Twitchin kinase interacts with MAPKAP kinase 2 in Caenorhabditis elegans striated muscle

Abstract

In Caenorhabditis elegans, twitchin is a giant polypeptide located in muscle A-bands. The protein kinase of twitchin is autoinhibited by 45 residues upstream (NL) and 60 residues downstream (CRD) of the kinase catalytic core. Molecular dynamics simulation on a twitchin fragment revealed that the NL is released by pulling force. However, it is unclear how the CRD is removed. To identify proteins that may remove the CRD, we performed a yeast two-hybrid screen using twitchin kinase as bait. One interactor is MAK-1, C. elegans orthologue of MAPKAP kinase 2. MAPKAP kinase 2 is phosphorylated and activated by p38 MAP kinase. We demonstrate that the CRD of twitchin is important for binding to MAK-1. mak-1 is expressed in nematode body wall muscle, and antibodies to MAK-1 localize between and around Z-disk analogues and to the edge of A-bands. Whereas unc-22 mutants are completely resistant, mak-1 mutants are partially resistant to nicotine. MAK-1 can phosphorylate twitchin NL-Kin-CRD in vitro. Genetic data suggest the involvement of two other mak-1 paralogues and two orthologues of p38 MAP kinase. These results suggest that MAK-1 is an activator of twitchin kinase and that the p38 MAP kinase pathway may be involved in the regulation of twitchin.

FIGURE 1:

Identification of MAK-1 as a binding partner for twitchin kinase. (A) Schematic representation of domains in twitchin, and segment used to search for interacting molecules. Purple, Ig domains; green, Fn3 domains. The Ig25-Fn31-kinase segment of twitchin was used to screen a yeast two-hybrid library, and among the positive preys recovered were two clones representing K08F8.1, later renamed MAK-1 (MAP kinase–associated kinase). (B) Domain organization of MAK-1 and homologous proteins in C. elegans and humans. MAK-1 is 521 aa, and the only recognizable domain is a protein kinase domain, most homologous (53% identical) to the protein kinase domain of human MAPKAP kinase 2. The two protein kinases most similar to MAK-1 from C. elegans are MAK-2 and MNK-1. The kinase domains of these proteins are, respectively, 52 and 31% identical in sequence to the kinase domain of MAK-1. (C) MAK-1 (81–405) is the minimum region for binding twitchin kinase, and the 2 MAK-1 paralogues do not interact by yeast two-hybrid assays. The indicated segments of MAK-1, MAK-2b, and MNK-1a were tested for interaction with the indicated region of twitchin. (D, E) MAK-1 interacts with twitchin kinase but not two related giant kinases, and the C-terminal region of twitchin kinase is important for binding to MAK-1. (D) By yeast two-hybrid analysis, interaction of MAK-1 with twitchin kinase is specific; the homologous region from the other giant proteins, TTN-1 and UNC-89 (PK2), fail to interact. In addition, the upstream Ig and Fn3 domains of twitchin are not required for interaction with MAK-1. (E) A series of twitchin/TTN-1 chimeras was constructed and assayed for interaction with MAK-1. As indicated, the C-terminal ∼140 residues of twitchin, including the end of the kinase catalytic core and most of the CRD, is crucial for this interaction. In C–E, to the right of each row are images of yeast growth of three independent colonies on plates lacking adenine (–Ade; C–E) and on plates lacking histidine (–His; E).

FIGURE 2:

Twitchin CRD is important for binding to MAK-1. (A) A far-Western blot was carried out in which the indicated His-tagged fragments of twitchin kinase were separated on a gel, transferred to a blot, and incubated with MBP-MAK-1 or MBP, with binding detected with antibodies to MBP. As shown, MBP-MAK-1 but not MBP binds to the twitchin fragments. However, binding is diminished when the CRD is missing. (B) A pull-down assay was conducted in which the indicated His-tagged fragments of twitchin kinase were incubated in solution with either MBP or MBP-MAK-1 and the proteins pulled down using anti-His antibody–conjugated beads; after separation on a gel and transfer to a membrane, MBP-MAK-1 or MBP was detected using anti-MBP conjugated to HRP and ECL. Note that only MBP-MAK-1 and not MBP was detected, and the amount of MBP-MAK-1 pulled down was diminished when CRD was missing from the twitchin fragments. Below the Western reaction blot is shown the blot stained with Ponceau S before incubation with antibody. The bands at 50 and 25 kDa represent the heavy and light chains of IgG, respectively.

FIGURE 3:

mak-1 expression pattern, mutations, and MAK-1 antibodies. (A) The mak-1 gene is expressed in the intestine and body wall muscle. To detect mak-1–expressing cells, we created transgenic worms expressing GFP from a 6.4-kb segment of genomic DNA upstream of the mak-1 predicted translational start. GFP was detected in the intestine (left) and body wall muscle cells (right) at the adult stage. Scale bar, 25 μm. (B, C) mak-1 mutation sites and effects on MAK-1 protein expression. (B) Schematic representation of the mak-1 gene, with boxes denoting exons, and introns denoting introns, the approximate spans of each deletion represented, and the sequence alterations shown below. mak-1(ok2987) is a 754–base pair deletion, essentially as depicted on WormBase. We determined that mak-1(tm3455) has both a 7–base pair insertion and a 267–base pair deletion extending from the 3′ end of exon 3 until the 5′ end of intron 4. (C) Location of immunogens used to generate antibodies to MAK-1 and Western blot analysis of wild-type and mak-1 mutants. Antibodies raised to either an N-terminal or a C-terminal region detect an ∼60-kDa protein, the size expected for MAK-1. For the anti–N-terminal antibody, note that on short exposure, MAK-1 cannot be detected from the two mutant alleles. On longer exposure, two novel bands are detected from tm3455, likely representing truncated MAK-1 polypeptides (indicated by arrows). On longer exposure, no protein is detected from ok2987, other than bands found from all three strains likely arising from cross-reactivity to bacterial products. For the anti–C-terminal antibody, MAK-1 proteins cannot be detected even after long exposure time.

FIGURE 4:

Localization of MAK-1 in wild type and in mak-1 and unc-22 mutants. (A) Portion of an adult body wall muscle cell from wild type and each of the mak-1 mutants immunostained with the anti–C-terminal MAK-1 antibody and costained with anti–PAT-6 (α-parvin), which marks M-lines (denoted by an arrow) and dense bodies (denoted by an arrow head). Anti–MAK-1 localizes between and around dense bodies (top) in striated muscle cells. This localization was not detected in mak-1 mutant animals (middle and bottom). (B) Portion of a wild-type body wall muscle cell immunostained with anti–MAK-1 and anti–MHC B. Anti–MAK-1 partially colocalizes with anti–MHC B at the outer edges of A-bands. (C) Portion of an adult body wall muscle cell from two unc-22 mutant alleles, e105 and e66, immunostained with anti–MAK-1 and anti–PAT-6. The localization of MAK-1 is not affected by the presence of a mutant form of UNC-22 (twitchin). Scale bars, 10 μm.

FIGURE 5:

Phenotype of mak-1 mutants. (A) A mak-1 mutant has slightly reduced locomotion. Results of swimming assays on adult nematodes of the indicated genotypes. Data are means and SEs with n = 20. Although mak-1(tm3455) animals show almost the same motility as wild-type animals, mak-1(ok2987) animals show slightly reduced motility that is statistically significant. The unc-22–null allele, ct37, and the unc-22 canonical allele, e66, are much slower than wild type. In contrast, unc-22(e105) shows normal or even increased locomotion in this assay. (B) mak-1(ok2987) shows normal sarcomeric structure. Images show part of a body wall muscle cell from either wild type or mak-1(ok2987) immunostained with the indicated antibodies. UNC-52 (perlecan), UNC-112, and UNC-95 all localize to M-lines and dense bodies; UNC-52 in the extracellular matrix; and UNC-112 and UNC-95 in the muscle cytoplasm close to the cell membrane. ATN-1 (α-actinin) localizes to the main, deeper portion of the dense bodies. UNC-89 (obscurin) is located at M-lines full depth, from muscle cell membrane, up through the deepest part of the myofilament lattice. Twitchin localizes to the outer portions of A-bands, and MHC A localizes to the middle of A-bands. For all these proteins, localization is no different in mak-1(ok2987) muscle than in wild-type muscle. Scale bar, 10 μm.

FIGURE 6:

Sarcomeres are disorganized in unc-22(e66) but normal in unc-22(e105). Each image shows part of a body wall muscle cell from wild type, unc-22(e66), or unc-22(e105). Some of the same antibodies described in Figure 8 were used, and, in addition, antibodies to MHC B, which localizes to the outer portions of A-bands. In unc-22(e66) muscle, all of these proteins show abnormal localization as compared with wild type. In unc-22(e105), all of these proteins show normal localization. Scale bar, 10 μm.

FIGURE 7:

Response of wild-type, unc-22, and mak-1 mutant strains to nicotine. A device called a WMicrotracker (DesignPlus) was used to monitor the locomotion of multiple worms per well in a microtiter dish over time during exposure to a solution of 0.1 or 0.05% nicotine. (A) Response of wild-type and three unc-22-mutant alleles. In 0.1% nicotine, wild-type animals nearly stop moving by 30 min. In contrast, unc-22–mutant animals, regardless of allele, continue moving even after 60 min. (B) Response of wild-type, unc-22(e105), and two mak-1–mutant alleles. In 0.1% nicotine, after 60 min of exposure, wild-type animals stop moving, whereas unc-22(e105) animals continue to move. mak-1(ok2987), the putative null allele of mak-1, but not mak-1 (tm3445), shows an intermediate response; that is, ok2987 is partially resistant to nicotine. (C) At a slightly lower concentration of nicotine (0.05%), even mak-1(tm3445) shows partial resistance to nicotine. (D) In 0.1% nicotine, wild-type animals fed bacteria with an empty RNAi plasmid stop moving, but wild-type animals fed bacteria with an unc-22(RNAi) plasmid continue to move after 60 min exposure. Whereas mak-1(ok2987) animals fed bacteria with an empty RNAi plasmid show an intermediate response, mak-1(ok2987) animals fed bacteria with an unc-22 RNAi plasmid continue to move. Therefore unc-22 is epistatic to mak-1.

FIGURE 8:

Adult expression patterns of mnk-1 and mak-2 promoters. (A) On WormBase there are two predicted mnk-1 splicing patterns, mnk-1a and mnk-1b. To detect mnk-1a–expressing cells, transgenic worms were created that express GFP from a 3.7-kb segment of genomic DNA upstream of the mnk-1a predicted translational start. GFP was detected in the pharynx, intestine, vulva muscle, and body wall muscle at the adult stage. To detect mnk-1b–expressing cells, transgenic worms were made that expressed GFP from a 4.8-kb segment of DNA upstream of the mnk-1b translational start. GFP was detected in the pharynx, including pharyngeal muscle, intestine, vulva muscle, and body wall muscle. (B) To detect mak-2–expressing cells, transgenic worms were made that expressed GFP from a 3.9-kb genomic segment upstream of the mak-2 predicted translational start. Expression was detected in pharynx, intestine, and body wall muscle. Scale bar, 25 μm.

FIGURE 9:

Response of mnk-1, mak-2, pmk-1, and pmk-3 mutants to nicotine. The WMicrotracker was used monitor the locomotion of the indicated strains to the indicated concentrations of nicotine. (A, B) Response of wild type, mak-1(ok2987), mnk-1(tm4266), and mak-2(gk1110) to 0.05 and 0.1% nicotine, respectively. Note that mnk-1 and mak-2 are partially resistant to nicotine, although to a lesser extent than mak-1. (C,D) Response of wild type and mak-1, mak-1; mak-2 and mak-1; mnk-1 double mutants to 0.05 and 0.1% nicotine, respectively. Note that the mak-1; mak-2 double mutant shows enhanced resistance compared with either mak-1 alone or the mak-1; mnk-1 double mutant. (E, F) Response of wild type, mak-1, the mak-1; mak-2 double, and unc-22(e105) to 0.05 and 0.1% nicotine, respectively. Although the mak-1; mak-2 double is more resistant than mak-1, it is less resistant than unc-22 (e105). (G, H) Response of wild type, mak-1, pmk-1(km25), and pmk-3(ok169) to 0.05 and 0.1% nicotine, respectively. pmk-1 and pmk-3 are also partially resistant but consistently less resistant than mak-1.

FIGURE 10:

MAK-1 phosphorylates twitchin kinase in vitro. (A) A protein kinase assay was conducted using MAK-1 (81–405) as the source of kinase and catalytically dead (K to A) twitchin NL-Kin-CRD as substrate. As expected, NL-Kin-CRD (K to A) does not show autophosphorylation (on autoradiogram, first lane from the left). However, MAK-1 (81–405) shows autophosphorylation (second lane from the left), and MAK-1 phosphorylates NL-Kin-CRD (K to A; third lane from the left, indicated with an asterisk). (B) Protein kinase assays were conducted using MAK-1 (81–405) as the source of kinase and various deletion derivatives of twitchin NL-Kin-CRD (K to A) as substrate. As shown by an asterisk, only NL-Kin-CRD was phosphorylated. For A and B the autoradiogram is shown on the left, and the Coomassie brilliant blue (CBB)–stained gel is shown on the right. (C) SDS–PAGE of 2 μg each of the proteins used in the kinase assays, stained with CBB. Each protein has an N-terminal hexahistidine tag. The positions of molecular weight markers are indicated.

FIGURE 11:

Speculative model for the function of MAK-1 in striated muscle cells. In vitro kinase assays of twitchin kinase show that NL and CRD each inhibits kinase activity by one-half. Molecular dynamic simulations indicate that the mechanically sensitive portion is NL, 45 residues lying between the Fn3 domain and the catalytic core, and that the CRD remains attached to the large lobe of the kinase even after the small lobe has been unwound (von Castelmur et al., 2012). Therefore MAK-1 might contribute to the removal of the CRD segment from the catalytic core by binding or by phosphorylation. The nematode MAK-1 paralogues, MNK-1 and MAK-2, are also expressed in muscle, and each alone and the two in combination show partial resistance to nicotine. Thus MNK-1 and MAK-2, in addition to MAK-1, may be involved in activation. MAPKAP kinase 2 in mammals is activated by phosphorylation by p38 MAP kinase. The nematode orthologues of p38 MAP kinase include PMK-1 and -3. Their null mutants show an intermediate resistance to nicotine. Therefore it is possible that PMK-1 and -3 are also upstream components of this relaxation pathway. Rather than full activation, it is possible that partial activation by force and partial activation by MAK-1 occur independently to meet different physiological requirements.