Secramine inhibits Cdc42-dependent functions in cells and Cdc42 activation in vitro

An Erratum to this article was published on 01 April 2006

This article has been updated

Abstract

Inspired by the usefulness of small molecules to study membrane traffic, we used high-throughput synthesis and phenotypic screening to discover secramine, a molecule that inhibits membrane traffic out of the Golgi apparatus by an unknown mechanism. We report here that secramine inhibits activation of the Rho GTPase Cdc42, a protein involved in membrane traffic, by a mechanism dependent upon the guanine dissociation inhibitor RhoGDI. RhoGDI binds Cdc42 and antagonizes its membrane association, nucleotide exchange and effector binding. In vitro, secramine inhibits Cdc42 binding to membranes, GTP and effectors in a RhoGDI-dependent manner. In cells, secramine mimics the effects of dominant-negative Cdc42 expression on protein export from the Golgi and on Golgi polarization in migrating cells. RhoGDI-dependent Cdc42 inhibition by secramine illustrates a new way to inhibit Rho GTPases with small molecules and provides a new means to study Cdc42, RhoGDI and the cellular processes they mediate.

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Figure 1: Screening of a library of galanthamine-like small molecules leading to the identification of secramine A as an inhibitor of Golgi-to–plasma membrane transport.
Figure 2: Secramine A inhibits PIP2 liposome–stimulated actin polymerization in X. laevis egg extract and acts upstream of Arp2/3 complex activation.
Figure 3: Secramine A inhibits Cdc42 activation in X. laevis egg extract.
Figure 4: Secramine A inhibits Cdc42 activation and PIP2 liposome association in vitro.
Figure 5: Secramine does not disrupt PIP2 liposomes.
Figure 6: Effect of secramine A on Cdc42-dependent events.

Change history

  • note made in HTML and Fig 3a; PDF appended; corrected online date added to PDF

Notes

  1. 1.

    *Note: In the version of this article initially published online, two labels in Figure 3a are incorrect. The label in the square below the arrow for step 2 should read 'Cdc42 GTP' instead of 'Cdc42 GDP', and ‘PI’ in step 4 should be ‘Pi’. These errors have been corrected in the PDF version of the article.

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Acknowledgements

We thank S.C. Harrison for comments on the manuscript. We also thank D.A. Annis, N. Nazef, G. Hoffman, R. Massol, A. Mata-Fink, and A. Griffin for technical assistance and discussions. This work was supported by US National Institutes of Health (NIH) grant GM62566-05 to T.K. and M.D.S. and NIH New England Regional Center of Excellence (NERCE) grant 5 U54 AI057159-03 to T.K. M.S. and J.C. were supported by NIH grant GM068674.

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Correspondence to Matthew D Shair or Tomas Kirchhausen.

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Supplementary information

Supplementary Fig. 1

Screening results for galanthamine-like library. (PDF 1035 kb)

Supplementary Fig. 2

Effects of secramine on the secretory pathway. (PDF 3147 kb)

Supplementary Fig. 3

Inhibition of Rac1 activation by secramine. (PDF 153 kb)

Supplementary Video 1

BS-C-1 cells expressing VSVGts-EGFP at 40°C were treated with cycloheximide and 1% DMSO (vehicle) for 10 min followed by transfer to 32°C and image acquisition. (MOV 3067 kb)

Supplementary Video 2

BS-C-1 cells expressing VSVGts-EGFP at 40°C were treated with cycloheximide and 10 μM secramine A for 10 min followed by transfer to 32°C and image acquisition. (MOV 3071 kb)

Supplementary Video 3

This movie is a continuation of Supplementary Video 2; it was recorded at 32°C and at the conclusion of movie 2. (MOV 3074 kb)

Supplementary Methods (PDF 2092 kb)

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Pelish, H., Peterson, J., Salvarezza, S. et al. Secramine inhibits Cdc42-dependent functions in cells and Cdc42 activation in vitro. Nat Chem Biol 2, 39–46 (2006). https://doi.org/10.1038/nchembio751

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