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 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.
Grishin
A V
AV
Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA 15213, USA. grishin@pitt.edu
Azhipa
O
O
Semenov
I
I
Corey
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D000943:Antigens, Differentiation; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001483:Base Sequence; D018797:Cell Cycle Proteins; D002460:Cell Line; D002645:Chickens; D004249:DNA Damage; D017931:DNA Primers / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009153:Mutagens / Q000633:toxicity; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D020798:Two-Hybrid System Techniques; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
D000943:Antigens, Differentiation; D018797:Cell Cycle Proteins; D017931:DNA Primers; D009153:Mutagens; D011506:Proteins; D011994:Recombinant Proteins; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; C087519:PPP1R15A protein, human; C513256:Ppp1r15a protein, mouse; D054645:Protein Phosphatase 1