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Interaction between growth arrest-DNA damage protein 34 and Src kinase Lyn negatively regulates genotoxic apoptosis
A V Grishin1, O Azhipa, I Semenov
1Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA 15213, USA. grishin@pitt.edu
Abstract:
Genotoxic stresses activate intracellular signaling molecules, which lead to growth arrest, DNA repair, and/or apoptosis. Among these molecules are the growth arrest and DNA damage protein 34 (GADD34) and the Src-related protein tyrosine kinase Lyn. Here, we report that these two proteins physically and functionally interact to regulate DNA damage-induced apoptosis. Multiple isolates of GADD34 and the related murine protein MyD116 were identified as binding partners of Lyn in a yeast two-hybrid screen. The specific interaction was confirmed by in vitro association of GADD34 with glutathione S-transferase fusion proteins containing the Src Homology 3 (SH3) domain of Lyn, as well as coimmunoprecipitation of GADD34 and Lyn from mammalian cells. GADD34 was tyrosine-phosphorylated in vivo in a Lyn-dependent manner. Lyn efficiently phosphorylated affinity-purified GADD34 in vitro. Lyn negatively regulated the proapoptotic function of GADD34 in a kinase-dependent manner. Expression of wild-type, but not kinase-inactive, Lyn weakened promotion of apoptosis by GADD34 following treatment with methyl-methanesulfonate or ionizing radiation in HEK293 and HeLa cells. In contrast, pretreatment of cells with the Src-specific tyrosine kinase inhibitor PP1 strengthened promotion of apoptosis by GADD34. We propose that Lyn regulates the proapoptotic function of GADD34 by binding and phosphorylating it.
Insights
Growth arrest and DNA damage protein 34 (GADD34) and Src-related protein tyrosine kinase Lyn interact to control DNA damage-induced apoptosis. Lyn binding and phosphorylating GADD34 negatively regulates its proapoptotic function.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Genotoxic stresses trigger signaling pathways involving growth arrest and DNA damage protein 34 (GADD34) and Src-related protein tyrosine kinase Lyn.
- These proteins are implicated in cellular responses to DNA damage, including apoptosis.
Purpose of the Study:
- To investigate the physical and functional interaction between GADD34 and Lyn in regulating DNA damage-induced apoptosis.
- To elucidate the mechanism by which Lyn influences GADD34's proapoptotic activity.
Main Methods:
- Yeast two-hybrid screening to identify binding partners of Lyn.
- In vitro association assays using glutathione S-transferase fusion proteins.
- Coimmunoprecipitation from mammalian cells (HEK293 and HeLa).
- In vitro kinase assays and analysis of apoptosis promotion following genotoxic stress (methyl-methanesulfonate, ionizing radiation).
Main Results:
- GADD34 and its murine homolog MyD116 were identified as Lyn binding partners.
- GADD34 physically interacts with Lyn, confirmed by in vitro and coimmunoprecipitation studies.
- Lyn phosphorylates GADD34 in a kinase-dependent manner, negatively regulating its proapoptotic function.
- Inhibition of Lyn kinase activity or expression of kinase-inactive Lyn enhanced GADD34-mediated apoptosis.
Conclusions:
- Lyn and GADD34 physically and functionally interact to regulate DNA damage-induced apoptosis.
- Lyn negatively modulates GADD34's proapoptotic role through binding and tyrosine phosphorylation.
- This interaction represents a novel regulatory mechanism in the cellular response to genotoxic stress.