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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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Structural and functional characterization of TK produced in E. coli. (a) Superposition of the crystal structures of TK expressed in bacteria (red) and insect cells (PDB entry 1TKI) (blue). The overall RMSD for Cα atoms is 0.29 Å. (b) Representation of the activated variant TKΔR2/Y170E, where the deleted fraction is in grey and the added loop is shown schematically in red. The sequence exchanges in this variant are shown below. (c) Identification of potential TK substrates in differentiating C2C12 cell extracts depleted of endogenous kinases by treatment with FSBA. Protein kinase A (PKA) was used as positive control. (i) Autoradiogram and (ii) densitogram of phosphor-image are provided. The data show no significant differences in labelling pattern when comparing cell extract alone or supplemented with activated forms of TK.
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RSOB140041F2: Structural and functional characterization of TK produced in E. coli. (a) Superposition of the crystal structures of TK expressed in bacteria (red) and insect cells (PDB entry 1TKI) (blue). The overall RMSD for Cα atoms is 0.29 Å. (b) Representation of the activated variant TKΔR2/Y170E, where the deleted fraction is in grey and the added loop is shown schematically in red. The sequence exchanges in this variant are shown below. (c) Identification of potential TK substrates in differentiating C2C12 cell extracts depleted of endogenous kinases by treatment with FSBA. Protein kinase A (PKA) was used as positive control. (i) Autoradiogram and (ii) densitogram of phosphor-image are provided. The data show no significant differences in labelling pattern when comparing cell extract alone or supplemented with activated forms of TK.

Mentions: As kinases present in insect cells’ preparations masked the potential catalysis of TK on Tcap, we established the over-production of TK in E. coli. To validate that the bacterial form of this sample was viable, we performed a mass spectrometry analysis of TK samples from E. coli and Sf21 cells. The data confirmed that neither of the proteins was truncated or otherwise chemically compromised (e.g. mass differences from full mass theoretical values of TKY170E from Sf21 and E. coli cells were +0.7 and +1.8 Da, respectively). The bacterially expressed TK had no phospho-transfer activity on Tcap, nor on the universal kinase substrates myelin basic protein and casein. This agrees with previous observations of inactivity of bacterial TK [16]. To test whether the lack of catalysis resulted from fold defects in the bacterial sample, we elucidated its crystal structure to 2.06 Å resolution (table 1). Crystallization used previous protocols for Sf9-expressed TK [22] and crystals reproduced the lattice parameters of the latter [13]. Following bias removal by simulated annealing, the resulting model of bacterially produced TK was in complete agreement with that of Sf9-expressed samples (RMSD = 0.29 Å for all Cα atoms, calculated with MUSTANG [23]; figure 2a). These data confirmed that there are no notable molecular differences between bacterial and eukaryotic forms of TK, and that the absence of catalysis on Tcap signifies that Tcap is not a substrate of TK.Table 1.


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)

Structural and functional characterization of TK produced in E. coli. (a) Superposition of the crystal structures of TK expressed in bacteria (red) and insect cells (PDB entry 1TKI) (blue). The overall RMSD for Cα atoms is 0.29 Å. (b) Representation of the activated variant TKΔR2/Y170E, where the deleted fraction is in grey and the added loop is shown schematically in red. The sequence exchanges in this variant are shown below. (c) Identification of potential TK substrates in differentiating C2C12 cell extracts depleted of endogenous kinases by treatment with FSBA. Protein kinase A (PKA) was used as positive control. (i) Autoradiogram and (ii) densitogram of phosphor-image are provided. The data show no significant differences in labelling pattern when comparing cell extract alone or supplemented with activated forms of TK.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4042850&req=5

RSOB140041F2: Structural and functional characterization of TK produced in E. coli. (a) Superposition of the crystal structures of TK expressed in bacteria (red) and insect cells (PDB entry 1TKI) (blue). The overall RMSD for Cα atoms is 0.29 Å. (b) Representation of the activated variant TKΔR2/Y170E, where the deleted fraction is in grey and the added loop is shown schematically in red. The sequence exchanges in this variant are shown below. (c) Identification of potential TK substrates in differentiating C2C12 cell extracts depleted of endogenous kinases by treatment with FSBA. Protein kinase A (PKA) was used as positive control. (i) Autoradiogram and (ii) densitogram of phosphor-image are provided. The data show no significant differences in labelling pattern when comparing cell extract alone or supplemented with activated forms of TK.
Mentions: As kinases present in insect cells’ preparations masked the potential catalysis of TK on Tcap, we established the over-production of TK in E. coli. To validate that the bacterial form of this sample was viable, we performed a mass spectrometry analysis of TK samples from E. coli and Sf21 cells. The data confirmed that neither of the proteins was truncated or otherwise chemically compromised (e.g. mass differences from full mass theoretical values of TKY170E from Sf21 and E. coli cells were +0.7 and +1.8 Da, respectively). The bacterially expressed TK had no phospho-transfer activity on Tcap, nor on the universal kinase substrates myelin basic protein and casein. This agrees with previous observations of inactivity of bacterial TK [16]. To test whether the lack of catalysis resulted from fold defects in the bacterial sample, we elucidated its crystal structure to 2.06 Å resolution (table 1). Crystallization used previous protocols for Sf9-expressed TK [22] and crystals reproduced the lattice parameters of the latter [13]. Following bias removal by simulated annealing, the resulting model of bacterially produced TK was in complete agreement with that of Sf9-expressed samples (RMSD = 0.29 Å for all Cα atoms, calculated with MUSTANG [23]; figure 2a). These data confirmed that there are no notable molecular differences between bacterial and eukaryotic forms of TK, and that the absence of catalysis on Tcap signifies that Tcap is not a substrate of TK.Table 1.

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