Twitchin kinase inhibits muscle activity

Abstract

Muscle sarcomeres contain giant polypeptides composed of multiple immunoglobulin and fibronectin domains and one or two protein kinase domains. Although binding partners for a number of this family's kinase domains have been identified, the catalytic necessity of these kinase domains remains unknown. In addition, various members of this kinase family are suspected pseudokinases with no or little activity. Here we address catalytic necessity for the first time, using the prototypic invertebrate representative twitchin (UNC-22) from Caenorhabditis elegans In in vitro experiments, change of a conserved lysine (K) that is involved in ATP coordination to alanine (A) resulted in elimination of kinase activity without affecting the overall structure of the kinase domain. The same mutation, unc-22(sf21), was generated in the endogenous twitchin gene. The unc-22(sf21) worms have well-organized sarcomeres. However, unc-22(sf21) mutants move faster than wild-type worms and, by optogenetic experiments, contract more. Wild-type nematodes exhibited greater competitive fitness than unc-22(sf21) mutants. Thus the catalytic activity of twitchin kinase has a role in vivo, where it inhibits muscle activity and is likely maintained by selection.

FIGURE 1:

Mutation of a lysine involved in ATP coordination/phosphotransfer to alanine (K6290A) eliminates catalytic activity but has no effect on the overall structure of twitchin kinase in vitro. (A) The wild-type (wt) and the K6290A mutant forms of the catalytic domain of twitchin kinase (TwcK) were each expressed and purified from E. coli. The ability to phosphorylate a model peptide was measured with [γ-³²P]ATP. As shown, the K6290A mutation almost entirely eliminates catalytic activity. (B) Size exclusion chromatograms of wt and mutant forms of a larger construct, TwcKR (Fn-NL-kin-CRD-Ig). TwcKR wt and TwcKR KtoA samples are coincident within experimental error, supporting the structural similarity of the samples. (C) SAXS analysis shows that the K6290A mutation does not affect the overall structure of the twitchin kinase region (TwcKR), so that the arrangement of assembled domains remains unaltered. Experimental scattering curves from TwcKR wt (black circle; capped error bars) and TwcKR K6290A (orange triangles; uncapped error bars) were scaled using DATADJUST (Petoukhov et al., 2012). The table below the graph lists molecular parameters calculated from the scattering data. MM, R_g, and D_max denote molecular mass, radius of gyration, and maximal particle size, respectively. MM^calc is the theoretical MM of the constructs computed from primary sequence. R_g was calculated using AUTORG (Petoukhov et al., 2007).

FIGURE 2:

Nematodes expressing twitchin with the K6290A mutation in its kinase domain show normal muscle sarcomere structure, including normal localization of twitchin, by immuno­fluorescence microscopy. Wild-type (WT) and unc-22(sf21) mutant nematodes were fixed and immunostained with antibodies to the indicated sarcomeric proteins: twitchin, myosin heavy chain A (MYO-3), and myosin heavy chain B (UNC-54) of the A-bands and UNC-95 of M-lines and dense bodies. Phalloidin staining of F-actin of I-bands is also shown. As indicated, unc-22(sf21) shows normal localization of each sarcomeric protein tested, including twitchin. Scale bars, 20 μm.

FIGURE 3:

The unc-22(sf21) mutant shows normal sarcomere structure by EM. Transmission EM of a cross-section of a body-wall muscle cell from a wild-type (WT) animal and from an unc-22(sf21) mutant animal. Top, across most of the width of a single cell near the vulva, there is uniform depth of the contractile apparatus along the body wall and regular spacing of the filament attachment structures, and the thick and thin filaments are all cut in cross-section, demonstrating correct orientation of the filaments parallel to the body axis. Bottom, higher-magnification views show proper morphology and organization of attachment structures and interdigitating thick and thin filaments. Arrows point to dense bodies, and arrowheads point to M-lines. The largest black dots are cross-sections of thick filaments in the A-bands; the smallest dots are cross-sections of thin filaments. Scale bars, 0.5 μm.

FIGURE 4:

Nematodes lacking twitchin kinase activity move faster and show a greater degree of muscle contraction. (A) Results of a swimming assay in which the number of times an animal moves back and forth in liquid are counted. The slower motility of animals with the loss-of-function allele unc-22(e66) is typical of unc-22 mutants. Both unc-22(sf21) and unc-22(e105) animals display, unexpectedly, faster motility. (B) Measurement of crawling velocity of nematodes on the surface of agar; results are normalized to the length of the animal. Results are similar to those obtained for swimming. (C) Typical results of an optogenetic experiment to measure contraction and relaxation of wild-type nematodes. Relative body area is a measure of the contraction state of body-wall muscle. (D) Optogenetic experiments on wild type and three unc-22 mutants. Compared with wild-type animals, unc-22(sf21) and unc-22(e105) worms display a greater degree of muscle contraction, whereas unc-22(e66) worms show less muscle contraction and an inability to maintain the contracted state. (E, F) Rate constants for contraction (“E” in C) and for relaxation (“F” in C) derived from fitting curves to data shown in D. (G) Relative body area at steady state, which is the average relative body area during 5 s before the relaxation (“G” in C). These numbers emphasize that unc-22(sf21) and unc-22(e105) nematodes contract more than wild type. For A, B, and E–G, n > 40; *p < 0.01, **p < 0.001, and ***p < 0.0001.

FIGURE 5:

A competition assay demonstrates that the unc-22(sf21) mutant has a selective disadvantage relative to wild type. (A) Competition assay results. Equal numbers of GFP-tagged wild-type (“tester” strain, JK2735) and the indicated strains were cultured on plates with E. coli food, and after four generations, the frequency of each strain was determined. (B) Relative brood sizes of wild-type strain N2, unc-22(sf21), and GFP-tagged strain (JK2735). *Significance with p < 0.05.