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A role for p38 MAPK in the regulation of ciliary motion in a eukaryote.

Ressurreição M, Rollinson D, Emery AM, Walker AJ - BMC Cell Biol. (2011)

Bottom Line: Western blotting and immunocytochemistry demonstrated that treatment of miracidia with the p38 MAPK activator anisomycin resulted in a rapid, sustained, activation of p38 MAPK, which was primarily localized to the cilia associated with the ciliated epidermal plates, and the tegument.Strikingly, anisomycin-mediated p38 MAPK activation rapidly attenuated swimming, reducing swim velocities by 55% after 15 min and 99% after 60 min.Finally, by inhibiting swimming, p38 MAPK activation resulted in early release of ciliated epidermal plates from the miracidium thus accelerating development to the post-miracidium larval stage.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Life Sciences, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, UK.

ABSTRACT

Background: Motile cilia are essential to the survival and reproduction of many eukaryotes; they are responsible for powering swimming of protists and small multicellular organisms and drive fluids across respiratory and reproductive surfaces in mammals. Although tremendous progress has been made to comprehend the biochemical basis of these complex evolutionarily-conserved organelles, few protein kinases have been reported to co-ordinate ciliary beat. Here we present evidence for p38 mitogen-activated protein kinase (p38 MAPK) playing a role in the ciliary beat of a multicellular eukaryote, the free-living miracidium stage of the platyhelminth parasite Schistosoma mansoni.

Results: Fluorescence confocal microscopy revealed that non-motile miracidia trapped within eggs prior to hatching displayed phosphorylated (activated) p38 MAPK associated with their ciliated surface. In contrast, freshly-hatched, rapidly swimming, miracidia lacked phosphorylated p38 MAPK. Western blotting and immunocytochemistry demonstrated that treatment of miracidia with the p38 MAPK activator anisomycin resulted in a rapid, sustained, activation of p38 MAPK, which was primarily localized to the cilia associated with the ciliated epidermal plates, and the tegument. Freshly-hatched miracidia possessed swim velocities between 2.17 - 2.38 mm/s. Strikingly, anisomycin-mediated p38 MAPK activation rapidly attenuated swimming, reducing swim velocities by 55% after 15 min and 99% after 60 min. In contrast, SB 203580, a p38 MAPK inhibitor, increased swim velocity by up to 15% over this duration. Finally, by inhibiting swimming, p38 MAPK activation resulted in early release of ciliated epidermal plates from the miracidium thus accelerating development to the post-miracidium larval stage.

Conclusions: This study supports a role for p38 MAPK in the regulation of ciliary-beat. Given the evolutionary conservation of signalling processes and cilia structure, we hypothesize that p38 MAPK may regulate ciliary beat and beat-frequency in a variety of eukaryotes.

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Sequence alignment and domain analysis of S. mansoni p38 MAPK. The S. mansoni putative p38 MAPK sequence (XP_002571000) was aligned with those for S. japonicum MAPK14a, Homo sapiens (p38 MAPKα, also known as MAPK14 isoform 1), Drosophila melanogaster (p38a MAPK and p38b MAPK), Caenorhabditis elegans (p38 MAPK family member, PMK1) and Danio rerio (MAPK 14a); accession numbers are shown. Multiple alignments were achieved using Geneious Pro 4.85 with Blosum62 cost matrix and default settings; shading of residues: black = 100% similar, dark grey = 80-100% similar, light grey = 60-80% similar and white < 60% similar. The ATP binding site, kinase interacting motif (KIM) docking site, and activation loop are indicated by coloured arrows. Within the activation loop, the conserved Thr-Gly-Tyr (TGY) phosphorylation motif is highlighted by the yellow box, with the phosphorylated residues (Thr and Tyr) central to kinase activation indicated with green asterisks; the substrate binding site is shown by the orange box. Sequence highlighted by the blue box is that used to generate the monoclonal anti-phospho p38 MAPK antibodies. The residues within the ATP binding site that are known to confer specificity and sensitivity of SB 230580 towards p38 MAPK are highlighted by the red box with the residue (Thr) most responsible for p38 MAPK inhibition indicated by the red asterisk; red crosses denote other known interaction sites between SB 203580 and p38 MAPK (see text for further details).
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Figure 1: Sequence alignment and domain analysis of S. mansoni p38 MAPK. The S. mansoni putative p38 MAPK sequence (XP_002571000) was aligned with those for S. japonicum MAPK14a, Homo sapiens (p38 MAPKα, also known as MAPK14 isoform 1), Drosophila melanogaster (p38a MAPK and p38b MAPK), Caenorhabditis elegans (p38 MAPK family member, PMK1) and Danio rerio (MAPK 14a); accession numbers are shown. Multiple alignments were achieved using Geneious Pro 4.85 with Blosum62 cost matrix and default settings; shading of residues: black = 100% similar, dark grey = 80-100% similar, light grey = 60-80% similar and white < 60% similar. The ATP binding site, kinase interacting motif (KIM) docking site, and activation loop are indicated by coloured arrows. Within the activation loop, the conserved Thr-Gly-Tyr (TGY) phosphorylation motif is highlighted by the yellow box, with the phosphorylated residues (Thr and Tyr) central to kinase activation indicated with green asterisks; the substrate binding site is shown by the orange box. Sequence highlighted by the blue box is that used to generate the monoclonal anti-phospho p38 MAPK antibodies. The residues within the ATP binding site that are known to confer specificity and sensitivity of SB 230580 towards p38 MAPK are highlighted by the red box with the residue (Thr) most responsible for p38 MAPK inhibition indicated by the red asterisk; red crosses denote other known interaction sites between SB 203580 and p38 MAPK (see text for further details).

Mentions: As with other MAPKs, p38 MAPK has been highly conserved during metazoan evolution [32]. Recently, the draft genomes for S. mansoni and Schistosoma japonicum were published [33,34] allowing S. mansoni p38 MAPK gene candidates to be identified and further assessed for similarity to p38 MAPKs from other organisms, including closely related schistosomes (Figure 1). For S. mansoni only a single putative p38 MAPK was identified and only partial cDNA reads were found; moreover, only one p38 MAPK was found in S. japonicum (Figure 1). Thus, in contrast to D. melanogaster and human which possess two and four p38 MAPKs respectively [32,35,36], it seems that schistosomes may possess one p38 MAPK orthologue only. Based on the partial sequence data (XP_002571000) spanning 74 amino acids, and supported by more complete data from S. japonicum, the identified S. mansoni p38 MAPK was most similar to p38α MAPK (MAPK 14) of humans, the most evolutionarily-conserved p38 MAPK [35]. In addition, there exist a number of putative exons on two separate scaffolds (Smp_scaff000038 and Smp_scaff06141) additional to the 74 amino acid fragment that, when translated, also match closely with the S. japonicum sequence; thus, we assume given the phylogenetic proximity between the two species that S. mansoni p38 MAPK is very similar to S. japonicum p38 MAPK. As for all other p38 MAPKs in the p38 subfamily, the dual phosphorylation site in the S. mansoni p38 MAPK activation loop reads Thr-Gly-Tyr (TGY)(Figure 1). The substrate binding site Ala-Thr-Arg-Trp (ATRW) is also conserved, as is the kinase interaction motif (KIM) docking site (Figure 1) which binds linear KIM sequences present in substrates and MAPK phosphatases [37]. Pair wise comparisons of the S. mansoni p38 MAPK fragment with corresponding sequences for other organisms revealed ~69-70% similarity with human, Drosophila melanogaster, Caenorhabditis elegans, or Danio rerio, and 86.5% with S. japonicum.


A role for p38 MAPK in the regulation of ciliary motion in a eukaryote.

Ressurreição M, Rollinson D, Emery AM, Walker AJ - BMC Cell Biol. (2011)

Sequence alignment and domain analysis of S. mansoni p38 MAPK. The S. mansoni putative p38 MAPK sequence (XP_002571000) was aligned with those for S. japonicum MAPK14a, Homo sapiens (p38 MAPKα, also known as MAPK14 isoform 1), Drosophila melanogaster (p38a MAPK and p38b MAPK), Caenorhabditis elegans (p38 MAPK family member, PMK1) and Danio rerio (MAPK 14a); accession numbers are shown. Multiple alignments were achieved using Geneious Pro 4.85 with Blosum62 cost matrix and default settings; shading of residues: black = 100% similar, dark grey = 80-100% similar, light grey = 60-80% similar and white < 60% similar. The ATP binding site, kinase interacting motif (KIM) docking site, and activation loop are indicated by coloured arrows. Within the activation loop, the conserved Thr-Gly-Tyr (TGY) phosphorylation motif is highlighted by the yellow box, with the phosphorylated residues (Thr and Tyr) central to kinase activation indicated with green asterisks; the substrate binding site is shown by the orange box. Sequence highlighted by the blue box is that used to generate the monoclonal anti-phospho p38 MAPK antibodies. The residues within the ATP binding site that are known to confer specificity and sensitivity of SB 230580 towards p38 MAPK are highlighted by the red box with the residue (Thr) most responsible for p38 MAPK inhibition indicated by the red asterisk; red crosses denote other known interaction sites between SB 203580 and p38 MAPK (see text for further details).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3040701&req=5

Figure 1: Sequence alignment and domain analysis of S. mansoni p38 MAPK. The S. mansoni putative p38 MAPK sequence (XP_002571000) was aligned with those for S. japonicum MAPK14a, Homo sapiens (p38 MAPKα, also known as MAPK14 isoform 1), Drosophila melanogaster (p38a MAPK and p38b MAPK), Caenorhabditis elegans (p38 MAPK family member, PMK1) and Danio rerio (MAPK 14a); accession numbers are shown. Multiple alignments were achieved using Geneious Pro 4.85 with Blosum62 cost matrix and default settings; shading of residues: black = 100% similar, dark grey = 80-100% similar, light grey = 60-80% similar and white < 60% similar. The ATP binding site, kinase interacting motif (KIM) docking site, and activation loop are indicated by coloured arrows. Within the activation loop, the conserved Thr-Gly-Tyr (TGY) phosphorylation motif is highlighted by the yellow box, with the phosphorylated residues (Thr and Tyr) central to kinase activation indicated with green asterisks; the substrate binding site is shown by the orange box. Sequence highlighted by the blue box is that used to generate the monoclonal anti-phospho p38 MAPK antibodies. The residues within the ATP binding site that are known to confer specificity and sensitivity of SB 230580 towards p38 MAPK are highlighted by the red box with the residue (Thr) most responsible for p38 MAPK inhibition indicated by the red asterisk; red crosses denote other known interaction sites between SB 203580 and p38 MAPK (see text for further details).
Mentions: As with other MAPKs, p38 MAPK has been highly conserved during metazoan evolution [32]. Recently, the draft genomes for S. mansoni and Schistosoma japonicum were published [33,34] allowing S. mansoni p38 MAPK gene candidates to be identified and further assessed for similarity to p38 MAPKs from other organisms, including closely related schistosomes (Figure 1). For S. mansoni only a single putative p38 MAPK was identified and only partial cDNA reads were found; moreover, only one p38 MAPK was found in S. japonicum (Figure 1). Thus, in contrast to D. melanogaster and human which possess two and four p38 MAPKs respectively [32,35,36], it seems that schistosomes may possess one p38 MAPK orthologue only. Based on the partial sequence data (XP_002571000) spanning 74 amino acids, and supported by more complete data from S. japonicum, the identified S. mansoni p38 MAPK was most similar to p38α MAPK (MAPK 14) of humans, the most evolutionarily-conserved p38 MAPK [35]. In addition, there exist a number of putative exons on two separate scaffolds (Smp_scaff000038 and Smp_scaff06141) additional to the 74 amino acid fragment that, when translated, also match closely with the S. japonicum sequence; thus, we assume given the phylogenetic proximity between the two species that S. mansoni p38 MAPK is very similar to S. japonicum p38 MAPK. As for all other p38 MAPKs in the p38 subfamily, the dual phosphorylation site in the S. mansoni p38 MAPK activation loop reads Thr-Gly-Tyr (TGY)(Figure 1). The substrate binding site Ala-Thr-Arg-Trp (ATRW) is also conserved, as is the kinase interaction motif (KIM) docking site (Figure 1) which binds linear KIM sequences present in substrates and MAPK phosphatases [37]. Pair wise comparisons of the S. mansoni p38 MAPK fragment with corresponding sequences for other organisms revealed ~69-70% similarity with human, Drosophila melanogaster, Caenorhabditis elegans, or Danio rerio, and 86.5% with S. japonicum.

Bottom Line: Western blotting and immunocytochemistry demonstrated that treatment of miracidia with the p38 MAPK activator anisomycin resulted in a rapid, sustained, activation of p38 MAPK, which was primarily localized to the cilia associated with the ciliated epidermal plates, and the tegument.Strikingly, anisomycin-mediated p38 MAPK activation rapidly attenuated swimming, reducing swim velocities by 55% after 15 min and 99% after 60 min.Finally, by inhibiting swimming, p38 MAPK activation resulted in early release of ciliated epidermal plates from the miracidium thus accelerating development to the post-miracidium larval stage.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Life Sciences, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, UK.

ABSTRACT

Background: Motile cilia are essential to the survival and reproduction of many eukaryotes; they are responsible for powering swimming of protists and small multicellular organisms and drive fluids across respiratory and reproductive surfaces in mammals. Although tremendous progress has been made to comprehend the biochemical basis of these complex evolutionarily-conserved organelles, few protein kinases have been reported to co-ordinate ciliary beat. Here we present evidence for p38 mitogen-activated protein kinase (p38 MAPK) playing a role in the ciliary beat of a multicellular eukaryote, the free-living miracidium stage of the platyhelminth parasite Schistosoma mansoni.

Results: Fluorescence confocal microscopy revealed that non-motile miracidia trapped within eggs prior to hatching displayed phosphorylated (activated) p38 MAPK associated with their ciliated surface. In contrast, freshly-hatched, rapidly swimming, miracidia lacked phosphorylated p38 MAPK. Western blotting and immunocytochemistry demonstrated that treatment of miracidia with the p38 MAPK activator anisomycin resulted in a rapid, sustained, activation of p38 MAPK, which was primarily localized to the cilia associated with the ciliated epidermal plates, and the tegument. Freshly-hatched miracidia possessed swim velocities between 2.17 - 2.38 mm/s. Strikingly, anisomycin-mediated p38 MAPK activation rapidly attenuated swimming, reducing swim velocities by 55% after 15 min and 99% after 60 min. In contrast, SB 203580, a p38 MAPK inhibitor, increased swim velocity by up to 15% over this duration. Finally, by inhibiting swimming, p38 MAPK activation resulted in early release of ciliated epidermal plates from the miracidium thus accelerating development to the post-miracidium larval stage.

Conclusions: This study supports a role for p38 MAPK in the regulation of ciliary-beat. Given the evolutionary conservation of signalling processes and cilia structure, we hypothesize that p38 MAPK may regulate ciliary beat and beat-frequency in a variety of eukaryotes.

Show MeSH
Related in: MedlinePlus