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Leucine 255 of Src couples intramolecular interactions to inhibition of catalysis

Nature Structural Biology volume 6, pages 760–764 (1999)Cite this article

Abstract

The activity of the c-Src tyrosine kinase is regulated through intramolecular interactions between the catalytic and SH2/SH3 domains. However, the exact mechanism by which this occurs remains obscure. In the crystal structure of c-Src, the peptide that links the SH2 and catalytic domain (SH2-CD linker) is sandwiched between the latter and the SH3 domain. A residue in the linker, Leu 255, inserts its side chain into a deep hydrophobic pocket present on the surface of the catalytic domain. To investigate the possible regulatory role of this prominent interaction, we mutated Leu 255 to different hydrophobic residues. We found that the length and 'bulkiness' of the side chain had a profound influence on c-Src regulation. Src-L255V was highly active but showed reduced SH3 accessibility in vitro as well as an altered localization in vivo when compared to other deregulated forms of Src. Our analyses lead us to suggest that the Leu 255–pocket interaction is a critical component of the intramolecular inhibition mechanism of Src family kinases.

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Figure 1: Structure of regulated Src (PDB entry 2PTK; see ref. 7) and of active Lck (PDB entry 3LCK; see ref. 14).
Figure 2: a, Growth curves of S. pombe cells expressing Src mutants from the inducible nmt1 promoter of the pRSP vector (as indicated) and pAU or pAU-Csk.
Figure 3: The activity of the various Src mutants expressed in human cells.
Figure 4: SH3 domain-binding experiment.
Figure 5: Immunofluorescence analysis of HeLa cells transfected with Src mutants (as indicated on top).

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Acknowledgements

We would like to thank J. Williams and R. Wierenga for helping us appreciate the importance of the hydrophobic pocket, A. Nelsbach and his colleagues at New England Biolabs for the very valuable anti-phospho-Y416 antibodies, E. Conti, M. Bottomley and Y. Modis for help with GRASP, R. Salgia and J. Griffin for paxillin cDNA, W. Pluk for the HA-paxillin construct, G. Cesareni for suggestions, E. Conti, A. Nebreda and members of the laboratory for critical reading of the manuscript. S.G. was supported by a fellowship from the Boncompagni-Ludovisi Foundation and F.F. is a recipient of an EMBL predoctoral fellowship.

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  1. European Molecular Biology Laboratory, Meyerhofstrasse 1, Hedeilberg, 69117, Germany

    Stefania Gonfloni, Friedrich Frischknecht, Michael Way & Giulio Superti-Furga

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  1. Stefania Gonfloni
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Correspondence to Giulio Superti-Furga.

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Gonfloni, S., Frischknecht, F., Way, M. et al. Leucine 255 of Src couples intramolecular interactions to inhibition of catalysis. Nat Struct Mol Biol 6, 760–764 (1999). https://doi.org/10.1038/11537

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