Specificity of Linear Motifs That Bind to a Common Mitogen-Activated Protein Kinase Docking Groove

May combine two claims.

Linear motif regions between consensus positions determine the binding mode in the docking groove and therefore the MAPK binding profile of a docking motif.

“We found that linear motif regions in-between consensus motif positions determine the binding mode in the docking groove and thus the MAPK binding profile of a given docking motif.”

May combine two claims.

MKK6 could phosphorylate p38α but not ERK2.

“These experiments also showed that MKK6 could phosphorylate p38α but not ERK2.”

May combine two claims.

These MAPKAPK-derived peptides competed with D-peptide reporters in binding assays, suggesting that they also bound the same MAPK docking groove.

“Nevertheless, these peptides competed with D-peptide reporters in binding assays, suggesting that they also bound to the same MAPK docking groove (fig. S1B).”

May combine two claims.

Binding affinities of 11 D-motif peptides to ERK2, p38α, and JNK1 fell into three groups: promiscuous peptides bound all three with 0-100 μM affinities, JNK-specific peptides bound only JNK1, and p38/ERK-selective peptides bound p38α and ERK2 similarly.

“We grouped peptides into three distinct sets based on their binding affinities: (i) promiscuous peptides bound with biologically relevant affinities (0-100 μM) to all three examined MAPKs; (ii) JNK-specific peptides bound only to JNK1; and (iii) p38/ERK selective peptides bound to p38α and ERK2 with similar affinities (Fig. 1B).”

May combine two claims.

pepRSK1 and pepMK2, which occur in ERK- and p38-specific MAPKAPKs, respectively, show more than tenfold preference for their cognate MAPK, with discrimination factors of about 20 and 400.

“PepRSK1 and pepMK2, which reside in ERK and p38-specific MAPKAPKs, respectively, show a more than tenfold preference (~20 and 400, respectively) for their cognate MAPK (Fig. 2E).”

May combine two claims.

The designed revD-peptide pepSynth-revD bound ERK2 and p38α but not JNK1, with similar affinities of about 10 μM for ERK2 and 30 μM for p38α.

“An artificially designed revD-peptide (pepSynth-revD) bound to both ERK2 and p38α (but not to JNK1) with similar affinities [KdERK2~10 μM, Kdp38α~30 μM].”

May combine two claims.

Residues forming the revD-motif contact the MAPK docking groove similarly, but the intervening regions between ΦU and ΦA differ between the two peptides.

“Amino acids that form the consensus revD-motif contact the MAPK docking groove similarly; however, the intervening regions between ΦU and ΦA differ in the two peptides.”

May combine two claims.

ERK2-pepRSK1 and ERK2-pepMNK1 peptides bound in a reversed N- to C-terminal orientation compared with D-motif peptides, similar to pepMK2 binding to p38α.

“These peptides bound in a reversed N- to C-terminal orientation compared to D-motif peptides (Fig. 3B-D), similar to the binding of pepMK2 to p38α (31).”

• cites [31] ter Haar E Prabhakar P Prabakhar P Liu X Lepre C Crystal structure of the p38 alpha-MAPKAP kinase 2 heterodimer J. Biol. Chem 2007 282 9733 9739 17255097 sourceNot checked yet

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May combine two claims.

Although the Cα positions of ERK2 and p38α superimpose well, some docking groove features in critical regions differed.

“Although the Cα positions of the two proteins superimpose well, some docking groove features in these critical regions differed (Fig. 4E).”

May combine two claims.

The JNK1 structures displayed two unrelated binding solutions: pepNFAT4 contacted the CD groove with an arginine side chain from a short alpha-helical region absent in pepJIP1, and its arginine side chain extended from a proline in the lower pocket.

“Thus, these structures displayed two unrelated solutions to bind to the JNK docking groove: pepNFAT4 contacted the CD groove with an arginine side-chain from a short α-helical region, which is not present in pepJIP1, and its arginine side chain directly extended from a proline residue located in the lower pocket.”

May combine two claims.

pepSynth-revD had no H-bond staples, pepMNK1 had one, and pepRSK1 and pepMK2 had at least two specific residues involved in H-bond stapling.

“The artificially designed pepSynth-revD peptide with an arbitrary intervening region did not have H-bond staples; the non-discriminatory pepMNK1 contains one H-bond staple, whereas at least two specific residues in pepRSK1 and pepMK2 are involved in H-bond stapling.”

May combine two claims.

D-motif peptides from proteins documented to interact with either p38α or ERK2 could bind both MAPKs and had less than four-fold discrimination factors.

“Unexpectedly, D-motif peptides from proteins with documented interactions with either p38α or ERK2 could bind both MAPKs and had modest (less than four-fold) discrimination factors, which is the ratio of binding affinities for cognate compared to non-cognate MAPKs.”

May combine two claims.

pepSynth-revD m1 had sub-micromolar affinity for p38α, but its affinity for ERK2 was not changed substantially.

“This new peptide had sub-micromolar affinity for p38α but its affinity for ERK2 was not changed substantially.”

May combine two claims.

pepNFAT4 bound JNK1 exclusively, whereas pepMKK4 bound all three tested MAPK paralogs.

“Our earlier analysis showed that pepNFAT4 bound JNK1 exclusively, whereas pepMKK4 bound all three tested MAPK paralogs.”

May combine two claims.

MAPK specificity decreased when H-bond staples were removed from MAPKAPK revD-motifs by alanine replacements and increased when they were introduced at new positions.

“MAPK specificity was decreased when H-bond staples were removed in MAPKAPK revD-motifs by alanine replacements and increased when they were introduced at new positions.”

May combine two claims.

The study successfully altered the MAPK binding profiles of naturally occurring motifs and designed novel peptides with tailored MAPK specificity.

“After elucidating the structural basis underlying biochemical specificity for MAPK-docking motif binding, we successfully altered the MAPK binding profiles of naturally occurring motifs and designed novel peptides with tailored MAPK specificity (Fig. 6).”

May combine two claims.

Amino acids in variable intervening regions form intra-motif hydrogen bonds and/or restrict peptide main chain flexibility via prolines.

“Amino acids located in variable intervening regions form intra-motif hydrogen bonds or limit peptide main chain flexibility through prolines (or both).”

May combine two claims.

pepSynth-revD m2 still bound ERK2 with similar affinity to the original peptide, but its p38α affinity increased by about 6,000-fold, from 30 μM to 5 nM.

“This second modified peptide still bound to ERK2 with similar affinity as the original Synth-revD motif containing peptide, but its binding affinity for p38α was increased by ~6,000 fold (from 30 μM to 5 nM) (Fig 6D and fig. S5D).”

May combine two claims.

pepMK2_R/A had lower affinity and specificity for p38α than pepMK2.

“The affinity and specificity of pepMK2_R/A for p38α was lower compared to pepMK2 and we replaced a solvent facing leucine (Leu384) with glutamine in this peptide (pepMK2_R/A,L/Q) to rebuild its original p38α specificity (fig. S4E).”

May combine two claims.

Although the binding affinity of pepMK2_R/A,L/Q did not increase substantially compared with pepMK2_R/A, its specificity for p38α over ERK2 increased to an extent comparable to pepMK2.

“Although the binding affinity of this latter peptide compared to pepMK2_R/A did not increase substantially, its specificity for p38α compared to ERK2 increased to an extent comparable to that of pepMK2.”

May combine two claims.

pepSynth-revD bound p38α and ERK2 with modest affinities of about 30 and 10 μM, respectively, compared with natural MAPKAPK revD-motifs that normally bind their cognate MAPK with sub-micromolar affinities.

“This artificially designed peptide bound to p38α and ERK2 with modest binding affinities (~30 and 10 μM, respectively) compared to other naturally occurring revD-motifs derived from MAPKAPKs, which normally bind with sub-micromolar affinities to their cognate MAPK.”

May combine two claims.

JIP1- and NFAT4-type D-motifs bind JNK, while MKK6-, HePTP-type D-motifs or revD-motifs bind ERK or p38 with specificity that varies with intervening region composition.

“pepJIP1- and pepNFAT4-type D-motifs bind to JNK, whereas pepMKK6-, pepHePTP-type D-motifs or revD-motifs bind to ERK or p38 with varying degree of specificity, depending on the composition of the intervening regions.”

May combine two claims.

The computational analysis identified 29 of the 30 benchmark proteins correctly and placed most of them into five motif classes correctly.

“Our in silico analysis correctly identified 29 of these benchmark proteins and mostly grouped them correctly into five motif classes, presumably binding MAPKs with different profiles: pepJIP1- and pepNFAT4-type D-motifs bind to JNK, whereas pepMKK6-, pepHePTP-type D-motifs or revD-motifs bind to ERK or p38 with varying degree of specificity, depending on the composition of the intervening regions.”

May combine two claims.

pepSynth-D and pepSynth-revD bound MAPKs with low-to-medium micromolar affinities of 1-30 μM, and their interaction profiles matched predictions: pepSynth-D was promiscuous and pepSynth-revD was ERK/p38-selective.

“Both peptides bound MAPKs with low-to-medium micromolar affinities (1-30 μM) and their MAPK interaction profiles fit well to what we predicted: pepSynth-D was promiscuous, whereas pepSynth-revD was ERK/p38-selective (fig. S5C,D).”

May combine two claims.

MAPKs have promiscuous active sites, so additional protein interaction sites are critical for determining MAPK pathway wiring.

“Similarly to many signaling enzymes, MAPKs contain promiscuous active sites. Therefore, additional protein interaction sites are critical for determining the wiring of MAPK pathways.”

• cites [7] Reményi A Good MC Lim WA Docking interactions in protein kinase and phosphatase networks Curr. Opin. Struct. Biol 2006 16 676 685 17079133 sourceNot checked yet

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

ERK1/2, JNK, and p38 are activated by dedicated MAP2Ks MKK1/2, MKK4/7, and MKK3/4/6, respectively.

“MAPKs are part of multi-tiered kinase cascades in which ERK1/2, JNK and p38 are activated by dedicated mitogen-activated protein kinase kinases (MAP2Ks) MKK1/2, MKK4/7 and MKK3/4/6, respectively (5).”

• cites [5] Johnson GL Lapadat R Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases Science 2002 298 1911 1912 12471242 sourceNot checked yet

  Not checked in the free run.

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

Linear binding motifs are usually defined by a consensus sequence and are less than 10-20 amino acids long.

“Linear binding motifs that play a crucial role in this process are normally defined based on their consensus motif sequence and are less than 10-20 amino acids in length (1).”

• cites [1] Neduva V Linding R Su-.Angrand I Stark A de Masi F Gibson TJ Lewis J Serrano L Russell RB Systematic discovery of new recognition peptides mediating protein interaction networks PLoS Biol 2005 3 e405 16279839 PMC1283537 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

A coherent structural model underlies MAPK docking specificity and explains how short linear motifs binding to a common kinase docking groove mediate diverse interaction patterns and contribute to correct MAPK partner selection.

“We present a coherent structural model underlying MAPK docking specificity that reveals how short linear motifs binding to a common kinase docking groove can mediate diverse interaction patterns and contribute to correct MAPK partner selection in signaling networks.”

May combine two claims.

Consensus positions in docking motifs act as anchor points that bind common MAPK surface features and mostly contribute to docking without discriminating among MAPKs.

“Although the consensus positions in the docking motifs served as anchor points that bound to common MAPK surface features and mostly contributed to docking in a non-discriminatory fashion, specificity was determined mainly by the conformation of the intervening region between the anchor points.”

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

MAPKs regulate diverse cellular processes and interact with proteins through linear binding motifs.

“Mitogen activated protein kinases (MAPKs) regulate diverse aspects of cellular life such as cell division, differentiation or apoptosis and interact with proteins through linear binding motifs (5-6).”

• cites [5] Johnson GL Lapadat R Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases Science 2002 298 1911 1912 12471242 sourceNot checked yet

  Not checked in the free run.

• cites [6] Tanoue T Nishida E Docking interactions in the mitogen-activated protein kinase cascades Pharmacol. Ther 2002 93 193 202 12191611 sourceNot checked yet

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Classified as probably not a substantial claim.

For protein kinases and phosphatases, the docking binding surface is separate from the active site.

“In this type of interaction, which is referred to as docking for protein kinases and phosphatases, the binding surface is separate from the active site (7).”

• cites [7] Reményi A Good MC Lim WA Docking interactions in protein kinase and phosphatase networks Curr. Opin. Struct. Biol 2006 16 676 685 17079133 sourceNot checked yet

  Not checked in the free run.

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

MAPKs phosphorylate serine and threonine followed by proline and bind D-motifs of about 8-12 amino acids that satisfy a loose consensus.

“For example, MAPKs phosphorylate serine and threonine residues that are followed by proline (called a S/TP target site or motif) and they also bind to docking (D)-motifs, which are ~8-12 amino acid long fragments satisfying a loose consensus sequence (9)”

• cites [9] Chang CI Xu B.-e Akella R Cobb MH Goldsmith EJ Crystal structures of MAP kinase p38 complexed to the docking sites on its nuclear substrate MEF2A and activator MKK3b Mol. Cell 2002 9 1241 1249 12086621 sourceNot checked yet

  Not checked in the free run.

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

MAPK docking is a well-characterized protein-peptide interaction system.

“At the structural level, MAPK docking is a well characterized protein-peptide interaction system (9, 12-16).”

• cites [9] Chang CI Xu B.-e Akella R Cobb MH Goldsmith EJ Crystal structures of MAP kinase p38 complexed to the docking sites on its nuclear substrate MEF2A and activator MKK3b Mol. Cell 2002 9 1241 1249 12086621 sourceNot checked yet

  Not checked in the free run.

• cites [12] Zhou T Sun L Humphreys J Goldsmith EJ Docking interactions induce exposure of activation loop in the MAP kinase ERK2 Structure 2006 14 1011 1019 16765894 sourceNot checked yet

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• cites [13] Heo Y-S Kim S-K Seo CI Kim YK Sung B-J Lee HS Lee JI Park S-Y Kim JH Hwang KY Hyun Y-L Jeon YH Ro S Cho JM Lee TG Yang CH Structural basis for the selective inhibition of JNK1 by the scaffolding protein JIP1 and SP600125 EMBO J 2004 23 2185 2195 15141161 PMC419904 sourceNot checked yet

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• cites [14] Liu S Sun J-P Zhou B Zhang Z-Y Structural basis of docking interactions between ERK2 and MAP kinase phosphatase 3 Proc. Natl. Acad. Sci. U.S.A 2006 103 5326 5331 16567630 PMC1459354 sourceNot checked yet

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• cites [15] Ma W Shang Y Wei Z Wen W Wang W Zhang M Phosphorylation of DCC by ERK2 is facilitated by direct docking of the receptor P1 domain to the kinase Structure 2010 18 1502 1511 21070949 sourceNot checked yet

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• cites [16] Reményi A Good MC Bhattacharyya RP Lim WA The role of docking interactions in mediating signaling input, output, and discrimination in the yeast MAPK network Mol. Cell 2005 20 951 962 16364919 sourceNot checked yet

  Not checked in the free run.

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

Docking motifs that bind directly to signaling enzyme catalytic domains are the simplest way to increase specificity of promiscuous active sites.

“Within the diverse repertoire of physical links observed in signaling networks, the use of linear motifs directly binding to signaling enzyme’s catalytic domains, known as docking motifs, is the simplest solution to increase the specificity of promiscuous active sites (8).”

• cites [8] Ubersax JA Ferrell JE Mechanisms of specificity in protein phosphorylation Nat. Rev. Mol. Cell Biol 2007 8 530 541 17585314 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The docking grooves of paralogous MAPKs are highly similar, likely because of duplication events and because ERK2, p38α, and JNK1 share about 40-50% sequence identity.

“The docking grooves of paralogous MAPKs show a high degree of similarity, likely because each major group of MAPKs in animals - typical MAPKs such as extracellular signal-regulated kinase (ERK) 1/2; c-Jun N-terminal kinases (JNKs); p38s; ERK5; and atypical MAPKs such as ERK3, 4, and 7 - emerged through whole genome or individual gene duplication events and because three members of this family - ER”

• cites [11] Caffrey DR O’Neill LA Shields DC The evolution of the MAP kinase pathways: coduplication of interacting proteins leads to new signaling cascades J. Mol. Evol 1999 49 567 582 10552038 sourceNot checked yet

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

Many MAPK docking motifs have been characterized in transcription factors whose activity is controlled by MAPK phosphorylation.

“Many MAPK docking motifs have been previously characterized in transcription factors whose activity is controlled by MAPK phosphorylation (20).”

• cites [20] Sharrocks AD Yang SH Galanis A A. Docking domains and substrate-specificity determination for MAP kinases Trends Biochem. Sci 2000 25 448 453 10973059 sourceNot checked yet

  Not checked in the free run.

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

Regulatory phosphorylation sites in transcription factors are often found in unstructured regions outside DNA-binding domains.

“The regulatory phosphorylation sites in transcription factors are often found in unstructured regions outside of the DNA-binding domains.”

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

A coherent structural model of SH2 domain binding specificity has been suggested.

“This is in contrast to the systematically studied interactions between SH2 domains and linear motifs for which a coherent structural model of SH2 domain binding specificity has been suggested (17-18).”

• cites [17] Kaneko T Huang H Zhao B Li L Liu H Voss CK Wu C Shiller MR Li SSC Loops Govern SH2 Domain Specificity by Controlling Access to Binding Pockets Science Signaling 2010 3 ra34 ra34 20442417 PMC6590088 sourceNot checked yet

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• cites [18] Kaneko T Sidhu SS Li SSC Evolving specificity from variability for protein interaction domains Trends Biochem. Sci 2011 36 183 190 21227701 sourceNot checked yet

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May combine two claims.

The C-terminal region of MAPKAPKs mediates binding to the MAPK docking groove on ERK or p38 and is indispensable for MAPKAPK activation in cells.

“This region mediates binding to the MAPK docking groove located on ERK or p38 and this association is indispensable for MAPKAPK activation in cells (25).”

• cites [25] Gavin AC Nebreda AR A MAP kinase docking site is required for phosphorylation and activation of p90(rsk)/MAPKAP kinase-1 Curr. Biol 1999 9 281 284 10074458 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The JNK docking groove is less charged and narrower, and these conserved differences in surface topography and charge distribution set JNK MAPKs apart from ERK and p38 enzymes.

“Therefore the JNK docking groove is less charged and narrower and these evolutionarily conserved differences in surface topography and charge distribution sets JNK MAPKs apart from ERK and p38 enzymes.”

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

MSKs can be activated by both p38 and ERK, whereas RSKs are generally thought to be ERK substrates.

“MSKs, similarly to MNKs, can also be activated by both MAPKs whereas RSKs are generally thought to be the substrate of ERKs (28) (Fig. 2B).”

• cites [28] Anjum R Blenis J The RSK family of kinases: emerging roles in cellular signalling Nat. Rev. Mol. Cell Biol 2008 9 747 758 18813292 sourceNot checked yet

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

MAPKAPKs were previously suggested to contain D-motifs that are indispensable for their activation by MAPKs.

“MAPKAPKs were previously suggested to contain D-motifs that are indispensable for their activation by MAPKs (29).”

• cites [29] Cargnello M Roux PP Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases Microbiol. Mol. Biol. Rev 2011 75 50 83 21372320 PMC3063353 sourceNot checked yet

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

The activity of prototypic MAPKAPKs depends on p38 MAPKs.

“The activity of prototypic MAPKAPKs depends upon p38 MAPKs (26).”

• cites [26] Gaestel M MAPKAP kinases - MKs - two’s company, three’s a crowd Nat. Rev. Mol. Cell Biol 2006 7 120 130 16421520 sourceNot checked yet

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May combine two claims.

The paper presents a coherent structural model for MAPK-docking motif specificity that explains how docking motifs can be specific or promiscuous and why previously characterized docking peptides bind MAPKs with their reported specificity.

“Here, we present a coherent structural model for MAPK-docking motif specificity that explains how docking motifs can be specific or promiscuous and why previously characterized docking peptides bind to MAPKs with their reported specificity.”

May combine two claims.

Phosphorylation of MEF2A Thr312 and Thr319 by p38α and presumably ERK2 stimulates MEF2A-mediated transactivation, while phosphorylation of NFAT4 Ser163 and Ser165 by JNK promotes its translocation from nucleus to cytosol.

“Phosphorylation of two residues in MEF2A (Thr312 and Thr319) by p38α and presumably ERK2 stimulates MEF2A-mediated transactivation in cells, whereas phosphorylation of Ser163 and Ser165 in NFAT4 by JNK promotes its translocation from the nucleus to the cytosol (22,24).”

• cites [22] Chow CW Rincón M Cavanagh J Dickens M Davis RJ Nuclear accumulation of NFAT4 opposed by the JNK signal transduction pathway Science 1997 278 1638 1641 9374467 sourceNot checked yet

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• cites [24] Potthoff MJ Olson EN MEF2: a central regulator of diverse developmental programs Development 2007 134 4131 4140 17959722 sourceNot checked yet

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May combine two claims.

Docking motifs facilitate signaling in MAP2K-mediated MAPK activation, although specificity here is also influenced by other factors.

“In summary, docking motifs facilitate signaling in MAP2K-mediated MAPK activation, although specificity here is also influenced by other factors.”

May combine two claims.

The MAPK interaction region in MAPKAPKs is located C-terminal to the CaMK-type kinase domain and determines the specificity of MAPK-mediated MAPKAPK activation.

“The MAPK interaction region in MAPKAPKs is located in their C-terminal region next to their (CaMK)-type protein kinase domain and this region determines the specificity of MAPK-mediated MAPKAPK activation (30).”

• cites [30] Smith JA Poteet-Smith CE Lanningan DA Freed TA Zoltoski AJ Sturgill TW Creation of a stress-activated p90 ribosomal S6 kinase. The carboxyl-terminal tail of the MAPK-activated protein kinases dictates the signal transduction pathway in which they function J. Biol. Chem 2000 275 31588 31593 10922375 sourceNot checked yet

  Not checked in the free run.

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

MAPKAPKs contain a conserved CaMK-type protein kinase domain and a C-terminal extension that enables activation by ERK, p38, or both.

“MAPKAPKs are MAPK activated protein kinases that are comprised of a conserved calcium/calmodulin-dependent (CaMK)-type protein kinase domain and a C-terminal extension that enables activation by various MAPKs (ERK, p38 or both) (25).”

• cites [25] Gavin AC Nebreda AR A MAP kinase docking site is required for phosphorylation and activation of p90(rsk)/MAPKAP kinase-1 Curr. Biol 1999 9 281 284 10074458 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

D-motif binding from non-cognate MAP2Ks may not necessarily cause illicit MAPK activation, because kinase catalytic domains may cooperate with independent linear binding motifs to formulate signaling specificity.

“Thus, our in vitro MAPK activation results suggest that D-motif binding from non-cognate MAP2Ks may not necessarily result in illicit MAPK activation: Kinase catalytic domains may cooperate with independent linear binding motifs to formulate the specificity of a given signaling event.”

May combine two claims.

MAP2K active site specificity is an important factor because MAPK activation loop sequences adjacent to the phosphorylated threonine and tyrosine are conserved within MAPK groups but differ among paralog groups.

“An important factor is the specificity of the MAP2K active site because the sequence of MAPK activation loops adjacent to the phosphorylated threonine and tyrosine residues are strictly conserved within specific MAPK groups but differ among MAPK paralog groups.”

• AssumptionRests on an assumption: MAPK activation loop sequences adjacent to the phosphorylated threonine and tyrosine are strictly conserved within specific MAPK groups but differ among MAPK paralog groups.

May combine two claims.

MKK4 appears unique in its ability to interact with the highly divergent docking grooves in JNK and ERK/p38, which agrees with its biological function of activating JNK and p38.

“It appears that MKK4 is unique in its ability to interact both with the highly divergent docking grooves in JNK and ERK/p38, which agrees with its biological function, which is to activate JNK and p38.”

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

Titrations were performed in triplicate, and the mean FP signal was used for curve fitting.

“Titration experiments were carried out in triplicates and the average FP signal was used for fitting the data with OriginPro7.”

May combine two claims.

Residues in variable intervening regions are mainly responsible for specificity, whereas residues in consensus motif positions bind general surface features and contribute in a non-discriminatory manner.

“Thus, these residues are mostly responsible for specificity, whereas residues in consensus motif positions bind the general surface features and mostly contribute to these interactions in a non-discriminatory fashion.”

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

JIP1 is a scaffold protein of the JNK MAPK module and recruits JNK through its D-motif.

“JIP1 is a scaffold protein of the JNK MAPK module and recruits JNK through its D-motif (pepJIP1) (34).”

• cites [34] Mooney LM Whitmarsh AJ Docking interactions in the c-Jun N-terminal kinase pathway J. Biol. Chem 2004 279 11843 11852 14699111 sourceNot checked yet

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May combine two claims.

Activation of p38α by MKK6 also depends on the substrate preference of the MKK6 active site even if the MKK6 D-motif autonomously appears to determine the binding affinity between these two proteins.

“For example, activation of p38α by MKK6 also depends on the substrate preference of the MKK6 active site even if the MKK6 D-motif autonomously appears to determine the binding affinity between these two proteins.”

May combine two claims.

Simple D-motif mediated interactions have limitations, although these may be efficiently mitigated by other binary interactions that also contribute to MAPK specificity.

“This latter observation highlights the limitations of simple D-motif mediated interactions, although these may be efficiently mitigated in the context of other binary interactions also contributing to MAPK specificity.”

May combine two claims.

D-motifs from the initial set that bound p38α also bound ERK2, presumably reflecting the highly similar groove topography of these two paralogs.

“D-motifs from the initial set that bound p38α also bound ERK2, which presumably reflected the highly similar groove topography in these two paralogs.”

May combine two claims.

Docking motifs are evolutionarily plastic solutions for forming new connections between signaling proteins because they can emerge in unstructured regions and their kinase binding specificity can be easily changed.

“They are evolutionarily plastic solutions for forming new connections between signaling proteins because they may freely emerge in unstructured protein regions and their binding specificity for kinases may also be easily changed (49).”

• cites [49] Neduva V Russell RB Linear motifs: evolutionary interaction switches FEBS Lett 2005 579 3342 3345 15943979 sourceNot checked yet

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May combine two claims.

FxFP motifs in certain MAPK substrates and a separate docking groove for such motifs on ERK suggest that two docking grooves on ERK may operate in combination; Elk1 contains both an FxFP and a D-motif.

“Moreover, the existence of FxFP (Phe-any amino acid-Phe-Pro) motifs in certain MAPK (especially ERK) substrates and a separate docking groove for such motifs on ERK suggest that in some cases two docking grooves on ERK may operate in combination: Elk1 for example contains both an FxFP and a D-motif (35-37).”

• cites [35] Lee T Hoofnagle AN Kabuyama Y Stroud J Min X Goldsmith EJ Chen L Resing KA Ahn NG Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry Mol. Cell 2004 14 43 55 15068802 sourceNot checked yet

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• cites [36] Dimitri CA Dowdle W MacKeigan JP Blenis J Murphy LO Spatially separate docking sites on ERK2 regulate distinct signaling events in vivo Curr. Biol 2005 15 1319 1324 16051177 sourceNot checked yet

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• cites [37] Jacobs D Glossip D Xing H Muslin AJ Kornfeld K Multiple docking sites on substrate proteins form a modular system that mediates recognition by ERK MAP kinase Genes Dev 1999 13 163 175 9925641 PMC316390 sourceNot checked yet

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May combine two claims.

H-bond staples are not unique to MAPKAPK docking regions bound to MAPKs and are also used in other linear motif based interaction systems such as TGFβ signaling and DNA repair regulation.

“H-bond staples, however, are not unique to MAPKAPK docking regions bound to MAPKs and are also used in other linear motif based interaction systems such as those operating in TGFβ signaling (the TGFβ effector SMAD3 interacts with a linear motif in the adaptor SARA) or in DNA repair regulation (the DNA recombinase RAD51 interacts with a short peptide segment from the scaffold protein BRCA2 ) (39-41”

• cites [39] Qin BY Lam SS Correia JJ Lin K Smad3 allostery links TGF-beta receptor kinase activation to transcriptional control Genes Dev 2002 16 1950 1963 12154125 PMC186427 sourceNot checked yet

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• cites [40] Chen CF Chen PL Zhong Q Sharp ZD Lee WH Expression of BRC repeats in breast cancer cells disrupts the BRCA2-Rad51 complex and leads to radiation hypersensitivity and loss of G(2)/M checkpoint control J. Biol. Chem 1999 274 32931 32935 10551859 sourceNot checked yet

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• cites [41] Pellegrini L Yu DS Lo T Anand S Lee MY Blundell TL Venkitaraman AR Insights into DNA recombination from the structure of a RAD51-BRCA2 complex Nature 2002 420 287 293 12442171 sourceNot checked yet

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Classified as probably not a substantial claim.

The D-motif consensus sequence was previously loosely defined and encompassed motif sequences belonging to different MAPK binding modes and specificity.

“The D-motif consensus sequence was previously loosely defined and it encompassed motif sequences that belonged to different MAPK binding modes and specificity.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

MAPKs represent a paradigm for understanding how specificity in cellular signaling can be achieved with enzymes that share a common origin.

“MAPKs represent a paradigm for understanding how specificity in cellular signaling can be achieved with enzymes that share a common origin (11).”

• cites [11] Caffrey DR O’Neill LA Shields DC The evolution of the MAP kinase pathways: coduplication of interacting proteins leads to new signaling cascades J. Mol. Evol 1999 49 567 582 10552038 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The intervening region composition of pepMKK4 is predicted to be compatible with two unrelated binding modes; it bound JNK1 similarly to pepNFAT4 and p38α similarly to pepMKK6.

“Unfortunately, our efforts to crystallize MAPK complexes with this peptide have failed so far but the intervening region composition of pepMKK4 is predicted to be compatible with two unrelated binding modes. It bound JNK1 in a similar manner to pepNFAT4 and p38α in a similar manner to pepMKK6.”

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

Every sequence was confirmed by DNA sequencing following sub-cloning into pET expression vectors.

“All sequences were verified by DNA sequencing after sub-cloning into pET expression vectors.”

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

The ED-site (also called the MAPK top region) helps determine MAPK-MAPKAPK wiring in cells.

“Similarly, the specificity of ERK2/p38 chimeras had already been tested (19), which showed that the “ED-site” (also known as the MAPK top region) helps to define MAPK-MAPKAPK wiring in cells.”

• cites [19] Tanoue T Maeda R Adachi M Nishida E Identification of a docking groove on ERK and p38 MAP kinases that regulates the specificity of docking interactions EMBO J 2001 20 466 479 11157753 PMC133461 sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Low sequence stringency in linear motif definitions normally hinders accurate assessment of their frequency in various proteomes.

“The low sequence stringency in linear motif definitions normally hinders an accurate assessment of their frequency in various proteomes.”

Probably not a substantial claim: may not pass S2 (checkable), S4 (based).

Current understanding of these interactions is rarely sufficient to segregate motifs into sub-groups representing different binding modes of the same global motif definition.

“Unfortunately, our current understanding of these interactions is rarely sufficient to segregate motifs into sub-groups representing different binding modes of the same global motif definition.”

May combine two claims.

pepSynth-revD could not bind JNK1, presumably because its helical structure around the basic motif was incompatible with the narrower CD groove in JNK1.

“pepSynth-revD could not bind to JNK1, presumably because its helical structure around the basic motif was not compatible with the narrower CD groove in JNK1.”

May combine two claims.

H-bond staples may favor binding to cognate MAPKs and may also prevent a peptide from adopting a conformation compatible with binding to non-cognate MAPKs.

“This could favor binding to cognate MAPKs but H-bond staples may also prevent a peptide from adopting a different conformation compatible with binding to non-cognate MAPKs.”

Classified as probably not a substantial claim.

Linear motifs are frequently found in unstructured regions.

“Because linear motifs often occur in unstructured regions (49), we used DisEMBL 1.5 (61) to computationally predict amino acids belonging to an unstructured or disordered protein region.”

• cites [49] Neduva V Russell RB Linear motifs: evolutionary interaction switches FEBS Lett 2005 579 3342 3345 15943979 sourceNot checked yet

  Not checked in the free run.

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

The final list underwent manual curation to eliminate remaining false positives.

“The final list was further manually curated to remove residual false positives consisting fof intralumenal, vesicular, mitochondrial or extracellular proteins (amounting to less than 5% of the final entries).”

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

To improve disorder prediction reliability, the study required more than 60% disordered residues for D-motifs and more than 90% for revD-motifs.

“To increase the reliability of disorder prediction, putative D-motifs were only considered if more than 60% of the amino acids spanning from the motif start site to the first S/TP target site were annotated as disordered (according to at least one of the DisEMBL disorder criteria); revD-motifs were only analyzed further if more than 90% of their amino acids were disordered.”

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

The enrichment analysis corrected p-values for multiple testing with FDR and treated q-values below 0.05 as significant.

“We adjusted the resulting p-values for multiple testing errors using the false discovery rate (FDR) (70). Only annotations with an adjusted p-value (q-value) of less than 0.05 were considered to be significantly enriched in the respective list.”

May combine two claims.

pepNFAT4 JNK1 specificity depended on a small helical turn in the intervening region between ΦL and Ψ, and a proline there disfavors the alpha-helical conformation needed for the JNK-specific binding mode.

“Because the ability of pepNFAT4 to bind specifically to JNK1 depended on the formation of a small helical turn at the intervening region between ΦL and Ψ, a proline at this region disfavors an alpha-helical main-chain conformation required to span the distance between ΦL and Ψ in a manner required for the JNK-specific pepNFAT4-type binding mode.”