Sarcomeres of probably all animals contain a number of extraordinarily large polypeptides (700 kDa - 4 MDa) composed of multiple Ig and Fn3 domains, one or two protein kinase domains, and in some cases, elastic regions. C. elegans muscle contains three such giant proteins: twitchin (754 kDa) in the polar regions of the A-band, UNC-89 (up to 900 kDa) in the middle of the A-band, and TTN-1 (2.2 MDa) in the I-band. Twitchin is encoded by
unc-22, which has a characteristic mutant phenotype: "twitching" of the worm''s surface that originates from the underlying muscle. When wild type animals are exposed to the acetylcholine agonist nicotine, they become paralyzed, whereas, when
unc-22 mutants twitch violently. The protein kinase domain shows protein kinase activity in vitro and is autoinhibited by the 60 residues C-terminal of the catalytic core. Recent molecular dynamics simulations and AFM experiments suggest that this normally autoinhibited kinase is activated by small forces that occur during muscle contraction/relaxation cycles (Greene et al. 2008). Despite this information, substrates or binding partners for the twitchin kinase domain were unknown. We screened a yeast 2-hybrid library using as bait, Ig25-Fn31-kinase of twitchin, and recovered clones representing K08F8.1. K08F8.1 encodes a protein homologous to mammalian MAP kinase activated protein kinase 2. There are, in fact, 3 MAPKAP kinase 2 homologs in C. elegans: K08F8.1, MAK-2 and MNK-1. Homology among the human and worm proteins resides only within the kinase domains; in this region, K08F8.1 is 53% identical to human MAPKAP kinase 2. SAGE data on WormBase indicates that only K08F8.1 is highly expressed in body wall muscle. Yeast 2-hybrid domain mapping shows that the twitchin / K08F8.1 interaction: (1) requires only the kinase domain of twitchin; and (2) residues 60-405 (essentially the kinase domain) of K08F8.1 are minimally required. Comparable regions of TTN-1 and UNC-89 do not interact with K08F8.1, and preliminarily, MAK-2 and MNK-1 fail to interact with twitchin. Luckily, two independently generated intragenic deletions of K08F8.1,
ok2987 and
tm3455, are available. By swimming assay, the motility of
ok2987 is 14% lower than wild type. Significantly, both
ok2987 and
tm3455 are hypersensitive to nicotine (they become paralyzed at a faster rate than wild type). The abnormal response to nicotine suggests that K08F8.1 acts in the same pathway as twitchin.