Cytoskeletal protein kinases: titin and its relations in mechanosensing
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Titin, the giant elastic ruler protein of striated muscle sarcomeres, contains a catalytic kinase domain related to a family of intrasterically regulated protein kinases. The most extensively studied member of this branch of the human kinome is the Ca2+–calmodulin (CaM)-regulated myosin light-chain kinases (MLCK). However, not all kinases of the MLCK branch are functional MLCKs, and about half lack a CaM binding site in their C-terminal autoinhibitory tail (AI). A unifying feature is their association with the cytoskeleton, mostly via actin and myosin filaments. Titin kinase, similar to its invertebrate analogue twitchin kinase and likely other “MLCKs”, is not Ca2+–calmodulin-activated. Recently, local protein unfolding of the C-terminal AI has emerged as a common mechanism in the activation of CaM kinases. Single-molecule data suggested that opening of the TK active site could also be achieved by mechanical unfolding of the AI. Mechanical modulation of catalytic activity might thus allow cytoskeletal signalling proteins to act as mechanosensors, creating feedback mechanisms between cytoskeletal tension and tension generation or cellular remodelling. Similar to other MLCK-like kinases like DRAK2 and DAPK1, TK is linked to protein turnover regulation via the autophagy/lysosomal system, suggesting the MLCK-like kinases have common functions beyond contraction regulation.
KeywordsSarcomere Mechanical strain sensor Mechanobiology Titin Connectin Twitchin Myosin light-chain kinase Autophagy Obscurin Myomesin Nbr1 p62/SQSTM1 MURF Telethonin/TCAP
Many cellular processes, from cell differentiation and migration during development to functional organ adaptation postnatally, involve the sensing and processing of mechanical stress to trigger cellular responses. Many tissues change their physiological properties rapidly in response to altered mechanical load, including skin, bone, connective tissue, vessels, and smooth and striated muscles. These responses include cell proliferation (e.g. skin callus formation), apoptosis and resorption, or functional remodelling of pre-existent cells by hypertrophy or atrophy. In striated muscle, remodelling on the cellular level plays a major role in the adaptation to changes in workload (reviewed in [35, 108]) especially in the heart, where cell proliferation plays a negligible role in short-term adaptation . Of clinical interest is the short-term adaptation of heart muscle to increased preload by the Frank Starling mechanism that modulates cardiac performance on a beat-to-beat basis, but cardiac growth and remodelling are also directly and indirectly mechanically controlled . For such control mechanisms to act, the muscle cell must contain sensors responding to changes in mechanical load. While mechanosensors have been identified at the cell membrane, e.g. the integrin receptor signalling pathway [22, 29], increasing evidence points to a pivotal role of sensing mechanisms in the contractile machinery itself.
Striated muscle contractility depends on ordered arrays of myosin and actin filaments in repetitive units, the sarcomeres (reviewed in ). Sarcomeres have emerged not only to generate force and motion, but also to integrate a host of signalling functions in muscle mechanotransduction. Both the transverse anchoring planes of actin and myosin filaments—the Z-disk and M-band, respectively—have been implicated in active signalling processes relaying information on mechanical strain to cellular systems that control gene expression, protein synthesis, and protein degradation (reviewed in [35, 36, 50, 60, 69]). Recently, combinations of structural biology, biochemical and cell biological analysis, molecular dynamics simulations [38, 68], and single-molecule force spectroscopy [12, 96, 125] have led to fundamental mechanistic insights into the function of some mechanosignalling complexes at the Z-disk and M-band anchoring planes.
Upon development of active force by myosin motors pulling on actin filaments, substantial mechanical stress acts on the various structures of the sarcomere, which needs to be counteracted by the cross-links of the actin and myosin filaments themselves. Electron microscopic analysis of isometrically contracting skeletal muscle fibres revealed that the resistance of Z-disks and M-bands to mechanical strain differ markedly: While Z-disks showed no appreciable deformation along the sarcomere axis, the M-bands buckled rapidly  up to the point of rupture. This selective buckling of the M-band is likely a result of the shear forces between adjacent myosin filaments and is on the order of 10 nm or more [1, 52]. At the same time, the myosin interfilament spacing does not change significantly under isometric contraction, suggesting that most contraction-induced changes seem to result in axial M-band strain and displacement of myosin . This is contrary to the Z-disk, where active contraction results predominantly in changes in lattice spacing (reviewed in ).
However, as we shall see, there are crucial differences in the TK structure and regulation mechanism that may lead to new perspectives also on other members of the MLCK branch of the human kinome.
An unusual active site: implications for regulation and activity of titin kinase
However, although the short amphipathic helix αR1 binds weakly to CaM as an isolated peptide , TK as well as the twitchin kinase domains from both Caenorhabditis and Aplysia fail to be activated by CaM [37, 45, 46, 58, 78], and CaM binding to the TK holoenzyme could only be detected using chemical cross-linking but not by size exclusion chromatography , unlike the stable CaM–DAPK complex . Similarly, the projectin kinase domain from Locusta migratoria is insensitive to CaM . Recent structural progress allows to compare the topology of the AI regions of these kinases: the ATP binding site in the canonical CaM-regulated kinases is blocked by a peptide segment which also forms the Ca2+–calmodulin binding site, in a region topologically equivalent to αR2 in TK and TwK (Fig. 2). Ca2+–calmodulin binding to TK AI peptides (and the amphipathic helix binding Ca2+–S100A1 in twitchin) resides further N-terminal, in αR1. These observations suggested that αR1, despite binding to CaM as an isolated peptide in a manner similar to but weaker than genuine CaM binding sites , has neither the right overall topology nor affinity for primary CaM regulation. TK and TwK are thus so far unique among the MLCK-like kinases in that CaM is not a primary activator of kinase activity.
Twitchin kinase could, however, be activated in vitro by the dimeric calcium-binding protein S100A1 in the presence of Ca2+ and low concentrations of Zn2+ ; this could not be confirmed for titin kinase . As the C. elegans genome does not contain an S100 gene, the physiological significance of S100 activation of giant muscle protein kinases remains unclear. Although a yet unidentified protein activator cannot be excluded, TwK and TK thus seem to share a non-canonical C-terminal autoinhibition mechanism that is primarily insensitive to Ca2+–calmodulin, despite possibly divergent physiological functions and further differences in the structure of their active sites.
The other unusual exchange in TK is the replacement of the aspartate in the conserved DFG motif at the beginning of the activation segment with glutamate, E147 (Figs. 3a and 4). Lastly, closer inspection of the active site reveals a further configuration suggestive of an inactive conformation of autoinhibited TK. E51 of the αC1 helix is a completely conserved residue engaged with K36 in a salt bridge in active kinases . In autoinhibited TK, these residues are separated beyond salt bridge distance by 4.91 Å (Fig. 3b), as opposed to, e.g. DAPK with 2.68 Å. However, whether this is supportive of an inactive conformation requiring further allosteric activation by αC1 helix movement is debatable, as the corresponding residues in the active, CaM-bound complexes of DAPK or CaMKIIδ are also separated by more than 4 Å (4.51 and 4.74 Å, respectively). Overall, the structure of the autoinhibited TK suggests that the catalytic site has accommodated these exchanges, and adopts a conformation consistent with an active kinase rather than a pseudokinase , as is also highlighted by the conservation of other crucial residues involved in catalysis (Fig. 4b). Indeed, cellular responses to TK discussed below require the presence of D127 .
A mechanically modulated activation mechanism?
The CaM-insensitive autoinhibited state of TK raises the question how an open state, capable of binding ATP and peptide substrates or potential scaffold proteins, could be achieved. For access of the autoinhibitory tyrosine, the C-terminal autoinhibitory tail needs to be partly removed, with no protein factor identified so far being able to do so. Relieving intramolecular autoinhibition can be regarded as a partial unfolding event of the autoinhibited conformation of the kinase. The folded and closed, and partially unfolded open states are separated by an energy barrier that can be overcome by ligand binding, e.g. CaM in the classical CaM kinases or phosphorylation. For a protein that is firmly integrated into the cytoskeleton and the contractile machinery and thus exposed to force, the conformational space is not only governed by thermal energy or ligand interactions, but also by the anisotropic effects of mechanical force . Could enzymatic functions in titin, whose elastic functions in the I-band are paradigmatic for force-induced conformational changes by reversible protein unfolding , also be modulated by mechanically induced conformational changes?
The C-terminus of titin is embedded into the M-band via interactions in a ternary complex with myomesin, the giant GTPase regulator and protein kinase obscurin [105, 139] and its small structural homologue obscurin-like 1 [33, 93]. The M-band, being much more compliant than the Z-disk [3, 49], is ideally placed as a strain sensor [1, 2]. As the M-band lattice is deformed only during active contraction due to the shear forces between adjacent myosin filaments, it is optimally placed for detecting the actual workload on the myofibril . Force-probe molecular dynamics simulations were thus used to test the hypothesis that conformational changes consistent with kinase activation could be induced mechanically in TK. Indeed, these simulations suggested that this could be the case and that forces acting at low velocities can lead to the sequential unfolding of the autoinhibitory tail, thus opening the active site while preserving the catalytic core . Experimental verification of these simulations, which were performed at pulling rates of between 0.4 to 5 m/s due to computational restraints, were performed using single-molecule atomic force spectroscopy at the much slower pulling rate of 0.72 μm/s. This experimental probing of the mechanical properties of TK confirmed that relief of autoinhibition is possible by partial unfolding of the C-terminal autoinhibitory domain by “gating” forces around 30 pN and displacements around 10 nm . Analysis of the force spectroscopy data and molecular dynamics simulations suggested that the open conformation is able to bind ATP, and to promote further steps in TK activation, by exposing the autoinhibitory Y170 for auto- or trans-phosphorylation. The experimentally determined forces of 30 pN at physiological temperature compare to the force generated by about five to six myosin motor domains (assuming a force of 6 pN each ), the myosin heads within one “crown” of the myosin filament. In the A-band, these crowns of 3 × 2 myosin heads are arranged every 14.3 nm on either side of the M-band. Therefore, a displacement of a myosin filament by just one 14.3 nm myosin repeat with respect to the Z-disks will lead to a theoretical maximal force imbalance of (3 × 2 × 2) × 6 pN or 72 pN. The measured gating force of 30 pN, and the gating distance of 9 nm for opening the TK active site are therefore within the predicted range of shear forces arising physiologically between myosin filaments.
Interestingly, local protein unfolding and refolding is emerging as a common mechanism in the activation of C-terminally autoinhibited kinases (Fig. 5). In the CaM-regulated CaMKIIδ, the entire autoinhibitory tail undergoes substantial unfolding with partial refolding of the CaM binding region in the complex with calmodulin . The autoinhibited form of the MLCK-like DAPK is not yet available, but its CaM-bound structure  suggests that similar major structural rearrangements and partial AI unfolding must occur during activation. These combined results suggest that TK—and possibly TwK—could indeed function as a force sensor by switching between a closed and open conformation by mechanically, rather than ligand-induced partial unfolding of its autoinhibitory tail.
Links to the protein turnover machinery
This signalling complex reveals links to the regulation of muscle protein turnover not only by the ubiquitin–proteasome pathway (Fig. 6) and is tightly regulated during development . P62/SQSTM1 is emerging as a central adaptor molecule involved in several pathways relevant in myogenic differentiation and stress response. P62/SQSTM1 interactions include MAP kinase p38 , the MAP kinase kinase MEK5  as well as its upstream MAPK kinase kinase, MEKK3  that are involved in ERK5 activation. Further interactions involve the TNF receptor-associated kinase RIP , atypical protein kinases-C (aPKC ), Src family tyrosine protein kinases like lck [55, 91], and insulin receptor/insulin-like growth factor-1 receptor signalling via Grb14 [17, 47]. P62/SQSTM1 links input from a number of these kinases to the activation of NFκB [84, 109, 110, 117, 136]. Some of these have multiple roles in cell survival and myogenic differentiation. For example, p38 activates the myogenic transcription factors MEF2 and MyoD by phosphorylation of MEF2C [99, 141], and the MEK5/ERK5 kinase cascade is crucially involved in myogenic differentiation and hypertrophic growth via MEF2 activation [8, 27, 85, 127]. P62/SQSTM1 also interacts with, and regulates the orphan hormone receptor COUP-TFII , which has been implicated in strain adaptation of cardiac gene expression and metabolic adaptation in skeletal muscle [20, 83, 106].
P62/SQSTM1 can also target ligands of its PB1 and ZZ domain region to polyubiquitin chains via its C-terminal ubiquitin-associated UBA domain. This may assemble larger signalosomes via lysine-63 linked polyubiquitin, in analogy to other ubiquitin-mediated kinase signalling pathways [40, 118]. Association with lysine-48 linked polyubiquitin chains, however, could target these complexes for proteasomal degradation  and, via the interaction of p62 with LC3, to the autophagy of ubiquitinated proteins . Nbr1 has recently emerged to be similarly implicated in autophagic protein turnover by recruiting polyubiquitinated proteins and binding to the autophagosomal membrane anchor LC3 [57, 128]. Similar to p62/SQSTM1, however, nbr1 also emerges as a scaffold for multiple protein kinase signalling pathways including PKCζ and p38 MAP kinase (Fig. 6) [131, 132], hinting at important generic and partly overlapping roles of theses adaptor proteins in cell signalling as well as protein turnover regulation.
Autophagy is increasingly recognised as a crucial protein degradation mechanism in muscle in addition to the ubiquitin–proteasome system , but is also emerging as a novel mechanism in regulating cellular signal transduction by removing activated signalling proteins [6, 54]. It is interesting to speculate whether the main role of nbr1 in TK signalling is in signal propagation or attenuation, a question that can only be answered by analysing appropriate knockout models. There may also be significant redundancy between p62/SQSTM1 and nbr1 due to the overlapping ligand spectrum. This may explain, for example, why p62 knockout animals do not seem to show an overt muscle phenotype [28, 59, 88] despite the involvement of p62/SQSTM1 in muscle autophagy , and why a truncated nbr1 mouse model (leaving the titin- and p62-binding PB1 domain region intact) shows a bone , but not muscle phenotype (MG and C. Whitehouse, unpublished observations).
The vital importance of TK in maintaining the turnover of muscle proteins in human via nbr1 and p62/SQSTM1 is highlighted by a point mutation in the αR1 helix, R279W, in the human kinase domain that abrogates nbr1 binding. This leads to a myopathy with failure of load-dependent protein turnover (human myopathy with early respiratory failure, HMERF) with the aberrant localisation, aggregation of p62/SQSTM1 and possibly vesicular accumulation of nbr and nuclear translocation of MURF . These observations suggest that TK acts as a strain-modulated sarcomeric “receptor” for proteins involved in cellular remodelling and thus contributes to the control of mechanical load-dependent remodelling of muscle. Interestingly, HMERF can also be caused by mutations in other proteins than titin kinase , and the identification of these additional disease loci should prove highly insightful for unravelling the disease mechanism and might also answer why in TK-associated HMERF, cardiac muscle appears to be spared in early disease.
It will be interesting to see whether strain activation of TK occurs continually to regulate sarcomere homeostasis and remodelling or only to sense and repair local mechanical damage. This remains to be tested and will require the combined use of animal models with biochemical and biophysical methods.
Substrates and scaffolds
Little is known about other physiological ligands and substrates of TK. The first in vitro substrate identified in developing myoblasts was the small, muscle-specific Z-disk protein telethonin (also known as TCAP), where constitutively active TK phosphorylates S157 in the C-terminus . As constitutively active TK disrupts myofibril formation in cultured myoblasts, it was suggested that TK-mediated telethonin regulation might play an important role in the control of ordered sarcomere assembly . However, a titin M-band deletion mouse model, where a larger part of the M-band including the TK domain was deleted, can form myofibrils even though these quickly become unstable . Although the ultimate disassembly of titin M-band-deficient sarcomeres would agree with the impaired communication to the protein turnover machinery via nbr1/SQSTM1/MURF, all of which are expressed in the heart from the earliest detectable stages onwards , the role of telethonin phosphorylation remains utterly enigmatic—as does, in fact, the protein overall. Although telethonin is a Z-disk protein, it interacts with a host of proteins including secreted growth factors (reviewed in ), and has also been observed at the M-band , similar to other Z-disk proteins like myotilin . Analysis of telethonin phosphorylation may be confounded by the observation that TK is not the only kinase, which, at least in vitro, can phosphorylate the C-terminus telethonin, as telethonin interacts also with protein kinase D  and is a substrate of this kinase of the CaM kinase branch. Redundant kinase pathways might therefore complicate the phenotype of PKD or TK knockout animals.
Point mutations inactivating the telethonin phosphorylation sites or short-term knockdown impair myofibril formation or maintenance in Xenopus ; in human, deletion of the C-terminal portion of telethonin  or mutations close to the TK phosphorylation site (R153H, ) cause hereditary limb-girdle muscular dystrophy (LGMD2G) or hypertrophic cardiomyopathy. This suggests an important yet uncharacterised function of the telethonin C-terminus in muscle maintenance. Surprisingly, a knockout mouse for telethonin shows only a mild, late onset myopathic phenotype with apparently normal myofibrils , despite the function of telethonin as a major cross-linker of titin filaments at the Z-disk [12, 142]. However, telethonin localisation is clearly sensitive to mechanical load, with the protein being a sensitive marker of neurogenic atrophy  and its mRNA being rapidly down-regulated under denervation atrophy . Understanding the physiological role of telethonin and its phosphorylation in load-dependent muscle remodelling will now likely require new approaches.
Both nbr1 and p62/SQSTM1 are in vitro substrates for TK , but as phosphorylation with recombinant titin kinase requires either mutagenesis on Y170 or C-terminal truncation, both of which may compromise catalytic activity or enzyme stability, the full impact and enzymatics of TK for these interacting proteins may be difficult to assess in vitro and may again require the use of more physiological settings. As both proteins also interact with many other signalling proteins and kinases [35, 73, 92, 131, 132], a scaffold function for the spatial integration of multiple signalling components is plausible, that could play a role in either signal propagation or attenuation (Fig. 6). In neonatal cardiomyocytes, the unloading-induced repression of SRF-dependent gene expression could be relieved by transfected constitutively active titin kinase , but not by a catalytically inactive D127A mutant, which also led to the reduction of cellular MURF. In this context, TK therefore acts as a brake on atrophic pathways, of which MURFs are one component. The exact mechanism of load-dependent muscle remodelling may again require a multidisciplinary approach to unravel completely.
The uneasy family relationships of the cytoskeletal “MLCK” kinases
Furthermore, it is now emerging that many MLCK-like kinases are not actual myosin light-chain kinases, but are involved in other regulatory processes: DAPK1 phosphorylates Beclin-1, a key protein involved in the initiation of autophagy, and is now recognised to regulate cell survival via the autophagy pathway [13, 140]. Similarly, “death-associated protein kinase-related apoptosis-inducing protein kinase 2” (DRAK2) phosphorylates p70S6 kinase  and is thus involved in metabolic flow regulation and, via the AKT-mTOR pathway, eventually also in the regulation of protein turnover . The full scope of regulators and downstream ligands for most other MLCK-like kinases is yet to emerge, but it is noteworthy that three of them are now linked to autophagy and protein turnover regulation.
Similarly, molluscan twitchin kinase is tightly attached to both actin and myosin filaments and seems to be stretch-activated, thereby possibly contributing to the phosphorylation-mediated maintenance of the catch state in response to stretch [5, 15, 16, 34]. Indeed, the simulated unfolding of C. elegans twitchin kinase and initial force spectroscopy data, showed similar patterns as for TK [38, 39]. The cytoskeletal association for other MLCK-like kinases is less well investigated, but it seems clear that TRIO and Kalirin are actin-associated [101, 113]. A better understanding of the molecular interactions and knowledge of the full scope of substrates will provide a significant advance in understanding these kinases at the crossroads of cellular mechanics and signalling, which might be better called cytoskeletal kinases.
Conclusions and perspectives
Over recent years, the notion that the sarcomere is purely a contractile machine of high order has been challenged by the discovery that multiple signalling pathways not only affect the assembly or function of this structure, but that the sarcomere is a source of active communication controlling muscle cell proliferation, growth, and remodelling. The kinase domain of the giant elastic protein titin highlights that classification by sequence similarity may not always come to the bottom of the functional complexity of the human kinome. Its analysis has suggested that kinase signalling and mechanosensing may be more tightly interwoven than previously assumed, with the opening of the TK active site being directly mechanically triggered. Important insight has also been gained from studies in C. elegans, underscoring the value of this model organism. The observation that TK itself and many of its interactors are targets of hereditary muscle diseases not only highlights their importance, but raises the hope that a detailed understanding of their functions may result in new therapeutic approaches to ameliorate certain acquired and hereditary muscle diseases. The involvement of TK and its ligands in load-dependent muscle turnover should now be studied in more clinically relevant models like left ventricular load-dependent remodelling or in skeletal muscle disuse atrophy. Although titin is abundant, kinases are well drugable targets, and the TK signalling pathway might offer new perspectives for targeting muscle atrophy or cardiac hypertrophy. However, to what extent it is the scaffolding function rather than the actual catalytic activity of titin kinase that plays a dominant role in its crucial biological function remains to be elucidated. In the future therefore, integrating atomic structures and molecular modelling with cellular and single-molecule AFM data will be required not only to address the mechanosignalling functions of titin and its disruptions in muscle disease, but also to understand related functions in other cytoskeletal systems. The direct mechanical activation of ATP binding by titin kinase may prove a paradigm for other cytoskeletal signalling domains, especially the kinases of the cytoskeletal MLCK-like family in vertebrates and invertebrates, whose tight cytoskeletal association makes such a regulation plausible.
The author gratefully acknowledges the generous support by the British Heart Foundation, the Medical Research Council and the Wellcome Trust. Many thanks to Ulf Hensen and Helmut Grubmüller for coordinates of mechanically opened TK.
Structure figures were generated with PyMOL Molecular Graphics System, version 1.3, Schrödinger, LLC.
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