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Nck adaptor proteins link nephrin to the actin cytoskeleton of kidney podocytes

Nature volume 440, pages 818–823 (2006)Cite this article

Abstract

The glomerular filtration barrier in the kidney is formed in part by a specialized intercellular junction known as the slit diaphragm, which connects adjacent actin-based foot processes of kidney epithelial cells (podocytes)1. Mutations affecting a number of slit diaphragm proteins, including nephrin (encoded by NPHS1)2, lead to renal disease owing to disruption of the filtration barrier and rearrangement of the actin cytoskeleton3, although the molecular basis for this is unclear. Here we show that nephrin selectively binds the Src homology 2 (SH2)/SH3 domain-containing Nck adaptor proteins4, which in turn control the podocyte cytoskeleton in vivo. The cytoplasmic tail of nephrin has multiple YDxV sites that form preferred binding motifs for the Nck SH2 domain once phosphorylated by Src-family kinases. We show that this Nck–nephrin interaction is required for nephrin-dependent actin reorganization. Selective deletion of Nck from podocytes of transgenic mice results in defects in the formation of foot processes and in congenital nephrotic syndrome. Together, these findings identify a physiological signalling pathway in which nephrin is linked through phosphotyrosine-based interactions to Nck adaptors, and thus to the underlying actin cytoskeleton in podocytes. Simple and widely expressed SH2/SH3 adaptor proteins can therefore direct the formation of a specialized cellular morphology in vivo.

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Figure 1: The kidney slit diaphragm protein nephrin has multiple YDxV motifs that bind the Nck SH2 domain.
Figure 2: Identification of a phosphotyrosine-dependent interaction between nephrin and the Nck SH2/SH3 adaptor.
Figure 3: Glomerulosclerosis and foot process fusion in mice lacking Nck expression in podocytes.
Figure 4: Nck recruitment to phosphorylated nephrin facilitates localized actin reorganization.

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Acknowledgements

We thank T. Huber, G. Rivera, B. Mayer, P. Mundel, P. Soriano, M. Kirschner, K. Tryggvason and D. Hedley for providing reagents and technical advice, D. Holmyard for electron microscopy, and N. Woody for technical contributions. N.J. was supported by fellowships from the Canadian Institutes for Health Research (CIHR) and the National Cancer Institute of Canada (NCIC), with funds from the Terry Fox Run. This work was supported by grants from the CIHR (to S.S.-C.L., T.T., S.E.Q. and T.P.), the NCIC (to S.S.-C.L., S.E.Q. and T.P.) and the Kidney Foundation of Canada (to T.T.). S.S.-C.L. is an NCIC Scientist with funds from the Canadian Cancer Society. L.L. and T.T. hold scholarships from the Fonds de la Recherche en Santé du Québec. S.E.Q. holds a Tier 2 Canada Research Chair. T.P. is a Distinguished Investigator of the CIHR.

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Author notes

  1. Ivan M. Blasutig and Vera Eremina: *These authors contributed equally to this work

Authors and Affiliations

  1. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Ontario, M5G 1X5, Toronto, Canada

    Nina Jones, Ivan M. Blasutig, Vera Eremina, Julie M. Ruston, Friedhelm Bladt, Susan E. Quaggin & Tony Pawson

  2. Department of Molecular and Medical Genetics, University of Toronto, 4388 Medical Sciences Building, 1 King's College Circle, Ontario, M5S 1A8, Toronto, Canada

    Ivan M. Blasutig & Tony Pawson

  3. Institute of Medical Science, University of Toronto, 7213 Medical Sciences Building, 1 King's College Circle, Ontario, M5S 1A8, Toronto, Canada

    Vera Eremina & Susan E. Quaggin

  4. Department of Medicine, McGill University Health Centre, 3775 University Street, Québec, H3A 2B4, Montreal, Canada

    Hongping Li, Louise Larose & Tomoko Takano

  5. Department of Biochemistry, University of Western Ontario, Ontario, N6A 5C1, London, Canada

    Haiming Huang & Shawn S.-C. Li

  6. Division of Nephrology, St. Michael's Hospital, 30 Bond Street, Ontario, M5B 1W8, Toronto, Canada

    Susan E. Quaggin

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Corresponding author

Correspondence to Tony Pawson.

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Supplementary information

Supplementary Figure 1

This figure shows expression of Nck1 and Nck2 in podocytes (by lacZ staining and immunoblotting) as well as the presence of mature foot processes in mice lacking either Nck1 or Nck2. The specificity of Podocin-Cre is shown using the Z/EG reporter strain. (PDF 12859 kb)

Supplementary Figure 2

This figure shows that weaning age mice lacking Nck1 and Nck2 in podocytes are less robust than littermates and display massive albuminuria. (PDF 3882 kb)

Supplementary Figure 3

This figure shows by RNA in situ hybridization analysis of glomeruli that podocytes are progressively lost in mice lacking Nck1 and Nck2 expression in podocytes. (PDF 4436 kb)

Supplementary Figure 4

This figure shows by immunoblotting that N-WASp is recruited to phosphorylated Nephrin containing Nck binding sites. (PDF 695 kb)

Supplementary Figure 5

This figure shows by immunoblotting that Src family kinase activity is required to induce tyrosine phosphorylation of Nephrin. (PDF 1719 kb)

Supplementary Methods

This file contains additional details of the methods used in this study. This file also contains additional references. (DOC 33 kb)

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Jones, N., Blasutig, I., Eremina, V. et al. Nck adaptor proteins link nephrin to the actin cytoskeleton of kidney podocytes. Nature 440, 818–823 (2006). https://doi.org/10.1038/nature04662

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