Titin kinase is an inactive pseudokinase scaffold that supports MuRF1 recruitment to the sarcomeric M-line.
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.
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.
Julijus
J
Bogomolovas
Department of Integrative Pathophysiology, Medical Faculty Mannheim, University of Heidelberg, Mannheim 68167, Germany.
Alexander
A
Gasch
Institute of Integrative Biology, Biosciences Building, University of Liverpool, Crown St., Liverpool L69 7ZB, UK.
Felix
http://orcid.org/0000-0003-3430-6828
F
Simkovic
Institute of Integrative Biology, Biosciences Building, University of Liverpool, Crown St., Liverpool L69 7ZB, UK.
Daniel J
DJ
Rigden
Department of Integrative Pathophysiology, Medical Faculty Mannheim, University of Heidelberg, Mannheim 68167, Germany labeit@medma.de.
Siegfried
S
Labeit
Institute of Integrative Biology, Biosciences Building, University of Liverpool, Crown St., Liverpool L69 7ZB, UK olga.mayans@liv.ac.uk.
Olga
O
Mayans
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D064211:Connectin / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D019143:Evolution, Molecular; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009124:Muscle Proteins / Q000378:metabolism*; D054792:Muscle, Striated / Q000378:metabolism*; D010802:Phylogeny; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012518:Sarcomeres / Q000378:metabolism*; D016415:Sequence Alignment; D061987:Sf9 Cells; D018411:Spodoptera; D054875:Ubiquitination
D064211:Connectin; D009124:Muscle Proteins
mutagenesis; phosphorylation assay; pseudokinase; titin