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Dissecting motility signaling through activation of specific Src-effector complexes

Nature Chemical Biology volume 10, pages 286–290 (2014)Cite this article

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A Corrigendum to this article was published on 18 July 2014

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Abstract

We describe an approach to selectively activate a kinase in a specific protein complex or at a specific subcellular location within living cells and within minutes. This reveals the effects of specific kinase pathways without time for genetic compensation. The new technique, dubbed rapamycin-regulated targeted activation of pathways (RapRTAP), was used to dissect the role of Src kinase interactions with FAK and p130Cas in cell motility and morphodynamics. The overall effects of Src activation on cell morphology and adhesion dynamics were first quantified, without restricting effector access. Subsets of Src-induced behaviors were then attributed to specific interactions between Src and the two downstream proteins. Activation of Src in the cytoplasm versus at the cell membrane also produced distinct phenotypes. The conserved nature of the kinase site modified for RapRTAP indicates that the technique can be applied to many kinases.

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Figure 1: RapRTAP design and Src-induced morphological changes.
Figure 2: Restricting the activation of Src to specific subcellular locations.
Figure 3: Activation of specific Src-protein complexes.
Figure 4: Model for the role of different Src-effector interactions.

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Change history

  • 07 April 2014

    In the version of this article initially published online, the colors of the purple (SH2 mutant + FAK-FRB) and green (SH2 mutant + p130Cas-FRB) curves in Figure 3d were swapped, leading to a mislabeling of the two experimental results. The error has been corrected for the PDF and HTML versions of this article.

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Acknowledgements

We thank B. Clarke for help with figures and are grateful to the US National Institutes of Health for funding (R21CA159179 to A.V.K., GM102924 and GM094663 to K.M.H. and GM079271 to T.C.E.).

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Author notes

  1. Andrei V Karginov

    Present address: Present address: Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois, USA.,

Authors and Affiliations

  1. Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Andrei V Karginov, Denis Tsygankov, Pei-Hsuan Chu, Evan D Trudeau, Jason J Yi, Timothy C Elston & Klaus M Hahn

  2. Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Matthew Berginski

  3. Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Shawn Gomez & Klaus M Hahn

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Contributions

A.V.K. and K.M.H. initiated the project, and A.V.K. carried out the bulk of the experimental studies. D.T. and T.C.E. developed and applied the new method used to analyze filopodia dynamics. M.B. and S.G. quantified cell adhesion dynamics. All of the other morphodynamic quantifications were by D.T. and A.V.K. E.D.T. and P.-H.C. assisted with kinase assays and cloning. J.J.Y. provided mCherry-stargazin. All of the authors contributed to the writing of the manuscript.

Corresponding author

Correspondence to Klaus M Hahn.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results and Supplementary Figures 1–31. (PDF 9898 kb)

Supplementary Video 1

Effect of RapR-Src activation on cell spreading. (MOV 4182 kb)

Supplementary Video 2

Activation of RapR-Src stimulates cell spreading and filopodia formation. (MOV 755 kb)

Supplementary Video 3

Effect of RapR-Src activation on focal adhesions (MOV 865 kb)

Supplementary Video 4

Effect of RapR-Src activation on focal adhesions (using different focal adhesion marker). (MOV 320 kb)

Supplementary Video 5

Effect of RapR-Src activation at the membrane (MOV 695 kb)

Supplementary Video 6

Effect of RapR-Src activation in the cytoplasm. (MOV 738 kb)

Supplementary Video 7

Effect of RapR-Src activation in complex with FAK on focal adhesions. (MOV 636 kb)

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Karginov, A., Tsygankov, D., Berginski, M. et al. Dissecting motility signaling through activation of specific Src-effector complexes. Nat Chem Biol 10, 286–290 (2014). https://doi.org/10.1038/nchembio.1477

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