Dataset Information

Chemical Genetic Identification of Cyclin-G-associated kinase (GAK) Substrates Reveals its Role in Regulating Na+/K+-ATPase and Sipa1L1 in Neurons

ABSTRACT: Recent genetic evidence implicates the serine/threonine kinase cyclin G-associated kinase (GAK) as a Parkinson’s disease risk. However, its role in neuronal function and many downstream effectors remain unclear. Employing a chemical genetics method, we show here that the sodium potassium pump (Na+/K+-ATPase) is a GAK target in the brain. We further show that GAK modulates Na+/K+-ATPase at a novel site affecting both pump localization and function. Whole-cell patch clamp recordings from CA1 pyramidal cells in GAK conditional knockout mice show a larger change in resting membrane potential when exposed to the Na+/K+-ATPase blocker, ouabain, indicating altered Na+/K+-ATPase function in GAK knockouts. Additionally, we show that GAK-deficient neurons have enlarged dendritic spines and we identify the spine associated protein Sipa1L1 (or SPAR) as a GAK target, which may contribute to this effect. Our results reveal novel functions of GAK in neurons.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Noreen Eder

LAB HEAD: Sila Ultanir

PROVIDER: PXD011319 | Pride | 2019-01-07

REPOSITORIES: Pride

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Publications

Chemical genetic identification of GAK substrates reveals its role in regulating Na<sup>+</sup>/K<sup>+</sup>-ATPase.

Lin Amy W AW Gill Kalbinder K KK Castañeda Marisol Sampedro MS Matucci Irene I Eder Noreen N Claxton Suzanne S Flynn Helen H Snijders Ambrosius P AP George Roger R Ultanir Sila K SK

Life science alliance 20181231 6

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 pota ...[more]

PMID: 30623173

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Project description:The Na/K-ATPase is the specific receptor for cardiotonic steroids (CTS) such as ouabain and digoxin. At pharmacological concentrations used in the treatment of cardiac conditions, CTS inhibit the ion-pumping function of Na/K-ATPase. At much lower concentrations, in the range of those reported for endogenous CTS in the blood, they stimulate hypertrophic growth of cultured cardiac myocytes through initiation of a Na/K-ATPase-mediated and reactive oxygen species (ROS)-dependent signaling. To examine a possible effect of endogenous concentrations of CTS on cardiac structure and function in vivo, we compared mice expressing the naturally resistant Na/K-ATPase α1 and age-matched mice genetically engineered to express a mutated Na/K-ATPase α1 with high affinity for CTS. In this model, total cardiac Na/K-ATPase activity, α1, α2 and β1 protein content remained unchanged, and the cardiac Na/K-ATPase dose-response curve to ouabain shifted to the left as expected. In males aged 3–6 months, increased α1 sensitivity to CTS resulted in a significant increase of cardiac carbonylated protein content, suggesting that ROS production was elevated. A moderate but significant increase of about 15% of the heart-weight-to-tibia-length ratio, accompanied by an increase of myocyte cross-sectional area was detected. Echocardiographic analyses did not reveal any change in cardiac function, and there was no fibrosis or re-expression of the fetal gene program. RNA sequencing analysis indicated that pathways related to energy metabolism were upregulated, while those related to extracellular matrix organization were downregulated. Consistent with a functional role of the latter, an angiotensin-II challenge that triggered fibrosis in the α1r/rα2s/s mouse failed to do so in the α1s/sα2s/s. Taken together, these results are indicative of a link between circulating CTS, Na/K-ATPase α1, ROS, and physiological cardiac hypertrophy in mice under baseline laboratory conditions.

Dataset Information

Chemical Genetic Identification of Cyclin-G-associated kinase (GAK) Substrates Reveals its Role in Regulating Na+/K+-ATPase and Sipa1L1 in Neurons

Publications

Chemical genetic identification of GAK substrates reveals its role in regulating Na<sup>+</sup>/K<sup>+</sup>-ATPase.

Similar Datasets

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