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A Functional Interaction Between Na,K-ATPase β2-Subunit/AMOG and NF2/Merlin Regulates Growth Factor Signaling in Cerebellar Granule Cells

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Abstract

The Na,K-ATPase, consisting of a catalytic α-subunit and a regulatory β-subunit, is a ubiquitously expressed ion pump that carries out the transport of Na+ and K+ across the plasma membranes of most animal cells. In addition to its pump function, Na,K-ATPase serves as a signaling scaffold and a cell adhesion molecule. Of the three β-subunit isoforms, β1 is found in almost all tissues, while β2 expression is mostly restricted to brain and muscle. In cerebellar granule cells, the β2-subunit, also known as adhesion molecule on glia (AMOG), has been linked to neuron–astrocyte adhesion and granule cell migration, suggesting its role in cerebellar development. Nevertheless, little is known about molecular pathways that link the β2-subunit to its cellular functions. Using cerebellar granule precursor cells, we found that the β2-subunit, but not the β1-subunit, negatively regulates the expression of a key activator of the Hippo/YAP signaling pathway, Merlin/neurofibromin-2 (NF2). The knockdown of the β2-subunit resulted in increased Merlin/NF2 expression and affected downstream targets of Hippo signaling, i.e., increased YAP phosphorylation and decreased expression of N-Ras. Further, the β2-subunit knockdown altered the kinetics of epidermal growth factor receptor (EGFR) signaling in a Merlin-dependent mode and impaired EGF-induced reorganization of the actin cytoskeleton. Therefore, our studies for the first time provide a functional link between the Na,K-ATPase β2-subunit and Merlin/NF2 and suggest a role for the β2-subunit in regulating cytoskeletal dynamics and Hippo/YAP signaling during neuronal differentiation.

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Funding

This work was supported by funds from NIGMS-8P20GM103464, the Nemours Foundation, and the DO Believe Foundation. The Biomolecular Core Lab is supported by grants NIGMS-P30GM114736 (COBRE) and NIGMS-P20GM103446 (INBRE).

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Authors and Affiliations

  1. Nemours Biomedical Research, Alfred I. duPont Hospital for Children, DuPont Experimental Station, Bldg 400, #4414, 200 Powder Mill Road, Wilmington, DE, 19803, USA

    Alisa Litan, Zhiqin Li, Patience Kelly & Sigrid A. Langhans

  2. Biological Sciences Graduate Program, University of Delaware, Newark, DE, 19716, USA

    Alisa Litan & Patience Kelly

  3. David Geffen School of Medicine, University of California, Los Angeles, and VA Greater Los Angeles Health Care System, Los Angeles, CA, 90073, USA

    Elmira Tokhtaeva & Olga Vagin

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  1. Alisa Litan
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  2. Zhiqin Li
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Correspondence to Sigrid A. Langhans.

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Supplemental Fig. 1

Soluble forms of Na,K-ATPase β1-subunit and β2-subunit. Immunoblot for the secreted forms of the β1- and β2-subunits in cell lysates (sec β1 lysate; sec β2 lysate) and in cell culture medium (sec β1 medium; sec β2 medium) using isoform-specific antibodies. Cell lysates and cell culture medium from untransfected cells are included for comparison. Only the glycosylated form of the β2-subunit was secreted into the medium (compare Sec β2 unglycosylated lysate with Sec β2 unglycosylated medium) (PNG 1200 kb)

High resolution image (TIF 237 kb)

Supplemental Fig. 2

Na,K-ATPase β2-subunit knockdown has no effect on DAOY cell proliferation. Cell proliferation of β2-subunit knockdown clones expressed as fold change in cell number at days 1, 2, 3, and 4 relative to cell number at day 0. The β2-subunit knockdown clones were generated by two independent transfections/selections. ShV – blue line is the corresponding control for clone 1 and ShV – green line for clone 2 of the knockdown cells (PNG 594 kb)

High resolution image (TIF 87 kb)

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Litan, A., Li, Z., Tokhtaeva, E. et al. A Functional Interaction Between Na,K-ATPase β2-Subunit/AMOG and NF2/Merlin Regulates Growth Factor Signaling in Cerebellar Granule Cells. Mol Neurobiol 56, 7557–7571 (2019). https://doi.org/10.1007/s12035-019-1592-4

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