Akt recruits Dab2 to albumin endocytosis in the proximal tubule

Abstract

Proximal tubule epithelial cells have a highly sophisticated endocytic machinery to retrieve the albumin in the glomerular filtrate. The megalin-cubilin complex and the endocytic adaptor disabled-2 (Dab2) play a pivotal role in albumin endocytosis. We previously demonstrated that protein kinase B (Akt) regulates albumin endocytosis in the proximal tubule through an interaction with Dab2. Here, we examined the nature of Akt-Dab2 interaction. The pleckstrin homology (PH) and catalytic domains (CD) of Akt interacted with the proline-rich domain (PRD) of Dab2 based on yeast-two hybrid (Y2H) experiments. Pull-down experiments utilizing the truncated constructs of Dab2 demonstrated that the initial 11 amino acids of Dab2-PRD were sufficient to mediate the interaction between Akt and Dab2. Endocytosis experiments utilizing Akt1- and Akt2-silencing RNA revealed that both Akt1 and Akt2 mediate albumin endocytosis in proximal tubule epithelial cells; therefore, Akt1 and Akt2 may play a compensatory role in albumin endocytosis. Furthermore, both Akt isoforms phosphorylated Dab2 at Ser residues 448 and 449. Ser-to-Ala mutations of these Dab2 residues inhibited albumin endocytosis and resulted in a shift in location of Dab2 from the peripheral to the perinuclear area, suggesting the physiological relevance of these phosphorylation sites in albumin endocytosis. We conclude that both Akt1 and Akt2 are involved in albumin endocytosis, and phosphorylation of Dab2 by Akt induces albumin endocytosis in proximal tubule epithelial cells. Further delineation of how Akt affects expression/phosphorylation of endocytic adaptors and receptors will enhance our understanding of the molecular network triggered by albumin overload in the proximal tubule.

Akt Links Insulin Signaling to Albumin Endocytosis in Proximal Tubule Epithelial Cells

“… In order to examine a possible direct interaction between cytoplasmic tail (CT) of megalin and AS160, in-vitro pull-down experiments were carried out. Cytoplasmic tail of megalin was cloned and GST-megalin fusion protein was generated as outlined in the methods section [ 21 ]. Approximately 100μg of GST, GST-megalin CT immobilized on GSH-Sepharose were incubated with HKC-8 cell lysate.…”

“…We reported that protein kinase B (Akt), a pivotal protein in insulin signaling, mediates albumin endocytosis in proximal tubule epithelial cells through its interaction with the endocytic proteins [ 20 – 21 ]. In this study, we examined the effect of insulin induced Akt activation on albumin endocytosis in proximal tubule epithelial cells.…”

Receptor-Mediated Endocytosis in the Proximal Tubule

“…Mutation of the phosphorylated serine residues on Dab2 caused its loss from the cell surface and reduced albumin uptake in human HKC-8 cells (94). Additionally, overexpression of Akt/PKB stimulated albumin uptake, whereas knockdown had the converse effect (94, 95).…”

“…Mutation of the phosphorylated serine residues on Dab2 caused its loss from the cell surface and reduced albumin uptake in human HKC-8 cells (94). Additionally, overexpression of Akt/PKB stimulated albumin uptake, whereas knockdown had the converse effect (94, 95). Angiotensin II, acting via the AT 2 receptor, also stimulates albumin endocytosis in LLC-PK1 cells by the activation of Akt/PKB via its upstream regulator phosphatidylinositol 3-kinase (PI3K) (96).…”

O-GlcNAcylation reduces proximal tubule protein reabsorption and promotes proteinuria in spontaneously hypertensive rats

“…Megalin was recently described as a fast-recycling, long-lived receptor, which completes its cycle through the different endosomal compartments in ϳ10 min (34). Different mechanisms were shown to regulate megalin trafficking, such as adaptor proteins ARH (35) and Dab2 (36), and GSK3␤-dependent phosphorylation in the PPPSP domain (37), explaining the fast cycling kinetics of this receptor. Here, we demonstrated, both in vitro and in vivo, that…”