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SH3-dependent stimulation of Src-family kinase autophosphorylation without tail release from the SH2 domain in vivo

Nature Structural Biology volume 9, pages 365–369 (2002)Cite this article

Abstract

Src family protein-tyrosine kinase activity is suppressed by two intramolecular interactions. These involve binding of the SH2 domain to the phosphorylated C-terminal tail and association of the SH3 domain with a polyproline type II helix formed by the SH2-kinase linker. Here we show that SH3-dependent activation of the Src family member Hck by HIV-1 Nef binding or by SH2-kinase linker mutation does not affect tail tyrosine phosphorylation in fibroblasts. Surprisingly, replacement of the wild type Hck tail with a high-affinity SH2 domain-binding sequence did not affect Hck activation or downstream signaling by these SH3-dependent mechanisms, suggesting that activation through SH3 occurs without SH2–tail dissociation. These results identify SH3–linker interaction as an independent mode of Hck kinase regulation in vivo and suggest that different mechanisms of Src kinase activation may generate distinct output signals because of differences in SH2 or SH3 domain accessibility.

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Figure 1: Structure of Hck kinases and expression in Sf-9 cells.
Figure 2: Tryptic phosphopeptide mapping of Hck proteins from 32P-labeled fibroblasts.
Figure 3: SH3-mediated activation of Hck does not affect tail tyrosine phosphorylation in vivo.
Figure 4: The enhanced SH2–tail interaction does not affect Hck activation by SH3-based mechanisms in vivo.
Figure 5: SH3-mediated activation does not affect tail tyrosine phosphorylation of Hck-YEEI mutants.

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Acknowledgements

Supported by grants from the National Cancer Institute, NIH. E.C.L. is the recipient of an NIH National Research Service Award. The authors would like to acknowledge the NIH AIDS Research and Reference Reagent Program for providing antibodies to HIV-1 Nef.

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  1. Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, 15261, Pennsylvania, USA

    Edwina C. Lerner & Thomas E. Smithgall

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  1. Edwina C. Lerner
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Correspondence to Thomas E. Smithgall.

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The authors declare no competing financial interests.

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Lerner, E., Smithgall, T. SH3-dependent stimulation of Src-family kinase autophosphorylation without tail release from the SH2 domain in vivo. Nat Struct Mol Biol 9, 365–369 (2002). https://doi.org/10.1038/nsb782

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