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Cdk5 targets active Src for ubiquitin-dependent degradation by phosphorylating Src(S75)

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Abstract

The non-receptor tyrosine kinase Src is a critical regulator of cytoskeletal contraction, cell adhesion, and migration. In normal cells, Src activity is stringently controlled by Csk-dependent phosphorylation of Src(Y530), and by Cullin-5-dependent ubiquitinylation, which affects active Src(pY419) exclusively, leading to its degradation by the proteosome. Previous work has shown that Src activity is also limited by Cdk5, a proline-directed kinase, which has been shown to phosphorylate Src(S75). Here we show that this phosphorylation promotes the ubiquitin-dependent degradation of Src, thus restricting the availability of active Src. We demonstrate that Src(S75) phosphorylation occurs in vivo in epithelial cells, and like ubiquitinylation, is associated only with active Src. Preventing Cdk5-dependent phosphorylation of Src(S75), by site-specific mutation of S75 or by Cdk5 inhibition or suppression, increases Src(Y419) phosphorylation and kinase activity, resulting in Src-dependent cytoskeletal changes. In transfected cells, ubiquitinylation of Src(S75A) is about 35% that of wild-type Src-V5, and its half-life is approximately 2.5-fold greater. Cdk5 suppression leads to a comparable decrease in the ubiquitinylation of endogenous Src and a similar increase in Src stability. Together, these findings demonstrate that Cdk5-dependent phosphorylation of Src(S75) is a physiologically significant mechanism of regulating intracellular Src activity.

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Acknowledgments

We thank Dr. John Reddan of Oakland University for the human lens epithelial cell line (FHL124), Drs. Xiao Dong Jiao and J. Fielding Hejtmancik for generously sequencing DNA clones, and Ms. Andrea Elfstrom for technical assistance. This work was supported by the National Eye Institute Intramural Research Program Z01-EY000238-25.

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Authors and Affiliations

  1. Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, 5635 Fishers Lane, Room 1127, Bethesda, MD, 20892, USA

    Q. Pan, F. Qiao, C. Gao, B. Norman & Peggy S. Zelenka

  2. Laboratory of Sensorimotor Research, NEI, NIH, 49 Convent Drive, Room 2A50, Bethesda, MD, 20892, USA

    L. Optican

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  1. Q. Pan
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  2. F. Qiao
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  6. Peggy S. Zelenka
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Correspondence to Peggy S. Zelenka.

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Pan, Q., Qiao, F., Gao, C. et al. Cdk5 targets active Src for ubiquitin-dependent degradation by phosphorylating Src(S75). Cell. Mol. Life Sci. 68, 3425–3436 (2011). https://doi.org/10.1007/s00018-011-0638-1

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  • DOI: https://doi.org/10.1007/s00018-011-0638-1

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