Aurora A regulation by reversible cysteine oxidation reveals evolutionarily conserved redox control of Ser/Thr protein kinase activity

Reactive oxygen species (ROS) are physiological mediators of cellular signaling and play potentially damaging roles in human diseases. In this study, we found that the catalytic activity of the Ser/Thr kinase Aurora A was inhibited by the oxidation of a conserved cysteine residue (Cys290) that lies adjacent to Thr288, a critical phosphorylation site in the activation segment. Cys is present at the equivalent position in ~100 human Ser/Thr kinases, a residue that we found was important not only for the activity of human Aurora A but also for that of fission yeast MAPK-activated kinase (Srk1) and PKA (Pka1). Moreover, the presence of this conserved Cys predicted biochemical redox sensitivity among a cohort of human CAMK, AGC, and AGC-like kinases. Thus, we predict that redox modulation of the conserved Cys290 of Aurora A may be an underappreciated regulatory mechanism that is widespread in eukaryotic Ser/Thr kinases. Given the key biological roles of these enzymes, these findings have implications for understanding physiological and pathological responses to ROS and highlight the importance of protein kinase regulation through multivalent modification of the activation segment.

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Oxidative stress inhibits MEKK1 by site-specific glutathionylation in the ATP-binding domain

Biochemical Journal ◽

10.1042/bj20040591 ◽

2004 ◽

Vol 381 (3) ◽

pp. 675-683 ◽

Cited By ~ 145

Author(s):

Janet V. CROSS ◽

Dennis J. TEMPLETON

Keyword(s):

Oxidative Stress ◽

Protein Kinase ◽

Cysteine Residue ◽

Binding Domain ◽

Mitogen Activated Protein Kinase ◽

Atp Binding ◽

Cysteine Oxidation ◽

Mitogen Activated Protein ◽

In Cells

Many intracellular signalling events are accompanied by generation of reactive oxygen species in cells. Oxidation of protein thiol groups is an emerging theme in signal-transduction research. We have found that MEKK1 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase kinase 1], an upstream activator of the SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase) pathway, is directly inhibited by cysteine alkylation using NEM (N-ethylmaleimide). The related kinase, ASK1 (apoptosis signal-regulating kinase 1), was not inhibited, but was instead activated by NEM. Inhibition of MEKK1 requires a single unique cysteine residue (Cys1238) in the ATP-binding domain of MEKK1. Oxidative stress induced by menadione (2-methyl-1,4-naphthoquinone) also inhibited MEKK1, but activated ASK1, in cells. MEKK1 inhibition by menadione also required Cys1238. Oxidant-inhibited MEKK1 was re-activated by dithiothreitol and glutathione, supporting reversible cysteine oxidation as a mechanism. Using various chemical probes, we excluded modification by S-nitrosylation or oxidation of cysteine to sulphenic acid. Oxidant-inhibited MEKK1 migrated normally on non-reducing gels, excluding the possibility of intra- or inter-molecular disulphide bond formation. MEKK1 was inhibited by glutathionylation in vitro, and MEKK1 isolated from menadione-treated cells was shown by MS to be modified by glutathione on Cys1238. Our results support a model whereby the redox environment within the cell selectively regulates stress signalling through MEKK1 versus ASK1, and may thereby participate in the induction of apoptosis by oxidative stress.

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ATR-Mediated Checkpoint Pathways Regulate Phosphorylation and Activation of Human Chk1

Molecular and Cellular Biology ◽

10.1128/mcb.21.13.4129-4139.2001 ◽

2001 ◽

Vol 21 (13) ◽

pp. 4129-4139 ◽

Cited By ~ 704

Author(s):

Hui Zhao ◽

Helen Piwnica-Worms

Keyword(s):

Protein Kinase ◽

Cell Cycle Progression ◽

Gel Filtration ◽

Genotoxic Stress ◽

Protein Kinase Activity ◽

Checkpoint Activation ◽

Cycle Progression ◽

Evolutionarily Conserved

ABSTRACT Chk1 is an evolutionarily conserved protein kinase that regulates cell cycle progression in response to checkpoint activation. In this study, we demonstrated that agents that block DNA replication or cause certain forms of DNA damage induce the phosphorylation of human Chk1. The phosphorylated form of Chk1 possessed higher intrinsic protein kinase activity and eluted more quickly on gel filtration columns. Serines 317 and 345 were identified as sites of phosphorylation in vivo, and ATR (the ATM- and Rad3-related protein kinase) phosphorylated both of these sites in vitro. Furthermore, phosphorylation of Chk1 on serines 317 and 345 in vivo was ATR dependent. Mutants of Chk1 containing alanine in place of serines 317 and 345 were poorly activated in response to replication blocks or genotoxic stress in vivo, were poorly phosphorylated by ATR in vitro, and were not found in faster-eluting fractions by gel filtration. These findings demonstrate that the activation of Chk1 in response to replication blocks and certain forms of genotoxic stress involves phosphorylation of serines 317 and 345. In addition, this study implicates ATR as a direct upstream activator of Chk1 in human cells.

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Redox priming promotes Aurora A activation during mitosis

Science Signaling ◽

10.1126/scisignal.abb6707 ◽

2020 ◽

Vol 13 (641) ◽

pp. eabb6707

Author(s):

Daniel C. Lim ◽

Vladimir Joukov ◽

T. Justin Rettenmaier ◽

Akiko Kumagai ◽

William G. Dunphy ◽

...

Keyword(s):

Cell Cycle ◽

Mammalian Cells ◽

Kinase Domain ◽

Cysteine Residue ◽

Covalent Modification ◽

Aurora A Kinase ◽

Aurora A ◽

Cysteine Oxidation ◽

Covalent Modifications ◽

Cycle Dependent

Cell cycle–dependent redox changes can mediate transient covalent modifications of cysteine thiols to modulate the activities of regulatory kinases and phosphatases. Our previously reported finding that protein cysteine oxidation is increased during mitosis relative to other cell cycle phases suggests that redox modifications could play prominent roles in regulating mitotic processes. The Aurora family of kinases and their downstream targets are key components of the cellular machinery that ensures the proper execution of mitosis and the accurate segregation of chromosomes to daughter cells. In this study, x-ray crystal structures of the Aurora A kinase domain delineate redox-sensitive cysteine residues that, upon covalent modification, can allosterically regulate kinase activity and oligomerization state. We showed in both Xenopus laevis egg extracts and mammalian cells that a conserved cysteine residue within the Aurora A activation loop is crucial for Aurora A activation by autophosphorylation. We further showed that covalent disulfide adducts of this residue promote autophosphorylation of the Aurora A kinase domain. These findings reveal a potential mechanistic link between Aurora A activation and changes in the intracellular redox state during mitosis and provide insights into how novel small-molecule inhibitors may be developed to target specific subpopulations of Aurora A.

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Nitric oxide negatively regulates abscisic acid signaling in guard cells by S-nitrosylation of OST1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1423481112 ◽

2014 ◽

Vol 112 (2) ◽

pp. 613-618 ◽

Cited By ~ 178

Author(s):

Pengcheng Wang ◽

Yanyan Du ◽

Yueh-Ju Hou ◽

Yang Zhao ◽

Chuan-Chih Hsu ◽

...

Keyword(s):

Nitric Oxide ◽

Abscisic Acid ◽

Protein Kinase ◽

Stomatal Closure ◽

Guard Cells ◽

Cysteine Residue ◽

Aba Signaling ◽

Related Protein ◽

Abscisic Acid Signaling ◽

Evolutionarily Conserved

The phytohormone abscisic acid (ABA) plays important roles in plant development and adaptation to environmental stress. ABA induces the production of nitric oxide (NO) in guard cells, but how NO regulates ABA signaling is not understood. Here, we show that NO negatively regulates ABA signaling in guard cells by inhibiting open stomata 1 (OST1)/sucrose nonfermenting 1 (SNF1)-related protein kinase 2.6 (SnRK2.6) through S-nitrosylation. We found that SnRK2.6 is S-nitrosylated at cysteine 137, a residue adjacent to the kinase catalytic site. Dysfunction in the S-nitrosoglutathione (GSNO) reductase (GSNOR) gene in the gsnor1-3 mutant causes NO overaccumulation in guard cells, constitutive S-nitrosylation of SnRK2.6, and impairment of ABA-induced stomatal closure. Introduction of the Cys137 to Ser mutated SnRK2.6 into the gsnor1-3/ost1-3 double-mutant partially suppressed the effect of gsnor1-3 on ABA-induced stomatal closure. A cysteine residue corresponding to Cys137 of SnRK2.6 is present in several yeast and human protein kinases and can be S-nitrosylated, suggesting that the S-nitrosylation may be an evolutionarily conserved mechanism for protein kinase regulation.

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Effect of Thrombin on Phosphorylation of Platelet Membrane Proteins

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647962 ◽

1976 ◽

Vol 35 (03) ◽

pp. 635-642 ◽

Cited By ~ 4

Author(s):

M Steiner

Keyword(s):

Protein Kinase ◽

Kinase Activity ◽

Qualitative Change ◽

Protein Kinase Activity ◽

Protein Substrates ◽

Phosphoprotein Phosphatase ◽

Progressive Loss ◽

Platelet Membranes ◽

Endogenous Protein ◽

Considerable Loss

SummaryThe effect of thrombin on the phosphorylating activity of platelet membranes was compared to that of trypsin. Preincubation of non-32P phosphorylated platelet membranes with or without either of these two enzymes resulted in a considerable loss of membrane protein kinase activity which was most severe when trypsin was used. Protein kinase activity and endogenous protein acceptors decreased in parallel. 32P-phosphorylated membranes showed a slow but progressive loss of label which was accelerated by trypsin. Thrombin under these conditions prevented the loss of 32P-phosphate. These results are interpreted to indicate a thrombin-induced destruction of a phosphoprotein phosphatase. The protein kinase activity of phosphorylated platelet membranes using endogenous or exogenous protein substrates showed a significant reduction compared to non-phosphorylated membranes suggesting a deactivation of protein kinase by phosphorylation of platelet membranes. Neither thrombin nor trypsin caused a qualitative change in the membrane polypeptides accepting 32P-phosphate but resulted in quantitative alterations of their ability to become phosphorylated.

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