Contents lists available at ScienceDirect BBA - Proteins and Proteomics journal homepage: www.elsevier.com/locate/bbapap Tyr-nitration in maize CDKA;1 results in lower affinity for ATP binding Andrea A.E. Méndez a , Irene C. Mangialavori a , Andrea V. Cabrera a , María P. Benavides a , Jorge M. Vázquez-Ramos b , Susana M. Gallego a, ⁎ a Universidad de Buenos Aires - Consejo Nacional de Investigaciones Científicas y Técnicas, Instituto de Química y Fisicoquímica Biológicas “Profesor Alejandro C. Paladini” (IQUIFIB), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina b Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, Mexico ARTICLE INFO Keywords: Protein nitration ATP Zea mays L. Maize cyclin-dependent kinase A;1 ABSTRACT Cyclin-dependent kinase A (CDKA) is a key component for cell cycle progression. The catalytic kinase activity depends on the protein's ability to form an active complex with cyclins and on phosphoregulatory mechanisms. 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). Among these, posttranslational modifications (PTMs) of key proteins such as CDKA;1 may be of significance. Herein, isolated maize embryo axes were subjected to sodium nitroprusside (SNP) as an inductor of nitrosative conditions to evaluate if CDKA;1 protein was a target for RNS. A high degree of protein nitration was detected; this included the specific Tyr-nitration of CDKA;1. Tyr15 and Tyr19, located at the ATP-binding site, were the selective targets for nitration according to both in silico analysis using the predictive software GPS-YNO 2 , and in vitro mass spectrometry studies of recombinant nitrated ZmCDKA;1. Spectrofluorometric measurements demonstrated a reduction of ZmCDKA;1-NO 2 affinity for ATP. From these results, we conclude that Tyr nitration in CDKA;1 could act as an active modulator of cell cycle progression during redox stress. 1. Introduction Cyclin-dependent kinases A (CDKA) are key proteins involved in the control of plant cell cycle progression by regulating both G1/S and G2/ M phase transitions [1–3]. The vast CDK protein family comprises eight main groups (A to F); each specific member of these groups is desig- nated by a number written after a semicolon, as proposed by Francis (2007) [4]. Plant CDKA;1 is phylogenetically related to other kinases such as CDK1/2 from animals, Cdc28 from Saccharomyces cerevisiae, and cdc2 from Schizosaccharomyces pombe [5]. As part of the kinase A protein family, its structure is characterized by highly conserved core sites among the different living organisms. 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 [6]. CDKA catalytic activity depends on the previous formation of a complex with its positive regulatory cyclin subunit, as well as on di- verse phosphoregulatory mechanisms [7]. Phosphorylation mediated by a CDK-activating kinase (CAK) on a conserved threonine residue (Thr160) in the T-loop is a prerequisite to allowing substrate entrance into the active site and full activation of the complex [8]. On the con- trary, the phosphorylation by WEE1-family kinases of two specific amino-acid residues in the ATP-binding site, Thr14 and Tyr15, leads to CDKA inactivation [9]. Reactive oxygen and nitrogen species (ROS and RNS) play a key role as signaling molecules in plants both under normal and stressful con- ditions [10,11]. Recently, a ROS-induced posttranslational protein modification (PTM) in the proline residue of the PSTAIRE cyclin binding motif has been described to be involved in the modulation of CDKA;1 affinity for its cyclin subunit [12]. Also, it has been reported that NO may modify the activity of several plant kinases [13–16]. In this regard, the molecular mechanism by which NO and other RNS exert their biological functions has been usually linked to redox NO-PTMs, such as S-nitrosylation and tyrosine nitration [17]. Over the last years, a growing body of information has accounted for the importance of NO- PTM not only during normal plant development but also in response to environmental stress [18–21]. In particular, the interplay between ROS –such as superoxide anion or O 2 •– – and NO has been associated with 3- nitro-tyrosine (3-Tyr-NO 2 ) formation [22]. Previous data obtained in our laboratory indicated that NO was involved in root growth inhibition under both abiotic stress and poly- amine treatments [23,24]. Besides, we corroborated that cell cycle ar- rest and post-germinative root growth inhibition in wheat seedlings https://doi.org/10.1016/j.bbapap.2020.140479 Received 28 March 2020; Received in revised form 17 June 2020; Accepted 22 June 2020 ⁎ Corresponding author at: Junín 956, 1° Piso, Buenos Aires C1113AAC, Argentina. E-mail address: sgallego@ffyb.uba.ar (S.M. Gallego). BBA - Proteins and Proteomics 1868 (2020) 140479 Available online 26 June 2020 1570-9639/ © 2020 Elsevier B.V. All rights reserved. T