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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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Tcap phosphorylation assays using TK preparations from insect cells. (a) Preparations of wild-type TK, the activated TKY170E phosphomimic and the constitutively inactive TKK36L phosphorylate Tcap comparably and stimulated by Ca2+/calmodulin. (i) SDS-PAGE and (ii) autoradiogram of catalysis by samples after Ni2+-NTA are shown. (b) Phosphorylation assay of TK sterically inhibited by immuno-complexation with an antibody raised against the P+1 loop vicinal to the active site. An antibody (anti-MuRF1) that does not complex TK is included for comparison. (c) Untransfected Sf21 cell extracts supplemented with Tcap (but not Ca2+/calmodulin) display phosphorylating activity (the stimulation of catalysis upon addition of calmodulin was approx. 25%, this modest activation is likely due to the presence of endogenous calmodulin in the extract). (i) SDS-PAGE and (ii) autoradiogram revealing Tcap phosphorylation. (d) (i) Chromatogram and (ii) corresponding SDS-PAGE of Sf21 cell crude extract containing recombinant TKK36L eluted from a Ni2+-NTA column. Segregation of phosphorylating activity (cyan) and TK (red) during purification is observed. Bound proteins were eluted with a linear gradient of imidazole (100% buffer B = 0.3 M imidazole; green line) and monitored by A280; the resultant chromatogram is in blue. The content of TKK36L in eluted fractions was determined by spot-blot immunoassay using anti-TK P+1 loop antibody. The amount of coloured product quantified densitometrically was proportional to the amount of TKK36L in each fraction (red). Phosphorylation of a Tcap-derived peptide substrate in the presence of calmodulin was quantified in each fraction densitometrically by our standard phosphorylation assay that used [γ-33P]ATP and spotting on P81 paper (cyan). The data show that Tcap phosphorylation segregated from TKK36L.
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RSOB140041F1: Tcap phosphorylation assays using TK preparations from insect cells. (a) Preparations of wild-type TK, the activated TKY170E phosphomimic and the constitutively inactive TKK36L phosphorylate Tcap comparably and stimulated by Ca2+/calmodulin. (i) SDS-PAGE and (ii) autoradiogram of catalysis by samples after Ni2+-NTA are shown. (b) Phosphorylation assay of TK sterically inhibited by immuno-complexation with an antibody raised against the P+1 loop vicinal to the active site. An antibody (anti-MuRF1) that does not complex TK is included for comparison. (c) Untransfected Sf21 cell extracts supplemented with Tcap (but not Ca2+/calmodulin) display phosphorylating activity (the stimulation of catalysis upon addition of calmodulin was approx. 25%, this modest activation is likely due to the presence of endogenous calmodulin in the extract). (i) SDS-PAGE and (ii) autoradiogram revealing Tcap phosphorylation. (d) (i) Chromatogram and (ii) corresponding SDS-PAGE of Sf21 cell crude extract containing recombinant TKK36L eluted from a Ni2+-NTA column. Segregation of phosphorylating activity (cyan) and TK (red) during purification is observed. Bound proteins were eluted with a linear gradient of imidazole (100% buffer B = 0.3 M imidazole; green line) and monitored by A280; the resultant chromatogram is in blue. The content of TKK36L in eluted fractions was determined by spot-blot immunoassay using anti-TK P+1 loop antibody. The amount of coloured product quantified densitometrically was proportional to the amount of TKK36L in each fraction (red). Phosphorylation of a Tcap-derived peptide substrate in the presence of calmodulin was quantified in each fraction densitometrically by our standard phosphorylation assay that used [γ-33P]ATP and spotting on P81 paper (cyan). The data show that Tcap phosphorylation segregated from TKK36L.

Mentions: In activity assays that used ATP[γ-33P], all three TK variants—including the inactive TKK36L—showed similar phospho-transfer activities on Tcap and were modestly stimulated by Ca2+/calmodulin (figure 1a). As an independent validation, we studied the activity of immuno-complexed wild-type TK where its active site had been blocked by a specific antibody directed against the P+1 loop (the efficient complexation of TK by this antibody is shown in the electronic supplementary material, figure S3). Immuno-complexed TK, non-complexed TK samples and non-treated TK controls showed similar levels of activity (figure 1b). Hence, activity data from either mutated or immuno-complexed samples together suggested that insect cell preparations catalysed Tcap phosphorylation in a TK-independent way, with kinase activity arising from other component(s) in the cell milieu.Figure 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)

Tcap phosphorylation assays using TK preparations from insect cells. (a) Preparations of wild-type TK, the activated TKY170E phosphomimic and the constitutively inactive TKK36L phosphorylate Tcap comparably and stimulated by Ca2+/calmodulin. (i) SDS-PAGE and (ii) autoradiogram of catalysis by samples after Ni2+-NTA are shown. (b) Phosphorylation assay of TK sterically inhibited by immuno-complexation with an antibody raised against the P+1 loop vicinal to the active site. An antibody (anti-MuRF1) that does not complex TK is included for comparison. (c) Untransfected Sf21 cell extracts supplemented with Tcap (but not Ca2+/calmodulin) display phosphorylating activity (the stimulation of catalysis upon addition of calmodulin was approx. 25%, this modest activation is likely due to the presence of endogenous calmodulin in the extract). (i) SDS-PAGE and (ii) autoradiogram revealing Tcap phosphorylation. (d) (i) Chromatogram and (ii) corresponding SDS-PAGE of Sf21 cell crude extract containing recombinant TKK36L eluted from a Ni2+-NTA column. Segregation of phosphorylating activity (cyan) and TK (red) during purification is observed. Bound proteins were eluted with a linear gradient of imidazole (100% buffer B = 0.3 M imidazole; green line) and monitored by A280; the resultant chromatogram is in blue. The content of TKK36L in eluted fractions was determined by spot-blot immunoassay using anti-TK P+1 loop antibody. The amount of coloured product quantified densitometrically was proportional to the amount of TKK36L in each fraction (red). Phosphorylation of a Tcap-derived peptide substrate in the presence of calmodulin was quantified in each fraction densitometrically by our standard phosphorylation assay that used [γ-33P]ATP and spotting on P81 paper (cyan). The data show that Tcap phosphorylation segregated from TKK36L.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB140041F1: Tcap phosphorylation assays using TK preparations from insect cells. (a) Preparations of wild-type TK, the activated TKY170E phosphomimic and the constitutively inactive TKK36L phosphorylate Tcap comparably and stimulated by Ca2+/calmodulin. (i) SDS-PAGE and (ii) autoradiogram of catalysis by samples after Ni2+-NTA are shown. (b) Phosphorylation assay of TK sterically inhibited by immuno-complexation with an antibody raised against the P+1 loop vicinal to the active site. An antibody (anti-MuRF1) that does not complex TK is included for comparison. (c) Untransfected Sf21 cell extracts supplemented with Tcap (but not Ca2+/calmodulin) display phosphorylating activity (the stimulation of catalysis upon addition of calmodulin was approx. 25%, this modest activation is likely due to the presence of endogenous calmodulin in the extract). (i) SDS-PAGE and (ii) autoradiogram revealing Tcap phosphorylation. (d) (i) Chromatogram and (ii) corresponding SDS-PAGE of Sf21 cell crude extract containing recombinant TKK36L eluted from a Ni2+-NTA column. Segregation of phosphorylating activity (cyan) and TK (red) during purification is observed. Bound proteins were eluted with a linear gradient of imidazole (100% buffer B = 0.3 M imidazole; green line) and monitored by A280; the resultant chromatogram is in blue. The content of TKK36L in eluted fractions was determined by spot-blot immunoassay using anti-TK P+1 loop antibody. The amount of coloured product quantified densitometrically was proportional to the amount of TKK36L in each fraction (red). Phosphorylation of a Tcap-derived peptide substrate in the presence of calmodulin was quantified in each fraction densitometrically by our standard phosphorylation assay that used [γ-33P]ATP and spotting on P81 paper (cyan). The data show that Tcap phosphorylation segregated from TKK36L.
Mentions: In activity assays that used ATP[γ-33P], all three TK variants—including the inactive TKK36L—showed similar phospho-transfer activities on Tcap and were modestly stimulated by Ca2+/calmodulin (figure 1a). As an independent validation, we studied the activity of immuno-complexed wild-type TK where its active site had been blocked by a specific antibody directed against the P+1 loop (the efficient complexation of TK by this antibody is shown in the electronic supplementary material, figure S3). Immuno-complexed TK, non-complexed TK samples and non-treated TK controls showed similar levels of activity (figure 1b). Hence, activity data from either mutated or immuno-complexed samples together suggested that insect cell preparations catalysed Tcap phosphorylation in a TK-independent way, with kinase activity arising from other component(s) in the cell milieu.Figure 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
Related in: MedlinePlus