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Overlapping roles of Drosophila Drak and Rok kinases in epithelial tissue morphogenesis.

Neubueser D, Hipfner DR - Mol. Biol. Cell (2010)

Bottom Line: Drak activity is largely redundant with that of the Drosophila ROCK orthologue, Rok, such that it is essential only when Rok levels are reduced.The lethality of drak/rok mutants can be rescued by restoring Sqh activity, indicating that Sqh is the critical common effector of these two kinases.These results provide the first evidence that DAPK family kinases regulate actin dynamics in vivo and identify Drak as a novel component of the signaling networks that shape epithelial tissues.

View Article: PubMed Central - PubMed

Affiliation: Institut de recherches cliniques de Montréal, Montreal, QC, Canada.

ABSTRACT
Dynamic regulation of cytoskeletal contractility through phosphorylation of the nonmuscle Myosin-II regulatory light chain (MRLC) provides an essential source of tension for shaping epithelial tissues. Rho GTPase and its effector kinase ROCK have been implicated in regulating MRLC phosphorylation in vivo, but evidence suggests that other mechanisms must be involved. Here, we report the identification of a single Drosophila homologue of the Death-associated protein kinase (DAPK) family, called Drak, as a regulator of MRLC phosphorylation. Based on analysis of mutants, we find that Drak broadly promotes proper morphogenesis of epithelial tissues during development. Drak activity is largely redundant with that of the Drosophila ROCK orthologue, Rok, such that it is essential only when Rok levels are reduced. We demonstrate that these two kinases synergistically promote phosphorylation of Spaghetti squash (Sqh), the Drosophila MRLC orthologue, in vivo. The lethality of drak/rok mutants can be rescued by restoring Sqh activity, indicating that Sqh is the critical common effector of these two kinases. These results provide the first evidence that DAPK family kinases regulate actin dynamics in vivo and identify Drak as a novel component of the signaling networks that shape epithelial tissues.

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Drak is the single Drosophila homologue of DAPK family kinases. (A) Multiple sequence alignment of Drosophila melanogaster Drak with human DRAK1 and DRAK2, ZIPK, and skeletal muscle MLCK (MYLK2). Purple, identical amino acids conserved in at least three of the five sequences. Black bar, limits of the conserved kinase domains. (B) Dendrogram showing relation of Drak and other Drosophila melanogaster MLCK-like proteins to human DAPK (blue) and MLCK family kinases, based on alignment of kinase domain sequences. Hs_MYLK1, 2, and 3 correspond to nonmuscle, skeletal muscle, and cardiac Myosin light chain kinases. (C and D) Developmental time course analysis of drak expression by RT-PCR. RNA was from staged embryos (C) and third instar larval (L3) and pupal (P) wing discs (D). Numbers in C represent ages of the embryos in hours after egg laying. drak primers amplified two bands representing unspliced (upper) and spliced mRNA. Reverse transcriptase was left out of one reaction to confirm that both products derived from RNA and not contaminating genomic DNA. α-tubulin84B served as a positive control. (E and F) In situ hybridization analysis of drak expression in wing/leg/haltere imaginal discs from wild-type (E) and drakdel mutant (F) animals. (G and H) Western blot analysis of lysates from S2 cells treated with dsRNAs targeting GFP (control) or three nonoverlapping regions of the drak transcript (exons 3–5, exon 6, and 3′ untranslated region), using anti-phospho-MRLC and anti-Sqh antibodies. Levels of phospho-Sqh, normalized to total Sqh levels, are plotted in H. Phospho-Sqh images are all from the same exposure of a single blot with intervening lanes removed, as are total Sqh images.
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Figure 1: Drak is the single Drosophila homologue of DAPK family kinases. (A) Multiple sequence alignment of Drosophila melanogaster Drak with human DRAK1 and DRAK2, ZIPK, and skeletal muscle MLCK (MYLK2). Purple, identical amino acids conserved in at least three of the five sequences. Black bar, limits of the conserved kinase domains. (B) Dendrogram showing relation of Drak and other Drosophila melanogaster MLCK-like proteins to human DAPK (blue) and MLCK family kinases, based on alignment of kinase domain sequences. Hs_MYLK1, 2, and 3 correspond to nonmuscle, skeletal muscle, and cardiac Myosin light chain kinases. (C and D) Developmental time course analysis of drak expression by RT-PCR. RNA was from staged embryos (C) and third instar larval (L3) and pupal (P) wing discs (D). Numbers in C represent ages of the embryos in hours after egg laying. drak primers amplified two bands representing unspliced (upper) and spliced mRNA. Reverse transcriptase was left out of one reaction to confirm that both products derived from RNA and not contaminating genomic DNA. α-tubulin84B served as a positive control. (E and F) In situ hybridization analysis of drak expression in wing/leg/haltere imaginal discs from wild-type (E) and drakdel mutant (F) animals. (G and H) Western blot analysis of lysates from S2 cells treated with dsRNAs targeting GFP (control) or three nonoverlapping regions of the drak transcript (exons 3–5, exon 6, and 3′ untranslated region), using anti-phospho-MRLC and anti-Sqh antibodies. Levels of phospho-Sqh, normalized to total Sqh levels, are plotted in H. Phospho-Sqh images are all from the same exposure of a single blot with intervening lanes removed, as are total Sqh images.

Mentions: As a first step toward testing the potential involvement of Drosophila DAPK family members in tissue morphogenesis, we sought to identify fly homologues of these kinases. In BLAST searches for annotated fly proteins similar to human DAPKs and MLCKs, four kinase-domain-containing proteins were consistently ranked among the most similar (Stretchin-MLCK, Bent, and the annotated gene products CG42347 and CG32666). Phylogenetic analyses of either whole protein or kinase domain sequences placed one of these proteins, CG32666 (which we renamed Drak), in the DAPK family branch, which includes DAPK1, DAPK2, ZIPK, and DRAK1 and 2 (Figure 1, A and B). Drak most closely resembles members of the DRAK-like subgroup of DAPKs, consisting of an amino-terminal kinase domain with a longer nonconserved C-terminal tail containing no recognizable domains (Figure 1A). Based on pairwise sequence comparisons, Drak is most closely related to DRAK1, with 50% sequence identity and 72% similarity in the kinase domain. We conclude that Drak is the single fly DAPK family homologue.


Overlapping roles of Drosophila Drak and Rok kinases in epithelial tissue morphogenesis.

Neubueser D, Hipfner DR - Mol. Biol. Cell (2010)

Drak is the single Drosophila homologue of DAPK family kinases. (A) Multiple sequence alignment of Drosophila melanogaster Drak with human DRAK1 and DRAK2, ZIPK, and skeletal muscle MLCK (MYLK2). Purple, identical amino acids conserved in at least three of the five sequences. Black bar, limits of the conserved kinase domains. (B) Dendrogram showing relation of Drak and other Drosophila melanogaster MLCK-like proteins to human DAPK (blue) and MLCK family kinases, based on alignment of kinase domain sequences. Hs_MYLK1, 2, and 3 correspond to nonmuscle, skeletal muscle, and cardiac Myosin light chain kinases. (C and D) Developmental time course analysis of drak expression by RT-PCR. RNA was from staged embryos (C) and third instar larval (L3) and pupal (P) wing discs (D). Numbers in C represent ages of the embryos in hours after egg laying. drak primers amplified two bands representing unspliced (upper) and spliced mRNA. Reverse transcriptase was left out of one reaction to confirm that both products derived from RNA and not contaminating genomic DNA. α-tubulin84B served as a positive control. (E and F) In situ hybridization analysis of drak expression in wing/leg/haltere imaginal discs from wild-type (E) and drakdel mutant (F) animals. (G and H) Western blot analysis of lysates from S2 cells treated with dsRNAs targeting GFP (control) or three nonoverlapping regions of the drak transcript (exons 3–5, exon 6, and 3′ untranslated region), using anti-phospho-MRLC and anti-Sqh antibodies. Levels of phospho-Sqh, normalized to total Sqh levels, are plotted in H. Phospho-Sqh images are all from the same exposure of a single blot with intervening lanes removed, as are total Sqh images.
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Related In: Results  -  Collection

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Figure 1: Drak is the single Drosophila homologue of DAPK family kinases. (A) Multiple sequence alignment of Drosophila melanogaster Drak with human DRAK1 and DRAK2, ZIPK, and skeletal muscle MLCK (MYLK2). Purple, identical amino acids conserved in at least three of the five sequences. Black bar, limits of the conserved kinase domains. (B) Dendrogram showing relation of Drak and other Drosophila melanogaster MLCK-like proteins to human DAPK (blue) and MLCK family kinases, based on alignment of kinase domain sequences. Hs_MYLK1, 2, and 3 correspond to nonmuscle, skeletal muscle, and cardiac Myosin light chain kinases. (C and D) Developmental time course analysis of drak expression by RT-PCR. RNA was from staged embryos (C) and third instar larval (L3) and pupal (P) wing discs (D). Numbers in C represent ages of the embryos in hours after egg laying. drak primers amplified two bands representing unspliced (upper) and spliced mRNA. Reverse transcriptase was left out of one reaction to confirm that both products derived from RNA and not contaminating genomic DNA. α-tubulin84B served as a positive control. (E and F) In situ hybridization analysis of drak expression in wing/leg/haltere imaginal discs from wild-type (E) and drakdel mutant (F) animals. (G and H) Western blot analysis of lysates from S2 cells treated with dsRNAs targeting GFP (control) or three nonoverlapping regions of the drak transcript (exons 3–5, exon 6, and 3′ untranslated region), using anti-phospho-MRLC and anti-Sqh antibodies. Levels of phospho-Sqh, normalized to total Sqh levels, are plotted in H. Phospho-Sqh images are all from the same exposure of a single blot with intervening lanes removed, as are total Sqh images.
Mentions: As a first step toward testing the potential involvement of Drosophila DAPK family members in tissue morphogenesis, we sought to identify fly homologues of these kinases. In BLAST searches for annotated fly proteins similar to human DAPKs and MLCKs, four kinase-domain-containing proteins were consistently ranked among the most similar (Stretchin-MLCK, Bent, and the annotated gene products CG42347 and CG32666). Phylogenetic analyses of either whole protein or kinase domain sequences placed one of these proteins, CG32666 (which we renamed Drak), in the DAPK family branch, which includes DAPK1, DAPK2, ZIPK, and DRAK1 and 2 (Figure 1, A and B). Drak most closely resembles members of the DRAK-like subgroup of DAPKs, consisting of an amino-terminal kinase domain with a longer nonconserved C-terminal tail containing no recognizable domains (Figure 1A). Based on pairwise sequence comparisons, Drak is most closely related to DRAK1, with 50% sequence identity and 72% similarity in the kinase domain. We conclude that Drak is the single fly DAPK family homologue.

Bottom Line: Drak activity is largely redundant with that of the Drosophila ROCK orthologue, Rok, such that it is essential only when Rok levels are reduced.The lethality of drak/rok mutants can be rescued by restoring Sqh activity, indicating that Sqh is the critical common effector of these two kinases.These results provide the first evidence that DAPK family kinases regulate actin dynamics in vivo and identify Drak as a novel component of the signaling networks that shape epithelial tissues.

View Article: PubMed Central - PubMed

Affiliation: Institut de recherches cliniques de Montréal, Montreal, QC, Canada.

ABSTRACT
Dynamic regulation of cytoskeletal contractility through phosphorylation of the nonmuscle Myosin-II regulatory light chain (MRLC) provides an essential source of tension for shaping epithelial tissues. Rho GTPase and its effector kinase ROCK have been implicated in regulating MRLC phosphorylation in vivo, but evidence suggests that other mechanisms must be involved. Here, we report the identification of a single Drosophila homologue of the Death-associated protein kinase (DAPK) family, called Drak, as a regulator of MRLC phosphorylation. Based on analysis of mutants, we find that Drak broadly promotes proper morphogenesis of epithelial tissues during development. Drak activity is largely redundant with that of the Drosophila ROCK orthologue, Rok, such that it is essential only when Rok levels are reduced. We demonstrate that these two kinases synergistically promote phosphorylation of Spaghetti squash (Sqh), the Drosophila MRLC orthologue, in vivo. The lethality of drak/rok mutants can be rescued by restoring Sqh activity, indicating that Sqh is the critical common effector of these two kinases. These results provide the first evidence that DAPK family kinases regulate actin dynamics in vivo and identify Drak as a novel component of the signaling networks that shape epithelial tissues.

Show MeSH
Related in: MedlinePlus