![DORN1 phosphorylates RBOHD-N at S22 and T24 sites in vitro and in vivo. (a) DORN1 phosphorylates the N-terminal domain of RBOHD. Purified His-RBOHD-N recombinant protein was incubated with GST-DORN1-KD kinase domain, GST-DORN1-KD-1 (kinase dead), GST-DORN1-KD-2 (kinase dead) or GST in an in vitro kinase assay. Autophosphorylation and trans-phosphorylation were measured by incorporation of γ-[32P]-ATP. MBP and GST-LYK5 were used as positive and negative controls, respectively. (b) DORN1 phosphorylates RBOHD-N at S22 and T24 sites in vitro. Purified GST or GST-DORN1-KD protein was incubated with His-RBOHD-N or the respective mutant proteins, S22A (His-S22A), T24A (His-T24A), S22AT24A (His-S22AT24A), followed by an in vitro kinase assay. (c) ATP-induced phosphorylation of RBOHD through S22 and T24 sites in vivo. The indicated constructs pGBW14-RBOHD (WT, S22A, T24A and S22AT24A) were transiently expressed in rbohd mutant protoplasts incubated with γ-[32P]-ATP overnight. After treating with 200 μM ATP for 30 min, total protein was extracted and subjected to immunoprecipitation. (d) RBOHD phosphosites are required for ATP-triggered ROS production. The indicated constructs were transiently expressed in rbohd mutant protoplasts and treated with or without 200 μM ATPγS. ROS production was measured after 30 min](https://nrms.kisti.re.kr/bitextimages/TRKO201800043375/TRKO201800043375_42_image_1.png "DORN1 phosphorylates RBOHD-N at S22 and T24 sites in vitro and in vivo. (a) DORN1 phosphorylates the N-terminal domain of RBOHD. Purified His-RBOHD-N recombinant protein was incubated with GST-DORN1-KD kinase domain, GST-DORN1-KD-1 (kinase dead), GST-DORN1-KD-2 (kinase dead) or GST in an in vitro kinase assay. Autophosphorylation and trans-phosphorylation were measured by incorporation of γ-[32P]-ATP. MBP and GST-LYK5 were used as positive and negative controls, respectively. (b) DORN1 phosphorylates RBOHD-N at S22 and T24 sites in vitro. Purified GST or GST-DORN1-KD protein was incubated with His-RBOHD-N or the respective mutant proteins, S22A (His-S22A), T24A (His-T24A), S22AT24A (His-S22AT24A), followed by an in vitro kinase assay. (c) ATP-induced phosphorylation of RBOHD through S22 and T24 sites in vivo. The indicated constructs pGBW14-RBOHD (WT, S22A, T24A and S22AT24A) were transiently expressed in rbohd mutant protoplasts incubated with γ-[32P]-ATP overnight. After treating with 200 μM ATP for 30 min, total protein was extracted and subjected to immunoprecipitation. (d) RBOHD phosphosites are required for ATP-triggered ROS production. The indicated constructs were transiently expressed in rbohd mutant protoplasts and treated with or without 200 μM ATPγS. ROS production was measured after 30 min")
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| 주관연구기관 | 가천대학교 Gachon University |
|---|---|
| 연구책임자 | 장호희 |
| 참여연구자 | 천민경 , 김요섭 , 손예원 , 장민경 , 이은별 , 강창호 , Gary Stacey , 이수인 , 박정훈 , 가네스 , 조성환 , 정미정 , 김진아 , 윤은경 |
| 보고서유형 | 최종보고서 |
| 발행국가 | 대한민국 |
| 언어 | 한국어 |
| 발행년월 | 2018-02 |
| 과제시작연도 | 2017 |
| 주관부처 | 농촌진흥청 Rural Development Administration(RDA) |
| 등록번호 | TRKO201800043375 |
| 과제고유번호 | 1395050070 |
| 사업명 | 차세대바이오그린21 |
| DB 구축일자 | 2019-05-18 |
| 키워드 | RNA-결합단백질.세포외ATP.스트레스.단백질체.식물.RNA-binding protein.Extracellular ATP.Stress.Proteomics.Plant. |
| DOI | https://doi.org/10.23000/TRKO201800043375 |
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