Phosphoinositide-3 kinase binds to a proline-rich motif in the Na+, K+-ATPase alpha subunit and regulates its trafficking

Abstract

Endocytosis of Na(+),K(+)-ATPase molecules in response to G protein-coupled receptor stimulation requires activation of class I(A) phosphoinositide-3 kinase (PI3K-I(A)) in a protein kinase C-dependent manner. In this paper, we report that PI3K-I(A), through its p85alpha subunit-SH3 domain, binds to a proline-rich region in the Na(+),K(+)-ATPase catalytic alpha subunit. This interaction is enhanced by protein kinase C-dependent phosphorylation of a serine residue that flanks the proline-rich motif in the Na(+),K(+)-ATPase alpha subunit and results in increased PI3K-I(A) activity, an effect necessary for adaptor protein 2 binding and clathrin recruitment. Thus, Ser-phosphorylation of the Na(+),K(+)-ATPase catalytic subunit serves as an anchor signal for regulating the location of PI3K-I(A) and its activation during Na(+),K(+)-ATPase endocytosis in response to G protein-coupled receptor signals.

Figure 1

Effect of Na⁺,K⁺-ATPase α subunit phosphorylation on the ability of DA to increase PI3K activity. (a) OK cells expressing the wild-type rat Na⁺,K⁺-ATPase α₁ subunit or this isoform carrying the S18A mutation were incubated in the presence or absence of 1 μM DA for 2.5 min at 23°C. PI3K activity was determined in immunoprecipitates (equal amounts of precipitated material as assessed by Western blotting were used in each condition) with a polyclonal antibody raised against the p85α subunit whose epitope corresponds to the amino acids 333–430 within the N-terminal SH2 domain (Santa Cruz Biotechnology). (b) The presence of Na⁺,K⁺-ATPase α₁ subunit in the immunoprecipitates was determined by Western blotting with a monoclonal antibody against the Na⁺,K⁺-ATPase α subunit. Experiments in a and b were repeated three and five times, respectively. Basal expression was similar in OK-Wt and S18A mutants as evidenced by Western blotting and Na⁺,K⁺-ATPase activity [Rb⁺ transport (nmol of Rb per mg of protein per min): OK-Wt, 9.8 ± 0.7 vs. S18A, 9.7 ± 1.0; hydrolytic activity in isolated membranes (μmol of Pi per mg of protein per min): OK-Wt, 0.305 ± 0.02 vs. S18A, 0.293 ± 0.01].

Figure 2

Role of p85α-SH3 domain in mediating the interaction of PI3K with the Na⁺,K⁺-ATPase α subunits. (a) The presence of Na⁺,K⁺-ATPase α₁ subunits (see Fig. 1b) in the immunoprecipitates with the p85α antibody was examined in OK cells incubated with 1 μM DA for 2.5 min at 23°C in the presence or absence of 10 μM SH3-binding peptide (Calbiochem). Transient cell permeabilization was performed to allow access of the peptide to the cells' interiors. (b) Rubidium transport (nmol of Rb per mg of protein per min) was used as an index of Na⁺,K⁺-ATPase transport activity. Experiments were performed with identical protocols as described in a. Data represent the mean ±SEM of three experiments performed in triplicate. ∗, P < 0.05 (Student's t test).

Figure 3

A proline-rich motif within the Na⁺,K⁺-ATPase α₁ subunit is necessary for binding and activation of PI3K. (a) Schematic representation of the rat Na⁺,K⁺-ATPase α₁ subunit N-terminal with the predicted amino acid (Gln⁹⁵) adjacent to the plasma membrane. A proline-rich motif (TPPPTTP⁸⁷) is located in the vicinity of the substrate (Ser¹⁸) for PKC. (b) Na⁺,K⁺-ATPase activity was determined in OK-Wt cells incubated with 1 μM DA in the presence or absence of a peptide (50 μM) comprising the proline motif (Pro-p) or of a scrambled peptide (Pro-s). Each bar represents the mean ±SEM of three experiments performed in triplicate. *, P < 0.02. (c) PI3K activity was determined as described (see legend to Fig. 1) in vehicle (V)- or DA-treated cells. (d) The presence of Na⁺,K⁺-ATPase α₁ subunits in the immunoprecipitates was determined by Western blotting with a monoclonal antibody against the Na⁺,K⁺-ATPase α₁ subunit as in Fig. 1. The data are representative of four experiments. (e) Na⁺,K⁺-ATPase activity was determined either as Rb⁺ transport (Left) or ATP hydrolysis (Right). Cells expressing the wild type (open bars) or the P83R mutant (filled bars) were incubated with vehicle (V; Hanks' buffer) or 1 μM DA. Each bar represents the mean ±SEM of five experiments performed in triplicate. **, P < 0.01. n.s., Not significant.

Figure 4

PI3K activity is required for recruitment and binding of AP-2 to the Na⁺,K⁺-ATPase α₁ subunit. (a) Na⁺,K⁺-ATPase α₁ subunit abundance in clathrin vesicles, early endosomes (EE) and late endosomes (LE) from OK cells expressing the wild type (OK-Wt) or P83R mutant. Cells were incubated at 23°C with vehicle (V; Hanks' buffer) or 1 μM DA for either 2.5 min (clathrin vesicles) or 10 min (EE and LE). Experiments were performed at least three times. (b) OK cells incubated with or without 1 μM DA (2.5 min at 23°C) were previously incubated (20 min at 23°C) in the presence or absence of either 100 nM wortmannin or 25 μM LY294002, two inhibitors of PI3K activity. The experiment shown was reproduced with identical results on three separate occasions. In both a and b, the material immunoprecipitated with an AP2αC antibody (Upstate Biotechnology, Lake Placid, NY) was analyzed by Western blotting using an antibody against the Na⁺,K⁺-ATPase α₁ subunit. (c) OK wild-type (OK Wt) cells and P83R mutants were incubated with or without 1 μM DA (2.5 min at 23°C). Experiments were repeated three times. (d) Organization of the GPCR signals promoting the internalization of Na⁺,K⁺-ATPase α₁ subunits (see Discussion for details).