Abstract
Cyclin G-associated kinase (GAK) is a ubiquitous serine/threonine kinase that facilitates clathrin uncoating during vesicle trafficking. GAK phosphorylates a coat adaptor component, AP2M1, to help achieve this function. GAK is also implicated in Parkinson's disease through genome-wide association studies. However, GAK's role in mammalian neurons remains unclear, and insight may come from identification of further substrates. Employing a chemical genetics method, we show here that the sodium potassium pump (Na+/K+-ATPase) α-subunit Atp1a3 is a GAK target and that GAK regulates Na+/K+-ATPase trafficking to the plasma membrane. Whole-cell patch clamp recordings from CA1 pyramidal neurons in GAK conditional knockout mice show a larger change in resting membrane potential when exposed to the Na+/K+-ATPase blocker ouabain, indicating compromised Na+/K+-ATPase function in GAK knockouts. Our results suggest a modulatory role for GAK via phosphoregulation of substrates such as Atp1a3 during cargo trafficking.
Highlights
Cyclin G–associated kinase (GAK), known as auxilin 2, is a member of the Numb-associated kinase (NAK) family
Using overexpression studies in neurons, COS7, and human embryonic kidney (HEK) 293T cells, we show that Atp1a3 T705 is phosphorylated by GAK and another NAK family kinase associated kinase 1 (AAK1), and it is important for NKA localization and function
We introduced mutations at a second aa site that comes into close contact with the N-6 position of ATP adenine (Eblen et al, 2003), V99 in GAK
Summary
Cyclin G–associated kinase (GAK), known as auxilin 2, is a member of the Numb-associated kinase (NAK) family. At its N-terminus, GAK has a highly conserved serine/ threonine kinase domain followed by a lipid-binding PTEN-like domain (Lee et al, 2006), a clathrin-binding domain (Kametaka et al, 2007), and a J-domain, the latter two domains being sufficient for Hsc and GAK-mediated uncoating of clathrin-coated vesicles (Greener et al, 2000; Umeda et al, 2000). A transgenic mouse expressing only the clathrinbinding and J-domains was able to rescue the lethality of GAK knockout in the liver and brain, showing that neither the PTEN-like domains nor the kinase domain was necessary for GAK function in these tissues (Park et al, 2015). The functional role of GAK is still not fully understood in neurons, and a number of unidentified GAK substrates likely remain
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