Cyclophilin A promotes cell migration via the Abl-Crk signaling pathway

Journal name:
Nature Chemical Biology
Volume:
12,
Pages:
117–123
Year published:
DOI:
doi:10.1038/nchembio.1981
Received
Accepted
Published online

Abstract

Cyclophilin A (CypA) is overexpressed in a number of human cancer types, but the mechanisms by which the protein promotes oncogenic properties of cells are not understood. Here we demonstrate that CypA binds the CrkII adaptor protein and prevents it from switching to the inhibited state. CrkII influences cell motility and invasion by mediating signaling through its SH2 and SH3 domains. CrkII Tyr221 phosphorylation by the Abl or EGFR kinases induces an inhibited state of CrkII by means of an intramolecular SH2-pTyr221 interaction, causing signaling interruption. We show that the CrkII phosphorylation site constitutes a binding site for CypA. Recruitment of CypA sterically restricts the accessibility of Tyr221 to kinases, thereby suppressing CrkII phosphorylation and promoting the active state. Structural, biophysical and in vivo data show that CypA augments CrkII-mediated signaling. A strong stimulation of cell migration is observed in cancer cells wherein both CypA and CrkII are greatly upregulated.

At a glance

Figures

  1. Binding between CypA and CrkII.
    Figure 1: Binding between CypA and CrkII.

    (a) Domain organization of human CrkII. Tyr221 and the Pro220 residues are highlighted. (b) Phosphorylation of Tyr2211 in CrkII results in an intramolecular interaction between pTyr221 and the SH2 domain of CrkII. (c) Sequence alignment of the region Pro216-Pro231 of CrkII from different species. (d) Overlaid 1H-15N HSQC NMR spectra of labeled CrkII (blue) and equimolar unlabeled CypA (orange). (e) Chemical shift mapping of the effect of CypA binding to CrkII. A stretch of approximately six residues flanking Pro220 and encompassing Tyr221 is most affected. CrkII is shown in a ribbon representation (PDB code 2EYZ). Tyr221 and Pro220 side chains are shown as sticks. (f) Chemical shift mapping of the effect of CrkII binding to CypA. CypA residues strongly affected by CrkII binding are colored green. CypA is shown as a solvent-accessible surface

  2. Specific interaction between CypA and CrkII in vitro and in vivo.
    Figure 2: Specific interaction between CypA and CrkII in vitro and in vivo.

    (a) Structure of CypA in complex with the CrkII peptide consisting of residues Ser216–Pro225. CypA is shown in a solvent-accessible surface representation, whereas CrkII is shown as a ball-and-stick model. CrkII residues Glu217–Gln223 are shown (CrkII residues Pro216, Pro224 and Pro225 do not interact with CypA). The backbone of the CrkII region interacting with CypA is buried inside the catalytic cleft of CypA. Two views related by a 90° rotation about the x axis are shown. CrkII is in the trans conformation. (b) Interacting interface between CypA and CrkII. CypA is shown as a light blue ribbon, and the side chains of the residues interacting with CrkII and CrkII are shown in green and yellow ball-and-stick formation, respectively. The magenta lines denote intermolecular polar contacts (hydrogen bonds, salt bridges or both). (c) FRET analysis of HeLa cells transiently transfected together with CrkII-YFP and CypA-CFP. FRET efficiency is color coded from 0–100% on the left. Images were acquired on live cells after confirming comparable expression of both proteins. An average FRET efficiency of 70% is observed between the two fluorophores. (d) FRET analysis of HeLa cells transiently transfected together with CrkII-YFP and CypA-CFP and treated with 25 μM of CsA before imaging. The average FRET efficiency drops to ~15%, indicating that the complex is disrupted in the cell. Scale bars in c,d, 10 μm.

  3. CypA attenuates CrkII Tyr221 phosphorylation in vitro and in vivo.
    Figure 3: CypA attenuates CrkII Tyr221 phosphorylation in vitro and in vivo.

    (a) Effect of CypA on CrkII Tyr221 phosphorylation by Abl kinase was investigated using an in vitro kinase assay (top). The intensity of the phosphorylated Tyr221 (pTyr221) bands were quantified and plotted (bottom). Full gels are shown in Supplementary Figure 13. Orange, without CypA; green, with CypA. IB, immunoblot. (b) The effect of CypA on CrkII Tyr221 phosphorylation in 293T cells was investigated by stably expressing scrambled shRNA or CypA shRNA and transiently transfected with Abl kinase and CrkII plasmids for 48 h. Top, lysates were prepared and analyzed by western blotting with the indicated antibodies. Bottom, the intensity of bands was quantified and plotted. The color code is as displayed in the figure panel. Full gels are shown in Supplementary Figure 13. (c) Effect of CrkII Tyr221 phosphorylation on CrkIIP218F in 293T cells transfected with the plasmids or treatment indicated for 48 h followed by immunoprecipitation of CrkII from lysate. Quantification of CrkII Tyr221 phosphorylation normalized to total CrkII (bottom) was performed after western blotting (top). Full gels are shown in Supplementary Figure 13. For all graphs, data represent the results from three independent experiments (± s.e.m.).

  4. CypA attenuates CrkII Tyr221 phosphorylation in MDA-MB-468 cancer cell line.
    Figure 4: CypA attenuates CrkII Tyr221 phosphorylation in MDA-MB-468 cancer cell line.

    (a) Effect of CypA on CrkII Tyr221 phosphorylation was measured upon EGF stimulation in MDA-MB-468 cells. Lysates were analyzed by western blotting with the antibodies indicated. The intensity of the bands (top) was quantified, and pTyr221 CrkII phosphorylation levels were normalized to overall CrkII expression and plotted (bottom). Full gels are shown in Supplementary Figure 13. Blue, scrambled shRNA; magenta, CypA-KD. IB, immunoblot. (b) The effect of CsA on CrkII Tyr221 phosphorylation was investigated in MDA-MB-468 cells pretreated with DMSO or CsA and stimulated with EGF for the times indicated, and lysates were analyzed for CrkII Tyr221 phosphorylation. The intensity of the bands (top) was quantified, and pY221 CrkII phosphorylation levels were normalized to overall CrkII expression and plotted (bottom). Full gels are shown in Supplementary Figure 13. Blue, without CsA; magenta, with CsA. (c) The effect of CypA on paxillin–CrkII complex formation was investigated in MDA-MB-468 cells stably expressing scrambled shRNA or CypA shRNA transiently transfected with Flag-paxillin and stimulated with EGF for 1 min or 2 min. Immunoprecipitation (IP) of CrkII from lysates was analyzed with anti-Flag. The intensity of the bands (top) representing paxillin were quantified, normalized against the paxillin level in an untreated cell line expressing CypA shRNA and plotted (bottom). Scrambled shRNA (blue) and CypA-KD (magenta). The two bands of paxillin represent differentially phosphorylated forms of paxillin. All of the experiments were analyzed by western blotting. Full gels are shown in Supplementary Figure 13. For all graphs, data represent the results from three independent experiments (± s.e.m.).

  5. Effect of the CypA-CrkII complex on cell migration.
    Figure 5: Effect of the CypA–CrkII complex on cell migration.

    (a) MDA-MB-468 cells stably expressing EYFP or CrkII were serum starved, and motility was analyzed towards EGF (25 ng ml−1) in real time using xCelligence technology. The Delta Cell Index is plotted on the y axis as an average of duplicate samples versus time on the x axis. (b) Wound healing assay of wild-type CrkII (left) and CrkIIP218F (right) in the presence and absence of CsA. Cells were cultured to near confluence, scratched using a pipette and imaged after 24 h. Scale bars, 500 μm. (c) Summary of cell migration measurements from the wound healing assay in b and Supplementary Figure 9c was quantified for each condition indicated. Data represent the mean of three independent experiments ± s.e.m. ****P ≤ 0.001; two-tailed t-test. (d) Focal contacts (Supplementary Fig. 10a) based on the signal from GFP-paxillin were quantified for each cell line and treatment using ImageJ and plotted. Light gray, without CsA; dark gray, with CsA. Data represent mean ± s.e.m. (n = 6). Two-tailed t-test, §§, P ≤ 0.05; § and ****, P ≤ 0.001. Detailed statistics are shown in Supplementary Figure 10b.

  6. Effect of CypA binding to CrkII in integrin signaling.
    Figure 6: Effect of CypA binding to CrkII in integrin signaling.

    (1) Integrin activation elicits paxillin and p130CAS phosphorylation by tyrosine kinases (TKs), and, as a result, Crk proteins are recruited. (2) Abl-induced phosphorylation of CrkII forces its dissociation from paxillin and p130CAS and thus results in signaling suppression. (3) Binding of CypA to Pro220 sterically inhibits CrkII Tyr221 phosphorylation, and CrkII remains in the active form. (4) Guanine nucleotide exchange actors (GEFs; for example, DOCK180 and C3G) associate with CrkII via its SH3N domain, giving rise to efficient localized activation of small GTPases at the membrane. (5) CrkI is a short alternatively spliced form of Crk that lacks the phosphorylation site and thus forms the active state constitutively.

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Referenced accessions

Protein Data Bank

References

  1. Ryffel, B. et al. Distribution of the cyclosporine binding protein cyclophilin in human tissues. Immunology 72, 399404 (1991).
  2. Nigro, P., Pompilio, G. & Capogrossi, M.C. Cyclophilin A: a key player for human disease. Cell Death Dis. 4, e888 (2013).
  3. Bonfils, C. et al. Cyclophilin A as negative regulator of apoptosis by sequestering cytochrome c. Biochem. Biophys. Res. Commun. 393, 325330 (2010).
  4. Bosco, D.A., Eisenmesser, E.Z., Pochapsky, S., Sundquist, W.I. & Kern, D. Catalysis of cis/trans isomerization in native HIV-1 capsid by human cyclophilin A. Proc. Natl. Acad. Sci. USA 99, 52475252 (2002).
  5. Brazin, K.N., Mallis, R.J., Fulton, D.B. & Andreotti, A.H. Regulation of the tyrosine kinase Itk by the peptidyl-prolyl isomerase cyclophilin A. Proc. Natl. Acad. Sci. USA 99, 18991904 (2002).
  6. Howard, B.R., Vajdos, F.F., Li, S., Sundquist, W.I. & Hill, C.P. Structural insights into the catalytic mechanism of cyclophilin A. Nat. Struct. Biol. 10, 475481 (2003).
  7. Göthel, S.F. & Marahiel, M.A. Peptidyl-prolyl cis-trans isomerases, a superfamily of ubiquitous folding catalysts. Cell. Mol. Life Sci. 55, 423436 (1999).
  8. Fischer, G., Tradler, T. & Zarnt, T. The mode of action of peptidyl prolyl cis/trans isomerases in vivo: binding vs. catalysis. FEBS Lett. 426, 1720 (1998).
  9. Lee, J. & Kim, S.S. An overview of cyclophilins in human cancers. J. Int. Med. Res. 38, 15611574 (2010).
  10. Li, Z. et al. Proteomics identification of cyclophilin a as a potential prognostic factor and therapeutic target in endometrial carcinoma. Mol. Cell. Proteomics 7, 18101823 (2008).
  11. Howard, B.A. et al. Stable RNA interference-mediated suppression of cyclophilin A diminishes non-small-cell lung tumor growth in vivo. Cancer Res. 65, 88538860 (2005).
  12. Obchoei, S. et al. Cyclophilin A enhances cell proliferation and tumor growth of liver fluke–associated cholangiocarcinoma. Mol. Cancer 10, 102 (2011).
  13. Stewart, T., Tsai, S.C., Grayson, H., Henderson, R. & Opelz, G. Incidence of de-novo breast cancer in women chronically immunosuppressed after organ transplantation. Lancet 346, 796798 (1995).
  14. Birge, R.B., Kalodimos, C., Inagaki, F. & Tanaka, S. Crk and CrkL adaptor proteins: networks for physiological and pathological signaling. Cell Commun. Signal. 7, 13 (2009).
  15. Linghu, H. et al. Involvement of adaptor protein Crk in malignant feature of human ovarian cancer cell line MCAS. Oncogene 25, 35473556 (2006).
  16. Nishihara, H. et al. Molecular and immunohistochemical analysis of signaling adaptor protein Crk in human cancers. Cancer Lett. 180, 5561 (2002).
  17. Miller, C.T. et al. Increased C-CRK proto-oncogene expression is associated with an aggressive phenotype in lung adenocarcinomas. Oncogene 22, 79507957 (2003).
  18. Watanabe, T. et al. Adaptor protein Crk induces Src-dependent activation of p38 MAPK in regulation of synovial sarcoma cell proliferation. Mol. Cancer Res. 7, 15821592 (2009).
  19. Takino, T. et al. CrkI adapter protein modulates cell migration and invasion in glioblastoma. Cancer Res. 63, 23352337 (2003).
  20. Kumar, S., Fajardo, J.E., Birge, R.B. & Sriram, G. Crk at the quarter century mark: perspectives in signaling and cancer. J. Cell. Biochem. 115, 819825 (2014).
  21. Fathers, K.E. et al. Crk adaptor proteins act as key signaling integrators for breast tumorigenesis. Breast Cancer Res. 14, R74 (2012).
  22. Shishido, T. et al. Crk family adaptor proteins trans-activate c-Abl kinase. Genes Cells 6, 431440 (2001).
  23. Sirvent, A., Benistant, C. & Roche, S. Cytoplasmic signalling by the c-Abl tyrosine kinase in normal and cancer cells. Biol. Cell 100, 617631 (2008).
  24. Petschnigg, J. et al. The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells. Nat. Methods 11, 585592 (2014).
  25. Sarkar, P., Reichman, C., Saleh, T., Birge, R.B. & Kalodimos, C.G. Proline cis-trans isomerization controls autoinhibition of a signaling protein. Mol. Cell 25, 413426 (2007).
  26. Sarkar, P., Saleh, T., Tzeng, S.R., Birge, R.B. & Kalodimos, C.G. Structural basis for regulation of the Crk signaling protein by a proline switch. Nat. Chem. Biol. 7, 5157 (2011).
  27. Kobashigawa, Y. et al. Structural basis for the transforming activity of human cancer-related signaling adaptor protein CRK. Nat. Struct. Mol. Biol. 14, 503510 (2007).
  28. Schmidpeter, P.A. & Schmid, F.X. Molecular determinants of a regulatory prolyl isomerization in the signal adapter protein c-CrkII. ACS Chem. Biol. 9, 11451152 (2014).
  29. Feller, S.M., Knudsen, B. & Hanafusa, H. c-Abl kinase regulates the protein binding activity of c-Crk. EMBO J. 13, 23412351 (1994).
  30. Hashimoto, Y. et al. Phosphorylation of CrkII adaptor protein at tyrosine 221 by epidermal growth factor receptor. J. Biol. Chem. 273, 1718617191 (1998).
  31. Rosen, M.K. et al. Direct demonstration of an intramolecular SH2-phosphotyrosine interaction in the Crk protein. Nature 374, 477479 (1995).
  32. Chodniewicz, D. & Klemke, R.L. Regulation of integrin-mediated cellular responses through assembly of a CAS/Crk scaffold. Biochim. Biophys. Acta 1692, 6376 (2004).
  33. Piotukh, K. et al. Cyclophilin A binds to linear peptide motifs containing a consensus that is present in many human proteins. J. Biol. Chem. 280, 2366823674 (2005).
  34. Handschumacher, R.E., Harding, M.W., Rice, J., Drugge, R.J. & Speicher, D.W. Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science 226, 544547 (1984).
  35. Ting, A.Y., Kain, K.H., Klemke, R.L. & Tsien, R.Y. Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells. Proc. Natl. Acad. Sci. USA 98, 1500315008 (2001).
  36. Filmus, J., Pollak, M.N., Cailleau, R. & Buick, R.N. MDA-468, a human breast cancer cell line with a high number of epidermal growth factor (EGF) receptors, has an amplified EGF receptor gene and is growth inhibited by EGF. Biochem. Biophys. Res. Commun. 128, 898905 (1985).
  37. Escalante, M. et al. Phosphorylation of c-Crk II on the negative regulatory Tyr222 mediates nerve growth factor–induced cell spreading and morphogenesis. J. Biol. Chem. 275, 2478724797 (2000).
  38. Yamada, S. et al. Overexpression of CRKII increases migration and invasive potential in oral squamous cell carcinoma. Cancer Lett. 303, 8491 (2011).
  39. Noren, N.K., Foos, G., Hauser, C.A. & Pasquale, E.B. The EphB4 receptor suppresses breast cancer cell tumorigenicity through an Abl-Crk pathway. Nat. Cell Biol. 8, 815825 (2006).
  40. Takino, T. et al. Tyrosine phosphorylation of the CrkII adaptor protein modulates cell migration. J. Cell Sci. 116, 31453155 (2003).
  41. Diemert, S. et al. Impedance measurement for real time detection of neuronal cell death. J. Neurosci. Methods 203, 6977 (2012).
  42. Deakin, N.O. & Turner, C.E. Paxillin comes of age. J. Cell Sci. 121, 24352444 (2008).
  43. Hu, Y.L. et al. FAK and paxillin dynamics at focal adhesions in the protrusions of migrating cells. Sci. Rep. 4, 6024 (2014).
  44. de Jong, R., ten Hoeve, J., Heisterkamp, N. & Groffen, J. Tyrosine 207 in CRKL is the BCR/ABL phosphorylation site. Oncogene 14, 507513 (1997).
  45. Hemmeryckx, B. et al. BCR/ABL P190 transgenic mice develop leukemia in the absence of Crkl. Oncogene 21, 32253231 (2002).
  46. Jankowski, W. et al. Domain organization differences explain Bcr-Abl's preference for CrkL over CrkII. Nat. Chem. Biol. 8, 590596 (2012).
  47. Bell, E.S. & Park, M. Models of Crk adaptor proteins in cancer. Genes Cancer 3, 341352 (2012).
  48. Bravo-Cordero, J.J., Hodgson, L. & Condeelis, J. Directed cell invasion and migration during metastasis. Curr. Opin. Cell Biol. 24, 277283 (2012).
  49. Klemke, R.L. et al. CAS/Crk coupling serves as a 'molecular switch' for induction of cell migration. J. Cell Biol. 140, 961972 (1998).
  50. Nath, P.R., Dong, G., Braiman, A. & Isakov, N. Immunophilins control T lymphocyte adhesion and migration by regulating CrkII binding to C3G. J. Immunol. 193, 39663977 (2014).
  51. Reichman, C. et al. Transactivation of Abl by the Crk II adapter protein requires a PNAY sequence in the Crk C-terminal SH3 domain. Oncogene 24, 81878199 (2005).
  52. Sriram, G. et al. Phosphorylation of Crk on tyrosine 251 in the RT loop of the SH3C domain promotes Abl kinase transactivation. Oncogene 30, 46454655 (2011).
  53. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277293 (1995).
  54. Güntert, P. Automated NMR structure calculation with CYANA. Methods Mol. Biol. 278, 353378 (2004).
  55. Shen, Y. & Bax, A. Protein backbone and side chain torsion angles predicted from NMR chemical shifts using artificial neural networks. J. Biomol. NMR 56, 227241 (2013).
  56. de Vries, S.J., van Dijk, M. & Bonvin, A.M. The HADDOCK web server for data-driven biomolecular docking. Nat. Protoc. 5, 883897 (2010).

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Author information

Affiliations

  1. Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, Minneapolis, Minnesota, USA.

    • Tamjeed Saleh,
    • Paolo Rossi &
    • Charalampos G Kalodimos
  2. Center for Integrative Proteomics Research, Rutgers University, Piscataway, New Jersey, USA.

    • Tamjeed Saleh,
    • Wojciech Jankowski,
    • Paolo Rossi &
    • Charalampos G Kalodimos
  3. Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey, USA.

    • Tamjeed Saleh,
    • Wojciech Jankowski,
    • Paolo Rossi,
    • Shreyas Shah,
    • Ki-Bum Lee &
    • Charalampos G Kalodimos
  4. Department of Biochemistry and Molecular Biology, Rutgers New Jersey Medical School, Newark, New Jersey, USA.

    • Ganapathy Sriram &
    • Raymond B Birge
  5. Department of Biological Sciences, Rutgers University, Newark, New Jersey, USA.

    • Lissette Alicia Cruz &
    • Alexis J Rodriguez

Contributions

T.S. and C.G.K. designed the study; T.S. and W.J. recorded and analyzed the NMR data; W.J. and P.R. determined the structure of the complex; T.S., G.S. and R.B.B. collected and analyzed the kinase phosphorylation and cell migration data; T.S., S.S. and K.-B.L. collected and analyzed the FRET and wound healing data; T.S., L.A.C. and A.J.R. collected and analyzed the fluorescence microscopy data; T.S. and C.G.K. wrote the manuscript.

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The authors declare no competing financial interests.

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    Supplementary Results, Supplementary Figures 1–13 and Supplementary Table 1.

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