A direct role for ATP1A1 in unconventional secretion of fibroblast growth factor 2

Abstract

Previous studies proposed a role for the Na/K-ATPase in unconventional secretion of fibroblast growth factor 2 (FGF2). This conclusion was based upon pharmacological inhibition of FGF2 secretion in the presence of ouabain. However, neither independent experimental evidence nor a potential mechanism was provided. Based upon an unbiased RNAi screen, we now report the identification of ATP1A1, the α1-chain of the Na/K-ATPase, as a factor required for efficient secretion of FGF2. As opposed to ATP1A1, down-regulation of the β1- and β3-chains (ATP1B1 and ATP1B3) of the Na/K-ATPase did not affect FGF2 secretion, suggesting that they are dispensable for this process. These findings indicate that it is not the membrane potential-generating function of the Na/K-ATPase complex but rather a so far unidentified role of potentially unassembled α1-chains that is critical for unconventional secretion of FGF2. Consistently, in the absence of β-chains, we found a direct interaction between the cytoplasmic domain of ATP1A1 and FGF2 with submicromolar affinity. Based upon these observations, we propose that ATP1A1 is a recruitment factor for FGF2 at the inner leaflet of plasma membranes that may control phosphatidylinositol 4,5-bisphosphate-dependent membrane translocation as part of the unconventional secretory pathway of FGF2.

Keywords: ATP1A1; Fibroblast Growth Factor (FGF); Heparan Sulfate; Membrane Recruitment; Membrane Translocation; Na+/K+-ATPase; Phosphoinositide; Plasma Membrane; Tec Kinase; Unconventional Secretion.

FIGURE 1.

Identification of ATP1A1 as a component of the machinery mediating unconventional secretion of FGF2. A, mean scores of a selected set of siRNAs used in large scale RNAi screening for gene products involved in FGF2 secretion. A stable HeLa cell line expressing FGF2-GFP in a doxycycline-dependent manner was used to quantify FGF2 secretion as described under “Experimental Procedures.” All isoforms of α- and β-chains of the Na/K-ATPase known in the human genome were targeted with three independent siRNAs. In addition, three siRNAs directed against FGF2 are shown. As a control, a validated siRNA against GFP was used to down-regulate the FGF2-GFP reporter itself. Error bars, S.D. (n = 4). B, expression analysis of all known α- and β-chains of the Na/K-ATPase complex in HeLa cells employing RT-PCR.

FIGURE 2.

Validation of ATP1A1 as a component of the machinery mediating unconventional secretion of FGF2. A flow cytometry assay was used to quantify unconventional secretion using a stable HeLa cell line expressing FGF2 and GFP in a doxycycline-dependent manner (18, 23, 24, 30, 31). A, FGF2 cell surface expression under control conditions and after down-regulation of ATP1A1. Validated siRNAs directed against GAPDH and FGF2 were used as negative and positive controls, respectively. Error bars, S.D. (n = 4). To test whether observed differences between experimental conditions were statistically significant, an unpaired two-tailed Student's t test was performed (ns, not significant; **, p value ≤ 0.01; ***, p value ≤ 0.001). B, Western blot analysis to test the efficiency of down-regulation by RNAi for the gene products indicated. The asterisk indicates a cross-reactivity of the anti-ATP1A1 antibody used in this analysis. C, FGF2 cell surface expression under control conditions and after down-regulation of ATP1B1 and ATP1B3, the β1- and β3-chains of the Na/K-ATPase expressed in HeLa cells. Validated siRNAs directed against GAPDH and FGF2 were used as negative and positive controls, respectively. Error bars, S.D. (n = 4). To test whether observed differences between experimental conditions were statistically significant, an unpaired two-tailed t test was performed (ns, not significant; ***, p ≤ 0.001). D, Western blot analysis to test the efficiency of down-regulation by RNAi for the gene products indicated. E, quantitative RT-PCR analysis to monitor RNAi-mediated down-regulation of ATP1B1 and ATP1B3, the β1- and β3-chains of the Na/K-ATPase.

FIGURE 3.

Analysis of cell proliferation following down-regulation of ATP1A1, ATP1B1, and ATP1B3. Cells were cultivated using exactly the conditions described for the FGF2 secretion experiments shown in Fig. 2, including doxycycline-dependent induction of FGF2 expression. Following siRNA-mediated down-regulation of gene products, as indicated, a kinetic analysis of cell proliferation was conducted by quantifying cell confluence using an Essen BioScience IncuCyte Zoom live cell imaging microscope. The starting density measured by cell confluence was set to 100% for each experimental condition. The results shown are representative for four independent biological replicates. Error bars, S.D. derived from three technical replicates.

FIGURE 4.

Inhibition of FGF2 secretion caused by down-regulation of ATP1A1 is not due to pleiotropic effects. A stable cell line expressing a GFP-CD4 fusion protein in a doxycycline-dependent manner was generated. This construct carries an N-terminal signal peptide and an extracellular GFP domain, followed by the transmembrane span and the cytoplasmic domain of CD4 (SP-GFP-CD4), and is transported to cell surfaces via the ER/Golgi-dependent secretory pathway. Following incubation of cells in the presence of doxycycline, GFP-CD4 was detected on cell surfaces using anti-GFP antibodies and flow cytometry. A, normalized cell surface expression of GFP-CD4 under control conditions and after down-regulation of ATP1A1 using three independent siRNAs. As a positive control, a validated siRNA directed against β-COP, a component of the coatomer complex that is essential for transport within the ER/Golgi-dependent pathway, was used (2). In addition, a siRNA directed against GFP was used to down-regulate the GFP-CD4 reporter itself. Error bars, S.D. (n = 3). B, Western blot analysis of the efficiency of down-regulation by RNA interference for ATP1A1 and GFP-CD4. C, Western blot analysis of the efficiency of down-regulation by RNA interference for β-COP.

FIGURE 5.

Direct interaction between FGF2 and the cytoplasmic domain of ATP1A1 as demonstrated by biochemical pull-down experiments. A, affinity beads containing FGF2 were incubated with various variant forms of the cytoplasmic domain of ATP1A1, as indicated. The latter were used as GST fusion proteins with GST alone as a negative control (lanes 1–3). The other constructs were GST-ATP1A1-CD1 (lanes 4–6), GST-ATP1A1-CD2 (lanes 7–9), GST-ATP1A1-CD3 (lanes 10–12), GST-ATP1A1-CD2–3 (lanes 13–15), and GST-ATP1A1-CD1–3 (lanes 16–18). Bound (50% of each fraction) and unbound material (5% of each fraction) was analyzed by SDS-PAGE and Coomassie Brilliant Blue protein staining. The results shown are representative for three independent experiments. B, affinity beads containing the various variant forms of the cytoplasmic domain of ATP1A1 were incubated with soluble recombinant FGF2, as indicated. GST alone was used as a negative control (lanes 1 and 2). The other constructs were GST-ATP1A1-CD1 (lanes 3 and 4), GST-ATP1A1-CD2 (lanes 5 and 6), GST-ATP1A1-CD3 (lanes 7 and 8), GST-ATP1A1-CD2–3 (lanes 9 and 10), and GST-ATP1A1-CD1–3 (lanes 11 and 12). Bound (50% of each fraction) and unbound material (5% of each fraction) was analyzed by SDS-PAGE and Coomassie Brilliant Blue protein staining. The results shown are representative for three independent experiments.

FIGURE 6.

FGF2 binds with submicromolar affinity to the cytoplasmic domain of ATP1A1. A quantitative protein-protein interaction assay based on AlphaScreen® technology (34) was used to determine the affinity of the interaction between FGF2 and ATP1A1-CD. As detailed under “Experimental Procedures,” AlphaScreen® protein-protein interaction signals were recorded for the various pairs of His- and GST-tagged proteins indicated. To determine affinity, an untagged, N-terminally truncated form of FGF2 (NΔ25-FGF2) was titrated into the binding reaction at the concentrations indicated. Error bars, S.D. (n = 3). The experimental data were fitted with a non-linear regression model (log (inhibitor) versus response − variable slope (four parameters)) using GraphPad Prism version 5.0c software to calculate IC₅₀ values. A, His₆-tagged FGF2 and GST-tagged ATP1A1-CD (blue spheres), His₆-tagged FGF2 and GST-tagged Tec kinase (red squares), and His₆-tagged CARP and GST-tagged titin (black triangles). B, comparison between the interactions of WT FGF2 (blue spheres) and FGF2-Y81pCMF (pink squares) with ATP1A1-CD. His₆-tagged CARP and GST-tagged titin (black triangles) were used as a negative control. C, analysis of a potential impact of ouabain on the interaction of FGF2 with ATP1A1-CD. Ouabain was titrated at the concentrations indicated. Green spheres, His₆-tagged FGF2 and GST-tagged ATP1A1-CD; black triangles, His₆-tagged CARP and GST-tagged titin.

FIGURE 7.

Proximity of ATP1A1 and FGF2 analyzed in cells. To test for proximity of ATP1A1 and FGF2 in cells, the Duolink® in situ proximity ligation immunoassay (PLA®; Sigma-Aldrich) was used. HeLa cells were fixed and permeabilized with acetone. The Duolink® assay was conducted as described under “Experimental Procedures” using pairs of primary antibodies (or single primary antibodies as controls) directed against the antigens indicated. Red dots, interaction/proximity events. To visualize the nuclei of cells, DNA was stained with SYTOX® green (Invitrogen), shown in blue. A, ATP1A1 and FGF2; B, transferrin receptor (TFR) and FGF2; C, cadherin and FGF2; D, GM130 and FGF2; E, control using just one primary antibody against FGF2; F, control using just one primary antibody directed against ATP1A1; G, statistical analysis of proximity events using the Duolink® image tool software (Olink Bioscience). Data are shown as absolute counts of proximity events per cell. Mean values were calculated from four independent experiments with 30–50 cells being analyzed per individual experiment and condition. Error bars, S.D. To test whether observed differences between experimental conditions were statistically significant, an unpaired two-tailed Student's t test was performed (ns, not significant; ***, p ≤ 0.001).

FIGURE 8.

Quantification of in-cell interactions of FGF2 and ATP1A1 following down-regulation of ATP1A1, ATP1B1, and ATP1B3. Following RNAi-mediated down-regulation of the gene products indicated, cells were processed for Duolink® in situ proximity assays as described under “Experimental Procedures” and in the legend to Fig. 7. A, Western analysis to monitor down-regulation of ATP1A1 and ATP1B1 under the experimental conditions indicated. B, Duolink® in situ proximity assay to quantify in-cell interactions between FGF2 and ATP1A1 following down-regulation of the gene products indicated. a, mock; b, siRNA ATP1A1; c, siRNA ATP1B1; d, siRNA ATP1B3; e, siRNA ATP1B1/ATP1B3; f, control 1 (mock; single antibody control) (ATP1A1); g, control 2 (mock; single antibody control) (FGF2); h, control 3 (mock; no primary antibodies against FGF2 and ATP1A1). C, quantification of in-cell interaction signals (proximity events/cell) under the experimental conditions shown in B. The blue dotted line indicates the background signal. Error bars, S.D. (n = 3). To test whether observed differences between experimental conditions were statistically significant, an unpaired two-tailed Student's t test was performed (*, p ≤ 0.05; **, p ≤ 0.01).

FIGURE 9.

A current model of FGF2 membrane translocation with ATP1A1 as a high affinity recruitment factor for FGF2 at the inner leaflet of plasma membranes. For details, see “Discussion.”