.103004.142647 17373907

31. Wallar BJ, Alberts AS. The formins: Active scaffolds that remodel the cytoskeleton. Trends Cell Biol. 2003;13:435–446. doi: 10.1016/s0962-8924(03)00153-3 12888296

32. Watanabe N, Higashida C. Formins: Processive cappers of growing actin filaments. Exp Cell Res. 2004;301:16–22. 15501440

33. Watanabe N, Madaule P, Reid T, Ishizaki T, Watanabe G, Kakizuka A, et al. p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for profilin. EMBO J. 1997;16:3044–3056. doi: 10.1093/emboj/16.11.3044 9214622

34. Faix J, Grosse R. Staying in shape with formins. Dev Cell. 2006;10:693–706. doi: 10.1016/j.devcel.2006.05.001 16740473

35. Suzuki T, Moriya K, Nagatoshi K, Ota Y, Ezure T, Ando E, et al. Strategy for comprehensive identification of human N-myristoylated proteins using an insect cell-free protein synthesis system. Proteomics. 2010;10:1780–1793. doi: 10.1002/pmic.200900783 20213681

36. Li Y, Zhu X, Zeng Y, Wang J, Zhang X, Ding YQ, et al. FMNL2 enhances invasion of colorectal carcinoma by inducing epithelial-mesenchymal transition. Mol Cancer Res 2010;8:1579–1590. doi: 10.1158/1541-7786.MCR-10-0081 21071512

37. Liang L, Li X, Zhang X, Lv Z, He G, Zhao W, et al. MicroRNA-137, an HMGA1 target, suppresses colorectal cancer cell invasion and metastasis in mice by directly targeting FMNL2. Gastroenterology. 2013;144:624–635.e4. doi: 10.1053/j.gastro.2012.11.033 23201162

38. Zhu X-L, Zeng Y-F, Guan J, Li Y-F, Deng Y-J, Bian X-W, et al. FMNL2 is a positive regulator of cell motility and metastasis in colorectal carcinoma. J Pathol. 2011;224:377–388. doi: 10.1002/path.2871 21506128

39. Utsumi T, Yoshinaga K, Koga D, Ide A, Nobori K, Okimasu E, et al. Association of myristoylated protein with biological membrane and its increased phosphorylation by protein kinase C. FEBS Lett. 1988;238:13–16. doi: 10.1016/0014-5793(88)80215-1 3169245

40. Taniguchi H. Protein myristoylation in protein–lipid and protein–protein interactions. Biophys Chem. 1999;82:129–137. doi: 10.1016/s0301-4622(99)00112-x 10631796

41. Nishikawa K, Toker A, Johannes F-J, Songyang Z, Cantley LC. Determination of the specific substrate sequence motifs of protein kinase C isozymes. J Biol Chem. 1997;272:952–960. doi: 10.1074/jbc.272.2.952 8995387

42. Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P, et al. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell. 2006;127:635–648. doi: 10.1016/j.cell.2006.09.026 17081983

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