Enrichment of PLC-γ1 and IP3R2 in the Caveolinassociated Na/K-ATPase Preparation
The most widely used procedure for purification of Na/K-ATPase involves the treatment of crude membranes with relatively low concentrations of SDS that leave most of the Na/K-ATPase within the membrane, but solubilize and remove many impurities (Jorgensen, 1988
). We used a modi- fication of this procedure combined with glycerol gradient centrifugation (see Materials and Methods
) to prepare two partially purified fractions (heavy and light) of the enzyme from rat kidney outer medulla and analyzed both fractions by Western blot for the presence of the signaling partners of Na/K-ATPase. The rat kidney was chosen for this study because the rat protein database is available for proteomic analysis. As shown in , both α1 and β1 subunits were coenriched in the heavy fraction as expected (Jorgensen, 1988
). When Na/K-ATPase activity was measured, a sevenfold increase in the specific activity was noted in comparison to that of the microsomal preparation. However, there was little coenrichment of Src and caveolin-1 (). In contrast, when the light fraction was analyzed, we found that Src and caveolin-1 were coenriched with the Na/K-ATPase. In addition, the soluble ERKs were also modestly enriched in this light fraction. These findings led us to speculate that this caveolin-enriched light fraction may contain most of the signaling Na/K-ATPase and its partners. To further test this possibility and identify other unknown partners of the signaling Na/K-ATPase, we separated the light fraction on SDS-PAGE. After silver staining, four discrete bands with apparent molecular mass of the β1 subunit (60 kDa), the α1 subunit (100 kDa) of Na/K-ATPase, 150 kDa and 240 kDa, were excised and subjected to in gel trypsindigestion. Subsequently, the masses of the resultant peptides were determined by MALDI-TOF analysis. As expected, data analysis identified the Na/K-ATPase β1 subunit from the 60-kDa band and the Na/K-ATPase α1 subunit from the 100-kDa band. We also identified Src kinase from the 60-kDa band (unpublished data), indicating that this method is sensitive for protein identification. MALDI-TOF analysis also revealed PLC-γ1 and IP3R2 from the 150- and the 240-kDa bands, respectively (Tables and ). These findings were confirmed by Western blot analysis (). Like Src and caveolin-1, PLC-γ1 and IP3 receptors (isoforms 2 and 3) were also coenriched with the Na/K-ATPase (), suggesting that these proteins could partner with the Na/K-ATPase to form a signalplex.
MS-identification of IP3 receptor (isoform 2)
Different Domain of Na/K-ATPase Interacts with PLC-γ1 and IP3 Receptors
To test the hypothesis that both PLC-γ1 and IP3 receptors are partners of the signaling Na/K-ATPase, we first deter- mined whether the α1 subunit of Na/K-ATPase possesses functional domains that can directly interact with PLC-γ1 and IP3 receptors. To do so, we expressed and purified GST-fused N-terminus (NT) and GST-fused central loop (CD3) of the Na/K-ATPase α1 subunit connecting the transmembrane helices 4 and 5 () and then performed GST pulldown assays. Analysis of the pulldown proteins by Western blot revealed that PLC-γ1 binds to GST-CD3, but not to GST-NT and GST (). On the other hand, we found that IP3R2 interacts with GST-NT, but not with GST (). We also observed the interaction between the GST-NT and IP3R3, which was reported previously (Miyakawa-Naito et al., 2003
). In addition, it appears that there was also a weak interaction between IP3R3 and GST-CD3 (). Thus, these findings suggest that the Na/K-ATPase α1 subunit can function as a scaffold, capable of tethering PLC-γ1 and its effector IP3 receptors together to form a signalplex via different domains.
Figure 2. Binding of PLC-γ1 and IP3R2 to different domains of Na/K-ATPase α1 subunit. (A) Schematic structure of Na/K-ATPase α1 subunit is showed with the indication of cytosolic and transmembrane domains. GST-fused N-terminus (amino acid (more ...)
Ouabain-activated Na/K-ATPase Signaling Complex Can Recruit and Activate PLC-γ1
Recently, we have demonstrated that ouabain activates the caveolar Na/K-ATPase signaling complex, resulting in tyrosine phosphorylation of multiple proteins and assembly of various signalplexes in LLC-PK1 cells (Wang et al., 2004
). It is important to note that the term “ouabain activates” was used to describe the activation of the signaling function, but not the ion pumping function of the Na/K-ATPase. Because PLC-γ1 interacts with the α1 subunit of Na/K-ATPase () and coenriched with the Na/K-ATPase in the light fraction prepared from rat kidney (), we tested if the signaling Na/K-ATPase interacts with and regulates PLC-γ1 in LLC-PK1 cells. As depicted in , LLC-PK1 cells were treated with 100 nM of ouabain for different times, and the cell lysates were immunoprecipitated with a polyclonal anti-Na/K-ATPase α1 antibody. Western blot analysis of the immunoprecipitates showed that PLC-γ1 was coprecipitated with the Na/K-ATPase α1 subunit in control LLC-PK1 cells, and ouabain significantly increased this interaction in a time-dependent manner. This ouabain effect was also dose-dependent. Significant changes were detected when the cells were exposed to 10 nM ouabain (). To corroborate the interaction, we repeated the time course experiments and immunoprecipitated the cell lysates with a monoclonal anti-PLC-γ1 antibody. Western blot analysis showed that ouabain increased the amount of coprecipitated Na/K-ATPase in a time-dependent manner (). We showed previously that Src activation and recruitment to the signaling Na/K-ATPase is essential for ouabain to evoke downstream cascades (Haas et al., 2002
). To address the role of Src in the ouabain-induced recruitment of PLC-γ1, we first probed for Src in the above immunoprecipitates, showing that ouabain stimulated the formation of the Na/K-ATPase/Src/PLC-γ1 complex (). These data suggest that the ouabain-induced recruitment of PLC-γ1 to the signaling Na/K-ATPase is likely due to the activation of Src. Therefore, in the second set of experiments, cells were pretreated with 1μM PP2, a Src inhibitor, and then exposed to ouabain. When cell lysates were immunoprecipitated with anti-Na/K-ATPase α1 antibody, we found that PP2 blocked the ouabain-induced increase in the amount of coprecipitated PLC-γ1 (). To further corroborate the role of Src, we repeated the above experiments in SYF cells that are derived from mouse embryos harboring functional null mutations in both alleles of the Src family kinases Src, Yes and Fyn. These experiments showed that ouabain failed to stimulate the interaction between Na/K-ATPase and PLC-γ1 (). On the other hand, ouabain was able to increase the interaction once Src is knocked back into the SYF cells (SYF+Src). Because mouse SYF and SYF+Src cells express ouabain-insensitive Na/K-ATPase α1, 100 μM ouabain was used in these experiments () as we previously reported (Haas et al., 2002
). These data indicate that activation of Src must make available additional binding sites for recruiting more PLC-γ1 to the Na/K-ATPase signaling complex.
Figure 3. Effects of ouabain on interaction between the Na/K-ATPase and PLC-γ1. CON, control; Oua, ouabain treatment. (A–C) LLC-PK1 cells were treated with 100 nM of ouabain for different times (A and B) or with different concentrations of ouabain (more ...)
Because we showed previously that ouabain-activated Na/K-ATPase/Src complex could transactivate EGFR (Haas et al., 2000
), we reasoned that ouabain might activate PLC-γ1 via either the active Src or transactivated EGFR. To test this hypothesis, we first determined if ouabain stimulates tyrosine phosphorylation of PLC-γ1. LLC-PK1 cells were treated with 100 nM of ouabain for different times, and the cell lysates were immunoprecipitated with a monoclonal anti-PLC-γ1 antibody. Immunoprecipitates were then probed for active PLC-γ1 using a polyclonal antibody raised against Tyr783
-phosphorylated PLC-γ1. These experiments demonstrated that ouabain could activate PLC-γ1 in a time-dependent manner in LLC-PK1 cells (). This result was confirmed when the cell lysates were immunoprecipitated by an anti-phosphotyrosine antibody, and then probed for PLC-γ1 (). As expected, inhibition of Src by PP2 blocked the ouabain-induced tyrosine phosphorylation of PLC-γ1 (). To further confirm that ouabain activates PLC-γ1, we measured the hydrolysis of PIP2 and the production of IP3 in LLC-PK1 cells in response to ouabain stimulation. To do so, we took the advantage of a newly developed assay based on a GFP-fused PLC-δ1 PH domain protein (Hirose et al., 1999
; Isshiki et al., 2004
). LLC-PK1 cells were transiently transfected with the expression vector. After 24 h, the cells were examined under confocal microscope. Consistent with the fact that the PH domain binds to PIP2, the expressed GFP-PH fusion protein appeared to be primarily associated with the plasma membrane in unstimulated cells (). Because the PH domain has at least equal binding affinity to IP3 compared with PIP2 (Hirose et al., 1999
), when PIP2 was hydrolyzed by PLC to produce IP3, the PH domain fusion protein would translocate into cytosolic compartments with IP3. This was demonstrated in control experiments in which the cells were treated with 10 μM ATP as previously reported (Isshiki et al., 2004
). When the cells were exposed to 100 nM of ouabain, the plasma membrane GFP signal decreased with a concomitant increase in cytosolic GFP signal, similar qualitatively to that observed after ATP treatment (). This suggests that the ouabain-activated PLC-γ1 can catalyze the hydrolysis of PIP2. However, when compared with ATP, the ouabain-induced changes appeared to be much smaller than that of ATP ().
Figure 4. Activation of PLC-γ1 by ouabain. (A and B) Cells were treated with 100 nM of ouabain for different times. Cell lysates were immunoprecipitated with anti-PLC-γ1 antibody and then analyzed by Western blot using an antibody raised against (more ...)
Figure 5. Effects of ouabain on PIP2 hydrolysis and IP3 production. Cells were transiently transfected with PLC-δ1 PH-GFP expression vector and then transfected cells were treated with 100 nM of ouabain, followed by 10 μM ATP. These cells were monitored (more ...)
Ouabain-activated Na/K-ATPase Signaling Complex Interacts with and Induces Tyrosine Phosphorylation of IP3 Receptors
There is evidence that the Na/K-ATPase interacts with IP3 receptors in kidney epithelial cells (Miyakawa-Naito et al., 2003
). The above data indicate that the ouabain-activated Na/K-ATPase signaling complex can recruit and activate PLC-γ1. Because the activated PLC-γ1 produces the ligand (IP3) of IP3 receptors, we tested whether the ouabain-activated complex could also recruit IP3 receptors in LLC-PK1 cells. Furthermore, we also tested whether the same ouabain-activated complex stimulates the tyrosine phosphorylation of IP3 receptors because Src family kinases can phosphorylate these receptors, resulting in increased sensitivity to IP3 (Jayaraman et al., 1996
; Yokoyama et al., 2002
; Cui et al., 2004
; Patterson et al., 2004
). As depicted in , we identified all three isoforms of IP3 receptors from LLC-PK1 cell lysates with commercially available antibodies. However, the anti-IP3R1 antibody produced a weak signal in comparison with other isoform-specific antibodies (). Control experiments also showed that the polyclonal anti-IP3R2 was a better choice for immunoprecipitation than the monoclonal anti-IP3R3. Therefore, the polyclonal antibody was used to immunoprecipitate IP3R in the following studies, whereas the monoclonal anti-IP3R3 antibody and the polyclonal anti-IP3R2 antibody were used to detect the coprecipitated IP3 receptors after immunoprecipitation with the polyclonal anti-Na/K-ATPase α1 antibody. As illustrated in , when cell lysates were immunoprecipitated by a polyclonal anti-Na/K-ATPase α1 antibody, we found that IP3R2 was coprecipitated. This interaction was regulated by ouabain in a time- and dose-dependent manner in LLC-PK1 cells (). Significant increases were observed when cells were exposed to 10 nM of ouabain. In addition, when cell lysates were immunoprecipitated by anti-IP3R2 antibody and probed for the Na/K-ATPase α1 subunit, we confirmed that ouabain increased the interaction between the Na/K-ATPase and the IP3R2 (). Because different isoforms of the IP3 receptor can form hetero-tetrameric channels (Monkawa et al., 1995
; Miyakawa-Naito et al., 2003
), it was not surprising that all three isoforms were coprecipitated using either anti-Na/K-ATPase α1 or anti-IP3R2 antibody (unpublished data).
Figure 6. Effects of ouabain on interaction between Na/K-ATPase and IP3 receptors. (A) Detection of IP3 receptor isoforms from 40 μg of LLC-PK1 cell lysates with commercially available antibodies. (B and C) Cells were treated with 1 μM ouabain for (more ...)
To test if ouabain stimulates the tyrosine phosphorylation of the IP3 receptors, LLC-PK1 cells were treated with 100 nM of ouabain for 5 min and cell lysates were immunoprecipitated with a polyclonal anti-phosphotyrosine antibody. As shown in , ouabain increased tyrosine phosphorylation of the IP3R2. This was confirmed when the ouabain-treated cell lysates were immunoprecipitated by a polyclonal anti-IP3R2 antibody and analyzed by a monoclonal anti-phosphotyrosine antibody (). To test if ouabain-induced increases in tyrosine phosphorylation of IP3R2 are due to the activation of Src, we pretreated the cells with PP2 for 15 min and then exposed the cells to ouabain. As shown in , inhibition of Src completely abolished ouabain-induced tyrosine phosphorylation of the IP3R2.
Figure 7. Effects of ouabain on tyrosine phosphorylation of IP3 receptors. (A and B) Cells were pretreated with 1 μM PP2 for 15 min, and both control and PP2-pretreated cells were then exposed to 100 nM of ouabain for 5 min. Cell lysates were immunoprecipitated (more ...)
To confirm that Src mediates ouabain-induced tyrosine phosphorylation of IP3 receptors, we performed the following two sets of experiments. As depicted in , ouabain failed to stimulate tyrosine phosphorylation of the IP3R2 in SYF cells. However, ouabain was able to stimulate tyrosine phosphorylation of the IP3R2 once the cells were rescued by Src (SYF+Src cells). In the second set of experiments the IP3R2 was immunoprecipitated from LLC-PK1 cells by an anti-IP3R2 polyclonal antibody. After the immunoprecipitates were washed with PBS, we added either Mg2+/ATP or recombinant active Src plus Mg2+/ATP into the phosphorylation buffer. After 5-min incubation at 30°C, the reactions were stopped by the addition of 2× Laemmli buffer and analyzed for tyrosine phosphorylation by Western blot. As illustrated in , addition of active Src in vitro was sufficient to stimulate the tyrosine phosphorylation of the IP3R2 in the presence of ATP. Taken together, these findings clearly demonstrated that the ouabain-activated Na/K-ATPase signaling complex could recruit and induce tyrosine phosphorylation of IP3R2 via Src in LLC-PK1 cells.
Figure 8. Src-mediated tyrosine phosphorylation of IP3 receptors. (A) Both SYF and SYF+Src cells were treated with 100 μM ouabain for 5 min as we previously reported (Wang et al., 2004 ). Cell lysates were immunoprecipitated by anti-IP3R2 antibody and then (more ...)
The Caveolar Na/K-ATPase Assembles the Complex in Response to Ouabain
We showed previously that caveolae played an essential role for the signaling Na/K-ATPase to interact with its partners (Wang et al., 2004
). To address the role of caveolae in ouabain-induced formation of the above signalplex, we treated LLC-PK1 cells with Mβ-CD to deplete cholesterol from plasma membrane. Depletion of cholesterol has been shown to reduce caveolar Na/K-ATPase and Src and to prevent the formation of the Na/K-ATPase/Src complex (Wang et al., 2004
). As depicted in , both control and Mβ-CD treated cells were exposed to 100 nM of ouabain for 5 min. Cell lysates were then immunoprecipitated with anti-Na/K-ATPase α1 antibody and probed for PLC-γ1 and IP3R2. As expected, depletion of cholesterol significantly reduced ouabain-induced increases in the interaction of Na/K-ATPase with both PLC-γ1 and IP3R2 ().
Figure 9. The signaling Na/K-ATPase tethers PLC-γ1 and IP3R2 together into a signalplex. (A) Cells were treated with methyl-β-cyclodextrin (Mβ-CD) as described in Materials and Methods. The control and cholesterol-depleted cells were then (more ...)
The Signaling Na/K-ATPase Tethers PLC-γ1 and IP3R2 Together into a Signalplex
The above findings indicate that the caveolar Na/K-ATPase may function as a scaffold, tethering PLC-γ1 and IP3R2 together, and thus facilitating the PLC-γ1-generated IP3 to act on IP3R2 in response to ouabain stimulation. However, because the immunoprecipitated Na/K-ATPase could contain two different pools that bind to either IP3R2 or PLC-γ1, we further examined this issue by immunoprecipitating the ouabain-treated cell lysates using an anti-IP3R2 antibody. As depicted in , the Na/K-ATPase, Src and PLC-γ1 were coprecipitated with IP3R2. Moreover, ouabain regulated these interactions (). Because ouabain-regulated interactions must involve the Na/K-ATPase, these data indicate that both PLC-γ1 and IP3R2 are brought together by the ouabain-activated Na/K-ATPase signaling complex.
Ouabain Stimulates Calcium Transients
To determine the functional role of the identified signalplex, we measured the effects of ouabain on [Ca2+
in LLC-PK1 cells. We found that ouabain-induced changes in [Ca2+
could be grouped into two types. Similar to the changes reported by Aperia's laboratory (Aizman et al., 2001
), we found that ouabain could stimulate calcium oscillations (unpublished data). However, oscillatory signals occurred in <1% of the cells. In contrast, ouabain induced single calcium transient in ~40% of the LLC-PK1 cells. The effects occurred 2–4 min after ouabain exposure () and at concentrations as low as 10 nM of ouabain (unpublished data). Significantly, in contrast to ouabain-induced calcium oscillations (Aizman et al., 2001
), ouabain elicited a single calcium transient even when the cells were incubated in Ca2+
-free medium (), indicating that ouabain could activate the release of Ca2+
from intracellular stores. Removal of extracellular Ca2+
did reduce the duration (1.79 ± 0.37 vs. 0.60 ± 0.05 min, n = 54, p < 0.01) of the Ca2+
transient, indicating that influx of Ca2+
is important for maintaining the transient. To test if the identified signalplex is involved in the ouabain-induced activation of calcium release, we determined whether inhibition of PLC or IP3 receptor could reduce ouabain-induced calcium transients. Pretreatment of LLC-PK1 cells with inhibitors of either PLC (U73122, 2 μM) or IP3 receptor (Xestospongin C, 10 μM) blocked ouabain-induced calcium transients in both Ca2+
-free and regular Ca2+
-containing medium (). These data are consistent with the notion that ouabain stimulates the calcium release via the activation of PLC and the subsequent increase in intracellular IP3. This notion is further supported by the fact that inhibition of Src by PP2 or depletion of cholesterol by Mβ-CD also reduced the effect of ouabain on [Ca2+
Figure 10. Effects of ouabain on [Ca2+]i. Cells were loaded with Fura-2/AM and changes in [Ca2+]i were recorded as a function of time after the chamber was perfused with 100 nM of ouabain. (A) The actual images of the cells in response to ouabain stimulation in (more ...)