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A potential role for protein palmitoylation and zDHHC16 in DNA damage response.

Cao N, Li JK, Rao YQ, Liu H, Wu J, Li B, Zhao P, Zeng L, Li J - BMC Mol. Biol. (2016)

Bottom Line: Inhibition of protein palmitoylation compromised DNA damage-induced activation of Atm, induction and activation of p53, cell cycle arrest at G2/M phase, and DNA damage foci assembly/disassembly in primary mouse embryonic fibroblasts.Furthermore, knockout of zDHHC16, a palmitoyltransferase gene identified as an interacting protein for c-Abl, a non-receptor tyrosine kinase involved in DNA damage response, reproduced most of the defects in DNA damage responses produced by the inhibition of protein palmitoylation.Our results revealed critical roles for protein palmitoylation and palmitoyltransferase zDHHC16 in early stages of DNA damage responses and in the regulation of Atm activation.

View Article: PubMed Central - PubMed

Affiliation: Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, 200240, China.

ABSTRACT

Background: Cells respond to DNA damage by activating the phosphatidylinositol-3 kinase-related kinases, p53 and other pathways to promote cell cycle arrest, apoptosis, and/or DNA repair. Here we report that protein palmitoylation, a modification carried out by protein acyltransferases with zinc-finger and Asp-His-His-Cys domains (zDHHC), is required for proper DNA damage responses.

Results: Inhibition of protein palmitoylation compromised DNA damage-induced activation of Atm, induction and activation of p53, cell cycle arrest at G2/M phase, and DNA damage foci assembly/disassembly in primary mouse embryonic fibroblasts. Furthermore, knockout of zDHHC16, a palmitoyltransferase gene identified as an interacting protein for c-Abl, a non-receptor tyrosine kinase involved in DNA damage response, reproduced most of the defects in DNA damage responses produced by the inhibition of protein palmitoylation.

Conclusions: Our results revealed critical roles for protein palmitoylation and palmitoyltransferase zDHHC16 in early stages of DNA damage responses and in the regulation of Atm activation.

No MeSH data available.


Related in: MedlinePlus

zDHHC16 deficiency impaired DNA damage-induced Atm-p53 activation. a Western blot analysis showed reduced Atm phosphorylation at S1981 in zDHHC16−/− MEFs. Primary zDHHC16−/− MEFs were isolated from zDHHC16−/− mice and treated with 1 μM Dox for different periods of time as indicated. b Quantitation data on p-Atm. Asterisk denoted significant difference (p < 0.05) between control and 2BP treated cells. c Activation and induction of p53 and p21. Cells were treated the same as above
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Fig6: zDHHC16 deficiency impaired DNA damage-induced Atm-p53 activation. a Western blot analysis showed reduced Atm phosphorylation at S1981 in zDHHC16−/− MEFs. Primary zDHHC16−/− MEFs were isolated from zDHHC16−/− mice and treated with 1 μM Dox for different periods of time as indicated. b Quantitation data on p-Atm. Asterisk denoted significant difference (p < 0.05) between control and 2BP treated cells. c Activation and induction of p53 and p21. Cells were treated the same as above

Mentions: We have previously reported the identification of one of the PATs, encoded by zDHHC16 gene. This protein, Aph2, was originally identified as a c-Abl interacting protein (Abl-philin2) [34]. c-Abl is involved in DNA damage response. In particular, it is required for Atm-p53 activation [35–38]. Biochemical and genetic studies have shown that at least one protein, phospholamban, a cardiac muscle specific protein, was palmitoylated by Aph2 [12]. In addition, MEFs overexpress zDHHC16 also showed increased total protein palmitoylation (Additional file 1: Figure S4a). Although Dox did not affect the expression of DHHC16 at the mRNA level (Additional file 1: Figure S2), Dox induced nuclear translocation of ectopically-expressed DHHC16 (Additional file 1: Figure S4b). We have tried to raise anti-DHHC16 antibodies but those antibodies could not recognize endogenous DHHC16. This could be due to the fact that DHHC16 is a membrane protein. The lack of anti-DHHC16 antibodies prevents us to study the expression, localization, and modification of endogenous DHHC16 at the moment. To test whether DHHC16 also plays a role in DDR, we treated primary MEFs deficient for zDHHC16 gene with Dox for different periods of time. Western blot analysis revealed that zDHHC16 deficiency, like inhibition of PATs with 2BP, compromised the activation of Atm and the induction of p53 and its target protein p21 (Fig. 6a–c).Fig. 6


A potential role for protein palmitoylation and zDHHC16 in DNA damage response.

Cao N, Li JK, Rao YQ, Liu H, Wu J, Li B, Zhao P, Zeng L, Li J - BMC Mol. Biol. (2016)

zDHHC16 deficiency impaired DNA damage-induced Atm-p53 activation. a Western blot analysis showed reduced Atm phosphorylation at S1981 in zDHHC16−/− MEFs. Primary zDHHC16−/− MEFs were isolated from zDHHC16−/− mice and treated with 1 μM Dox for different periods of time as indicated. b Quantitation data on p-Atm. Asterisk denoted significant difference (p < 0.05) between control and 2BP treated cells. c Activation and induction of p53 and p21. Cells were treated the same as above
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Fig6: zDHHC16 deficiency impaired DNA damage-induced Atm-p53 activation. a Western blot analysis showed reduced Atm phosphorylation at S1981 in zDHHC16−/− MEFs. Primary zDHHC16−/− MEFs were isolated from zDHHC16−/− mice and treated with 1 μM Dox for different periods of time as indicated. b Quantitation data on p-Atm. Asterisk denoted significant difference (p < 0.05) between control and 2BP treated cells. c Activation and induction of p53 and p21. Cells were treated the same as above
Mentions: We have previously reported the identification of one of the PATs, encoded by zDHHC16 gene. This protein, Aph2, was originally identified as a c-Abl interacting protein (Abl-philin2) [34]. c-Abl is involved in DNA damage response. In particular, it is required for Atm-p53 activation [35–38]. Biochemical and genetic studies have shown that at least one protein, phospholamban, a cardiac muscle specific protein, was palmitoylated by Aph2 [12]. In addition, MEFs overexpress zDHHC16 also showed increased total protein palmitoylation (Additional file 1: Figure S4a). Although Dox did not affect the expression of DHHC16 at the mRNA level (Additional file 1: Figure S2), Dox induced nuclear translocation of ectopically-expressed DHHC16 (Additional file 1: Figure S4b). We have tried to raise anti-DHHC16 antibodies but those antibodies could not recognize endogenous DHHC16. This could be due to the fact that DHHC16 is a membrane protein. The lack of anti-DHHC16 antibodies prevents us to study the expression, localization, and modification of endogenous DHHC16 at the moment. To test whether DHHC16 also plays a role in DDR, we treated primary MEFs deficient for zDHHC16 gene with Dox for different periods of time. Western blot analysis revealed that zDHHC16 deficiency, like inhibition of PATs with 2BP, compromised the activation of Atm and the induction of p53 and its target protein p21 (Fig. 6a–c).Fig. 6

Bottom Line: Inhibition of protein palmitoylation compromised DNA damage-induced activation of Atm, induction and activation of p53, cell cycle arrest at G2/M phase, and DNA damage foci assembly/disassembly in primary mouse embryonic fibroblasts.Furthermore, knockout of zDHHC16, a palmitoyltransferase gene identified as an interacting protein for c-Abl, a non-receptor tyrosine kinase involved in DNA damage response, reproduced most of the defects in DNA damage responses produced by the inhibition of protein palmitoylation.Our results revealed critical roles for protein palmitoylation and palmitoyltransferase zDHHC16 in early stages of DNA damage responses and in the regulation of Atm activation.

View Article: PubMed Central - PubMed

Affiliation: Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, 200240, China.

ABSTRACT

Background: Cells respond to DNA damage by activating the phosphatidylinositol-3 kinase-related kinases, p53 and other pathways to promote cell cycle arrest, apoptosis, and/or DNA repair. Here we report that protein palmitoylation, a modification carried out by protein acyltransferases with zinc-finger and Asp-His-His-Cys domains (zDHHC), is required for proper DNA damage responses.

Results: Inhibition of protein palmitoylation compromised DNA damage-induced activation of Atm, induction and activation of p53, cell cycle arrest at G2/M phase, and DNA damage foci assembly/disassembly in primary mouse embryonic fibroblasts. Furthermore, knockout of zDHHC16, a palmitoyltransferase gene identified as an interacting protein for c-Abl, a non-receptor tyrosine kinase involved in DNA damage response, reproduced most of the defects in DNA damage responses produced by the inhibition of protein palmitoylation.

Conclusions: Our results revealed critical roles for protein palmitoylation and palmitoyltransferase zDHHC16 in early stages of DNA damage responses and in the regulation of Atm activation.

No MeSH data available.


Related in: MedlinePlus