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Na,K-ATPase 1-subunit dephosphorylation by protein phosphatase 2A is necessary for its recruitment to the plasma membrane

Emilia Lecuona^*,1, Laura A. Dada^*, Haiying Sun^*, Maria L. Butti^*, Guofei Zhou^*, Teng-Leong Chew and Jacob I. Sznajder^*

^* Division of Pulmonary and Critical Care Medicine, Department of Medicine and

Cell Imaging Facility, Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois, USA

¹Correspondence: Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, 240 E. Huron, McGaw M410, Chicago, IL 60611, USA. E-mail: e-lecuona{at}northwestern.edu

SPECIFIC AIMS

G-protein coupled-receptor (GPCR) agonists increase lung edema clearance by stimulating Na,K-ATPase recruitment to the plasma membrane, which results in increased Na,K-ATPase activity, a process mediated by protein phosphatase 2A (PP2A). The present study was undertaken to define the role that PP2A plays in the GPCR-mediated regulation of the Na,K-ATPase in alveolar epithelial cells (AEC). We focused on the role that dephosphorylation of the Na,K-ATPase has in its trafficking from intracellular compartments to the plasma membrane as well as in defining the interactions between the Na,K-ATPase 1-subunit and the catalytic subunit of PP2A.

PRINCIPAL FINDINGS

1. G-protein coupled-receptor agonists promote Na,K-ATPase 1-subunit dephosphorylation by PP2A at Serine 18 in alveolar epithelial cells
To determine whether the GPCR-mediated up-regulation of the Na,K-ATPase involved a change in the phosphorylation status of its 1-subunit, we used a backphosphorylation technique. A549 cells expressing the rat Na,K-ATPase 1-subunit with a PV5 tag (P1V5-A549) were incubated with 10 µM dopamine (DA) for 5 min, and the Na,K-ATPase 1-subunit was immunoprecipitated and subjected to an in vitro phosphorylation reaction with purified PKC and [ -³²P]ATP. We found that more ³²P-labeled phosphate was incorporated into the Na,K-ATPase 1-subunit during the in vitro phosphorylation in cells treated with DA than in control cells, suggesting that DA promotes the dephosphorylation of the Na,K-ATPase 1-subunit in AEC.

It has been described that in steady-state conditions there is baseline level of phosphorylation at Ser residues of the Na,K-ATPase 1-subunit and in particular at the Ser-18. To determine whether this residue was dephosphorylated by DA in AEC, we used an antibody (Ab)-based assay described by Feschenko and Sweadner, using the monoclonal antibody (mAb) Mck1 that binds the amino acid sequence DKKS¹⁸KK only when Ser-18 is unphosphorylated. We found that DA increased the binding of the Mck1 Ab after 1 and 5 min of incubation, suggesting dephosphorylation of the Na,K-ATPase 1-subunit at Ser-18. The increased dephosphorylation of the Na,K-ATPase 1-subunit was paralleled by increased Na,K-ATPase recruitment to the plasma membrane. To determine whether PP2A was involved in the DA-induced dephosphorylation of the Na,K-ATPase 1-subunit at Ser-18, we preincubated AEC with 5 nM okadaic acid (OA) and found that it prevented the DA-mediated dephosphorylation of the 1-Na,K-ATPase by two different methods: backphosphorylation and Mck1 Ab binding (Fig. 1 A, B). The prevention of dephosphorylation by OA paralleled the inhibition of the DA-mediated recruitment of Na,K-ATPase to the plasma membrane (Fig. 1C ). Taken together these data suggest that dephosphorylation of Ser-18 by PP2A is necessary for GPCR-induced translocation of the Na,K-ATPase to the plasma membrane.

View larger version (12K): [in this window] [in a new window]   Figure 1. Dephosphorylation of Na,K-ATPase 1-subunit at Ser-18 by PP2A is necessary for its recruitment to the plasma membrane. A) In vitro backphosphorylation assay performed on the immunoprecipitated Na,K-ATPase 1-subunit from 1V5-A549 cells exposed for 5 min to 10 µM dopamine in the presence or absence of 5 nM OA. Upper panel) Shows a representative autoradiography. Lower panel) Depicts a representative Western blot. Graph represents the mean ± SEM of three different experiments. B) ATII cells were exposed for 5 min to 10 µM dopamine in the presence or absence of 5 nM OA, the 1% Triton X-100 soluble fraction was isolated and S18 phosphorylation was studied using the Mck1 Ab (upper panel). Equal loading was demonstrated by stripping the membrane and probing with another Ab against the Na,K-ATPase 1-subunit (lower panel). Graph represents the mean ± SEM of three different experiments. C) ATII cells were exposed for 5 min to 10 µM dopamine in the presence or absence of 5 nM OA, and the amount of Na,K-ATPase abundance at the plasma membrane was studied by biotin-streptavidin pull down and subsequent Western blot. Graph represents the mean ± SEM of three different experiments DA: dopamine; OA: OA; i.b.: immunoblot; i.p.: immunoprecipitation. *P < 0.05, **P < 0.01.

2. The Na,K-ATPase is a substrate for PP2A in vitro
Several protein phosphatases have been shown to regulate the Na,K-ATPase activity: protein phosphatase type 1 (PP1), PP2A, and calcineurin. Two of them, PP1 and calcineurin, have been shown to dephosphorylate the Na,K-ATPase 1-subunit, but direct dephosphorylation of the Na,K-ATPase 1-subunit by PP2A has not been reported previously. To determine whether the Na,K-ATPase 1-subunit is a substrate for PP2A, Na,K-ATPase 1-subunit immunoprecipitates where subjected to in vitro kinase assay followed by in vitro phosphatase assay, which revealed that recombinant PP2A dephosphorylated the Na,K-ATPase 1-subunit.

3. The PP2A catalytic subunit interacts directly with the N-terminus of the Na,K-ATPase 1-subunit
PP2A is often found in complexes with its substrates, and many proteins have been described to be directly associated with the phosphatase. To determine whether there is a direct interaction between PP2A and the Na,K-ATPase 1-subunit, we generated glutathione S-transferase (GST)-fusion proteins containing the six intracellular domains of the rat Na,K-ATPase 1-subunit and incubated them with 1 mg of AEC lysate and performed in vitro GST-pull down assays. We found that the catalytic subunit of PP2A was pulled down with the GST fusion protein that contained the first 90 amino acids of the Na,K-ATPase 1-subunit, but it was not recovered in the pull-downs performed with the other GST-fusion proteins nor with GST alone. Mutation of Ser-18 to alanine did not affect the binding of the catalytic subunit of PP2A to the first intracellular domain of the Na,K-ATPase 1-subunit.

4. GPCR agonists promote PP2A translocation from the cytosol to the membrane fraction and coimmunoprecipitates and colocalizes with the Na,K-ATPase
It has been suggested that PP2A can be rapidly translocated to the membrane fraction after stimulation. To determine whether DA induced the translocation of PP2A to a membrane compartment, AEC were incubated with DA and the amount of the catalytic subunit of PP2A in the 1% Nonidet P-40 soluble fraction was analyzed by Western blot. We found that GPCR-mediated translocation of PP2A to the membrane compartment occurred within 15 s (Fig. 2 A). The translocation correlated with the increased coimmunoprecipitation (Fig. 2B ) and colocalization of the Na,K-ATPase 1-subunit and the catalytic subunit of PP2A after DA treatment.

View larger version (16K): [in this window] [in a new window]   Figure 2. GPCR agonists induce the translocation of PP2A to the membrane fraction and its coimmunoprecipitation with the Na,K-ATPase 1-subunit. A) ATII cells were incubated with 10 µM dopamine for the indicated times, 1% Nonidet P-40 soluble fraction was isolated and a Western blot using an Ab against the catalytic subunit of PP2A was performed. Upper panel) Shows a composite graph of four different experiments. Lower panel) Depicts a representative Western blot. B) GFP1-A549 and GFP-S18A1-A549 cells were exposed for 0 and 15 s to 10 µM dopamine. An immunoprecipiation with and anti-GFP Ab and Western blot against PP2Ac were performed (upper panel). Equal loading was confirmed stripping the membrane and performing a Western blot against GFP (lower panel). DA: dopamine; i.b.: immunoblot. *P < 0.05.

CONCLUSIONS AND SIGNIFICANCE

The phosphorylation-dephosphorylation status of proteins plays an important role in their sorting and trafficking to the different compartments in the cells, and thus modulate their function. This is the case for the Na,K-ATPase, where regulation by phosphorylation-dephosphorylation has been proposed as the mechanism for its trafficking to and from the plasma membrane. Here, we provide first evidence that dephosphorylation of the Na,K-ATPase 1-subunit at the Ser-18 residue by PP2A is necessary for its recruitment from intracellular compartments to the plasma membrane which is known to correlate with an increased Na,K-ATPase activity. Moreover, we provide evidence for a direct interaction between PP2A and the Na,K-ATPase 1-subunit which occurs within the first 90 amino acids of the N-terminus.

We provide evidence that the Na,K-ATPase 1-subunit is dephosphorylated after GPCR agonists treatment and that dephosphorylation is necessary for its recruitment into the plasma membrane. Dephosphorylation was demonstrated by two approaches: 1) a backphosphorylation approach and 2) an Ab-based assay. The combination of both approaches allowed us not only to demonstrate that the GPCR agonists mediated the dephosphorylation of the Na,K-ATPase 1-subunit but also that it occurs at the Ser-18 residue by using a construct with the Ser-18 mutated to an alanine. We found that dephosphorylation of the Na,K-ATPase 1-subunit at Ser-18 was necessary for recruitment of the Na,K-ATPase to the plasma membrane. Sweeney et al. reported that in HEK-293 cells overexpressing a rat Na,K-ATPase 1-subunit, the insulin-mediated dephosphorylation of the 1-subunit increased by 30% and correlated with the increase of the Na⁺ pump at the cell plasma membrane. We provide evidence that PP2A is important for the dephosphorylation and trafficking of the Na,K-ATPase (Fig. 1) . Moreover, performing an in vitro dephosphorylation assay we demonstrated that the Na,K-ATPase 1-subunit is a substrate for PP2A.

PP2A is often found in complexes with its substrates, and several proteins have been described to interact directly with the phosphatase. We performed GST-binding assays and found a direct interaction of the catalytic subunit of PP2A and the first 90 amino acids of the 1-subunit. We did not find Ser-18 to be necessary for the in vitro binding of the catalytic subunit of PP2A and the N-terminus of the Na,K-ATPase 1-subunit. However, it was necessary for their in vivo interaction as we were unable to find increased interaction of the catalytic subunit of PP2A and the Na,K-ATPase 1-subunit in the coimmunoprecipitation assays when a cell expressing the S18A mutation was used, suggesting that phosphorylation of Ser-18 is necessary for PP2A binding during GPCR agonists stimulation.

In sum, we found that the catalytic subunit of PP2A and the Na,K-ATPase interact after GPCR stimulation similarly to previously reported interactions of proteins that bind the Na,K-ATPase such as PI3K, Polycystin-1 and Src.

Taken together, our data suggest a model where GPCR stimulation in alveolar epithelial cells induces the translocation and activation of PP2A to membrane compartments where it interacts with the Na,K-ATPase 1-subunit, dephosphorylating it and triggering the recruitment to the plasma membrane which results in increased Na,K-ATPase function (Fig. 3 ).

View larger version (7K): [in this window] [in a new window]   Figure 3. Proposed model of PP2A-mediated Na,K-ATPase exocytosis by GPCR agonists in alveolar epithelial cells.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6503fje

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