Analysis of oxidative posttranslational modifications in cell cycle ZmCDKA;1 protein

AAE MENDEZ; SM GALLEGO; JM VAZQUEZ RAMOS; LB PENA

CABA

Conferencia; Frontiers in Bioscience 3; 2018

IBIOBA-MPSP

Complex formed between cyclin dependent kinase A (CDKA) and cyclin type D is responsible for cell cycle G1-S transition. Its activity is regulated by a complex interaction network with other proteins and by posttranslational modifications (PTM). Previous research in our laboratory showed that abiotic stress in plants causes redox imbalance and growth detention. More specifically, it was verified that under these conditions cell cycle proteins are target of oxidative PTMs, which may be involved in cell cycle arrest. In order to assess the molecular sites of oxidative PTMs, CDKA;1 from Zea Mays was cloned and produced in E. coli for further purification and in vitro oxidation assays with 1% (v/v) H2O2. Western blot for protein carbonylation and an electrophoretic mobility shift assay for methionine sulfoxide were performed. Further analysis was done by mass spectrometry, where results showed that under mild oxidation conditions, a specific proline (P45) undergoes irreversible carbonylation and that a methionine (M58) suffers reversible oxidation to methionine sulfoxide. Computational homology modeling of ZmCDKA;1 showed that P45 is in the PSTAIRE cyclin binding motif, and that M58 is nearby the interaction zone between this kinase and its cyclin. We therefore propose that these PTMs could affect the kinase-cyclin complex activity, thus affecting G1-S transition and progression through S phase in abiotic stress conditions.