Interaction between growth arrest-DNA damage protein 34 and Src kinase Lyn negatively regulates genotoxic apoptosis

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 MyD1 16 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.