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A novel PKC-regulated mechanism controls CD44–ezrin association and directional cell motility

Nature Cell Biology volume 4, pages 399–407 (2002)Cite this article

An Erratum to this article was published on 01 July 2002

Abstract

The dynamic assembly and disassembly of membrane–cytoskeleton junctional complexes is critical in cell migration. Here we describe a novel phosphorylation mechanism that regulates the hyaluronan receptor CD44. In resting cells, CD44 is constitutively phosphorylated at a single serine residue, Ser325. After protein kinase C is activated, a switch in phosphorylation results in CD44 being phosphorylated solely at an alternative residue, Ser291. Using fluorescence resonance energy transfer (FRET) monitored by fluorescence lifetime imaging microscopy (FLIM) and chemotaxis assays we show that phosphorylation of Ser291 modulates the interaction between CD44 and the cytoskeletal linker protein ezrin in vivo, and that this phosphorylation is critical for CD44-dependent directional cell motility.

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Figure 1: Generation and characterization of mAbs specific for CD44 phosphorylated at Ser325.
Figure 2: Phorbol ester stimulation results in dephosphorylation of CD44 at Ser325.
Figure 3: Phorbol ester stimulation results in serine phosphorylation of CD44 at a non-325 site(s).
Figure 4: In vitro phosphorylation of CD44 by PKC.
Figure 5: Substituting aspartic acid for Ser291 reduces CD44–ezrin association in vitro.
Figure 6: Phorbol ester stimulation reduces CD44-ezrin association in vivo.
Figure 7: Phosphorylation of Ser291 is required for CD44-mediated directional cell motility.

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Acknowledgements

We thank S. Mittnacht for the phosphoamino acid analysis, A. Sullivan for the anti-ezrin antibody, M. Arpin for the pCB6EzTAG VSVG-ezrin construct, R. Lamb for the CD44-GFP construct, M.-T. Webster for generation of the GST-CD44(Ala291) construct and H. Yarwood and P. Townsend for many useful discussions. C.L. was funded by a Cancer Research Campaign studentship, J.L. was funded by an Arthritis Research Council studentship, T.N. is the recipient of an MRC Clinician Scientist grant. This work was supported by funding from the Arthritis Research Council, Breakthrough Breast Cancer, Cancer Research Campaign and the Medical Research Council.

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Author notes

  1. James W. Legg

    Present address: Keratinocyte Laboratory, Cancer Research UK, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK

  2. James W. Legg and Charlotte A. Lewis: These authors contributed equally to the work

Authors and Affiliations

  1. Department of Biological Sciences, Imperial College of Science, Technology and Medicine, Sir Alexander Fleming Building, Imperial College Road, London, SW7 2AZ, UK

    James W. Legg & Charlotte A. Lewis

  2. Richard Dimbleby ICRF Department of Cancer Research, St Thomas' Hospital, Lambeth Palace Road, London, SE1 7EH, UK

    Maddy Parsons & Tony Ng

  3. The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Mary-Jean Mitchell Green Building, Chester Beatty Laboratories, 237 Fulham Road, London, SW3 6JB, UK

    Clare M. Isacke

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Correspondence to Clare M. Isacke.

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Legg, J., Lewis, C., Parsons, M. et al. A novel PKC-regulated mechanism controls CD44–ezrin association and directional cell motility. Nat Cell Biol 4, 399–407 (2002). https://doi.org/10.1038/ncb797

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