Immunolocalization of the Na⁺,K⁺-ATPase and arrestin in the presence of PP2A in COS cells.

<p>COS cells were transfected with H85N plus Na⁺,K⁺-ATPase β-subunit (A), with H85N, Na⁺,K⁺-ATPase β-subunit and flag tagged arrestin (B-D), with H85N, Na⁺,K⁺-ATPase β-subunit and HA tagged PP2A C-subunit (E-G), or with H85N plus Na⁺,K⁺-ATPase β-subunit and flag tagged arrestin plus HA tagged PP2A C-subunit (H-M). Cells were stained with HK9 (A, B, E, H and K), anti-flag for arrestin (C and I), and anti-HA for PP2A C-subunit (F and L) antibodies. Overlay patterns are shown in D, G, J and M. (×200 magnification) A large fraction of the H85N was found in intracellular compartments when cells expressed arrestin in the absence of PP2A C-subunit. This effect was not observed when arrestin was expressed together with the PP2A C-subunit. Typical results from one of three experiments are shown.</p

Deletion constructs of the large cytoplasmic loop of the Na⁺,K⁺-ATPase α-subunit and GST pull down of PP2A with GST constructs.

<p>A. HA tagged PP2A C-subunit was expressed in COS cells and cell lysates were incubated with GST fusion proteins. PP2A C-subunit was detected by Western blot with anti-HA antibody (upper panel) and GST fusion proteins were detected by CBB staining. Not only N-terminal segments but also the C-terminal half of the large cytoplasmic loop binds to PP2A C-subunit. Typical results from one of four experiments are shown. B. Flag tagged PP2A A-subunit was expressed in COS cells and cell lysates were incubated with GST fusion proteins. PP2A A-subunit was detected by Western blot with anti-flag antibody (upper panel) and GST fusion proteins were detected by CBB staining. The PP2A A-subunit co-precipitated with the A-domain of the Na⁺,K⁺-ATPase α-subunit. Typical results from one of three experiments are shown.</p

In vitro translation of PP2A and pull down with a GST construct incorporating the large cytoplasmic loop of the Na⁺,K⁺-ATPase α-subunit.

<p>PP2A A (flag tagged)- and C (HA tagged)- subunit proteins were prepared by in vitro translation and GST pull down was performed with GST alone or GST-Na⁺,K⁺-ATPase large cytoplasmic loop (Na,K-GST). A. GST proteins used for pull down were detected by coomassie brilliant blue (CBB) staining. PP2A C-subunit and A-subunit were detected by Western blot with anti-HA (B) and anti-flag (C) antibodies, respectively. The PP2A C-subunit, but not the A-subunit, specifically co-precipitate with the GST-Na⁺,K⁺-ATPase. Typical results from one of three experiments are shown.</p

Immunolocalization of Na⁺,K⁺-ATPase and PP2A in situ.

<p>Mouse kidney sections were stained with the Na⁺,K⁺-ATPase antibody (A) and PP2A C-subunit (PP2A) antibody (B). Merged images are shown in C. (×40 magnification) The Na⁺,K⁺-ATPase and PP2A partially co-localize within the basolateral infoldings of proximal tubule epithelial cells. Typical results from one of three experiments are shown.</p

In vitro phosphorylation of the large cytoplasmic loop of the Na⁺,K⁺-ATPase α-subunit.

<p>A. GST construct incorporating the large cytoplasmic loop of the Na⁺,K⁺-ATPase α-subunit was phosphorylated with [γ-³²P]-ATP by GRKs prepared by immunoprecipitation from COS cell lysates. Phosphorylation was performed in the presence or absence of PP2A at 22°C for 30 min. Reactions were stopped by adding SDS-PAGE sample buffer and samples were separated by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue, dried, and analyzed by autoradiography (upper panel). The Coomassie Brilliant Blue staining demonstrating the expression levels of the GST proteins that were used for in vitro phosphorylation is shown in the lower panel. GRK 2 and GRK 3 phosphorylated the large cytoplasmic loop α-subunit, and PP2A partially reversed this phosphorylation. Typical results from one of four experiments are shown.</p

Competition between PP2A and arrestin for binding to the large cytoplasmic loop of the Na⁺,K⁺-ATPase.

<p>COS cell lysates expressing flag tagged arrestin 2 or HA tagged PP2A C-subunit were prepared. A. Coomassie Brilliant Blue staining demonstrating that the same quantities of GST fusion protein incorporating the large cytoplasmic loop of the Na⁺,K⁺-ATPase were used in each lane of the experiments depicted in panels B and C. B. GST fusion protein incorporating the large cytoplasmic loop of the Na⁺,K⁺-ATPase was incubated with 500 µl of lysate from arrestin2-expressing cells and varying amounts of lysate from PP2A C-subunit-expressing cells. Arrestin 2 (upper panel) and PP2A C-subunit (lower panel) were detected by western blotting using an anti-flag and anti-HA antibodies, respectively. C. GST fusion protein incorporating the large cytoplasmic loop of the Na⁺,K⁺-ATPase was incubated with 500 µl of lysate from PP2A C-subunit-expressing cells and varying amounts of lysate from arrestin 2-expressing cells. PP2A C-subunit (upper panel) and arrestin 2 (lower panel) were detected by western blotting with an anti-HA and anti-flag antibodies, respectively. Arrestin 2 binding to the Na⁺,K⁺-ATPase large loop was strongly inhibited by the PP2A C-subunit. Typical results from one of three experiments are shown.</p

Immunoprecipitation of Na⁺,K⁺-ATPase and PP2A from rat kidney.

<p>Rat kidney lysate was incubated with antibodies directed against the PP2A C-subunit, the PP2A A-subunit or the HA epitope (control) followed by protein A beads. As an additional control for the fact that the Na,K-ATPase α-subunit migrates in SDS-PAGE in close proximity to the band corresponding to IgG heavy chain dimers, the antibodies directed against the PP2A A-subunit or C-subunit were incubated with lysis buffer without the addition of tissue lysate. Immune complexes were separated by SDS-PAGE and Western blotting was performed with biotinylated anti Na⁺,K⁺-ATPase antibody, 6H. The Na⁺,K⁺-ATPase was co-precipitated with both the C- and A-subunits of PP2A. Typical results from one of three experiments are shown.</p

Co-immunoprecipitation of PP2A and the Na⁺,K⁺-ATPase or the H⁺,K⁺-ATPase expressed in COS cells.

<p>A. COS cells were transfected with HA tagged PP2A C-subunit alone, H85N plus HA tagged PP2A C-subunit, or H85N, flag tagged PP2A A- and HA tagged C-subunits. Immunoprecipitation was performed with HK9 antibody directed against the N terminus of H85N, and PP2A C-subunit was detected by Western blotting with anti-HA antibody. The quantity of H85N present in the cell lysates is detected by blotting with HK9 in the bottom panel. B. COS cells were transfected with flag tagged PP2A A-subunit alone, H85N plus flag tagged PP2A A-subunit, or H85N, flag tagged PP2A A- and HA tagged C-subunits. Immunoprecipitation was performed with HK9 antibody and PP2A A-subunit was detected by Western blotting with anti-flag antibody. The quantity of H85N present in the cell lysates is detected by blotting with HK9 in the bottom panel. Both the A- and C-subunits co-precipitated specifically with the Na⁺,K⁺-ATPase α-subunit. C. COS cells were transfected with HA tagged PP2A C-subunit alone, H⁺,K⁺-ATPase α- and β-subunits plus HA tagged PP2A C-subunit, or H85N plus HA tagged PP2A C-subunit. Immunoprecipitation was performed with HK9 antibody and PP2A C-subunit was detected by Western blotting with anti-HA antibody. The quantities of H85N and H⁺,K⁺-ATPase α-subunit present in the cell lysates are detected by blotting with HK9 in the bottom panel. The PP2A C-subunit was not immunoprecipitated with the H⁺,K⁺-ATPase. Typical results from one of five experiments are shown.</p