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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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The genomic organizations and the protein sequences of zebrafish GAK and auxilin. (A) The zGAK locus contains 28 exons and spans 78 kb of genomic DNA. The corresponding cDNA contains a 3834 bp ORF, encoding a polypeptide of 1278 amino acids with an Ark kinase domain (blue), a PTEN-homologous region (orange), a clathrin-binding motif (CBM), and a J-domain (green). Two morpholinos, GAK-MO1 and GAK-MO2, (red boxes) were designed to disrupt the splicing of zGAK mRNA by blocking the splice acceptors of exon3 and exon19, respectively. The amino acids deleted by GAK-MO1 injection are boxed in red. The premature stop codon (after Lys679) generated by GAK-MO2 injection is indicated by a red line. (B) The zAux locus is smaller, as it contains 17 exons and spans across 40 kb of genomic DNA. The zAux protein, encoded by a 2619 bp ORF, contains a PTEN-related region (orange), CBM, and J-domain (green). (C) A schematic drawing comparing the domain composition of zebrafish auxilin-related genes. The kinase, PTEN, and the J-domains are represented by blue, orange, and green boxes, respectively.
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Figure 1: The genomic organizations and the protein sequences of zebrafish GAK and auxilin. (A) The zGAK locus contains 28 exons and spans 78 kb of genomic DNA. The corresponding cDNA contains a 3834 bp ORF, encoding a polypeptide of 1278 amino acids with an Ark kinase domain (blue), a PTEN-homologous region (orange), a clathrin-binding motif (CBM), and a J-domain (green). Two morpholinos, GAK-MO1 and GAK-MO2, (red boxes) were designed to disrupt the splicing of zGAK mRNA by blocking the splice acceptors of exon3 and exon19, respectively. The amino acids deleted by GAK-MO1 injection are boxed in red. The premature stop codon (after Lys679) generated by GAK-MO2 injection is indicated by a red line. (B) The zAux locus is smaller, as it contains 17 exons and spans across 40 kb of genomic DNA. The zAux protein, encoded by a 2619 bp ORF, contains a PTEN-related region (orange), CBM, and J-domain (green). (C) A schematic drawing comparing the domain composition of zebrafish auxilin-related genes. The kinase, PTEN, and the J-domains are represented by blue, orange, and green boxes, respectively.

Mentions: The zGAK locus encodes a polypeptide of 1278 amino acids and, like other GAK orthologs, contains an N-terminal Ark kinase, a PTEN domain, a CBM region and a C-terminal J domain (Figure 1A). Alignment of protein sequences shows that zGAK is 65% identical to human GAK overall, with the sequence conservations concentrated in the kinase, PTEN and the J-domain. The kinase, PTEN and J-domain of zGAK are 82.4%, 84.7%, and 78.8% identical to the corresponding domains of human GAK respectively. In contrast, the CBM domains of zGAK and human GAK are more divergent, sharing only 31.7% amino acid sequence identity.


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)

The genomic organizations and the protein sequences of zebrafish GAK and auxilin. (A) The zGAK locus contains 28 exons and spans 78 kb of genomic DNA. The corresponding cDNA contains a 3834 bp ORF, encoding a polypeptide of 1278 amino acids with an Ark kinase domain (blue), a PTEN-homologous region (orange), a clathrin-binding motif (CBM), and a J-domain (green). Two morpholinos, GAK-MO1 and GAK-MO2, (red boxes) were designed to disrupt the splicing of zGAK mRNA by blocking the splice acceptors of exon3 and exon19, respectively. The amino acids deleted by GAK-MO1 injection are boxed in red. The premature stop codon (after Lys679) generated by GAK-MO2 injection is indicated by a red line. (B) The zAux locus is smaller, as it contains 17 exons and spans across 40 kb of genomic DNA. The zAux protein, encoded by a 2619 bp ORF, contains a PTEN-related region (orange), CBM, and J-domain (green). (C) A schematic drawing comparing the domain composition of zebrafish auxilin-related genes. The kinase, PTEN, and the J-domains are represented by blue, orange, and green boxes, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: The genomic organizations and the protein sequences of zebrafish GAK and auxilin. (A) The zGAK locus contains 28 exons and spans 78 kb of genomic DNA. The corresponding cDNA contains a 3834 bp ORF, encoding a polypeptide of 1278 amino acids with an Ark kinase domain (blue), a PTEN-homologous region (orange), a clathrin-binding motif (CBM), and a J-domain (green). Two morpholinos, GAK-MO1 and GAK-MO2, (red boxes) were designed to disrupt the splicing of zGAK mRNA by blocking the splice acceptors of exon3 and exon19, respectively. The amino acids deleted by GAK-MO1 injection are boxed in red. The premature stop codon (after Lys679) generated by GAK-MO2 injection is indicated by a red line. (B) The zAux locus is smaller, as it contains 17 exons and spans across 40 kb of genomic DNA. The zAux protein, encoded by a 2619 bp ORF, contains a PTEN-related region (orange), CBM, and J-domain (green). (C) A schematic drawing comparing the domain composition of zebrafish auxilin-related genes. The kinase, PTEN, and the J-domains are represented by blue, orange, and green boxes, respectively.
Mentions: The zGAK locus encodes a polypeptide of 1278 amino acids and, like other GAK orthologs, contains an N-terminal Ark kinase, a PTEN domain, a CBM region and a C-terminal J domain (Figure 1A). Alignment of protein sequences shows that zGAK is 65% identical to human GAK overall, with the sequence conservations concentrated in the kinase, PTEN and the J-domain. The kinase, PTEN and J-domain of zGAK are 82.4%, 84.7%, and 78.8% identical to the corresponding domains of human GAK respectively. In contrast, the CBM domains of zGAK and human GAK are more divergent, sharing only 31.7% amino acid sequence identity.

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