Structural Mechanisms of Allostery and Autoinhibition in JNK Family Kinases

May combine two claims.

The αD shift measured from N158 was about 1.6 Å and was highly significant (p < 0.01).

“The shift in αD as measured by the distance from N158 was ~1.6 Å and was highly significant (p < 0.01) (Figures 2I and S2C).”

May combine two claims.

In kinase assays with ATF2 and SAB protein substrates, the peptides inhibited 33P incorporation more effectively than when competing against their peptide counterparts in the FRET assay, but JIP1 still showed higher affinity.

“Here, the peptides inhibited incorporation of 33P into the protein substrates better than they did when competing against their peptide counterparts in the FRET assay for each peptide, but showed similar higher affinity for JIP1.”

May combine two claims.

SAB Arg343, corresponding to JIP1 Arg160 and ATF2 His49, forms an electrostatic interaction with JNK3 Glu367 that shifts the L16 loop and mediates the αL16 and N-lobe shift.

“In the peptide, SAB Arg343 (JIP Arg160, ATF2 His49) forms an electrostatic interaction with JNK3 Glu367 that induced a shift in the L16 loop (Figures 2F and S2B), mediating the shift in αL16 and associated shift of the N-lobe (Figure 2E).”

May combine two claims.

The inhibitory helix oriented Thr221 toward the peptide-docking site, and Tyr223 hydrogen-bonded to Lys191.

“The inhibitory helix oriented the phosphoacceptor Thr221 toward the peptide-docking site, while Tyr223 H-bonds to Lys191 (Figure 1B).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The ATF2 peptide did not saturate the ITC curves, so thermodynamic parameters were not obtained.

“ATF2 peptide did not saturate the ITC curves, and the thermodynamic parameters were not attained.”

May combine two claims.

The ordered L16 loop region had no crystal contact in the peptide-bound structures and appeared in two different space groups, indicating that its ordering and shift were peptide-induced.

“There was no crystal contact for the ordered region of the L16 loop found in all the peptide bound structures, and it occurred in two different space groups, demonstrating that the ordering and shift in position of the L16 loop was peptide induced.”

May combine two claims.

The L16 loop was fully ordered in all peptide-bound structures and in none of the peptide-free structures, showing a class-specific structural phenotype.

“Importantly, the L16 loop was fully ordered in all of the peptide bound structures, and none of the peptide-free structures, demonstrating a class-specific structural phenotype.”

May combine two claims.

In JNK3 structures bound to JIP1, SAB, and ATF2 peptides, the activation loop coiled into a helix that docked into the ATP-binding site and did not participate in crystal packing.

“When these three peptide structures were superimposed, they showed that the A-loop coiled into a helix, docked into the ATP binding site (Figure 1A), and did not participate in crystal packing.”

May combine two claims.

Pro161 in pepJIP1 corresponds to Glu50 in pepATF2, which flips out into solvent and allows the adjacent Met51 to dock against JNK3 in place of JIP1 Pro161.

“Pro161 in pepJIP1 corresponds to Glu50 in pepATF2, which was flipped out into the solvent, allowing the side chain of the adjacent Met51 to dock against JNK3 in place of JIP1 Pro161 (Figure 3B).”

May combine two claims.

In all cases, the FRET signal increased dramatically with active enzymes, which could reflect a conformational change between the FRET pairs or an increased population of JNK molecules in a peptide-binding-competent conformation.

“In all cases, there was also a dramatic increase in the FRET signal with the active enzymes, which either represents a conformational change between the FRET pairs, or an increase in the population of JNK molecules that are in a conformation that allows peptide binding.”

May combine two claims.

The location of Phe55 in pepATF2 instead of pepJIP1 Leu166 caused significant differences in the peptide backbones and in the docking of ATF2/JIP1 Leu53/Leu164.

“The location of Phe55 in pepATF2 in place of pepJIP1 Leu166 induced significant differences in the peptide backbones (Figure 3C), which caused differences in the docking of ATF2/JIP1 Leu53/Leu164.”

May combine two claims.

JNK1 Thr183Glu hydrogen-bonded on the opposite side of αC from ERK phospho-Thr183, reversing the peptide-induced lobe opening but mis-positioning αC for catalysis.

“In contrast, JNK1 Thr183Glu H-bonded on the other side of αC compared to ERK phospho-Thr183, where it reversed the peptide-induced lobe opening, but mis-positioned αC for catalysis (Figures 4C and 4D).”

May combine two claims.

Structure class analysis uncovered the subtle allosteric mechanism by which peptide binding controls the JNK catalytic site, with both peptide ends modulating N-lobe position.

“To get around limitations of single structure analysis we utilized structure class analysis which revealed the subtle allosteric mechanism through which peptide binding can control the JNK catalytic site, as both ends of the peptide modulate N-lobe position like reins on a horse.”

May combine two claims.

The αD counter-shift is caused by differential positioning of activation-loop Tyr191, which forms a tight hydrogen bond with Glu217 in αF, which in turn maintains a close van der Waals contact with Ile119 in αD.

“The counter-shift in αD was caused by the differential positioning of A-loop Tyr191, which formed a tight hydrogen bond with Glu217 in αF, which in turn maintained a close VDW contact with Ile119 in αD (Figure 5B).”

May combine two claims.

Phosphoacceptor mutant JNK1 structures show closure of the peptide-induced interlobe motion but no restoration of rotation, which explains why these mutants are not constitutively active.

“Though there is no structure of active JNK, the series of phosphoacceptor mutant JNK1 structures show a closing of the peptide-induced interlobe motion, but not a restoration of the rotation motion, explaining why these mutants are not constitutively active (Figures 4A–4C).”

May combine two claims.

The SAB peptide is similar to the JIP1 sequence but still has 21-fold lower affinity.

“The SAB peptide is similar to the JIP1 sequence, but still has 21-fold lower affinity (Figure 3D).”

May combine two claims.

ITC showed that the pepSAB glycine in the middle of the sequence is important, because the Thr162Gly mutant pepJIP1 peptide had 6-fold lower affinity.

“ITC data showed that the pepSAB glycine in the middle of the sequence was an important difference, as the Thr162Gly mutant pepJIP1 peptide showed 6-fold lower affinity (Table 1).”

May combine two claims.

Sulfate ions in the JNK1 structures occupy identical locations to the phosphates in the ERK structure, showing conservation of activation-loop control of αC.

“The conservation of the control of αC by the activation loop was shown by the presence of sulfate ions in the JNK1 structures in the identical locations as the phosphates in the ERK structure (red arrows, Figure 4D).”

May combine two claims.

Without bound inhibitor, the interlobe movement permits the A-loop to occupy the catalytic site and form an inhibitory helix.

“In the absence of bound inhibitor, this interlobe motion further allowed the A-loop to bind in the catalytic site, forming an inhibitory helix (Figure 1).”

May combine two claims.

Different D-motif peptides can directly control the A-loop through αD positioning, showing that small sequence changes contribute to allosteric control.

“Comparison of the different D-motif peptides also showed that they can directly control the A-loop via positioning of αD (Figures 3C–3E), demonstrating how small changes in D-motif sequence can contribute to allosteric control.”

May combine two claims.

Mutation of the phosphoacceptor residues to glutamate reverses the peptide-induced hinge motion but not the interlobe rotation, which explains why these mutations are not activating.

“Remarkably, mutation of the phosphoacceptor residues to glutamate reverses the peptide-induced hinge motion, but not the interlobe rotation (Figures 4A and 4B), explaining why these mutations are not activating.”

May combine two claims.

The Km of SAB (1-390) for JNK3α1 was 0.77 ± 0.09 μM, compared with the previously reported ATF2 Km of 0.16 ± 0.04 μM.

“Lastly, we measured the Km (0.77 ± 0.09 μM) of SAB (1–390) for JNK3α1, compared to Km for ATF2 = 0.16 ± 0.04 μM from our previous work (Ember et al., 2008).”

• cites Ember et al., 2008 sourceNot checked yet

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May combine two claims.

Peptide binding induced interlobe opening and rotation in JNK1 and remodeled L16 and αC to produce this change.

“The peptide-induced interlobe opening and rotation of JNK1 (Figures 4A and 4B), and remodeled L16 and αC to effect this change.”

May combine two claims.

pepJIP1 Arg160 also forms hydrogen bonds with JNK3 Tyr168, the Trp362 backbone, and a network of ordered water.

“pepJIP1 Arg160 also formed H-bonds with JNK3 Tyr168, the backbone of Trp362 and a network of ordered water.”

May combine two claims.

JIP1 forms unique interactions that account for its much higher affinity than SAB and ATF2, including distinct hydrogen bond networks and a different interaction with αD.

“We show here that JIP1 makes a number of unique interactions that explain its much higher affinity compared to SAB and ATF2, including both unique hydrogen bond networks, and differential interaction with αD (Figure 3).”

• AssumptionRests on an assumption: The D-motif peptides used in the structural and biochemical experiments are adequate proxies for the corresponding full-length JNK-binding proteins.

May combine two claims.

Except for SAB binding to JNK3α1, which was very weak, the peptides had lower IC50 values for active JNKs, suggesting tighter binding to the active enzymes.

“Except for SAB binding to JNK3α1 (where the binding was very weak), in all other cases the peptides displayed lower IC50s, suggesting tighter binding to the active JNKs.”

May combine two claims.

His49 in pepATF2 binds in the same general site as pepJIP1 Arg160, but the interaction differences suggest this is a significant determinant of the lower ATF2 affinity.

“His49 in pepATF2 binds in the same general site as pepJIP1 Arg160 (Figure 3A), but there are numerous differences in the interaction that suggest this is a significant determinant of the lower affinity of pepATF2.”

May combine two claims.

Peptide binding causes a large opening between lobes and a rotation that displaces αC from its catalytic position.

“Peptide binding induced a substantial interlobe opening, and a rotation as well, which moved αC out of the position required for catalysis (Figures 1 and 2).”

May combine two claims.

SAB induced a similar αD shift as ATF2, which was transmitted to P222 in the JNK3 activation loop and stabilized the well-ordered inhibitory helix.

“SAB induced a similar shift in αD as seen with ATF2, which again was transmitted to P222 in the A-loop of JNK3, stabilizing the well-ordered inhibitory helix (Figure 3E).”

May combine two claims.

Allostery among the catalytic site, activation loop, and peptide-docking site is well documented for ERK and p38 MAPKs but is less understood for JNK.

“Allostery among the catalytic site, A-loop, and peptide-docking site of ERK and p38 MAPKs has been well documented (Chang et al., 2002; Goldsmith, 2011; Lee et al., 2004; Wang et al., 1997; Zhang et al., 1994; Zhou et al., 2006a, 2006b), but much less is known about these processes for JNK.”

• cites Chang et al., 2002 sourceNot checked yet

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• cites Goldsmith, 2011 sourceNot checked yet

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• cites Lee et al., 2004 sourceNot checked yet

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May combine two claims.

Binding to JIP1, ATF2, or SAB D-motifs directs JNK to different cellular locations and thereby affects signaling specificity.

“Thus interaction with these three types of D-motif will drive JNK to different cellular locations, directly affecting JNK signaling specificity.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The JNK1 structure bound to a JIP1 peptide showed peptide-induced rotation of the two lobes that distorted the active site, which was proposed to explain JIP1-mediated inhibition.

“The structure of JNK1 bound to a peptide from JIP1 protein showed that the peptide induced a rotation of the two lobes that distorted the active site, which was proposed as a mechanism for JIP1-mediated inhibition of JNK signaling (Heo et al., 2004).”

• cites Heo et al., 2004 sourceNot checked yet

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Probably not a substantial claim: may not pass S4 (based).

Structure class analysis assumes that comparing groups of structures exposes subtle differences that individual structures do not reveal.

“In this work we use an approach that we call structure class analysis, which postulates that examining groups of structures reveals subtle differences that are not apparent in an individual structure.”

Classified as probably not a substantial claim. May combine two claims.

SAB is a mitochondrial scaffold protein linked to JNK-mediated ROS generation and acetaminophen-induced liver injury, and it is also a JNK substrate.

“SAB is a more recently identified scaffold protein localized to the mitochondria, accounting for JNK-mediated ROS generation (Chambers and LoGrasso, 2011), and acetaminophen-induced liver injury (Win et al., 2011), and is also a JNK substrate.”

• cites Chambers and LoGrasso, 2011Not checked yet

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• cites Win et al., 2011 sourceNot checked yet

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Classified as probably not a substantial claim.

MAPKs are activated by dual phosphorylation of a TPY motif in the activation loop, which connects the two kinase lobes.

“The MAPKs are activated via dual phosphorylation of a TPY motif found in the activation loop (A-loop), which connects the two lobes common to the kinase domain (Zhang et al., 1994) (Figure 1A).”

• cites Zhang et al., 1994 sourceNot checked yet

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May combine two claims.

Structure class analysis permits statistical comparison of structural differences and reduces the problem of crystal-packing artifacts by comparing different space groups.

“It allows for statistical analysis of structural differences, and overcomes the difficulty in interpreting the role of crystal packing by comparing different space groups (Schulze-Gahmen et al., 1993).”

• cites Schulze-Gahmen et al., 1993 sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim. May combine two claims.

The D-motif contains basic residues, a short linker, and a C-terminal hydrophobic Φ-x-Φ motif, and its sequence determines specificity among MAPK modules.

“The motif contains basic residues, a short linker, and a more C-terminal Φ-x-Φ hydrophobic motif, with the specific sequence determining specificity for the different MAPK signaling modules (Bardwell et al., 2009).”

• cites Bardwell et al., 2009 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

JIP1 is a cytoplasmic scaffold that interacts with JNK-activating kinases and is needed for JNK activity in several settings, including obesity-induced insulin resistance.

“JIP1 is a cytoplasmic scaffold protein that interacts with the upstream activating kinases for JNK, and is required for activity in a number of different contexts, including regulation of obesity-induced insulin resistance (Jaeschke et al., 2004; Morel et al., 2010; Weston and Davis, 2007; Whitmarsh et al., 2001).”

• cites Jaeschke et al., 2004 sourceNot checked yet

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• cites Morel et al., 2010 sourceNot checked yet

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• cites Weston and Davis, 2007 sourceNot checked yet

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May combine two claims.

More physiological studies show that JIP1 is required for JNK activation and is inhibitory only when overexpressed.

“However, it is now clear from more physiologically relevant studies that JIP1 is required for activation of JNK (Jaeschke et al., 2004; Morel et al., 2010; Whitmarsh et al., 2001), and is only inhibitory when overexpressed.”

• cites Jaeschke et al., 2004 sourceNot checked yet

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• cites Morel et al., 2010 sourceNot checked yet

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• cites Whitmarsh et al., 2001 sourceNot checked yet

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May combine two claims.

Peptide binding to unphosphorylated JNK induces two distinct autoinhibitory mechanisms: interlobe hinge motion and formation of the activation loop into an inhibitory helix.

“Thus peptide binding to unphosphorylated JNK induced two distinct mechanisms of autoinhibition including interlobe hinge motion and the formation of the A-loop into an inhibitory helix.”

May combine two claims.

Structure class analysis suggests that A-loop phosphorylation stabilizes the αC helix in the catalytic conformer and reverses peptide-induced autoinhibition.

“Our structure class analysis suggests that the function of A-loop phosphorylation is to stabilize the αC helix in the conformer required for catalysis, and in doing so reverse the peptide-induced autoinhibitory mechanisms.”

May combine two claims.

Subtle differences in D-motifs determine the ensemble of interacting proteins and the nature of the signal.

“Thus subtle differences in D-motifs determine the ensemble of interacting proteins, and the nature of the signal.”

May combine two claims.

With JIP1 bound to JNK3, the slight shift of Pro222 toward αD was associated with partial disordering of the inactivation helix, suggesting that docking-motif differences may affect activation-loop positioning or dynamics.

“With JIP1 bound JNK3, this slight shift in Pro222 toward αD was associated with a partial disordering of the inactivation helix, suggesting that differences in the docking motifs may affect the positioning or dynamics of the A-loop.”

May combine two claims.

The ordered activation loop packs against αC but also docks into the peptide-binding site of an adjacent symmetry-related molecule, a site blocked in peptide-bound structures, so the loop is likely not disordered by peptide binding itself.

“The A-loop packed up against αC, but also docked into the peptide-binding site of the adjacent symmetry related molecule (Figure S4A), which was blocked in the peptide bound structure (Figure S4B), and thus likely not rendered disordered by the peptide per se.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Retroactivity is a counter-intuitive feature of D-motif binding in which signaling is bidirectional because sequestration of the enzyme changes the ensemble of interacting proteins.

“A counter-intuitive feature of D-motif binding is called retroactivity, which is that signaling is bidirectional as sequestration of the enzyme alters the ensemble of interacting proteins (Del Vecchio et al., 2008).”

• cites Del Vecchio et al., 2008 sourceNot checked yet

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May combine two claims.

This interaction pulls αC but does not reverse the peptide-induced interlobe rotation.

“This interaction pulls αC but does not reverse the peptide-induced interlobe rotation.”

May combine two claims.

Both JNK1 and JNK3 datasets show that the activation loop affects αD conformation, and the JNK1 data also show a direct effect on peptide binding.

“Both the JNK1 and JNK3 sets showed that the A-loop affected the conformation of αD, while the JNK1 data also showed a direct impact on peptide binding.”

Classified as probably not a substantial claim.

The duration and timing of JNK signals can affect whether cells live or die.

“In fact, subtle changes in timing and duration of JNK signaling can determine cell fate decisions (Lin, 2006), highlighting the critical importance of tight regulation.”

• cites Lin, 2006 sourceNot checked yet

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May combine two claims.

The original JIP1-bound JNK structure showed interlobe rotation but not the larger lobe opening or the allosteric signal transduction mechanism, because a single structure has limitations.

“In the initial publication of JIP1-bound JNK (Heo et al., 2004), the interlobe rotation was apparent, but not the more substantial lobe opening, nor the mechanism of allosteric signal transduction, due to the inherent limitations of examining a single structure.”

• cites Heo et al., 2004 sourceNot checked yet

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May combine two claims.

JNK1 structures with phosphoacceptor-to-glutamate mutations do not have constitutive activity but do adopt a different activation-loop conformer.

“While these mutations do not yield constitutive activity (Zheng et al., 1999), they induce a different conformer for the A-loop, allowing us to ask how changing the A-loop conformer impacts the catalytic and peptide-binding sites.”

• cites Zheng et al., 1999 sourceNot checked yet

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Probably not a substantial claim: may not pass S1 (asserted), S4 (based). May combine two claims.

If peptide binding to inactive JNK inhibits catalysis, then phosphorylation of the activation loop must reverse the autoinhibitory allostery.

“To begin this analysis, we reasoned that if peptide binding to inactive JNK inhibits catalysis, which implies that phosphorylation of the A-loop must reverse the autoinhibitory allostery.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The D-motif is also present in phosphatases, substrates, and scaffold proteins.

“The D-motif is also found in phosphatases, substrates, and scaffold proteins (Raman et al., 2007).”

• cites Raman et al., 2007 sourceNot checked yet

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May combine two claims.

αD acts as a key allosteric sensor linking the activation loop to the peptide-binding site and mediating interlobe communication.

“Thus αD functions as a key allosteric sensor that links the A-loop to the peptide-binding site, and mediates interlobe communication.”

May combine two claims.

The D-motif is the primary determinant of MAPK signaling outcomes and is found in the N-terminus of upstream activating kinases, where it is required for MAPK binding and activation.

“As the protein motif responsible for docking onto the MAPK, the D-motif is the primary determinant of signaling outcomes, as it is found in the N-terminus of upstream activating kinases, where it is required for binding and activation of MAPKs.”

May combine two claims.

Structural analyses suggest the A-loop can directly control αD positioning and alter the peptide-binding site.

“In addition to A-loop control of interlobe conformation, our structural analyses suggest that the A-loop can more directly control the positioning of αD, and alter the peptide-binding site (Figure 5).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

IC50 values are from a single experiment with standard error, but each experiment was repeated at least three times with similar results.

“Reported IC50 values represent a single experiment ± standard error; however, each experiment was repeated at least three times and similar results were observed.”

May combine two claims.

Tyr phosphorylation adds 25% activity by binding the C-lobe and further locking the active interlobe conformer.

“Phosphorylation of Tyr adds an additional 25% activity by binding to the C-lobe and further locking in the active interlobe conformer.”

• cites Lisnock et al., 2000 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Ngoei et al. also found greater affinity for pepJIP with JNK1α1.

“These results are consistent with those seen by Ngoei et al. who also observed greater affinity for pepJIP with JNK1α1 (Ngoei et al., 2011).”

• cites Ngoei et al., 2011 sourceNot checked yet

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May combine two claims.

Comparing classes of structures allowed identification of common features across many structures and space groups and statistical characterization of subtle features within single-structure noise.

“Comparison of classes of structures enabled identification of common features across many structures and space groups, and allowed statistical analysis and characterization of subtle features within the noise of a single structure, as we previously used for dissecting estrogen receptor signaling (Bruning et al., 2010; Nettles et al., 2004, 2008).”

• cites Bruning et al., 2010 sourceNot checked yet

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• cites Nettles et al., 2004 sourceNot checked yet

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• cites Nettles et al., 2008 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Several best models were combined and validated with PHENIX and MolProbity.

“Elements of several of the best models were combined and validated using PHENIX and MolProbity (Chen et al., 2010).”

• cites Chen et al., 2010 sourceNot checked yet

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May combine two claims. The quote only partly states this claim.

PhosphoThr stabilizes N-lobe αC, which largely limits conformational heterogeneity between the kinase lobes.

“This occurs largely via stabilization of N-lobe αC by the phosphoThr residue.”

Probably not a substantial claim: may not pass S3 (load-bearing).

After Thr phosphorylation, a movement makes Tyr accessible for phosphorylation by MKK7.

“Indeed, this has been shown biochemically for JNK3 because it was suggested that once Thr is phosphorylated, a movement occurred so that Tyr is now accessible for phosphorylation by MKK7 (Lisnock et al., 2000).”

• cites Lisnock et al., 2000 sourceNot checked yet

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May combine two claims.

Biochemical observations from inactive and active JNK support the structural findings and have implications for signaling regulation through active and inactive kinases.

“Second, the biochemical observations form both inactive and active forms of JNK support the structural findings and again have implications on how signaling is regulated through active and inactive kinases.”

May combine two claims.

The structures explain why JIP binds more potently than the other peptides and suggest how this binding may regulate signaling to affect cell death and survival.

“As previously mentioned, the structures reveal why JIP binds more potently than the other peptides, and provides insights into how this binding may regulate signaling to influence cell death and survival.”

• AssumptionRests on an assumption: The D-motif peptides used in the structural and biochemical experiments are adequate proxies for the corresponding full-length JNK-binding proteins.