Titins in C. elegans with Unusual Features: Coiled-coil Domains, Novel Regulation of Kinase Activity and Two New Possible Elastic Regions

Abstract

We report that there are previously unrecognized proteins in Caenorhabditis elegans that are similar to the giant muscle proteins called titins, and these are encoded by a single ∼90 kb gene. The gene structure was predicted by GeneMark.hmm and then experimentally verified. The Ce titin gene encodes polypeptides of 2.2 MDa, 1.2 MDa and 301 kDa. The 2.2 MDa isoform resembles twitchin and UNC-89 in that it contains multiple Ig (56) and FnIII (11) domains, and a single protein kinase domain. In addition, however, the 2.2 MDa isoform contains four classes of short, 14–51 residue, repeat motifs arranged mostly in many tandem copies. One of these tandem repeat regions is similar to the PEVK regions of vertebrate and fly titins. As the PEVK region is one of the main elastic elements of the titins and is also composed of short tandem repeats, this suggests that the repeat motifs in the Ce titins may have a similar elastic function. An interesting aspect of the two largest Ce titin isoforms, is that in contrast to other members of the twitchin/titin family, there are multiple regions which are likely to form coiled-coil structure. In transgenic animals, the first ∼100 residues of the largest isoforms targets to dense bodies, the worm analogs of Z-discs. Anti-Ce titin antibodies show localization to muscle I-bands beginning at the L2–L3 larval stages and this pattern continues into adult muscle. Ce titins may not have a role in early myofibril assembly: (1) Ce titins are too short to span half a sarcomere, and the onset of their expression is well after the initial assembly of thick filaments. (2) Ce titins are not localized to I-bands in embryonic or L1 larval muscle. The Ce titin protein kinase domain is most similar to the kinase domains of the twitchins and projectin. The Ce titin kinase has protein kinase activity in vitro, and this activity is regulated by a novel mechanism.

Introduction

In vertebrate striated muscle, the giant polypeptide titin,¹ also known as connectin,² appears to function both in myofibril assembly and in providing passive tension for muscle. Single titin polypeptides are 3–4 MDa, are 1.2 μm long at rest, and span half a sarcomere, with N termini anchored at the Z-line and C termini anchored at the M-line.3., 4. The complete sequence of human cardiac titin was shown to contain approximately 166 copies of immunoglobulin (Ig) and 132 copies of fibronectin type III (FnIII) domains, a single protein kinase domain, and a “PEVK region”.⁵ The Ig and FnIII domains are arranged into different patterns or super-repeats, in different regions of the sarcomere. The I-band region contains up to 90 Ig domains in tandem. The A-band portion contains super-repeats of Ig and FnIII domains in different patterns. The A-band portion of titin is tightly associated with the shaft of the thick filament, and specific regions of titin have been shown to interact with myosin, thick filament accessory proteins, M-line proteins⁶ and MURF-1. MURF-1 interacts with a region near the titin kinase domain and might have a role in regulating muscle gene expression.⁷ Most of these other A-band proteins, like titin itself, are members of the intracellular branch of the Ig superfamily. Furthermore, the A-band portion of titin has been hypothesized to determine the length of a thick filament as it is being assembled from myosin.⁸ This “molecular ruler” hypothesis for titin has not been tested experimentally.

Although encoded by a single gene in humans, differential splicing results in multiple isoforms varying from 2970 kDa to 3700 kDa.⁹ Most of this variation is in the I-band portion of titin created by varying numbers of tandem Ig domains and the length of the PEVK domain. Most of the passive tension of muscle arises from the I-band portion of titin, which is elastic. Both of the regions that vary (poly(Ig) and PEVK) are considered distinct spring elements, based upon several different types of experiments.10., 11. In skeletal muscle titins, the best interpretation of these experiments is that the poly(Ig) region straightens at modest sarcomere stretch (probably without unfolding of Ig domains), and the PEVK region extends at higher physiological stretch. In cardiac titins, there is a third spring element formed by the “N2B unique sequence” which extends together with the PEVK region upon higher physiological stretch.

The PEVK domain, so far only found in the titins and stretchin/MLCK (MLCK, myosin light-chain kinase),¹² is a region consisting of ∼70% of just the four amino acid residues, P, E, V, and K. The PEVK region was originally considered to be non-repetitive in sequence, and to have a random coil structure.⁵ However, during the past year, three papers13., 14., 15. report that the PEVK region consists of tandem repeats of modules that are ∼28 amino acid residues long. These motifs occur in groups of 2–12 that are separated by regions rich in glutamic acid (∼45%) called polyE segments.¹⁵ Moreover, Gutierrez-Cruz et al.¹⁴ obtained CD spectral data on a 469 aa PEVK segment that indicates that the PEVK region is an open and flexible chain containing ∼13% stable structural elements, probably consisting of polyproline II helices. Indeed, 2D-NMR on one representative 28 amino acid (aa) module shows three short stretches of polyproline II helices of four, five and six aa interrupted by unordered, presumably flexible, spacer regions.¹⁶ Even more surprising, the PEVK region was shown to interact at micromolar affinity to both actin and some segments of nebulin.¹⁴ It has been suggested that transient interaction with thin filaments might provide another mechanism to modulate titin's elasticity. In addition, experiments suggest that interaction of titin, through its PEVK region, with the thin filament, might regulate the actin–myosin interaction: in vitro binding was detected at physiological ionic strength between F-actin and a PEVK region of cardiac titin and this was inhibited by Ca²⁺/S100A1. This PEVK region retarded myosin-based F-actin sliding velocity, in vitro.¹⁷

There have been a number of reports of phenotypes resulting from mutations in both vertebrate and Drosophila titins, and yet the true functions of titin in muscle remain unclear. Mutations in zebrafish titin are recessive embryonic lethal, in which the major defect is in the embryonic heart.¹⁸ Electron microscopy indicated that a few myofibrils were formed at first, but later, no myofibrils could be detected. This is consistent with a role for titin in either assembly or maintenance of myofibrils. Mutations in human titin result in either some cases of autosomal dominant dilated cardiomyopathy,19., 20. or a tibial muscular dystrophy, a late-onset, autosomal dominant distal myopathy without cardiac involvement.²¹ It is currently unclear why in humans titin mutations cause both heart muscle-specific and skeletal muscle-specific diseases. In contrast to vertebrate titins, which span half of a sarcomere, Drosophila titin (∼2 MDa) seems to reside only in the I-band.²² Most mutations in Drosophila titin result in embryonic lethality with a muscle phenotype characterized by failure of myoblasts to fuse, and in some alleles, no Z-discs are formed.²³ But some alleles make it through embryonic and larval development and die at the larval-pupal transition. In these late-stage larvae, mitotically active cells have defects in chromosome condensation, chromosome integrity and sister chromatid cohesion.²³ Indeed, antibodies to D-titin localize not only to muscle Z-discs, but also to the nuclei of many cells, specifically to condensed chromosomes.²²

Until recently, the closest homologs to titin in Caenorhabditis elegans were considered to be twitchin and UNC-89, both located in A-bands. Twitchin is a 754,000 Da polypeptide encoded by the unc-22 gene,24., 25. and consists of a single MLCK-like protein kinase domain and multiple copies of FnIII and Ig domains. unc-22 mutants have a characteristic “twitching” phenotype and variably disorganized muscle structure.²⁶ Twitchin is localized to the outer portions of the muscle A-bands.²⁷ Studies on Aplysia and Mytilus twitchin suggest that twitchin inhibits the rate of relaxation and that cAMP-dependent phosphorylation of twitchin relieves this inhibition.28., 29. The second titin-like protein in nematode muscle is UNC-89, a 732,000 Da polypeptide composed of many Ig domains, a KSP-containing phosphorylation domain, and near its N terminus, several domains (SH3, DH and PH) that are implicated in signal transduction.³⁰ Mutants in unc-89 move nearly as fast as wild-type, but are thinner and more transparent.²⁶ Muscle structure is disorganized, and for most mutant alleles, there are no M-lines.26., 31. Antibodies to UNC-89 localize the protein to the middle of A-bands,³⁰ a region that contains the M-line.

We now report that there is a third set of molecules, encoded by the same gene in C. elegans, that are more similar to mammalian titin, and these molecules are located in the I-band. The largest isoform of Ce titin, approximately 2.2 MDa, resembles twitchin and UNC-89 in that it contains multiple Ig (56) and FnIII (11) domains, and a single MLCK-like protein kinase. But, in addition, the 2.2 MDa Ce titin contains four classes of short repeat motifs, three of which are arranged in many tandem copies. One of these tandem repeat regions is similar to the elastic PEVK domain of vertebrate titin. This suggests that the repeat motifs in Ce titin are also elastic. An interesting feature of the two largest Ce titins, is that in contrast to other members of the twitchin/titin family, there are multiple regions that are predicted to form coiled-coil structure. Anti-Ce titin antibodies localize to muscle I-bands beginning at the L2–L3 larval stages and this pattern continues into adult muscle. Our data indicate that Ce titins have no role in the initial assembly of thick filaments, and either a minor role or no role in the assembly of I-bands. The Ce titin protein kinase domain is most similar to the kinase domains of the twitchins and projectin. The Ce titin kinase has protein kinase activity in vitro, and this activity is regulated by an unusual mechanism.