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Titin kinase is an inactive pseudokinase scaffold that supports MuRF1 recruitment to the sarcomeric M-line.

Bogomolovas J, Gasch A, Simkovic F, Rigden DJ, Labeit S, Mayans O - Open Biol (2014)

Bottom Line: Inactivity is the result of two atypical residues in TK's active site, M34 and E147, that do not appear compatible with canonical kinase patterns.While not mediating stretch-dependent phospho-transfers, TK binds the E3 ubiquitin ligase MuRF1 that promotes sarcomeric ubiquitination in a stress-induced manner.Finally, we suggest that an evolutionary dichotomy of kinases/pseudokinases has occurred in TK-like kinases, where invertebrate members are active enzymes but vertebrate counterparts perform their signalling function as pseudokinase scaffolds.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Pathophysiology, Medical Faculty Mannheim, University of Heidelberg, Mannheim 68167, Germany Institute of Integrative Biology, Biosciences Building, University of Liverpool, Crown St., Liverpool L69 7ZB, UK.

ABSTRACT
Striated muscle tissues undergo adaptive remodelling in response to mechanical load. This process involves the myofilament titin and, specifically, its kinase domain (TK; titin kinase) that translates mechanical signals into regulatory pathways of gene expression in the myofibril. TK mechanosensing appears mediated by a C-terminal regulatory tail (CRD) that sterically inhibits its active site. Allegedly, stretch-induced unfolding of this tail during muscle function releases TK inhibition and leads to its catalytic activation. However, the cellular pathway of TK is poorly understood and substrates proposed to date remain controversial. TK's best-established substrate is Tcap, a small structural protein of the Z-disc believed to link TK to myofibrillogenesis. Here, we show that TK is a pseudokinase with undetectable levels of catalysis and, therefore, that Tcap is not its substrate. Inactivity is the result of two atypical residues in TK's active site, M34 and E147, that do not appear compatible with canonical kinase patterns. While not mediating stretch-dependent phospho-transfers, TK binds the E3 ubiquitin ligase MuRF1 that promotes sarcomeric ubiquitination in a stress-induced manner. Given previous evidence of MuRF2 interaction, we propose that the cellular role of TK is to act as a conformationally regulated scaffold that functionally couples the ubiquitin ligases MuRF1 and MuRF2, thereby coordinating muscle-specific ubiquitination pathways and myofibril trophicity. Finally, we suggest that an evolutionary dichotomy of kinases/pseudokinases has occurred in TK-like kinases, where invertebrate members are active enzymes but vertebrate counterparts perform their signalling function as pseudokinase scaffolds.

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Comparison of TK and TwcK active sites. (a) Structural superposition of TK and ceTwcK (PDB entry 3UTO). Ribbon thickness and colouring indicate the RMSD values of the superposition as given in the accompanying scale (minimum, maximum and average values are shown). The structural agreement is excellent overall, including active site regions; divergences only occur in peripheral loop areas. (b) Detailed comparison of the ATP-binding pockets of TK (green) and ceTwcK (pink) (numbering corresponds to TK). Boxed labels indicate TK residues that were trans-engineered into ceTwcK. (c) Structure-based sequence alignment of the catalytic domains of human TK and ceTwcK corresponding to the superposition displayed in (a,b). Identical residues are highlighted in black and closest conservation is in grey. The canonical composition of functional motifs is shown in blue, the P+1 loop is boxed and the tyrosine residue undergoing phosphorylation in TK is indicated with an asterisk. The CRD is in red. (d) Comparative autoradiogram of catalysis by ecTwck and its variants TwcKA34M, TwcKD147E and TwcKA34M/D147E carrying TK residues in their ATP-binding pockets. The time course shows phosphorylation of a MLC-derived peptide. (e) Comparative autoradiogram of the catalysis from ecTwck and TwcKA34V. The latter carries the non-inactivating valine residue commonly found in the ATP-binding pocket of TwcK from molluscs.
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RSOB140041F4: Comparison of TK and TwcK active sites. (a) Structural superposition of TK and ceTwcK (PDB entry 3UTO). Ribbon thickness and colouring indicate the RMSD values of the superposition as given in the accompanying scale (minimum, maximum and average values are shown). The structural agreement is excellent overall, including active site regions; divergences only occur in peripheral loop areas. (b) Detailed comparison of the ATP-binding pockets of TK (green) and ceTwcK (pink) (numbering corresponds to TK). Boxed labels indicate TK residues that were trans-engineered into ceTwcK. (c) Structure-based sequence alignment of the catalytic domains of human TK and ceTwcK corresponding to the superposition displayed in (a,b). Identical residues are highlighted in black and closest conservation is in grey. The canonical composition of functional motifs is shown in blue, the P+1 loop is boxed and the tyrosine residue undergoing phosphorylation in TK is indicated with an asterisk. The CRD is in red. (d) Comparative autoradiogram of catalysis by ecTwck and its variants TwcKA34M, TwcKD147E and TwcKA34M/D147E carrying TK residues in their ATP-binding pockets. The time course shows phosphorylation of a MLC-derived peptide. (e) Comparative autoradiogram of the catalysis from ecTwck and TwcKA34V. The latter carries the non-inactivating valine residue commonly found in the ATP-binding pocket of TwcK from molluscs.

Mentions: To study the role of the atypical, conserved methionine and glutamate residues in the active site of TK, we substituted these for their canonical equivalents in the variants TKE147D, TKE147D/M34A/R129K and TKE147D/M34A/R129K/Y170E (R129 is normally a conserved lysine residue in the catalytic loop of active kinases of the titin-like family; we speculated that in TK, the exchange to arginine might be coupled to other divergences in the catalytic motifs of this kinase). Of these variants, only TKE147D could be produced soluble in E. coli. However, it was not active when assayed on Tcap or the generic kinase substrates myelin basic protein and casein. Therefore, we performed the reverse experiment, mutating the TK residues into ceTwcK. The latter is a close homologue of human TK that is well characterized structurally and biochemically [25–27]. ceTwcK (bacterially expressed) exhibits high levels of catalysis when assayed on a model peptide substrate derived from myosin light chain (MLC) protein. The catalytic domains of ceTwcK and TK share 40% sequence identity and 65% conservation (figure 4c), and, accordingly, high structural similarity (RMSD for Cα atoms = 1.29 Å when comparing the structure of TK in this work and PDB entry 3UTO using MUSTANG [23]; figure 4a). The active sites of these two kinases are in close structural agreement, particularly their ATP-binding pockets, their β3 strands and the D/EFG motifs (figure 4b). Thus, we concluded that ceTwcK is a suitable template to investigate the effect of the unusual motifs of TK on catalysis.Figure 4.


Titin kinase is an inactive pseudokinase scaffold that supports MuRF1 recruitment to the sarcomeric M-line.

Bogomolovas J, Gasch A, Simkovic F, Rigden DJ, Labeit S, Mayans O - Open Biol (2014)

Comparison of TK and TwcK active sites. (a) Structural superposition of TK and ceTwcK (PDB entry 3UTO). Ribbon thickness and colouring indicate the RMSD values of the superposition as given in the accompanying scale (minimum, maximum and average values are shown). The structural agreement is excellent overall, including active site regions; divergences only occur in peripheral loop areas. (b) Detailed comparison of the ATP-binding pockets of TK (green) and ceTwcK (pink) (numbering corresponds to TK). Boxed labels indicate TK residues that were trans-engineered into ceTwcK. (c) Structure-based sequence alignment of the catalytic domains of human TK and ceTwcK corresponding to the superposition displayed in (a,b). Identical residues are highlighted in black and closest conservation is in grey. The canonical composition of functional motifs is shown in blue, the P+1 loop is boxed and the tyrosine residue undergoing phosphorylation in TK is indicated with an asterisk. The CRD is in red. (d) Comparative autoradiogram of catalysis by ecTwck and its variants TwcKA34M, TwcKD147E and TwcKA34M/D147E carrying TK residues in their ATP-binding pockets. The time course shows phosphorylation of a MLC-derived peptide. (e) Comparative autoradiogram of the catalysis from ecTwck and TwcKA34V. The latter carries the non-inactivating valine residue commonly found in the ATP-binding pocket of TwcK from molluscs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4042850&req=5

RSOB140041F4: Comparison of TK and TwcK active sites. (a) Structural superposition of TK and ceTwcK (PDB entry 3UTO). Ribbon thickness and colouring indicate the RMSD values of the superposition as given in the accompanying scale (minimum, maximum and average values are shown). The structural agreement is excellent overall, including active site regions; divergences only occur in peripheral loop areas. (b) Detailed comparison of the ATP-binding pockets of TK (green) and ceTwcK (pink) (numbering corresponds to TK). Boxed labels indicate TK residues that were trans-engineered into ceTwcK. (c) Structure-based sequence alignment of the catalytic domains of human TK and ceTwcK corresponding to the superposition displayed in (a,b). Identical residues are highlighted in black and closest conservation is in grey. The canonical composition of functional motifs is shown in blue, the P+1 loop is boxed and the tyrosine residue undergoing phosphorylation in TK is indicated with an asterisk. The CRD is in red. (d) Comparative autoradiogram of catalysis by ecTwck and its variants TwcKA34M, TwcKD147E and TwcKA34M/D147E carrying TK residues in their ATP-binding pockets. The time course shows phosphorylation of a MLC-derived peptide. (e) Comparative autoradiogram of the catalysis from ecTwck and TwcKA34V. The latter carries the non-inactivating valine residue commonly found in the ATP-binding pocket of TwcK from molluscs.
Mentions: To study the role of the atypical, conserved methionine and glutamate residues in the active site of TK, we substituted these for their canonical equivalents in the variants TKE147D, TKE147D/M34A/R129K and TKE147D/M34A/R129K/Y170E (R129 is normally a conserved lysine residue in the catalytic loop of active kinases of the titin-like family; we speculated that in TK, the exchange to arginine might be coupled to other divergences in the catalytic motifs of this kinase). Of these variants, only TKE147D could be produced soluble in E. coli. However, it was not active when assayed on Tcap or the generic kinase substrates myelin basic protein and casein. Therefore, we performed the reverse experiment, mutating the TK residues into ceTwcK. The latter is a close homologue of human TK that is well characterized structurally and biochemically [25–27]. ceTwcK (bacterially expressed) exhibits high levels of catalysis when assayed on a model peptide substrate derived from myosin light chain (MLC) protein. The catalytic domains of ceTwcK and TK share 40% sequence identity and 65% conservation (figure 4c), and, accordingly, high structural similarity (RMSD for Cα atoms = 1.29 Å when comparing the structure of TK in this work and PDB entry 3UTO using MUSTANG [23]; figure 4a). The active sites of these two kinases are in close structural agreement, particularly their ATP-binding pockets, their β3 strands and the D/EFG motifs (figure 4b). Thus, we concluded that ceTwcK is a suitable template to investigate the effect of the unusual motifs of TK on catalysis.Figure 4.

Bottom Line: Inactivity is the result of two atypical residues in TK's active site, M34 and E147, that do not appear compatible with canonical kinase patterns.While not mediating stretch-dependent phospho-transfers, TK binds the E3 ubiquitin ligase MuRF1 that promotes sarcomeric ubiquitination in a stress-induced manner.Finally, we suggest that an evolutionary dichotomy of kinases/pseudokinases has occurred in TK-like kinases, where invertebrate members are active enzymes but vertebrate counterparts perform their signalling function as pseudokinase scaffolds.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Pathophysiology, Medical Faculty Mannheim, University of Heidelberg, Mannheim 68167, Germany Institute of Integrative Biology, Biosciences Building, University of Liverpool, Crown St., Liverpool L69 7ZB, UK.

ABSTRACT
Striated muscle tissues undergo adaptive remodelling in response to mechanical load. This process involves the myofilament titin and, specifically, its kinase domain (TK; titin kinase) that translates mechanical signals into regulatory pathways of gene expression in the myofibril. TK mechanosensing appears mediated by a C-terminal regulatory tail (CRD) that sterically inhibits its active site. Allegedly, stretch-induced unfolding of this tail during muscle function releases TK inhibition and leads to its catalytic activation. However, the cellular pathway of TK is poorly understood and substrates proposed to date remain controversial. TK's best-established substrate is Tcap, a small structural protein of the Z-disc believed to link TK to myofibrillogenesis. Here, we show that TK is a pseudokinase with undetectable levels of catalysis and, therefore, that Tcap is not its substrate. Inactivity is the result of two atypical residues in TK's active site, M34 and E147, that do not appear compatible with canonical kinase patterns. While not mediating stretch-dependent phospho-transfers, TK binds the E3 ubiquitin ligase MuRF1 that promotes sarcomeric ubiquitination in a stress-induced manner. Given previous evidence of MuRF2 interaction, we propose that the cellular role of TK is to act as a conformationally regulated scaffold that functionally couples the ubiquitin ligases MuRF1 and MuRF2, thereby coordinating muscle-specific ubiquitination pathways and myofibril trophicity. Finally, we suggest that an evolutionary dichotomy of kinases/pseudokinases has occurred in TK-like kinases, where invertebrate members are active enzymes but vertebrate counterparts perform their signalling function as pseudokinase scaffolds.

Show MeSH