Mardiyyah/TAPT-PDBE-V1 · Datasets at Hugging Face

PMCID stringclasses 30 values	Title stringclasses 30 values	Sentences stringlengths 2 1.94k
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	One side of the β-sheet is covered with two α-helices and the other is covered with four α-helices (Fig. 3b).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The catalytic domain of MKP7 interacts with JNK1 through a contiguous surface area that is remote from the active site.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	MKP7-CD is positioned onto the JNK1 molecule so that the active site of the phosphatase faces towards the activation segment.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	In an alignment of the structure of MKP7-CD with that of VHR25, an atypical ‘MKP' consisting of only a catalytic domain, 119 of 147 MKP7-CD residues could be superimposed with a r.m.s.d. (
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	root mean squared deviation) of 1.05 Å (Fig. 3c).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The most striking difference is that helix α0 and loop α0–β1 of VHR are absent in MKP7-CD.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Another region that cannot be aligned with VHR is found in loop β3–β4.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	This loop is shortened by nine residues in MKP7-CD compared with that in VHR.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Since helix α0 and the following loop α0–β1 are known for a substrate-recognition motif of VHR and other phosphatases, the absence of these moieties implicates a different substrate-binding mode of MKP7.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The active site of MKP7 consists of the phosphate-binding loop (P-loop, Cys244-Leu245-Ala246-Gly247-Ile248-Ser249-Arg250), and Asp213 in the general acid loop (Fig. 3b and Supplementary Fig. 1b).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The MKP7-CD structure near the active site exhibits a typical active conformation as found in VHR and other PTPs25.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The catalytic residue, Cys244, is located just after strand β5 and optimally positioned for nucleophilic attack262728.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Asp213 in MKP7 also adopts a position similar to that of Asp92 in VHR (Supplementary Fig. 1c), indicating that Asp213 is likely to function as the general acid in MKP7.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	We also observed the binding of a chloride ion in the active site of MKP7-CD.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	It is located 3.36 Å from the Cys244 side chain and makes electrostatic interactions with the dipole moment of helix α3 and with several main-chain amide groups.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The side chain of strictly conserved Arg250 is oriented towards the negatively charged chloride, similar to the canonical phosphate-coordinating conformation.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Thus this chloride ion is a mimic for the phosphate group of the substrate, as revealed by a comparison with the structure of PTP1B in complex with phosphotyrosine29 (Supplementary Fig. 1d).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Although the catalytically important residues in MKP7-CD are well aligned with those in VHR, the residues in the P-loop of MKP7 are smaller and have a more hydrophobic character than those of VHR (Cys124-Arg125-Glu126-Gly127-Tyr128-Gly129-Arg130; Fig. 3b,c).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The difference in the polarity/hydrophobicity of the surface may also point to the origin of the differences in the substrate-recognition mechanism for these two phosphatases (Supplementary Fig. 1e,f).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	In the complex, MKP7-CD and JNK1 form extensive protein–protein interactions involving the C-terminal helices of MKP7-CD and C-lobe of JNK1 (Fig. 3d,e).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	As a result, the buried solvent-accessible surface area is ∼1,315 Å. In the C-terminal domain, JNK1 has an insertion after the helix αG. This insertion consists of two helices (α1L14 and α2L14) that are common to all members of the MAPK family.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The interactive surface in JNK1, formed by the helices αG and α2L14, displays a hydrophobic region, centred at Trp234 (Fig. 3d).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The MKP7-docking region includes two helices, α4 and α5, and the general acid loop.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The aromatic ring of Phe285 on MKP7 α5-helix is nestled in a hydrophobic pocket on JNK1, formed by side chains of Ile197, Leu198, Ile231, Trp234, Val256, Tyr259, Val260 and the aliphatic portion of His230 (Fig. 3d,f and Supplementary Fig. 1g).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	In addition, there are hydrogen bonds between Ser282 and Asn286 of MKP7 and His230 and Thr255 of JNK1, and the main chain of Phe215 in the general acid loop of MKP7 is hydrogen-bonded to the side chain of Gln253 in JNK1.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The second interactive area involves the α4 helix of MKP7 and charged/polar residues of JNK1 (Fig. 3e).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The carboxylate of Asp268 in MKP7 forms a salt bridge with side chain of Arg263 in JNK1, and Lys275 of MKP7 forms a hydrogen bond and a salt bridge with Thr228 and Asp229 of JNK1, respectively.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	To assess the importance of the aforementioned interactions, we generated a series of point mutations on the MKP7-CD and examined their effect on the MKP7-catalysed JNK1 dephosphorylation (Fig. 4a).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	When the hydrophobic residues Phe285 and Phe287 on the α5 of MKP7-CD were replaced by Asp or Ala, their phosphatase activities for JNK1 dephosphorylation decreased ∼10-fold.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	In comparison, replacement of the other residues (Phe215, Asp268, Lys275, Ser282, Asn286 and Leu292) with an Ala or Asp individually led to a modest decrease in catalytic efficiencies, suggesting that this position may only affect some selectivity of MKP.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Mutation of Leu288 markedly reduced its solubility when expressed in Escherichia coli, resulting in the insoluble aggregation of the mutant protein.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Gel filtration analysis further confirmed the key role of Phe285 in the MKP7–JNK1 interaction: no F285D–JNK1 complex was detected when 3 molar equivalents of MKP7-CD (F285D) were mixed with 1 molar equivalent of JNK1 (Fig. 4b).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Interestingly, mutation of Phe287 results in a considerable loss of activity against pJNK1 without altering the affinity of MKP7-CD for JNK1 (Supplementary Fig. 2a).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	We also generated a series of point mutations in the JNK1 and assessed the effect on JNK1 binding using the GST pull-down assay (Fig. 4c).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Substitution at Asp229, Trp234, Thr255, Val256, Tyr259 and Val260 significantly reduced the binding affinity of MKP7-CD for JNK.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	To determine whether the deficiencies in their abilities to bind partner proteins or carry out catalytic function are owing to misfolding of the purified mutant proteins, we also examined the folding properties of the JNK1 and MKP7 mutants with circular dichroism.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The spectra of these mutants are similar to the wild-type proteins, indicating that these mutants fold as well as the wild-type proteins (Fig. 4d,e).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Taken together, these results are consistent with the present crystallographic model, which reveal the hydrophobic contacts between the MKP7 catalytic domain and JNK1 have a predominant role in the enzyme–substrate interaction, and hydrophobic residue Phe285 in the MKP7-CD is a key residue for its high-affinity binding to JNK1.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	It has previously been reported that several cytosolic and inducible nuclear MKPs undergo catalytic activation upon interaction with the MAPK substrates15.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	This allosteric activation of MKP3 has been well-documented in vitro using pNPP, a small-molecule phosphotyrosine analogue of its normal substrate3031.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	We then assayed pNPPase activities of MKP7ΔC304 and MKP7-CD in the presence of JNK1.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Incubation of MKP7 with JNK1 did not markedly stimulate the phosphatase activity, which is consistent with previous results that MKP7 solely possesses the intrinsic activity (Supplementary Fig. 2b).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The small pNPP molecule binds directly at the enzyme active site and can be used to probe the reaction mechanism of protein phosphatases.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	We therefore examined the effects of the MKP7-CD mutants on their pNPPase activities.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	As shown in Fig. 4f, all the mutants, except F287D/A, showed little or no activity change compared with the wild-type MKP7-CD.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	In the JNK1/MKP7-CD complex structure, Phe287 of MKP7 does not make contacts with JNK1 substrate.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis32333435 (Supplementary Fig. 2c).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Kinase-associated phosphatase (KAP), a member of the DUSP family, plays a crucial role in cell cycle regulation by dephosphorylating the pThr160 residue of CDK2 (cyclin-dependent kinase 2).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The crystal structure of the CDK2/KAP complex has been determined at 3.0 Å (Fig. 5a)36.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The interface between these two proteins consists of three discontinuous contact regions.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Biochemical results suggested that the affinity and specificity between KAP and CDK2 results from the recognition site comprising CDK2 residues from the αG helix and L14 loop and the N-terminal helical region of KAP (Fig. 5b).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	There is a hydrogen bond between the main-chain nitrogen of Ile183 (KAP) and side chain oxygen of Glu208 (CDK2), and salt bridges between Lys184 of KAP and Asp235 of CDK2.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Structural analysis and sequence alignment reveal that one of the few differences between MKP7-CD and KAP in the substrate-binding region is the presence of the motif FNFL in MKP7-CD, which corresponds to IKQY in KAP (Fig. 5c).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The substitution of the two hydrophobic residues with charged/polar residues (F285I/N286K) seriously disrupts the hydrophobic interaction required for MKP7 binding on JNK1 (Fig. 4a).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	In addition, His230 and Val256 in JNK1 are replaced by the negatively charged residues Glu208 and Asp235 in CDK2 (Fig. 5d), and the charge distribution on the CDK2 interactive surface is quite different from that of JNK.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	These data indicated that a unique hydrophobic pocket formed between the MAPK insert and αG helix plays a major role in the substrate recognition by MKPs.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	JNK is activated following cellular exposure to a number of acute stimuli such as anisomycin, H2O2, ultraviolet light, sorbitol, DNA-damaging agents and several strong apoptosis inducers (etoposide, cisplatin and taxol)373839.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	To assess the effects of MKP7 and its mutants on the activation of endogenous JNK in vivo, HEK293T cells were transfected with blank vector or with HA-tagged constructs for full-length MKP7, MKP7ΔC304 and MKP7-CD or MKP7 mutants, and stimulated with ultraviolet or etoposide treatment.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	As shown in Fig. 6a–c, immunobloting showed similar expression levels for the different MKP7 constructs in all the cells.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Overexpressed full-length MKP7, MKP7ΔC304 and MKP7-CD significantly reduced the endogenous level of phosphorylated JNK compared with vector-transfected cells.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Parallel experiments showed clearly that the D-motif mutants (R56A/R57A and V63A/I65A) dephosphorylated JNK as did the wild type under the same conditions, further confirming that the MKP7-KBD is not required for the JNK inactivation in vivo.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Consistent with the in vitro data, the level of phosphorylated JNK was not or little altered in MKP7 FXF-motif mutants (F285D, F287D and L288D)-transfected cells, and the MKP7 D268A and N286A mutants retained the ability to reduce the phosphorylation levels of JNK.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	We next tested in vivo interactions between JNK1 mutants and full-length MKP7 by coimmunoprecipitation experiments under unstimulated conditions.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	When co-expressed in HEK293T cells, wild-type (HA)-JNK1 was readily precipitated with (Myc)-MKP7 (Fig. 6d), indicating that MKP7 binds dephosphorylated JNK1 protein in vivo.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	In agreement with the in vitro pull-down results, the mutants D229A, W234D and Y259D were not co-precipitated with MKP7, and the I231D mutant had only little effect on the JNK1–MKP7 interaction (Fig. 6d and Supplementary Fig. 3a).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Activation of the JNK signalling pathway is frequently associated with apoptotic cell death, and inhibition of JNK can prevent apoptotic death of multiple cells640414243.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	To examine whether the inhibition of JNK activity by MKP7 would provide protections against the apoptosis, we analysed the rate of apoptosis in ultraviolet-irradiated cells transfected with MKP7 (wild type or mutants) by flow cytometry.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The results showed similar apoptotic rates between cells transfected with blank vector or with MKP7 (wild type or mutants) under unstimulated conditions (Supplementary Fig. 3b), while ultraviolet-irradiation significantly increased apoptotic rate in cells transfected with blank vector (Fig. 6e).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Expressions of wild-type MKP7, MKP7ΔC304 and MKP7-CD significantly decreased the proportion of apoptotic cells after ultraviolet treatment.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Moreover, treatment of cells expressing MKP7-KBD mutants (R56A/R57A and V63A/I65A) decreased the apoptosis rates to a similar extent as MKP7 wild type did.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	In contrast, cells transfected with the MKP7 FXF-motif mutants (F285D, F287D and L288D) showed little protective effect after ultraviolet treatment and similar levels of apoptosis rates were detected to cells transfected with control vectors (Fig. 6e,f).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Taken together, our results suggested that FXF-motif-mediated, rather than KBD-mediated, interaction is essential for MKP7 to block ultraviolet-induced apoptosis.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	MKP5 belongs to the same subfamily as MKP7.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	MKP5 is unique among the MKPs in possessing an additional domain of unknown function at the N-terminus44 (Fig. 7a).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The KBD of MKP5 interacts with the D-site of p38α to mediate the enzyme–substrate interaction.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Deletion of the KBD in MKP5 leads to a 280-fold increase in Km for p38α substrate23.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	In contrast to p38α substrate, deletion of the MKP5-KBD had little effects on the kinetic parameters for the JNK1 dephosphorylation, indicating that the KBD of MKP5 is not required for the JNK1 dephosphorylation (Fig. 7b).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The substrate specificity constant kcat /Km value for MKP5-CD was calculated as 1.0 × 10 M s, which is very close to that of MKP7-CD (1.07 × 10 M s).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The crystal structure of human MKP5-CD has been determined45.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Comparisons between catalytic domains structures of MKP5 and MKP7 reveal that the overall folds of the two proteins are highly similar, with only a few regions exhibiting small deviations (r.m.s.d.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	of 0.79 Å; Fig. 7c).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Given the distinct interaction mode revealed by the crystal structure of JNK1–MKP7-CD, one obvious question is whether this is a general mechanism used by all members of the JNK-specific MKPs.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	To address this issue, we first examined the docking ability of JNK1 to the KBD and CD of MKP5 using gel filtration analysis and pull-down assays.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	It can be seen from gel filtration experiments that JNK1 can forms a stable heterodimer with MKP5-CD in solution, but no detectable interaction was found with the KBD domain (Fig. 7d).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Pull-down assays also confirmed the protein–protein interactions observed above.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The catalytic domain of MKP5, but not its KBD, was able to pull-down a detectable amount of JNK1 (Fig. 7e), implicating a different substrate-recognition mechanisms for p38 and JNK MAPKs.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	To further test our hypothesis, we generated forms of MKP5-CD bearing mutations corresponding to the changes we made on MKP7-CD on the basis of sequence and structural alignment and examined their effects on the phosphatase activity.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	As shown in Fig. 7f, the T432A and L449F MKP5 mutant showed little or no difference in phosphatase activity, whereas the other mutants showed reduced specific activities of MKP5.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	As in the case of MKP7, all the mutants, except F451D/A, showed no pNPPase activity changes compared with the wild-type MKP5-CD (Fig. 7g), and the point mutations in JNK1 also reduced the binding affinity of MKP5-CD for JNK1 (Fig. 7h).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	In addition, there were no significant differences in the CD spectra between wild-type and mutant proteins, indicating that the overall structures of these mutants did not change significantly from that of wild-type MKP5 protein (Supplementary Fig. 4a).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Taken together, our results suggest that MKP5 binds JNK1 in a docking mode similar to that in the JNK1–MKP7 complex, and the detailed interaction model can be generated using molecular dynamics simulation based on the structure of JNK1–MKP7-CD complex (Supplementary Fig. 4b,c).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	In this model, the MKP5-CD adopts a conformation nearly identical to that in its unbound form, suggesting that the conformation of the catalytic domain undergoes little change, if any at all, upon JNK1 binding.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	In particular, Leu449 of MKP5, which is equivalent to the key residue Phe285 of MKP7, buried deeply within the hydrophobic pocket of JNK1 in the same way as Phe285 in the JNK1–MKP7-CD complex (Supplementary Fig. 4d).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Despite the strong similarities between JNK1–MKP5-CD and JNK1–MKP7-CD, however, there are differences.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The JNK1–MKP7-CD interaction is better and more extensive.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Asp268 of MKP7-CD forms salt bridge with JNK1 Arg263, whereas the corresponding residue Thr432 in MKP5-CD may not interact with JNK1.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	This is consistent with the experimental observation showing that JNK1 binds to MKP7-CD much more tightly than MKP5-CD (Km value of MKP5-CD for pJNK1 substrate is ∼20-fold higher than that of MKP7-CD).
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	The MAPKs p38, ERK and JNK, are central to evolutionarily conserved signalling pathways that are present in all eukaryotic cells.
PMC4802042	A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.	Each MAPK cascade is activated in response to a diverse array of extracellular signals and culminates in the dual-phosphorylation of a threonine and a tyrosine residue in the MAPK-activation loop2.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

One side of the β-sheet is covered with two α-helices and the other is covered with four α-helices (Fig. 3b).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The catalytic domain of MKP7 interacts with JNK1 through a contiguous surface area that is remote from the active site.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

MKP7-CD is positioned onto the JNK1 molecule so that the active site of the phosphatase faces towards the activation segment.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

In an alignment of the structure of MKP7-CD with that of VHR25, an atypical ‘MKP' consisting of only a catalytic domain, 119 of 147 MKP7-CD residues could be superimposed with a r.m.s.d. (

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

root mean squared deviation) of 1.05 Å (Fig. 3c).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The most striking difference is that helix α0 and loop α0–β1 of VHR are absent in MKP7-CD.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Another region that cannot be aligned with VHR is found in loop β3–β4.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

This loop is shortened by nine residues in MKP7-CD compared with that in VHR.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Since helix α0 and the following loop α0–β1 are known for a substrate-recognition motif of VHR and other phosphatases, the absence of these moieties implicates a different substrate-binding mode of MKP7.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The active site of MKP7 consists of the phosphate-binding loop (P-loop, Cys244-Leu245-Ala246-Gly247-Ile248-Ser249-Arg250), and Asp213 in the general acid loop (Fig. 3b and Supplementary Fig. 1b).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The MKP7-CD structure near the active site exhibits a typical active conformation as found in VHR and other PTPs25.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The catalytic residue, Cys244, is located just after strand β5 and optimally positioned for nucleophilic attack262728.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Asp213 in MKP7 also adopts a position similar to that of Asp92 in VHR (Supplementary Fig. 1c), indicating that Asp213 is likely to function as the general acid in MKP7.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

We also observed the binding of a chloride ion in the active site of MKP7-CD.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

It is located 3.36 Å from the Cys244 side chain and makes electrostatic interactions with the dipole moment of helix α3 and with several main-chain amide groups.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The side chain of strictly conserved Arg250 is oriented towards the negatively charged chloride, similar to the canonical phosphate-coordinating conformation.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Thus this chloride ion is a mimic for the phosphate group of the substrate, as revealed by a comparison with the structure of PTP1B in complex with phosphotyrosine29 (Supplementary Fig. 1d).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Although the catalytically important residues in MKP7-CD are well aligned with those in VHR, the residues in the P-loop of MKP7 are smaller and have a more hydrophobic character than those of VHR (Cys124-Arg125-Glu126-Gly127-Tyr128-Gly129-Arg130; Fig. 3b,c).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The difference in the polarity/hydrophobicity of the surface may also point to the origin of the differences in the substrate-recognition mechanism for these two phosphatases (Supplementary Fig. 1e,f).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

In the complex, MKP7-CD and JNK1 form extensive protein–protein interactions involving the C-terminal helices of MKP7-CD and C-lobe of JNK1 (Fig. 3d,e).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

As a result, the buried solvent-accessible surface area is ∼1,315 Å. In the C-terminal domain, JNK1 has an insertion after the helix αG. This insertion consists of two helices (α1L14 and α2L14) that are common to all members of the MAPK family.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The interactive surface in JNK1, formed by the helices αG and α2L14, displays a hydrophobic region, centred at Trp234 (Fig. 3d).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The MKP7-docking region includes two helices, α4 and α5, and the general acid loop.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The aromatic ring of Phe285 on MKP7 α5-helix is nestled in a hydrophobic pocket on JNK1, formed by side chains of Ile197, Leu198, Ile231, Trp234, Val256, Tyr259, Val260 and the aliphatic portion of His230 (Fig. 3d,f and Supplementary Fig. 1g).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

In addition, there are hydrogen bonds between Ser282 and Asn286 of MKP7 and His230 and Thr255 of JNK1, and the main chain of Phe215 in the general acid loop of MKP7 is hydrogen-bonded to the side chain of Gln253 in JNK1.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The second interactive area involves the α4 helix of MKP7 and charged/polar residues of JNK1 (Fig. 3e).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The carboxylate of Asp268 in MKP7 forms a salt bridge with side chain of Arg263 in JNK1, and Lys275 of MKP7 forms a hydrogen bond and a salt bridge with Thr228 and Asp229 of JNK1, respectively.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

To assess the importance of the aforementioned interactions, we generated a series of point mutations on the MKP7-CD and examined their effect on the MKP7-catalysed JNK1 dephosphorylation (Fig. 4a).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

When the hydrophobic residues Phe285 and Phe287 on the α5 of MKP7-CD were replaced by Asp or Ala, their phosphatase activities for JNK1 dephosphorylation decreased ∼10-fold.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

In comparison, replacement of the other residues (Phe215, Asp268, Lys275, Ser282, Asn286 and Leu292) with an Ala or Asp individually led to a modest decrease in catalytic efficiencies, suggesting that this position may only affect some selectivity of MKP.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Mutation of Leu288 markedly reduced its solubility when expressed in Escherichia coli, resulting in the insoluble aggregation of the mutant protein.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Gel filtration analysis further confirmed the key role of Phe285 in the MKP7–JNK1 interaction: no F285D–JNK1 complex was detected when 3 molar equivalents of MKP7-CD (F285D) were mixed with 1 molar equivalent of JNK1 (Fig. 4b).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Interestingly, mutation of Phe287 results in a considerable loss of activity against pJNK1 without altering the affinity of MKP7-CD for JNK1 (Supplementary Fig. 2a).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

We also generated a series of point mutations in the JNK1 and assessed the effect on JNK1 binding using the GST pull-down assay (Fig. 4c).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Substitution at Asp229, Trp234, Thr255, Val256, Tyr259 and Val260 significantly reduced the binding affinity of MKP7-CD for JNK.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

To determine whether the deficiencies in their abilities to bind partner proteins or carry out catalytic function are owing to misfolding of the purified mutant proteins, we also examined the folding properties of the JNK1 and MKP7 mutants with circular dichroism.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The spectra of these mutants are similar to the wild-type proteins, indicating that these mutants fold as well as the wild-type proteins (Fig. 4d,e).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Taken together, these results are consistent with the present crystallographic model, which reveal the hydrophobic contacts between the MKP7 catalytic domain and JNK1 have a predominant role in the enzyme–substrate interaction, and hydrophobic residue Phe285 in the MKP7-CD is a key residue for its high-affinity binding to JNK1.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

It has previously been reported that several cytosolic and inducible nuclear MKPs undergo catalytic activation upon interaction with the MAPK substrates15.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

This allosteric activation of MKP3 has been well-documented in vitro using pNPP, a small-molecule phosphotyrosine analogue of its normal substrate3031.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

We then assayed pNPPase activities of MKP7ΔC304 and MKP7-CD in the presence of JNK1.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Incubation of MKP7 with JNK1 did not markedly stimulate the phosphatase activity, which is consistent with previous results that MKP7 solely possesses the intrinsic activity (Supplementary Fig. 2b).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The small pNPP molecule binds directly at the enzyme active site and can be used to probe the reaction mechanism of protein phosphatases.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

We therefore examined the effects of the MKP7-CD mutants on their pNPPase activities.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

As shown in Fig. 4f, all the mutants, except F287D/A, showed little or no activity change compared with the wild-type MKP7-CD.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

In the JNK1/MKP7-CD complex structure, Phe287 of MKP7 does not make contacts with JNK1 substrate.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

It penetrates into a pocket formed by residues from the P-loop and general acid loop and forms hydrophobic contacts with the aliphatic portions of side chains of Arg250, Glu217 and Ile219, suggesting that Phe287 in MKP7 would play a similar role to that of its structural counterpart in the PTPs (Gln266 in PTP1B) and VHR (Phe166 in VHR) in the precise alignment of active-site residues in MKP7 with respect to the substrate for efficient catalysis32333435 (Supplementary Fig. 2c).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Kinase-associated phosphatase (KAP), a member of the DUSP family, plays a crucial role in cell cycle regulation by dephosphorylating the pThr160 residue of CDK2 (cyclin-dependent kinase 2).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The crystal structure of the CDK2/KAP complex has been determined at 3.0 Å (Fig. 5a)36.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The interface between these two proteins consists of three discontinuous contact regions.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Biochemical results suggested that the affinity and specificity between KAP and CDK2 results from the recognition site comprising CDK2 residues from the αG helix and L14 loop and the N-terminal helical region of KAP (Fig. 5b).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

There is a hydrogen bond between the main-chain nitrogen of Ile183 (KAP) and side chain oxygen of Glu208 (CDK2), and salt bridges between Lys184 of KAP and Asp235 of CDK2.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Structural analysis and sequence alignment reveal that one of the few differences between MKP7-CD and KAP in the substrate-binding region is the presence of the motif FNFL in MKP7-CD, which corresponds to IKQY in KAP (Fig. 5c).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The substitution of the two hydrophobic residues with charged/polar residues (F285I/N286K) seriously disrupts the hydrophobic interaction required for MKP7 binding on JNK1 (Fig. 4a).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

In addition, His230 and Val256 in JNK1 are replaced by the negatively charged residues Glu208 and Asp235 in CDK2 (Fig. 5d), and the charge distribution on the CDK2 interactive surface is quite different from that of JNK.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

These data indicated that a unique hydrophobic pocket formed between the MAPK insert and αG helix plays a major role in the substrate recognition by MKPs.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

JNK is activated following cellular exposure to a number of acute stimuli such as anisomycin, H2O2, ultraviolet light, sorbitol, DNA-damaging agents and several strong apoptosis inducers (etoposide, cisplatin and taxol)373839.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

To assess the effects of MKP7 and its mutants on the activation of endogenous JNK in vivo, HEK293T cells were transfected with blank vector or with HA-tagged constructs for full-length MKP7, MKP7ΔC304 and MKP7-CD or MKP7 mutants, and stimulated with ultraviolet or etoposide treatment.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

As shown in Fig. 6a–c, immunobloting showed similar expression levels for the different MKP7 constructs in all the cells.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Overexpressed full-length MKP7, MKP7ΔC304 and MKP7-CD significantly reduced the endogenous level of phosphorylated JNK compared with vector-transfected cells.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Parallel experiments showed clearly that the D-motif mutants (R56A/R57A and V63A/I65A) dephosphorylated JNK as did the wild type under the same conditions, further confirming that the MKP7-KBD is not required for the JNK inactivation in vivo.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Consistent with the in vitro data, the level of phosphorylated JNK was not or little altered in MKP7 FXF-motif mutants (F285D, F287D and L288D)-transfected cells, and the MKP7 D268A and N286A mutants retained the ability to reduce the phosphorylation levels of JNK.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

We next tested in vivo interactions between JNK1 mutants and full-length MKP7 by coimmunoprecipitation experiments under unstimulated conditions.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

When co-expressed in HEK293T cells, wild-type (HA)-JNK1 was readily precipitated with (Myc)-MKP7 (Fig. 6d), indicating that MKP7 binds dephosphorylated JNK1 protein in vivo.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

In agreement with the in vitro pull-down results, the mutants D229A, W234D and Y259D were not co-precipitated with MKP7, and the I231D mutant had only little effect on the JNK1–MKP7 interaction (Fig. 6d and Supplementary Fig. 3a).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Activation of the JNK signalling pathway is frequently associated with apoptotic cell death, and inhibition of JNK can prevent apoptotic death of multiple cells640414243.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

To examine whether the inhibition of JNK activity by MKP7 would provide protections against the apoptosis, we analysed the rate of apoptosis in ultraviolet-irradiated cells transfected with MKP7 (wild type or mutants) by flow cytometry.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The results showed similar apoptotic rates between cells transfected with blank vector or with MKP7 (wild type or mutants) under unstimulated conditions (Supplementary Fig. 3b), while ultraviolet-irradiation significantly increased apoptotic rate in cells transfected with blank vector (Fig. 6e).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Expressions of wild-type MKP7, MKP7ΔC304 and MKP7-CD significantly decreased the proportion of apoptotic cells after ultraviolet treatment.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Moreover, treatment of cells expressing MKP7-KBD mutants (R56A/R57A and V63A/I65A) decreased the apoptosis rates to a similar extent as MKP7 wild type did.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

In contrast, cells transfected with the MKP7 FXF-motif mutants (F285D, F287D and L288D) showed little protective effect after ultraviolet treatment and similar levels of apoptosis rates were detected to cells transfected with control vectors (Fig. 6e,f).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Taken together, our results suggested that FXF-motif-mediated, rather than KBD-mediated, interaction is essential for MKP7 to block ultraviolet-induced apoptosis.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

MKP5 belongs to the same subfamily as MKP7.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

MKP5 is unique among the MKPs in possessing an additional domain of unknown function at the N-terminus44 (Fig. 7a).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The KBD of MKP5 interacts with the D-site of p38α to mediate the enzyme–substrate interaction.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Deletion of the KBD in MKP5 leads to a 280-fold increase in Km for p38α substrate23.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

In contrast to p38α substrate, deletion of the MKP5-KBD had little effects on the kinetic parameters for the JNK1 dephosphorylation, indicating that the KBD of MKP5 is not required for the JNK1 dephosphorylation (Fig. 7b).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The substrate specificity constant kcat /Km value for MKP5-CD was calculated as 1.0 × 10 M s, which is very close to that of MKP7-CD (1.07 × 10 M s).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The crystal structure of human MKP5-CD has been determined45.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Comparisons between catalytic domains structures of MKP5 and MKP7 reveal that the overall folds of the two proteins are highly similar, with only a few regions exhibiting small deviations (r.m.s.d.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

of 0.79 Å; Fig. 7c).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Given the distinct interaction mode revealed by the crystal structure of JNK1–MKP7-CD, one obvious question is whether this is a general mechanism used by all members of the JNK-specific MKPs.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

To address this issue, we first examined the docking ability of JNK1 to the KBD and CD of MKP5 using gel filtration analysis and pull-down assays.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

It can be seen from gel filtration experiments that JNK1 can forms a stable heterodimer with MKP5-CD in solution, but no detectable interaction was found with the KBD domain (Fig. 7d).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Pull-down assays also confirmed the protein–protein interactions observed above.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The catalytic domain of MKP5, but not its KBD, was able to pull-down a detectable amount of JNK1 (Fig. 7e), implicating a different substrate-recognition mechanisms for p38 and JNK MAPKs.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

To further test our hypothesis, we generated forms of MKP5-CD bearing mutations corresponding to the changes we made on MKP7-CD on the basis of sequence and structural alignment and examined their effects on the phosphatase activity.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

As shown in Fig. 7f, the T432A and L449F MKP5 mutant showed little or no difference in phosphatase activity, whereas the other mutants showed reduced specific activities of MKP5.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

As in the case of MKP7, all the mutants, except F451D/A, showed no pNPPase activity changes compared with the wild-type MKP5-CD (Fig. 7g), and the point mutations in JNK1 also reduced the binding affinity of MKP5-CD for JNK1 (Fig. 7h).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

In addition, there were no significant differences in the CD spectra between wild-type and mutant proteins, indicating that the overall structures of these mutants did not change significantly from that of wild-type MKP5 protein (Supplementary Fig. 4a).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Taken together, our results suggest that MKP5 binds JNK1 in a docking mode similar to that in the JNK1–MKP7 complex, and the detailed interaction model can be generated using molecular dynamics simulation based on the structure of JNK1–MKP7-CD complex (Supplementary Fig. 4b,c).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

In this model, the MKP5-CD adopts a conformation nearly identical to that in its unbound form, suggesting that the conformation of the catalytic domain undergoes little change, if any at all, upon JNK1 binding.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

In particular, Leu449 of MKP5, which is equivalent to the key residue Phe285 of MKP7, buried deeply within the hydrophobic pocket of JNK1 in the same way as Phe285 in the JNK1–MKP7-CD complex (Supplementary Fig. 4d).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Despite the strong similarities between JNK1–MKP5-CD and JNK1–MKP7-CD, however, there are differences.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The JNK1–MKP7-CD interaction is better and more extensive.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Asp268 of MKP7-CD forms salt bridge with JNK1 Arg263, whereas the corresponding residue Thr432 in MKP5-CD may not interact with JNK1.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

In addition, the key interacting residues of MKP7-CD, Phe215, Leu267 and Leu288, are replaced by less hydrophobic residues, Asn379, Met431 and Met452 in MKP5-CD (Fig. 5c), respectively, which may result in weaker hydrophobic interactions between MKP5-CD and JNK1.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

This is consistent with the experimental observation showing that JNK1 binds to MKP7-CD much more tightly than MKP5-CD (Km value of MKP5-CD for pJNK1 substrate is ∼20-fold higher than that of MKP7-CD).

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

The MAPKs p38, ERK and JNK, are central to evolutionarily conserved signalling pathways that are present in all eukaryotic cells.

PMC4802042

A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation.

Each MAPK cascade is activated in response to a diverse array of extracellular signals and culminates in the dual-phosphorylation of a threonine and a tyrosine residue in the MAPK-activation loop2.