Characterization of the EF-Hand Calcium-Binding Domains of Human Plastins

  • Oleg Khassan
  • Katharine V. Jensen
  • Andrew G. Woodman
  • Hans J. VogelEmail author
  • Hiroaki Ishida
Part of the Methods in Molecular Biology book series (MIMB, volume 1929)


The three human plastins (L-plastin, T-plastin, and I-plastin) are important regulatory Ca2+-binding proteins that belong to the family of actin-binding proteins. Plastins are involved in the regulation of the actin cytoskeleton as well as the cross-linking of actin filaments. In addition to four calponin-homology (CH) domains, all three plastins contain two N-terminal EF-hand Ca2+-binding motifs which together are homologous to a single lobe of the well-known calcium-regulatory protein calmodulin. This part of the protein allows for the regulation of the actin bundling activity in response to elevated calcium levels. In this protocol, we describe the purification of the EF-hand headpiece domains of all three plastins, as well as SPR studies, ITC studies, and NMR interaction studies with different peptides and calcium. In combination, these three experimental techniques provide detailed insights into a novel regulatory mechanism, involving the linker region between the EF-hand domain and the first CH domain of the plastins.

Key words

L-plastin T-plastin I-plastin Fimbrin Calcium-binding domain Actin bundling EF-hand motifs NMR spectroscopy SPR ITC 



This research is currently supported by a Discovery grant from the Natural Sciences and Engineering Research Council of Canada.


  1. 1.
    Delanote V, Vandekerckhove J, Gettemans J (2005) Plastins: versatile modulators of actin organization in (patho)physiological cellular processes. Acta Pharmacol Sin 26:769–779CrossRefGoogle Scholar
  2. 2.
    Shinomiya H (2012) Plastin family of actin-bundling proteins: its functions in leukocytes, neurons, intestines, and cancer. Int J Cell Biol 2012:213492CrossRefGoogle Scholar
  3. 3.
    Lin CS, Aebersold RH, Kent SB, Varma M, Leavitt J (1988) Molecular cloning and characterization of plastin, a human leukocyte protein expressed in transformed human fibroblasts. Mol Cell Biol 8:4659–4668CrossRefGoogle Scholar
  4. 4.
    Miyakawa T, Shinomiya H, Yumoto F, Miyauchi Y, Tanaka H, Ojima T, Kato YS, Tanokura M (2012) Different Ca2+−sensitivities between the EF-hands of T- and L-plastins. Biochem Biophys Res Commun 429:137–141CrossRefGoogle Scholar
  5. 5.
    Dos Remedios CG, Chhabra D, Kekic M, Dedova IV, Tsubakihara M, Berry DA, Nosworthy NJ (2003) Actin binding proteins: regulation of cytoskeletal microfilaments. Physiol Rev 83:433–473CrossRefGoogle Scholar
  6. 6.
    Morley SC (2012) The actin-bundling protein L-plastin: a critical regulator of immune cell function. Int J Cell Biol 2012:935173CrossRefGoogle Scholar
  7. 7.
    Morley SC (2013) The actin-bundling protein L-plastin supports T-cell motility and activation. Immunol Rev 256:48–62CrossRefGoogle Scholar
  8. 8.
    Wang C, Morley SC, Donermeyer D, Peng I, Lee WP, Devoss J, Danilenko DM, Lin Z, Zhang J, Zhou J, Allen PM, Brown EJ (2010) Actin-bundling protein L-plastin regulates T cell activation. J Immunol 185:7487–7497CrossRefGoogle Scholar
  9. 9.
    Todd EM, Deady LE, Morley SC (2011) The actin-bundling protein L-plastin is essential for marginal zone B cell development. J Immunol 187:3015–3025CrossRefGoogle Scholar
  10. 10.
    Kell MJ, Riccio RE, Baumgartner EA, Compton ZJ, Pecorin PJ, Mitchell TA, Topczewski J, LeClair EE (2018) Targeted deletion of the zebrafish actin-bundling protein L-plastin (lcp1). PLoS One 13:e0190353CrossRefGoogle Scholar
  11. 11.
    Chen H, Mocsai A, Zhang H, Ding R-X, Morisaki JH, White M, Rothfork JM, Heiser P, Colucci-Guyon E, Lowell CA, Gresham HD, Allen PM, Brown EJ (2003) Role for plastin in host defense distinguishes integrin signaling from cell adhesion and spreading. Immunity 19:95–104CrossRefGoogle Scholar
  12. 12.
    Park T, Chen ZP, Leavitt J (1994) Activation of the leukocyte plastin gene occurs in most human cancer cells. Cancer Res 54:1775–1781PubMedGoogle Scholar
  13. 13.
    Lin CS, Park T, Chen ZP, Leavitt J (1993) Human plastin genes. Comparative gene structure, chromosome location, and differential expression in normal and neoplastic cells. J Biol Chem 268:2781–2792PubMedGoogle Scholar
  14. 14.
    Riplinger SM, Wabnitz GH, Kirchgessner H, Jahraus B, Lasitschka F, Schulte B, van der Pluijm G, van der Horst G, Hämmerling GJ, Nakchbandi I, Samstag Y (2014) Metastasis of prostate cancer and melanoma cells in a preclinical in vivo mouse model is enhanced by L-plastin expression and phosphorylation. Mol Cancer 13:10CrossRefGoogle Scholar
  15. 15.
    Koide N, Kasamatsu A, Endo-Sakamoto Y, Ishida S, Shimizu T, Kimura Y, Miyamoto I, Yoshimura S, Shiiba M, Tanzawa H, Uzawa K (2017) Evidence for critical role of lymphocyte cytosolic protein 1 in oral cancer. Sci Rep 7:43379CrossRefGoogle Scholar
  16. 16.
    Foran E, McWilliam P, Kelleher D, Croke DT, Long A (2006) The leukocyte protein L-plastin induces proliferation, invasion and loss of E-cadherin expression in colon cancer cells. Int J Cancer 118:2098–2104CrossRefGoogle Scholar
  17. 17.
    Klemke M, Rafael MT, Wabnitz GH, Weschenfelder T, Konstandin MH, Garbi N, Autschbach F, Hartschuh W, Samstag Y (2007) Phosphorylation of ectopically expressed L-plastin enhances invasiveness of human melanoma cells. Int J Cancer 120:2590–2599CrossRefGoogle Scholar
  18. 18.
    Delanote V, Vanloo B, Catillon M, Friederich E, Vandekerckhove J, Gettemans J (2010) An alpaca single-domain antibody blocks filopodia formation by obstructing L-plastin-mediated F-actin bundling. FASEB J 24:105–118CrossRefGoogle Scholar
  19. 19.
    Zheng J, Rudra-Ganguly N, Powell WC, Roy-Burman P (1999) Suppression of prostate carcinoma cell invasion by expression of antisense L-plastin gene. Am J Pathol 155:115–122CrossRefGoogle Scholar
  20. 20.
    Samstag Y, Klemke M (2007) Ectopic expression of L-plastin in human tumor cells: diagnostic and therapeutic implications. Adv Enzym Regul 47:118–126CrossRefGoogle Scholar
  21. 21.
    Lommel MJ, Trairatphisan P, Gäbler K, Laurini C, Muller A, Kaoma T, Vallar L, Sauter T, Schaffner-Reckinger E (2016) L-plastin Ser5 phosphorylation in breast cancer cells and in vitro is mediated by RSK downstream of the ERK/MAPK pathway. FASEB J 30:1218–1233CrossRefGoogle Scholar
  22. 22.
    Hasselblatt M, Böhm C, Tatenhorst L, Dinh V, Newrzella D, Keyvani K, Jeibmann A, Buerger H, Rickert CH, Paulus W (2006) Identification of novel diagnostic markers for choroid plexus tumors: A microarray-based approach. Am J Surg Pathol 30:66–74CrossRefGoogle Scholar
  23. 23.
    Li J, Zhao R (2011) Expression and clinical significance of l-plastin in colorectal carcinoma. J Gastrointest Surg 15:1982–1988CrossRefGoogle Scholar
  24. 24.
    Harris LD, De La Cerda J, Tuziak T, Rosen D, Xiao L, Shen Y, Sabichi AL, Czerniak B, Grossman HB (2008) Analysis of the expression of biomarkers in urinary bladder cancer using a tissue microarray. Mol Carcinog 47:678–685CrossRefGoogle Scholar
  25. 25.
    Öztürk VÖ, Emingil G, Osterwalder V, Bostanci N (2015) The actin-bundling protein L-plastin: a novel local inflammatory marker associated with periodontitis. J Periodontal Res 50:337–346CrossRefGoogle Scholar
  26. 26.
    Oprea GE, Kröber S, McWhorter ML, Rossoll W, Müller S, Krawczak M, Bassell GJ, Beattie CE, Wirth B (2008) Plastin 3 is a protective modifier of autosomal recessive spinal muscular atrophy. Science 320:524–527CrossRefGoogle Scholar
  27. 27.
    Ralser M, Nonhoff U, Albrecht M, Lengauer T, Wanker EE, Lehrach H, Krobitsch S (2005) Ataxin-2 and huntingtin interact with endophilin-A complexes to function in plastin-associated pathways. Hum Mol Genet 14:2893–2909CrossRefGoogle Scholar
  28. 28.
    van Dijk FS, Zillikens MC, Micha D, Riessland M, Marcelis CLM, de Die-Smulders CE, Milbradt J, Franken AA, Harsevoort AJ, Lichtenbelt KD, Pruijs HE, Rubio-Gozalbo ME, Zwertbroek R, Moutaouakil Y, Egthuijsen J, Hammerschmidt M, Bijman R, Semeins CM, Bakker AD, Everts V, Klein-Nulend J, Campos-Obando N, Hofman A, te Meerman GJ, Verkerk AJMH, Uitterlinden AG, Maugeri A, Sistermans EA, Waisfisz Q, Meijers-Heijboer H, Wirth B, Simon MEH, Pals G (2013) PLS3 mutations in X-linked osteoporosis with fractures. N Engl J Med 369:1529–1536CrossRefGoogle Scholar
  29. 29.
    Mäkitie RE, Kämpe AJ, Taylan F, Mäkitie O (2017) Recent discoveries in monogenic disorders of childhood bone fragility. Curr Osteoporos Rep 15:303–310CrossRefGoogle Scholar
  30. 30.
    Higuchi Y, Kita K, Nakanishi H, Wang XL, Sugaya S, Tanzawa H, Yamamori H, Sugita K, Yamaura A, Suzuki N (1998) Search for genes involved in UV-resistance in human cells by mRNA differential display: Increased transcriptional expression of nucleophosmin and T-plastin genes in association with the resistance. Biochem Biophys Res Commun 248:597–602CrossRefGoogle Scholar
  31. 31.
    Suzuki N (1984) A UV-resistant mutant without an increased repair synthesis activity, established from a UV-sensitive human clonal cell line. Mutat Res 125:55–63CrossRefGoogle Scholar
  32. 32.
    Yokobori T, Iinuma H, Shimamura T, Imoto S, Sugimachi K, Ishii H, Iwatsuki M, Ota D, Ohkuma M, Iwaya T, Nishida N, Kogo R, Sudo T, Tanaka F, Shibata K, Toh H, Sato T, Barnard GF, Fukagawa T, Yamamoto S, Nakanishi H, Sasaki S, Miyano S, Watanabe T, Kuwano H, Mimori K, Pantel K, Mori M (2013) Plastin3 is a novel marker for circulating tumor cells undergoing the epithelial-mesenchymal transition and is associated with colorectal cancer prognosis. Cancer Res 73:2059–2069CrossRefGoogle Scholar
  33. 33.
    Ueo H, Sugimachi K, Gorges TM, Bartkowiak K, Yokobori T, Müller V, Shinden Y, Ueda M, Ueo H, Mori M, Kuwano H, Maehara Y, Ohno S, Pantel K, Mimori K (2015) Circulating tumour cell-derived plastin3 is a novel marker for predicting long-term prognosis in patients with breast cancer. Br J Cancer 112:1519–1526CrossRefGoogle Scholar
  34. 34.
    Grimm-Gunter E-MS, Revenu C, Ramos S, Hurbain I, Smyth N, Ferrary E, Louvard D, Robine S, Rivero F (2009) Plastin 1 binds to keratin and is required for terminal web assembly in the intestinal epithelium. Mol Biol Cell 20:2549–2562CrossRefGoogle Scholar
  35. 35.
    Taylor R, Bullen A, Johnson SL, Grimm-Günter EM, Rivero F, Marcotti W, Forge A, Daudet N (2015) Absence of plastin 1 causes abnormal maintenance of hair cell stereocilia and a moderate form of hearing loss in mice. Hum Mol Genet 24:37–49CrossRefGoogle Scholar
  36. 36.
    Krey JF, Krystofiak ES, Dumont RA, Vijayakumar S, Choi D, Rivero F, Kachar B, Jones SM, Barr-Gillespie PG (2016) Plastin 1 widens stereocilia by transforming actin filament packing from hexagonal to liquid. J Cell Biol 215:467–482CrossRefGoogle Scholar
  37. 37.
    Bretscher A, Weber K (1980) Fimbrin, a new microfilament-associated protein present in microvilli and other cell surface structures. J Cell Biol 86:335–340CrossRefGoogle Scholar
  38. 38.
    Stradal T, Kranewitter W, Winder SJ, Gimona M (1998) CH domains revisited. FEBS Lett 431:134–137CrossRefGoogle Scholar
  39. 39.
    Mattila PK, Lappalainen P (2008) Filopodia: molecular architecture and cellular functions. Nat Rev Mol Cell Biol 9:446–454CrossRefGoogle Scholar
  40. 40.
    Van Audenhove I, Denert M, Boucherie C, Pieters L, Cornelissen M, Gettemans J (2016) Fascin rigidity and L-plastin flexibility cooperate in cancer cell invadopodia and Filopodia. J Biol Chem 291:9148–9160CrossRefGoogle Scholar
  41. 41.
    Klein MG, Shi W, Ramagopal U, Tseng Y, Wirtz D, Kovar DR, Staiger CJ, Almo SC (2004) Structure of the actin crosslinking core of fimbrin. Structure 12:999–1013CrossRefGoogle Scholar
  42. 42.
    Namba Y, Ito M, Zu Y, Shigesada K, Maruyama K (1992) Human T cell L-plastin bundles actin filaments in a calcium-dependent manner. J Biochem 112:503–507CrossRefGoogle Scholar
  43. 43.
    Ishida H, Jensen KV, Woodman AG, Hyndman ME, Vogel HJ (2017) The calcium-dependent switch helix of L-plastin regulates actin bundling. Sci Rep 7:40662CrossRefGoogle Scholar
  44. 44.
    Schwebach CL, Agrawal R, Lindert S, Kudryashova E, Kudryashov DS (2017) The roles of actin-binding domains 1 and 2 in the calcium-dependent regulation of actin filament bundling by human plastins. J Mol Biol 429:2490–2508CrossRefGoogle Scholar
  45. 45.
    Prassler J, Stocker S, Marriott G, Heidecker M, Kellermann J, Gerisch G (1997) Interaction of a dictyostelium member of the plastin/fimbrin family with actin filaments and actin-myosin complexes. Mol Biol Cell 8:83–95CrossRefGoogle Scholar
  46. 46.
    Kapust RB, Tözsér J, Fox JD, Anderson DE, Cherry S, Copeland TD, Waugh DS (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng Des Sel 14:993–1000CrossRefGoogle Scholar
  47. 47.
    SPR-Pages Thiol coupling. Accessed June 4 2018

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Oleg Khassan
    • 1
  • Katharine V. Jensen
    • 1
  • Andrew G. Woodman
    • 1
  • Hans J. Vogel
    • 1
    Email author
  • Hiroaki Ishida
    • 1
  1. 1.Biochemistry Research Group, Department of Biological SciencesUniversity of CalgaryCalgaryCanada

Personalised recommendations