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Disruption of zebrafish cyclin G-associated kinase (GAK) function impairs the expression of Notch-dependent genes during neurogenesis and causes defects in neuronal development.

Bai T, Seebald JL, Kim KE, Ding HM, Szeto DP, Chang HC - BMC Dev. Biol. (2010)

Bottom Line: Like mammals, zebrafish has two distinct auxilin-like molecules, auxilin and cyclin G-associated kinase (GAK), differing in their domain structures and expression patterns.Still, they are not completely redundant, as morpholino-mediated knockdown of the ubiquitously expressed GAK alone can increase the specification of neuronal cells, a known Notch-dependent process, and decrease the expression of Her4, a Notch target gene.In addition, our analysis suggests that zebrafish GAK has at least two functions during the development of neural tissues: an early Notch-dependent role in neuronal patterning and a late role in maintaining the survival of neural cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Purdue University, 915 W, State St,, West Lafayette, Indiana 47907-2054, USA.

ABSTRACT

Background: The J-domain-containing protein auxilin, a critical regulator in clathrin-mediated transport, has been implicated in Drosophila Notch signaling. To ask if this role of auxilin is conserved and whether auxilin has additional roles in development, we have investigated the functions of auxilin orthologs in zebrafish.

Results: Like mammals, zebrafish has two distinct auxilin-like molecules, auxilin and cyclin G-associated kinase (GAK), differing in their domain structures and expression patterns. Both zebrafish auxilin and GAK can functionally substitute for the Drosophila auxilin, suggesting that they have overlapping molecular functions. Still, they are not completely redundant, as morpholino-mediated knockdown of the ubiquitously expressed GAK alone can increase the specification of neuronal cells, a known Notch-dependent process, and decrease the expression of Her4, a Notch target gene. Furthermore, inhibition of GAK function caused an elevated level of apoptosis in neural tissues, resulting in severe degeneration of neural structures.

Conclusion: In support of the notion that endocytosis plays important roles in Notch signaling, inhibition of zebrafish GAK function affects embryonic neuronal cell specification and Her4 expression. In addition, our analysis suggests that zebrafish GAK has at least two functions during the development of neural tissues: an early Notch-dependent role in neuronal patterning and a late role in maintaining the survival of neural cells.

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zGAK and zAux arose from gene duplication. (A) A phylogenetic analysis of the amino acid sequences of auxilins and GAKs without their kinase domains. The alignment was performed using Geneious software (Biomatters). (B) Alignment of intron positions in the PTEN region of auxilin and GAK orthologs. The residues shared by all are shaded in yellow and the residues conserved in mammals and vertebrates are shaded in orange. The presence of an intron is indicated by a solid line, and the phase is indicated by a number inside the triangle. ^ indicates an intron that is conserved in honeybee but not in Drosophila, whereas * indicates an intron that is conserved in fly but not in honeybee. Am: Apis mellifera, Dm: Drosophila melanogaster, Gg: Gallus gallus, Hs: Homo sapiens, Mm: Mus musculus, and Dr: Danio rerio.
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Figure 2: zGAK and zAux arose from gene duplication. (A) A phylogenetic analysis of the amino acid sequences of auxilins and GAKs without their kinase domains. The alignment was performed using Geneious software (Biomatters). (B) Alignment of intron positions in the PTEN region of auxilin and GAK orthologs. The residues shared by all are shaded in yellow and the residues conserved in mammals and vertebrates are shaded in orange. The presence of an intron is indicated by a solid line, and the phase is indicated by a number inside the triangle. ^ indicates an intron that is conserved in honeybee but not in Drosophila, whereas * indicates an intron that is conserved in fly but not in honeybee. Am: Apis mellifera, Dm: Drosophila melanogaster, Gg: Gallus gallus, Hs: Homo sapiens, Mm: Mus musculus, and Dr: Danio rerio.

Mentions: The zAux protein, on the other hand, is an 873 amino acid long polypeptide, which contains a PTEN region, a CBM and a J-domain, but lacks the N-terminal kinase (Figure 1B). The protein sequence of zAux is 59.3% identical overall to the human auxilin. Again, the conservations are higher in the PTEN and the J-domain, as these domains share 67.5% and 94.5% amino acid identity respectively. These domains of zAux are also highly related to the PTEN and the J-domain of zGAK (50.8% and 89.4% protein sequence identity respectively), suggesting that zAux and zGAK are derived from a common ancestral gene. However, while zAux is similar to zGAK, it is more similar to human and mouse auxilin proteins (Figure 2A), suggesting that the divergence of GAK and auxilin occurred prior to the divergence of fish and mammals during vertebrate evolution.


Disruption of zebrafish cyclin G-associated kinase (GAK) function impairs the expression of Notch-dependent genes during neurogenesis and causes defects in neuronal development.

Bai T, Seebald JL, Kim KE, Ding HM, Szeto DP, Chang HC - BMC Dev. Biol. (2010)

zGAK and zAux arose from gene duplication. (A) A phylogenetic analysis of the amino acid sequences of auxilins and GAKs without their kinase domains. The alignment was performed using Geneious software (Biomatters). (B) Alignment of intron positions in the PTEN region of auxilin and GAK orthologs. The residues shared by all are shaded in yellow and the residues conserved in mammals and vertebrates are shaded in orange. The presence of an intron is indicated by a solid line, and the phase is indicated by a number inside the triangle. ^ indicates an intron that is conserved in honeybee but not in Drosophila, whereas * indicates an intron that is conserved in fly but not in honeybee. Am: Apis mellifera, Dm: Drosophila melanogaster, Gg: Gallus gallus, Hs: Homo sapiens, Mm: Mus musculus, and Dr: Danio rerio.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2821301&req=5

Figure 2: zGAK and zAux arose from gene duplication. (A) A phylogenetic analysis of the amino acid sequences of auxilins and GAKs without their kinase domains. The alignment was performed using Geneious software (Biomatters). (B) Alignment of intron positions in the PTEN region of auxilin and GAK orthologs. The residues shared by all are shaded in yellow and the residues conserved in mammals and vertebrates are shaded in orange. The presence of an intron is indicated by a solid line, and the phase is indicated by a number inside the triangle. ^ indicates an intron that is conserved in honeybee but not in Drosophila, whereas * indicates an intron that is conserved in fly but not in honeybee. Am: Apis mellifera, Dm: Drosophila melanogaster, Gg: Gallus gallus, Hs: Homo sapiens, Mm: Mus musculus, and Dr: Danio rerio.
Mentions: The zAux protein, on the other hand, is an 873 amino acid long polypeptide, which contains a PTEN region, a CBM and a J-domain, but lacks the N-terminal kinase (Figure 1B). The protein sequence of zAux is 59.3% identical overall to the human auxilin. Again, the conservations are higher in the PTEN and the J-domain, as these domains share 67.5% and 94.5% amino acid identity respectively. These domains of zAux are also highly related to the PTEN and the J-domain of zGAK (50.8% and 89.4% protein sequence identity respectively), suggesting that zAux and zGAK are derived from a common ancestral gene. However, while zAux is similar to zGAK, it is more similar to human and mouse auxilin proteins (Figure 2A), suggesting that the divergence of GAK and auxilin occurred prior to the divergence of fish and mammals during vertebrate evolution.

Bottom Line: Like mammals, zebrafish has two distinct auxilin-like molecules, auxilin and cyclin G-associated kinase (GAK), differing in their domain structures and expression patterns.Still, they are not completely redundant, as morpholino-mediated knockdown of the ubiquitously expressed GAK alone can increase the specification of neuronal cells, a known Notch-dependent process, and decrease the expression of Her4, a Notch target gene.In addition, our analysis suggests that zebrafish GAK has at least two functions during the development of neural tissues: an early Notch-dependent role in neuronal patterning and a late role in maintaining the survival of neural cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Purdue University, 915 W, State St,, West Lafayette, Indiana 47907-2054, USA.

ABSTRACT

Background: The J-domain-containing protein auxilin, a critical regulator in clathrin-mediated transport, has been implicated in Drosophila Notch signaling. To ask if this role of auxilin is conserved and whether auxilin has additional roles in development, we have investigated the functions of auxilin orthologs in zebrafish.

Results: Like mammals, zebrafish has two distinct auxilin-like molecules, auxilin and cyclin G-associated kinase (GAK), differing in their domain structures and expression patterns. Both zebrafish auxilin and GAK can functionally substitute for the Drosophila auxilin, suggesting that they have overlapping molecular functions. Still, they are not completely redundant, as morpholino-mediated knockdown of the ubiquitously expressed GAK alone can increase the specification of neuronal cells, a known Notch-dependent process, and decrease the expression of Her4, a Notch target gene. Furthermore, inhibition of GAK function caused an elevated level of apoptosis in neural tissues, resulting in severe degeneration of neural structures.

Conclusion: In support of the notion that endocytosis plays important roles in Notch signaling, inhibition of zebrafish GAK function affects embryonic neuronal cell specification and Her4 expression. In addition, our analysis suggests that zebrafish GAK has at least two functions during the development of neural tissues: an early Notch-dependent role in neuronal patterning and a late role in maintaining the survival of neural cells.

Show MeSH
Related in: MedlinePlus