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. 2001 Aug 28;98(18):10172-7.
doi: 10.1073/pnas.191130798. Epub 2001 Aug 21.

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

A V Grishin  1 , O Azhipa, I Semenov, S J Corey
Affiliations

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

A V Grishin et al. Proc Natl Acad Sci U S A. .

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.

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Figures

Figure 1
Figure 1
Yeast two-hybrid interaction between GADD34 and Lyn. (Upper) Block diagram of Lyn's domains. Unique, SH3, SH2, and catalytic domains as well as the portion of Lyn used as bait are shown. (Lower) YRG-2 yeast transformants harboring pGBT9-Lyn, the human bone marrow library GADD34 plasmid, and control plasmids p53 and pSV40 as indicated were streaked on a plate lacking leucine, tryptophan, and histidine. The plate was incubated at 30°C for 3 days. Histidine-independent growth indicates the two-hybrid interaction between plasmid-encoded proteins.
Figure 2
Figure 2
GADD34–Lyn interaction in vitro and in mammalian cells. (A) Alignment of human GADD34 (h) and mouse MyD116 (m) proline-rich clusters with Lyn SH3 binding site consensus sequence (c). Single-letter amino acid code is used. Residue numbers are shown. (B) GST pull-down assay with GST or GST fusions with unique (U), unique SH3 (U3), unique SH2 (U2), and unique SH3-SH2 (U32) domains of Lyn, and lysates of HEK293 cells transiently transfected with pFLAG-CMV2-GADD34. Proteins from lysate bound to GST fusion beads were analyzed by using immunoblot (IB). (Upper) Immunoblot probed with the anti-FLAG antibody. (Lower) Blot stained with Ponceau S to show equal load of GST fusions. L, total cell lysate lane shown to indicate position of FLAG-GADD34. Size markers on the left are in kilodaltons. The minor band migrating slightly faster than FLAG-GADD34 is a result of partial degradation. (C) Immunoblot of Daudi cell lysate, anti-GADD34 (IP GADD), and control (IP Cdc42) immunoprecipitates probed with the monoclonal anti-Lyn antibody H-6. The diffuse bands at 46 kDa in immunoprecipitate lanes are because of the presence of crossreacting Ig heavy chain. Position of Lyn doublet is indicated. (D) Immunoblot of the lysate of 293 cells transfected with pFlag-CMV2-GADD34, anti-Lyn, and anti-Src immunoprecipitates probed with the anti-GADD34 antibody. Position of GADD34 is indicated.
Figure 3
Figure 3
Lyn-mediated phosphorylation of GADD34. (A) Antiphosphotyrosine (PY) and anti-GADD34 (GADD) immunoblots of anti-FLAG immunoprecipitates of HEK293 cells transfected with empty vectors, pFLAG-CMV2-GADD34 (5 μg), and pcDNA3-Lyn constructs (5 μg) as indicated. Vector DNA was added to make 10 μg total whenever required. Equal amounts of GADD34 protein were recovered in the immunoprecipitates of GADD34-transfected cells. (B) Antiphosphotyrosine (PY) and anti-GADD34 (GADD) immunoblots of anti-GADD34 immunoprecipitates of BaF-3 cells untreated or treated with 50 μg/ml MMS for 1 h. (C) Antiphosphotyrosine (PY) and anti-FLAG (FLAG) immunoblots of anti-FLAG immunoprecipitates of DT40 and D33 cells transfected with pFLAG-CMV2-GADD34 (5 μg) and pcDNA3-Lyn (5 μg) and treated with 50 μg/ml MMS as indicated. (D) Anti-Lyn immune kinase assays with the exogenous substrates BSA, FLAG-GADD34 (GADD), and acid-denatured enolase. Lysates were prepared from DT40 wild-type or D33 Lyn-deficient cells and immunoprecipitated with the polyclonal anti-Lyn antibody. Equal amounts of protein from immunoprecipitates were used. Asterisks indicate positions of phosphorylated substrates. Lyn is a prominent doublet of 53 and 56 kDa. BSA is expected to appear as a 68-kDa band. Size markers on the left are in kilodaltons.
Figure 4
Figure 4
DNA fragmentation apoptosis assay following DNA damage in cells expressing GADD34 and Lyn. Agarose gel electropherograms of DNA from HEK293 cells transfected with 5 μg each of pcDNA3.1 and pFLAG-CMV2 (lanes 1 and 2), pFLAG-CMV2-GADD34 + pcDNA3.1 (lane 3), pFLAG-CMV2 + pcDNA3-Lyn (lane 4), pFLAG-CMV2-GADD34 + pcDNA3-Lyn (lane 5), and either left untreated (A) or treated with MMS (B) or IR (C). FLAG-GADD34 and Lyn expression levels were equal in double and single transfectants, as judged by Western blots (data not shown). DNA was extracted 12 h following treatment because at this time point, DNA laddering is most pronounced (data not shown). M, molecular size marker lane; position of 1-kb band is indicated. Two other repeats of the same experiment produced similar results (data not shown).
Figure 5
Figure 5
Nuclear condensation and DNA content assays for genotoxic apoptosis in cells expressing GADD34 and Lyn. (Upper) HEK293 cells transfected with 5 μg each of pFLAG-CMV2, pcDNA3, pFLAG-CMV2-GADD34, and pcDNA3-Lyn as indicated were treated with MMS for 2 h and stained with Hoechst 33342. Cells with bright compact nuclei and total cells were counted by using epifluorescence and differential interference contrast illumination, respectively. (Lower) HeLa cells transfected as above were treated with MMS for 12 h, fixed, and stained with propidium iodide. Percentage of cells with sub-G1 DNA content was determined by flow cytometry. The data shown are average ± standard error of three independent experiments.
Figure 6
Figure 6
Inhibition of Lyn augments apoptosis in cells expressing GADD34. HEK293 cells were transfected with 5 μg each of constructs indicated. Western blots showed equal levels of FLAG-GADD34 in transfectants (data not shown). Cells were left untreated or treated with MMS as indicated for 12 h. Where indicated,10 μM PP1 was added to cells 10 min before MMS. DNA was extracted and analyzed by electrophoresis through agarose gel. Results are representative of three independent experiments.
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