GROWTH ARREST AND DNA DAMAGE INDUCIBLE GENE GADD153-- REGULATION BY DNA DAMAGE

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gadd153 is a mammalian gene whose expression is increased in response to a variety of stresses including DNA damage and growth arrest. It encodes a 19 kd protein with homology to the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. This family consists of at least 5 members which contain a conserved bZIP carboxyl-terminal domain. The bZIP domain consists of a basic region involved in DNA recognition and binding, and an adjacent leucine zipper region, that mediates subunit dimerization. gadd153 is unique among C/EBP-related proteins in that it cannot bind to DNA. However, it can form stable heterodimers with C/EBP proteins and prevent their DNA-binding, suggesting that gadd153 acts as an inhibitor of these transcriptional activators. The functional significance of these interactions and their relationship to elevated gadd153 expression following stress is unclear, but we have recently shown that two other C/EBP-related proteins, C/EBPa, and C/EBP6, are also induced by DNA damage. We have examined the role of calcium as a second messenger in regulating gadd153 expression and have found that treatment of cells with the calcium ionophore A23187 leads to the rapid induction of gadd153 mRNA. Elevated mRNA levels result from both an increase in the rate of gadd153 transcription and an enhanced stability of gadd153 mRNA. Buffering intracellular calcium by treatment with BAPTA-AM prevents gadd153 induction by both A23187 and the DNA alkylating agent MMS. Thus, it is likely that intracellular calcium plays a role in controlling gadd153 expression following DNA damage. Inhibitors of various protein kinase and phosphatases have also been tested for their ability to influence gadd153 expression following DNA damage. Protein kinase C inhibitors failed to inhibit gadd153 mRNA induction as well as gadd153 promoter activation by MMS. Tyrosine kinase inhibitors (genestein and vanadate) were also without effect. However, two general kinase inhibitors, 2-aminopurine and H7, both blocked gadd153 mRNA expression. Okadaic acid and calyculin, specific inhibitors of phosphatases 1 and 2A, induced gadd153 mRNA expression and enhanced gadd153 promoter activity. Thus, gadd153 appears to be under control of both protein kinase(s) and phosphatase(s), although the specific enzymes remain to be identified.