Editors' ChoiceCell Biology

Surviving DNA Damage Through Golgi Dispersal

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Science Signaling  25 Feb 2014:
Vol. 7, Issue 314, pp. ec58
DOI: 10.1126/scisignal.2005213

DNA damage triggers nuclear events, such as DNA repair, changes in transcription, and cell cycle arrest, and in some cases damage can cause apoptosis. DNA-damaging agents are common therapeutic strategies for cancer. Farber-Katz et al. found that DNA damage induced changes in the Golgi. In human cancer cell lines, primary human umbilical vein endothelial cells (HUVECs), and mouse primary fibroblasts and hepatocytes, treatment with camptothecin (CPT), doxorubicin (DOX), or ionizing radiation (IR) induced time- and dose-dependent (but apoptosis-independent) morphological changes in the Golgi: The Golgi went from a ribbon-like perinuclear stack to dispersed fragments. Although the Golgi was dispersed and fragmented, Golgi fragments maintained markers for either cis or trans faces. Golgi phosphoprotein 3 (GOLPH3), which is increased in abundance in some cancers, regulates Golgi morphology by applying tensile force to the Golgi membrane through the myosin MYO18A and the actin cytoskeleton. Depletion of either GOLPH3 or MYO18A using small-interfering RNA (siRNA) in HeLa cells prevented Golgi dispersal but not the formation of nuclear DNA damage foci in response to CPT. Reconstitution of GOLPH3-deficient HeLa cells with siRNA-resistant wild-type GOLPH3, a phosphomimetic mutant, or an unphosphorylatable mutant indicated that CPT induced the phosphorylation of GOLPH3 at a TQ site, a preferred motif for the DNA repair kinases ATM and DNA-PK. GOLPH3 coimmunoprecipitated with Ku80 (a regulatory subunit of DNA-PK) in HeLa cell lysates in the absence of any DNA-damaging stimulus, and DNA-PK directly phosphorylated GOLPH3 at the TQ site in vitro. Golgi dispersal induced by DNA damage was not substantially affected in ATM-deficient cells or by addition of an ATM inhibitor, whereas DNA-PK depletion or pharmacological inhibition prevented Golgi dispersal caused by DOX or CPT. CPT or DOX increased the interaction between GOLPH3 and MYO18A, and a phosphomimetic-mutant GOLPH3 exhibited increased interaction with MYO18A in vitro. The abundance of cleaved caspase 3, an apoptotic marker, was increased in GOLPH3- or MYO18A-deficient HeLa cells or HUVECs after treatment with DOXO or CPT, respectively. Likewise, compared with wild-type cells, proliferation was decreased in DOX-treated GOLPH3- or MYO18A-deficient HeLa cells. In HeLa cells engineered to inducibly coexpress GFP with GOLPH3, CPT enriched the proportion of cells coexpressing GFP and GOLPH3 but not a GOLPH3 mutant incapable of localizing to the Golgi, suggesting that the mutant-expressing cells were more likely to die. Thus, the DNA repair protein DNA-PK induced changes in the Golgi through GOLPH3 to enable cells to survive DNA damage, indicating that targeting this cytoplasmic response may improve the cytotoxicity of DNA-damaging drugs.

S. E. Farber-Katz, H. C. Dippold, M. D. Buschman, M. C. Peterman, M. Xing, C. J. Noakes, J. Tat, M. M. Ng, J. Rahajeng, D. M. Cowan, G. J. Fuchs, H. Zhou, S. J. Field, DNA damage triggers Golgi dispersal via DNA-PK and GOLPH3. Cell 156, 413–427 (2014). [PubMed]