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

Abstract

BackgroundThe 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.ResultsLike 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.ConclusionIn 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.

Review: The role of vitamin D in nervous system health and disease

“…GWAS have identified an increasing number of candidate risk genes in PD, several of which have VDR-binding sites closely associated with them raising the possibility that vitamin D may influence their expression. The biological relevance of a subset of these susceptibility genes with associated VDR binding on brain function has been well delineated with evidence for roles in nigrostriatal dopaminergic neurotransmission, neurogenesis and neurite outgrowth, and neural ectodermal expression (especially within the marginal and subventricular zones) (see Table 2) [140][141][142][143][144]. (ii) Amyotrophic lateral sclerosis Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting both the central and peripheral nervous systems [145].…”

The clathrin-binding and J-domains of GAK support the uncoating and chaperoning of clathrin by Hsc70 in the brain

“…When morpholinos are used to excise a small region of the kinase domain of GAK in zebrafish, the brain has developmental defects due to impairment of notch signaling (Bai et al, 2010), just as observed in Drosophila (Eun et al, 2008;Hagedorn et al, 2006). Surprisingly, a similar phenotype in the zebrafish is obtained when morpholinos are used to generate a nonsense mutation in the PTEN-like domain of GAK (Bai et al, 2010).…”

“…When morpholinos are used to excise a small region of the kinase domain of GAK in zebrafish, the brain has developmental defects due to impairment of notch signaling (Bai et al, 2010), just as observed in Drosophila (Eun et al, 2008;Hagedorn et al, 2006). Surprisingly, a similar phenotype in the zebrafish is obtained when morpholinos are used to generate a nonsense mutation in the PTEN-like domain of GAK (Bai et al, 2010). However, there has been no attempt to rescue the zebrafish defects with a fragment comprising just the clathrinbinding and J-domains of GAK so it is unclear whether, as in Drosophila (Eun et al, 2008;Kandachar et al, 2008), these domains are sufficient for GAK function in zebrafish.…”

“…Functional analysis of GAK in zebrafish has been performed by injecting morpholinos into the zygote (Bai et al, 2010). When 20 residues are excised from the kinase domain of GAK, there are defects in Notch signaling during early zebrafish development, whereas later in development, there is an increased level of apoptosis in neuronal tissues, resulting in neurodegeneration.…”

Cyclin G–Associated Kinase Is Necessary for Osteosarcoma Cell Proliferation and Receptor Trafficking

“…GAK knockdown by siRNA in HeLa cells resulted in increased activation of ERK1/2, ERK5, and AKT (38), whereas introduction of dominant-negative dynamin, another protein involved in receptor trafficking, inhibited MAPK activation (41). GAK depletion has been linked to decrease in expression of Her4, a Notch target gene, in zebrafish models (42). Two receptors (IGF-IR and EGFR) that have been repeatedly described in osteosarcoma were analyzed to assess the effect of GAK depletion on their expression.…”