BECAS
MENDEZ andrea Analia Elena
congresos y reuniones científicas
Título:
Tyr-nitration in maize CDKA;1 results in lower affinity for ATP binding
Autor/es:
AAE MENDEZ; IC MANGIALAVORI; AV CABRERA; MP BENAVIDES; JM VAZQUEZ RAMOS; SM GALLEGO
Reunión:
Congreso; Reunión conjunta SAIB-SAMIGE; 2020
Resumen:
The cyclin-dependent kinase A (CDKA) is a protein closely involved in the control of eukaryotic cell cycle G1/S and G2/M phase transitions. As part of the protein kinase family, its structure is characterized for presenting highly conserved core sites among species. These sequences include the ATP binding site, the Ser/Thr kinase active site, the PSTAIRE cyclin binding motif and the T-loop or activation loop. Cell cycle arrest and plant growth impairment under abiotic stress have been linked to different molecular processes triggered by increased levels of reactive oxygen and nitrogen species (ROS and RNS). As part of a project to determine possible sites of CDKA protein nitration, embryo axes isolated from Zea mays L (maize) were subjected to sodium nitroprusside (SNP) as an inductor of nitrosative conditions. After detecting an increased level of whole protein nitrotyrosination, immunoprecipitation of the CDKA;1 protein revealed that the basal level of nitrotyrosine detected in endogenous CDKA;1 was increased, confirming that CDKA;1 protein is target for RNS. To decipher specific Tyr-nitration sites, recombinant CDKA;1 was cloned, expressed in E. coli and exposed in vitro to peroxynitrite (ONOO-). As well as the endogenous CDKA;1, the recombinant maize ZmCDKA;1 was target of tyrosine nitration. The primary sequence of CDKA;1, identified as P23111 (CDC2_MAIZE, cell division control protein 2 homolog - Zea mays) in UniProt database, contains 294 amino acids, 12 of which are Tyr residues. Analysis using GPS-YNO2 software predicted Y4 and Y15 as target of nitration, both with high scores, but also predicted Y19 and Y270, with medium score. Mass spectrometry analysis of recombinant nitrated CDKA;1 (ZmCDKA;1-NO2) showed Tyr 15 and Tyr 19, located at the ATP-binding site, as selective targets for nitration. Kinase-ATP interaction of nitrated and non-nitrated recombinant protein was analyzed by using TNT-ATP fluorescent analog. Comparative spectrofluorometric measurements demonstrated a reduction of ZmCDKA;1-NO2 affinity for ATP. The difference in ATP affinity between the two forms -ZmCDKA;1 and ZmCDKA;1-NO2- could be attributable to steric restrictions since nitrotyrosine is more spacious than tyrosine. Nonetheless, other physico-chemical properties of Tyr are modified by the introduction of the nitro group into its aromatic ring that can also contribute to the functional change of the CDKA;1.From these results, we conclude that Tyr-nitration in CDKA;1 could act as an active and rapid modulator of cell cycle progression during redox stress. In this sense, this mechanism could contribute to plant growth arrest caused by abiotic stress conditions, allowing them to trigger adaptive responses to cope with the stress.