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

Matsunaga Y, Qadota H, Furukawa M, Choe HH, Benian GM - Mol. Biol. Cell (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Emory University, Atlanta, GA 30322.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: Although SAGE data indicate that mak-1 is expressed in body wall muscle (Meissner et al., 2009; Supplemental Table S1), we sought additional evidence for this expression pattern. A 6.4-kb genomic fragment that includes the putative promoter sequence upstream of mak-1, a putative 5′-untranslated region, initiator ATG, and the first 12 nucleotides of coding sequence of mak-1 was fused to green fluorescent protein (GFP), and transgenic worms carrying this segment were generated. This mak-1 promoter reporter is expressed in body wall muscle and intestine (Figure 3A), as well as in hypodermis (unpublished data).


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

Matsunaga Y, Qadota H, Furukawa M, Choe HH, Benian GM - Mol. Biol. Cell (2015)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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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.
Mentions: Although SAGE data indicate that mak-1 is expressed in body wall muscle (Meissner et al., 2009; Supplemental Table S1), we sought additional evidence for this expression pattern. A 6.4-kb genomic fragment that includes the putative promoter sequence upstream of mak-1, a putative 5′-untranslated region, initiator ATG, and the first 12 nucleotides of coding sequence of mak-1 was fused to green fluorescent protein (GFP), and transgenic worms carrying this segment were generated. This mak-1 promoter reporter is expressed in body wall muscle and intestine (Figure 3A), as well as in hypodermis (unpublished data).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Emory University, Atlanta, GA 30322.

No MeSH data available.


Related in: MedlinePlus