Albumin endocytosis in proximal tubule cells is modulated by angiotensin II through an AT2 receptor-mediated protein kinase B activation
- *Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS-Bloco G, 21949-900, Rio de Janeiro, Brazil; and †Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205
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Edited by Maurice B. Burg, National Institutes of Health, Bethesda, MD, and approved October 8, 2005 (received for review August 19, 2005)
Abstract
Albumin endocytosis in renal proximal tubule cells is a clathrin- and receptor-mediated mechanism that, in several pathophysiological conditions, is involved in initiating or promoting tubule-interstitial disease. Although much work has been done on this pathway, the regulation of albumin endocytosis in proximal tubule cells is not well understood. Here, we study the modulation by angiotensin II (Ang II) of albumin endocytosis in LLC-PK1, a model of proximal tubule cells. We observed that Ang II increases albumin endocytosis by ≈100% at 10-9 M. This effect is completely reversed by 10-9 M PD123319, a specific AT2 receptor antagonist, but not by losartan, a specific AT1 receptor antagonist, at concentrations up to 10-7 M. The Ang II effect on albumin endocytosis is also reversed by: phosphoinositide 3-kinase inhibitors LY294002 (2.5 × 10-6 M) or wortmannin (10-7 M), the protein kinase B inhibitor (2 × 10-5 M), and staurosporine (2 × 10-6 M), an inhibitor of 3′-phosphoinositide-dependent kinase 1. Ang II induced the selective phosphorylation of protein kinase B (PKB) at the Thr-308 residue without a change in Ser-473 phosphorylation, a combination that leads to an increase in PKB activity. These effects were completely abolished by 3 × 10-6 M staurosporine or 10-8 M PD123319. Our experiments also showed that PKB is present in the membrane fraction in overnight-starved LLC-PK1 cells. Taken together, these data show that Ang II increases albumin endocytosis through an AT2 receptor mediated by activation of PKB in the plasma membrane, which depends on the basal activity of the phosphatidyl-inositol 3-kinase.
In the normal human adult, ≈7 g of albumin daily is filtered by the glomerulus, most of it effectively reabsorbed in the proximal tubule (1). Albuminuria caused by increased glomerular filtration or tubular injury is a well known marker for renal disease with direct evidence for its involvement in the progression of chronic kidney disease to end-stage renal failure (2). In more physiological conditions, it has been observed in vitro that albumin inhibits apoptosis and promotes the survival of primary cultures of mouse proximal tubular epithelial cells (3). These data suggest that normal amounts of albumin filtration and absorption is important for health.
Albumin reabsorption by proximal tubule cells occurs by clathrin- and receptor-mediated endocytosis, where the binding of albumin involves at least the complex of two proteins, megalin and cubilin (1). Under normal circumstances, endocytosis prevents the loss of albumin in urine. However, pathophysiological conditions can lead to disease in two ways. Overstressing this endocytic system with excess albumin after glomerular injury can either initiate or promote tubule-interstitial disease (2). On the other hand, a reduction in albumin endocytosis in proximal tubule cells in Dent's disease leads to low molecular weight proteinuria and nephrolithiasis (4).
The importance of albumin endocytosis in proximal tubule cells for human health suggests that it must be regulated carefully. However, little is known about whether albumin endocytosis is regulated by hormones and, if so, which signal transduction pathways are coupled to specific hormones. The observation that albumin endocytosis in proximal tubule cells is modulated by heterotrimeric GTP-binding proteins indicates that this process could be modulated by hormones acting through G protein-coupled receptors (1), but which specific hormone is unknown. It is known that albumin endocytosis is modulated by different kinases among them phosphoinositide 3-kinase (PI3K) (5). This enzyme catalyzes the phosphorylation of the 3′ position of the inositol ring of phosphoinositides (PIs) (6). One well-known pathway activated downstream of PI3K is protein kinase B (PKB) which belongs to the AGC kinase family (7). Two phosphorylation sites in PKB are crucial for its activity: serine 473 (Ser-473) and threonine 308 (Thr-308) (7). PKB binds to 3′-phosphorylated phosphoinositides formed by PI3K at the plasma membrane. Immediately after binding at the plasma membrane, PKB is phosphorylated on the Ser-473 residue of the regulatory domain. Subsequently, phosphorylation of the Thr-308 residue in the activation loop by phosphoinositide-dependent kinase 1 (PDK1) then leads to activation of PKB. Although each residue is phosphorylated by different pathways, maximal activation of PKB occurs only when both residues are phosphorylated (7). Although it is well known that PI3K is part of the signal transduction pathway involved in regulating albumin endocytosis, how this signal transduction pathway is coupled to hormonal control is unknown.
Ang II receptors AT1 and AT2 (8) are located on both basolateral and luminal sides of the proximal tubule. Proximal tubule cells produce and secrete Ang II into the lumen where its concentration could reach as high as 6-10 nmol/liter (9). This concentration is at least 10 times higher than the concentration found in the plasma. The location of receptors on both membranes and the high concentration of Ang II in the lumen are consistent with the regulation of proximal tubule function via receptors on both membranes. Interestingly, in patients with proteinuria, treatment with angiotensin-converting enzyme inhibitors and/or angiotensin (AT1) receptor antagonists is a very effective way of ameliorating urinary loss of albumin (10). Likewise, in a mouse model, overexpression of the AT2 receptor decreases albuminuria-induced glomerular injury (11). Given these data, angiotensin II (Ang II) is a likely candidate as a regulator of albumin endocytosis.
Angiotensin receptors have seven transmembrane domains and belong to the family of G protein-coupled receptors (8, 12). The AT2 receptor shares 34% amino acid sequence homology with the AT1 receptor. The AT2 receptor is expressed more abundantly during fetal development (8, 12). However, studies in adults indicate that the AT2 receptor could be up-regulated during specific conditions such as sodium depletion or glomerular injury (11, 12). The physiological role for AT2 is not well known. It has been suggested that it plays an important role in the adult animal, probably by counteracting the physiological and pathophysiological effects mediated by the AT1 receptor (8, 11, 12).
Despite the importance of angiotensin-converting enzyme inhibitors and AT1 antagonists to modulate renal albumin loss in patients with proteinuria, little is known about the direct effect of Ang II in regulating albumin endocytosis in the proximal tubule. The aim of this work is to investigate the modulation of albumin endocytosis in the proximal tubule cell by Ang II and identify which molecular pathway is involved. We used the LLC-PK1 cell line as a model of the proximal tubular epithelium. We found that Ang II increases albumin endocytosis through an AT2 receptor, mediated by activation of a resident pool of PKB in the plasma membrane. These results elucidate a mechanism of activation of PKB and opens possibilities to understand the physiological and pathophysiological actions of Ang II through the AT2 receptor and the mechanisms of regulation of PKB.
Materials and Methods
Materials and Reagents. Angiotensin II, PD12319, albumin, FITC-Albumin, wortmannin, LY294002, genistein and staurosporine were purchased from Sigma. AT1 selective antagonist losartan was purchased from Merck. PKB inhibitor (PKBi) was purchased from Calbiochem. Polyclonal PKB, polyclonal phospho-PKB (Ser-473), and monoclonal phospho-PKB (Thr-308) antibodies and PKB kinase assay kit were purchased from Cell Signaling Technology.
Cell Culture. LLC-PK1 cells, a well characterized porcine proximal tubule cell line (American Type Culture Collection), were maintained in DMEM F/12 with 10% FBS, 1% penicillin and streptomycin (37°C and 5% CO2). For albumin endocytosis experiments, cells were grown on coverslips. Cells were used 1 day after confluence, typically 3 days after seeding.
Albumin Endocytosis. The albumin endocytosis assay was performed as described (13). After treatment with the indicated compounds, the cells were washed with PBS2+ (PBS supplemented with 1.8 mM CaCl2 and 1 mM MgCl2) and exposed to FITC-albumin at 37°C for 15 min, except when indicated. Parallel experiments were performed on cells held at 4°C to abolish endocytosis. After washing, the cells were fixed with 4% paraformaldehyde at 25°C for 15 min, permeabilized with 0.1% Triton X-100 for 1-2 min, and blocked with 3% nonfat milk in PBS2+ for 30 min. To stain the plasma membrane, the cells were incubated with specific mouse primary antibody, ZO-1 (Zymed), and cy3-anti-mouse secondary antibody (Jackson ImmunoResearch). Then, the cells were mounted in Vectashield medium (Vector Lab, CA). To quantify albumin endocytosis, the images of fluorescent-labeled markers were acquired by a Zeiss Axiovert fluorescence microscope using a ×40 oil immersion objective lens (Achrostigmat) and the average intensity per cell was calculated by using iplab software from multiple images after background subtraction. The endocytic curve consisting of average total intensity per cell versus time was plotted by using origin. Fluorescent labeling was visualized with Ultraview confocal microscopy by using temporal software (PerkinElmer) using a ×63 oil immersion objective lens (Zeiss).
Phosphorylation of PKB. After overnight removal of serum, serum-starved LLC-PK1 cells were preincubated with different compounds, described in Figs. 4 and 5. The cells were washed with PBS2+, harvested, and incubated for 30 min in lysis buffer containing 20 mM Hepes (pH 7.4), 2 mM EGTA, 1% Triton X-100, 400 μM PMSF, 50 mM NaF, 2 μM Microcystin LR, 2× complete protease inhibitor (Roche), 10 ng/μl leupeptin, 10 ng/μl aprotinin, 4 ng/μl elastatinal, 2.5 mM 1,10-phenatholine, and 100 μM TPCK, and cleared by centrifugation at 4°C for 10 min at 14,000 rpm (15,000 × g). The supernatant was retained and the protein concentration was determined by using the Bio-Rad Protein Assay kit. Proteins were resolved on SDS/10% PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia). Western blot for phospho-PKB (Ser-473), phospho-PKB (Thr-308), and total PKB was performed according to instructions from the suppliers. After antibody labeling, detection was performed with ECL (Amersham Pharmacia). The images were acquired with Fuji Image Reader (LAS-1000 Lite), and quantification was done with imagegauge 4.0.
PKB Activity Assay. PKB activity was measured by the phosphorylation of glycogen synthase kinase-3 (GSK-3) according the instructions of the manufacturer (Cell Signaling Technology). Briefly, after incubation with the reagents indicated in Fig. 4, cells were lysed and cleared by centrifugation at 14,000 rpm (15,000 × g) for 10 min at 4°C. The supernatant was retained, and PKB was pulled down by using immobilized PKB primary antibody incubated with gentle rocking overnight at 4°C. The cell lysate was centrifuged at 14,000 rpm (15,000 × g) for 10 min at 4°C, washed two times with lysis buffer and two times with kinase buffer. GSK-3 (1 μg) fusion protein was added for 30 min at 30°C. The reaction was stopped by addition of 3× SDS sample buffer. Protein was resolved on SDS/10% PAGE gels and transferred to polyvinylidene difluoride membranes. Western blot for phospho-GSK-3 was performed. The images were acquired with Fuji Image Reader, and quantification was done with imagegauge 4.0.
Cell Fractionation. Cells was washed with PBS2+ and harvested in buffer containing 20 mM Hepes (pH 7.4), 2 mM EGTA, 400 μM PMSF, 50 mM NaF, 2 μM Microcystin LR, 2× complete protease inhibitor (Roche), 10 ng/μl leupeptin, 10 ng/μl aprotinin, 4 ng/μl elastatinal, 2.5 mM 1,10-phenathroline, and 100 μM TPCK and lysed by 30 strokes in a Dounce homogenizer. Nuclei were removed by centrifugation for 10 min at 1,000 × g at 4°C. The membrane and cytosol fractions were obtained by centrifugation at 100,000 × g for 1 h at 4°C. The pellet was resuspended in buffer described above.
Statistical Analysis. Results are expressed as means ± SE. Statistical significance was assessed by Student's unpaired t test. Significance was determined as P < 0.05.
Results
Effect of Ang II on Albumin Endocytosis. To determine the effect of Ang II on albumin endocytosis, LL-CPK1 cells were preincubated for 4 h with different concentrations of Ang II ranging from 10-12 to 10-7 M. After this procedure, cells were washed free of Ang II and measurements of albumin endocytosis were carried out. Endocytosis of FITC-albumin was calculated as described in Materials and Methods. Images from these experiments obtained by dual wavelength confocal microscopy in permeabilized cells using an antibody against ZO-1 as a marker for the apical plasma membrane are shown Fig. 1A. Ang II increased albumin endocytosis in a dose-dependent manner with a maximal effect observed at 10-8 M (Fig. 1B). In this condition, albumin endocytosis was two times higher when compared to the control. In contrast, no albumin endocytosis either in the absence or after preincubation with Ang II occurred in parallel experiments at 4°C. The maximal activation observed at 10-8 M Ang II, occurs after 3 h of preincubation and remains the same up to 24 h of preincubation (Fig. 1C).
Ang II increases albumin endocytosis. (A) Albumin endocytosis of confluent LLC-PK1 cells is shown. Red (anti ZO-1 antibody) represents tight junction and green is FITC-albumin. (a) The albumin endocytosis measured at 4°C. (b) Albumin endocytosis at 37°C (control). (c) Albumin endocytosis measured at 37°C after the preincubation of the cells with 10-8 M Ang II for 4 h. (B) Dose-response for Ang II on albumin endocytosis. The cells were preincubated with different concentrations of Ang II, and the albumin endocytosis assay was carried out after washing. The quantification of albumin endocytosis was performed as described in Materials and Methods (n = 6). (C) Time course of the preincubation of the LLC-PK1 cells with 10-8 M Ang II (n = 4).
The time course of albumin endocytosis in LLC-PK1 cells in the presence or in the absence of 10-8 M Ang II was performed (Fig. 6A, which is published as supporting information on the PNAS web site). Albumin uptake increases quickly over time, reaching a maximal level in 20 min. Prior incubation of cells with 10-8 M Ang II for 4 h at 37°C increases albumin endocytosis at all time points studied with a maximal stimulation observed as early as 1 min. In our experiments, the apparent affinity for albumin is 0.6 ± 0.2 mg/ml in control cells, and it was not changed by the preincubation with 10-8 M Ang II for 4 h (0.5 ± 0.2 mg/ml) (Fig. 6A). Our measured affinity is within the range of the concentration of albumin in the proximal tubule during pathophysiological conditions, which ranges from 0.1 to 1.0 mg/ml (1).
To investigate which Ang II receptor is involved in the modulation of the albumin endocytosis, LLC-PK1 cells were treated with losartan and PD123319, specific antagonists for AT1 and AT2 receptors, respectively (Fig. 2). Losartan in concentrations ranging from 10-11 to 10-7 M did not change the stimulation of albumin endocytosis by Ang II (Fig. 2 A). In sharp contrast, Ang II simulation was completely reversed by PD123319 at a concentration as low as 10-9 M (Fig. 2B). The addition of 10-7 M PD123319 or 10-7 M losartan alone did not affect basal albumin endocytosis. These data indicate that Ang II stimulates albumin endocytosis in LLC-PK1 cells through AT2 receptors.
AT2 receptor mediated Ang II effect on the albumin endocytosis. The cells were preincubated for 30 min with losartan (A) or PD123319 (B) for 30 min before the preincubation for 4 h with 10-8 M Ang II. After treatment, albumin endocytosis was carried out as described in Materials and Methods.
PI3K Is Involved in the Effect of Ang II. It was shown that albumin endocytosis in OK cells, a model of proximal tubule cells, depends on PI3K activity (5). Two selective, chemically unrelated inhibitors, LY294002 and wortmannin, are widely used to study the involvement of PI3K in different cellular events (6). To study the involvement of PI3K on Ang II stimulation of albumin endocytosis, LLC-PK1 cells were treated for 0.5 h with 2.5 × 10-6 M LY294002 or 10-7 M wortmannin followed by 4 h incubation with 10-8 M Ang II in the presence of the inhibitors. Again, the cells were washed and endocytosis measured. Under these circumstances, both LY294002 and wortmannin abolished the stimulatory effect of 10-8 M Ang II on albumin endocytosis (Fig. 3A).
PI3K/PKB pathway is involved in the effect of Ang II on albumin endocytosis. The cells were treated for 30 min with indicated concentrations of PI3K inhibitors wortmannin and LY294002 (A) or PKB inhibitor PKBi (B) before the preincubation for 4 h with 10-8 M Ang II. After treatment, albumin endocytosis was carried out as described in Materials and Methods.
PKB Involvement. One of the well known downstream pathways involved in PI3K activation is PKB (7). Fig. 3B shows the effect of the PKBi on the modulation of albumin endocytosis by 10-8 M Ang II. This inhibitor was recently described as a PI analogue that abolishes PKB activation without affecting PDK1 or mitogen activated protein kinases (14). Five-hour preincubation with 2 × 10-5 M PKBi alone did not change albumin endocytosis. In contrast, 1-h preincubation with 2 × 10-5 M PKBi followed by 4 h incubation with Ang II plus the inhibitor abolished completely the stimulation of albumin endocytosis by Ang II.
To study in more depth the mechanism of action of Ang II on PKB, the effect of 10-8 M Ang II on the phosphorylation of the Ser-473 and Thr-308 residues was studied in overnight serum-starved LLC-PK1 cells (Fig. 7, which is published as supporting information on the PNAS web site). Ang II increases the phosphorylation of the Thr-308 residue in a time-dependent manner with a maximal effect observed after only 10 min of treatment with Ang II. This effect disappeared after 30 min of incubation with Ang II, suggesting that the phosphorylation of Thr-308 is an early event in the stimulation of albumin endocytosis by Ang II. On the other hand, the phosphorylation of the Ser-473 residue was not changed by 10-8 M Ang II.
Thr-308 phosphorylation is mediated by PDK1, which can be inhibited by staurosporine (7, 15). Fig. 4A shows the effect of 3 × 10-6 M staurosporine on Ang II-induced, PKB-Thr-308 phosphorylation. When cells were preincubated with staurosporine for 30 min followed by incubation with 10-8 M Ang II plus staurosporine for 10 min, the stimulatory effect of Ang II on Thr-308 phosphorylation was completely reversed. Moreover, preincubation with staurosporine alone did not change Ser-473 phosphorylation, but drastically reduced the basal level of Thr-308 phosphorylation. To take these experiments further, we ascertained whether Ang II-induced phosphorylation of Thr-308 (Fig. 4B) leads to activation of PKB activity by using glycogen synthase kinase-3 as a substrate and as a measure of PKB activity. Importantly, Ang II dramatically increased PKB activity by 100%. This effect is completely abolished by incubation of cells with 3 × 10-6 M staurosporine or 10-8 M PD123319. The addition of staurosporine alone decreased the basal PKB activity by 50%. On the other hand, 10-8 M PD123319 did not change basal PKB activity. To correlate the effect of Ang II on Thr-308 phosphorylation with its effect on albumin endocytosis, we performed experiments on albumin endocytosis in the presence of 3 × 10-6 M staurosporine (Fig. 4C). Preincubation with staurosporine for 30 min abolished the stimulatory effect of Ang II on albumin endocytosis. In addition, staurosporine alone decreased albumin endocytosis by 40%.
Ang II induces the selective phosphorylation of the Thr-308 residue in PKB. The cells were incubated with 10-8 M Ang II for the indicated period time and the cells lysed. (A) The immunoblotting with anti-phosphoThr308, anti-phosphoSer473, or total PKB antibodies. Equal amounts of protein were loaded. Immunoblots are representative of six independent experiments. (B and C) Densitometry determinations of the level of the phosphorylated PKB residues, calculated as the ratio between the phosphor-residue and total PKB. phosphoSer473 (B) and phosphoThr308 (C) are shown.
The above data show that Ang II-induced the activation of PKB through selective phosphorylation of the Thr-308 residue. The addition of 10-7 M wortmannin eliminated the stimulatory effect of Ang II on Thr-308 phosphorylation (Fig. 5A). Furthermore, wortmannin inhibited basal phosphorylation of both Ser-473 and Thr-308 phosphorylation. Thr-308 phosphorylation requires lipid binding of both PKB and PDK1, providing a condition whereby PDK1 can access Thr-308 (7, 15, 16). With this in mind, we determined where PKB is located in the membrane fraction and whether Ang II has an effect on PKB location by cellular fractionation (Fig. 5B). The results show that most of the PKB is found in the cytosolic fraction but a significant level of PKB is located in the membrane fraction in the absence of Ang II. The addition of 10-8 M Ang II did not change ratio of PKB in cytosol vs. membrane fractions (Fig. 5B). At 10-8 M, Ang II increased the magnitude of Thr-308 phosphorylation in the membrane fraction by ≈50%, but left Ser-473 phosphorylation unchanged (data not shown).
PKB is present in the membrane. (A) Modulation of the effect of 10-8 M Ang II on the phosphorylation of the PKB Thr-308 and Ser-473 residues by 10-7 M wortmannin. The cells were treated for 30 min with 10-7 M wortmannin before the preincubation with 10-8 M Ang II for 10 min. The level the phospho-residue was determined as described in Fig. 4. (B) Determination of PKB in the cytosol and membrane fraction. Each fraction was immunoblotted with anti-total-PKB antibody.
Discussion
Albumin endocytosis in the proximal tubule is very important and therefore should be under fine regulation by hormones (1). It is well known that albuminuria is a major risk factor for progression of renal diseases (17). Albuminuria may be a by-product of renal disease or by itself promote tubular injury, with interstitial inflammation and fibrosis through a mechanism mediated by albumin endocytosis (17). Here, we show that Ang II through the AT2 receptor increases albumin endocytosis in LLC-PK1 cells at concentrations as low as 10-9 M and within range of concentrations of Ang II present in the lumen of the proximal tubule (≈10-30 nM) (9). Ang II does not change the apparent affinity for albumin endocytosis in LLC-PK1 cells.
AT2 receptors are expressed in the luminal membrane of the proximal tubule of adult animals (8, 12) where albumin endocytosis occurs. We show that Ang II is operating to stimulate endocytosis via an AT2 receptor. Our data are consistent with published work (11) showing that overexpression of the AT2 receptor in mice with glomerular injury reduces urinary albumin excretion and that this effect is completely abolished by the AT2 antagonist, PD123319. In view of the published work and our experiments, we postulate that AT2 receptors play an important role in the reabsorption of albumin through an increase of albumin endocytosis in proximal tubule cells.
The AT2 receptor is an atypical G protein-coupled receptor. Consensus regarding the exact nature of the signaling mechanisms involved in its mechanism of action is still lacking (12). Our work resolves some of this controversy by showing that the early events in the AT2-mediated Ang II stimulation of albumin endocytosis involve the activation of PKB.
In contrast to our work, activation of PKB by Ang II mediated through the AT1 receptor (18, 19) has been observed in other cell types. Activation by AT1 and tyrosine receptors (7, 15, 18, 19) involves the translocation of PKB to the plasma membrane and the phosphorylation of its two regulatory sites, Ser-473 in the hydrophobic C-terminal regulatory domain and Thr-308 in the activation loop of the kinase domain (20).
In contrast, we showed that Ang II increases PKB activity via the AT2 receptor in LLC-PK1 cells by a selective and transient increase in phosphorylation of the Thr-308 residue above basal levels without a change in Ser-473 phosphorylation. These data indicate that the mechanisms of signal transduction involved in AT2-mediated activation of PKB observed in our studies are very different from those reported for AT1-medited activation (18, 19).
In our studies, staurosporine reduced basal levels of Thr-308 phosphorylation and inhibited the stimulatory effect of Ang II on Thr-308 phosphorylation and PKB activity, indicating an involvement of PDK1. Staurosporine inhibits other kinases besides PDK1, although the involvement of these kinases could be ruled out here because these kinases are not able to phosphorylate the Thr-308 residue (7, 21). In LLC-PKI cells, staurosporine did not change the basal phosphorylation of Ser-473, in agreement with prior studies showing that phosphorylation of Ser-473 occurs via autophosphorylation by PKB itself or kinases other than PDK1 (22, 23).
Activation of PKB by selective phosphorylation of Thr-308 was observed after β-adrenergic receptor activation by isoproterenol in fat cells (24), by Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK) in NG108 neuroblastoma cells (25) and by cAMP in PKB-transfected 293 cells (26). However, in all of these studies, it was observed that phosphorylation of the Thr-308 residue is independent of wortmannin. In sharp contrast, our results show that both LY294002 and wortmannin, two compounds known to effectively reduce PI3K activity (5, 6), abolished Ang II-induced stimulation of albumin endocytosis and reduced basal and Ang II-induced increases in Thr-308 phosphorylation, indicating an involvement of PI3K in the activation of PKB.
It is well known that increasing the activity of PI3K increases translocation of PKB to the plasma membrane and, consequently, phosphorylation of Ser-473 (7, 15). Thus, the phosphorylation of Ser-473 is indicative of PKB binding to the plasma membrane. Our data show that inhibition of PI3K by wortmannin reduces the basal phosphorylation of Ser-473, suggesting that there is a resident pool of PKB in the membrane fraction that is regulating the basal PI3K activity. The observation that Ang II did not change the phosphorylation of the Ser-473 residue suggests that Ang II did not induce further increases in PI3K activity above basal levels or enhance PKB translocation to the plasma membrane.
Taken together, our data show that Ang II increases Thr-308 phosphorylation of a resident pool of PKB in the membrane fraction in LLC-PK1 cells. Watton and Downward (27) showed that, in the polarized epithelial MDCK cell line, a resident pool of PKB localizes to sites of cell-matrix and cell-cell contact even in the absence of activation by serum.
Other proteins belonging to AGC kinases family, such as protein kinase A (PKA) and protein kinase C (PKC), modulate albumin endocytosis in OK cells (1, 28). Both kinases inhibit albumin endocytosis through the modulation of a different step in endocytosis. For example, PKA modulates the early phase whereas PKC modulates the late phase of endocytosis (1, 28). In our studies, the maximal effect of Ang II to stimulate endocytosis was observed after 3 h of preincubation, indicating that it takes several hours to elicit the full effect of Ang II on the signal transduction pathways regulating albumin endocytosis. In our experimental approach, we studied the time course of albumin endocytosis over a period of 1-30 min. We found that, once fully activated by Ang II, the early events in albumin endocytosis that are observed after only 1 min of treatment with albumin are increased compared with control.
Albumin overloading in proximal tubule cells is associated with tubular cell injury and apoptosis. This process involves the release of cytokines such as TGF-β1 (29). During albumin overload in proximal tubule cells, the organism faces a dilemma between avoiding the loss to albumin, a very abundant protein in serum that contributes to many physiological functions, and preventing the toxic effects of albumin. PKB induces cell survival and blocks programmed cell death or apoptosis (16). Therefore, it is plausible that AT2-mediated Ang II stimulation, which induces PKB activation in proximal tubule cells, could decrease the toxic effects of albumin and avoid the loss of albumin through an increase in albumin reabsorption. This hypothesis is made stronger by the observation that, in a glomerular injury-induced mouse model, overexpression of AT2 decreases the expression of TGF-β1 and urinary albumin excretion (11).
In summary, we have shown that low-dose of Ang II increases albumin endocytosis through an AT2 receptor located on the luminal side and triggers the activation of PKB. The demonstration of coupling between the AT2 receptor and PKB activation involving a transient increase in phosphorylation of the Thr-308 residue without a change in the magnitude of Ser-473 phosphorylation suggests that Ang II activates a plasma membrane resident pool of PKB. This represents a previously undescribed mechanism of PKB activation and opens several possibilities to understand PKB regulation and the cellular signaling coupled to AT2 receptors
Acknowledgments
We thank Drs. Doug Murphy and Yinghong Wang for their help with the microscopy. This work was funded by National Institutes of Health Grant DK R01 32753.
Footnotes
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↵ ‡ To whom correspondence should be addressed. E-mail: wguggino{at}jhmi.edu.
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Conflict of interest statement: No conflicts declared.
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This paper was submitted directly (Track II) to the PNAS office.
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Abbreviations: PI3K, phosphoinositide 3-kinase; PI, phosphoinositides; PDK1, phosphoinositide-dependent kinase 1; AT1, angiotensin receptor type 1; AT2, angiotensin receptor type 2; Ang II, angiotensin II; PKB, protein kinase B; PKBi, PKB inhibitor.
- Copyright © 2005, The National Academy of Sciences
