International Journal of Molecular Sciences Review Phosphorylation Dynamics of JNK Signaling: Effects of Dual-Specificity Phosphatases (DUSPs) on the JNK Pathway Jain Ha , Eunjeong Kang, Jihye Seo and Sayeon Cho * Laboratory of Molecular and Pharmacological Cell Biology, College of Pharmacy, Chung-Ang University, Seoul 06974, Korea; [email protected] (J.H.); [email protected] (E.K.); [email protected] (J.S.) * Correspondence: [email protected] Received: 29 October 2019; Accepted: 4 December 2019; Published: 6 December 2019 Abstract: Protein phosphorylation affects conformational change, interaction, catalytic activity, and subcellular localization of proteins. Because the post-modification of proteins regulates diverse cellular signaling pathways, the precise control of phosphorylation states is essential for maintaining cellular homeostasis. Kinases function as phosphorylating enzymes, and phosphatases dephosphorylate their target substrates, typically in a much shorter time. The c-Jun N-terminal kinase (JNK) signaling pathway, a mitogen-activated protein kinase pathway, is regulated by a cascade of kinases and in turn regulates other physiological processes, such as cell differentiation, apoptosis, neuronal functions, and embryonic development. However, the activation of the JNK pathway is also implicated in human pathologies such as cancer, neurodegenerative diseases, and inflammatory diseases. Therefore, the proper balance between activation and inactivation of the JNK pathway needs to be tightly regulated. Dual specificity phosphatases (DUSPs) regulate the magnitude and duration of signal transduction of the JNK pathway by dephosphorylating their substrates. In this review, we will discuss the dynamics of phosphorylation/dephosphorylation, the mechanism of JNK pathway regulation by DUSPs, and the new possibilities of targeting DUSPs in JNK-related diseases elucidated in recent studies. Keywords: mitogen-activated protein kinase pathway; c-Jun N-terminal kinase pathway; dual-specificity phosphatase; dephosphorylation 1. Phosphorylation-Dephosphorylation: The Scope of Thermodynamics Cellular regulatory mechanisms respond specifically and robustly to extracellular stimuli. Post-translational modification (PTM) indicates covalent modifications of proteins after translation, such as protein methylation, glycosylation, acetylation, sumoylation, and ubiquitination. PTM plays a vital role in the control of protein activity, stability, and subcellular localization, thereby contributing to intracellular regulation. Among these modifications, protein phosphorylation is one of the most studied PTMs. Based on phosphoproteomics approaches, phosphorylation at serine was reported to be most abundant (86.4%), followed by threonine (11.8%), and tyrosine (1.8%) in HeLa cells [1]. In addition, more than 30% of eukaryotic proteins were observed to be phosphorylated [1]. As phosphorylation alters protein function, the reversible regulation of phosphorylation is essential for maintaining cellular physiology within a normal range (Figure1)[2,3]. Int. J. Mol. Sci. 2019, 20, 6157; doi:10.3390/ijms20246157 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 6157 2 of 19 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 2 of 19 FigureFigure 1. ReversibleReversible phosphorylation phosphorylation by by kinase kinase and ph phosphatase.osphatase. Phosphorylation Phosphorylation is is an an essential essential post-translationalpost-translational modification modification that that is mediated by kinases.kinases. Reversible Reversible phosphorylation phosphorylation induces induces conformationalconformational changechange withinwithin the the protein protein or or provides provides a platform a platform for phospho-binding for phospho-binding proteins, proteins, which whichin turn in triggers turn triggers alterations alterations in protein in protein stability, stabilit activity,y, activity, interaction, interaction, or subcellular or subcellular localization. localization. Because Becausephosphorylation phosphorylation regulates regulates diverse diverse protein protein functions, functions, it should it beshould tightly be controlledtightly controlled by the reverseby the reversereaction—dephosphorylation reaction—dephosphorylation catalyzed catalyzed by phosphatases. by phosphatases. X represents X represents a protein a that protein is reversibly that is reversiblyphosphorylated phosphorylated and dephosphorylated, and dephosphorylated, and p stands and for p a stands phosphate. for a ATP,phosphate. adenosine ATP, triphosphate; adenosine triphosphate;ADP, adenosine ADP, diphosphate. adenosine diphosphate. ToTo further understand the balance between phosphorylation and dephosphorylation, the the thermodynamicsthermodynamics of of Gibbs Gibbs free free energy energy will will be bebriefly briefly discussed. discussed. A change A change in Gibbs in Gibbs free energy free energy (∆G), an(D G),indicator an indicator of the ofdirection the direction of chemical of chemical reaction reactions,s, is dependent is dependent on the on temperature the temperature and molar and molarratio ofratio the of reactants the reactants and andproducts. products. A negative A negative value value of of∆GD Gmeans means that that the the reaction reaction proceeds proceeds spontaneouslyspontaneously [4]. [4]. Most Most phosphodiester phosphodiester hydrolysis hydrolysis reactions reactions have have negative negative ∆DGG values, values, which which means means thatthat the the dissociation dissociation of phosphate isis proneprone toto occuroccur [[5].5]. However,However, “spontaneous” “spontaneous” dephosphorylation dephosphorylation is ischallenging challenging to identifyto identify in biologicalin biological systems systems even ev thoughen though it has it a negativehas a negative net change net change in energy in becauseenergy becauseit proceeds it proceeds at a slow at rate a dueslow to rate a high due activation to a high energy activation (Figure energy2). Acid (Figure hydrolysis 2). Acid of phosphoamino hydrolysis of phosphoaminoacids in 1 N HCl acids results in 1 inN approximately HCl results in 40% approximately of phosphoserine 40% of and phosphoserine 60% of phosphothreonine and 60% of phosphothreonineremaining after 24 hremaining [6,7], indicating after that24 h non-enzymatic [6,7], indicating hydrolysis that non-en of phosphoproteinszymatic hydrolysis in neutral of phosphoproteinssolutions would requirein neutral a much solutions longer time.would When require a protein a much phosphatase longer istime. present, When it dramatically a protein phosphataseshortens the reactionis present, time it dramatically by lowering the shortens activation the reaction energy. Thetime catalytic by loweri activityng the of activation all other enzymes, energy. Theincluding catalytic phosphatases, activity of all arises other from enzymes, this ability including to lower phosphatases, the activation arises energy from of this the ability reaction. to lower In the thecase activation of catalysis energy by vaccinia of the H1-related reaction. (VHR)In the dual-specificitycase of catalysis phosphatase, by vaccinia the H1-related calculated (VHR) energy dual-specificitybarrier was 16.4 phosphatase, kcal/mol, which the wascalculated less than ener halfgy thebarrier energy was barrier 16.4 kcal/mol, of non-catalytic which was hydrolysis less than [8]. halfThe the change energy in activation barrier of energy non-cata barrierlytica hydrolysisffects the reaction [8]. The rate change [9]. Denu in activation et al. reported energy that barrier the turnover affects theof p reaction-nitrophenyl rate [9]. phosphate Denu et (pNPP) al. reported by wild-type that the (WT)turnover VHR of phosphatase p-nitrophenyl was phosphate more than (pNPP) 6000-fold by wild-typeof that of the(WT) VHR VHR S131A phosphatase/D92N inactive was more mutant than [9]. 6,000-fold In addition, of athat simulation of the studyVHR byS131A/D92N Kolmodin inactiveand Aqvist mutant suggested [9]. In that addition, hydrolysis a simulation of the phosphoenzyme study by Kolmodin intermediate and by Aqvist low-molecular-weight suggested that hydrolysisprotein tyrosine of the phosphatase phosphoenzyme (LM-PTP) intermediate lowered the activation by low-molecular-weight energy by approximately protein ~15 tyrosine kcal/mol phosphatasecompared to (LM-PTP) non-enzymatic lowered hydrolysis the activation [10]. energy by approximately ~15 kcal/mol compared to non-enzymatic hydrolysis [10]. Int. J. Mol. Sci. 2019, 20, 6157 3 of 19 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 19 Figure 2. Dephosphorylation reaction with or without phosphatase. Because a phosphorylated substrateFigure 2. (p-X) Dephosphorylation is at a higher free reaction energy thanwith the or unphosphorylatedwithout phosphatase. form (X),Because it is thermodynamically a phosphorylated pronesubstrate to lose (p-X) a phosphate is at eventually.a higher However,free energy for thethan p-X the to lose unphosphorylated its phosphate and becomeform (X), X, energyit is isthermodynamically required. Due to this prone high-energy to lose a barrierphosphate (indicated eventually. as a blue However, line), non-catalytic for the p-X to reactions lose its takephosphate a long time.and become With a catalyticX, energy enzyme is required. (in this case,Due ato phosphatase), this high-energy the activation barrier (indic energyated (indicated as a blue as aline), red dashednon-catalytic line) of reactions the enzymatic take a reactionlong time. is greatly With a reduced