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

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.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.The conserved Notch pathway participates in diverse aspects of animal development, and has been implicated in human diseases and cancers [1-3]. Notch encodes a transmembrane receptor, which, upon ligand binding, undergoes proteolytic processing and releases an intracellular fragment capable of acting as a transcription co-regulator. As both Notch and its ligands (also transmembrane proteins) are widely expressed, their activities need to be tightly regulated. One such important regulation appears to be ligand internalization, which plays a critical role in activating Notch receptors [4,5].Notch ligand internalization utilizes an ubiquitin-dependent endocytic pathway, as two structurally unrelated E3 ubiquitin ligases, neuralized (neur) and mind bomb (dMib), can append ubiquitin to DSL (Delta, Serrate, Lag2) ligands [6-13]. Epsin/lqf (liqu