Podocalyxin Activates RhoA and Induces Actin Reorganization through NHERF1 and Ezrin in MDCK Cells

Abstract

Abstract. Podocalyxin (PC) is the major sialoglycoprotein expressed on the apical membrane of the podocyte. Previously it was shown that PC is connected to actin through the PC/ NHERF2/ezrin complex, and this connection is disrupted in the nephrotic syndrome. For assessing whether expression of PC affects the organization of the actin cytoskeleton, MDCK cell lines stably expressing either full-length PC or a PC mutant lacking the NHERF binding site was established. It was found that full-length PC but not the PC mutant is connected to actin, induces redistribution of actin toward the apical membrane, and leads to increased RhoA activity. By immunofluorescence redistribution of RhoA and RhoGDI was observed in the presence of both full-length PC and the PC mutant. With the use of pulldown assays, PC and ezrin were found to interact directly and the ezrin binding site was mapped to the juxtamembrane region of PC's cytoplasmic tail. It is concluded that PC binds to ezrin both directly and indirectly. PC activates RhoA through NHERF and ezrin, leading to redistribution of actin filaments. These results suggest that in podocytes, PC may also regulate foot process architecture through RhoA.The unique foot process and filtration slit architecture of podocytes is essential for maintaining glomerular filtration. Disruption of this organization is characteristically seen in glomerular diseases associated with the nephrotic syndrome. Although the molecular mechanisms that lead to these derangements are not yet fully understood, it has been shown that the dense actin network present in the foot processes is normally anchored to apical and basal membrane proteins as well as to proteins in the slit diaphragm region (1) and undergoes substantial changes in the nephrotic syndrome (2-6).We have previously shown that podocalyxin (PC), the major apical sialoglycoprotein of the podocyte, serves as an antiadhesin that maintains the filtration slits between the foot processes open (7) and is connected to the actin cytoskeleton indirectly through NHERF2 (8), a PDZ protein, and ezrin, a member of the ERM (ezrin-radixin-moesin) family of actinbinding proteins (9). Moreover, we found that the connection of PC to the actin cytoskeleton is disrupted in puromycin aminonucleoside nephrosis (8), a rat model in which there is a dramatic reorganization of the actin cytoskeleton (5,6).In this article, we investigate how the association of PC with actin is regulated, focusing on the small GTPase RhoA, because RhoA has been shown to activate ezrin (10) and ezrin connects various plasma membrane proteins, including PC, to actin filaments in its open or active conformation (11). Activated ERM proteins also bind and sequester RhoGDI (12-15), a negative regulator of Rho GTPases, thereby initiating the activation of RhoA (13) and maintaining ERM activation (16). Moreover, it is clear that proper regulation of Rho GTPase is required for maintaining the differentiation of podocytes, as RhoGDI␣ Ϫ/Ϫ mice, lacking RhoGDI␣, show massive proteinuria and loss o...

Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis

“…The formation of a complex of podocalyxin-EBP50-ezrin facilitates the activation of Rho-family GTPases. 18 Previously, we identified a novel interaction between EBP50 and b-PIX, a specific GEF for Rac1 and Cdc42, and proposed that the podocalyxin-EBP50-ezrin complex recruited b-PIX and, thereby, enhanced the metastatic potential of renal cell carcinoma. 19 To confirm the role of EBP50 in Rac1 activation in living cells, we measured Rac1 activity in EBP50 knockdown MDCK cells in the presence of MRC-5 conditioned medium, which induces Rac1 activation via bioactive hepatocyte growth factor (HGF).…”

“…18,19 Interactions with plasma membrane lipid PIP2 converts ezrin from an auto-inhibitory into an open active conformation, and subsequent PKCmediated phosphorylation of ezrin further stabilizes the activated state of ezrin. 36 Active ezrin then sequesters RhoGDI to release small GTPases for activation by exchanging GDP for GTP.…”

“…17 EBP50 can activate Rho-family small GTPases by recruiting ezrin, which is thought to cause the dissociation of GDI from the small GTPase, and b-PIX, a Rac1 GEF that contains a PDZ-binding motif. 18,19 EBP50 is a serine/threonine-phosphorylated protein. 3 The phosphorylation state of EBP50 regulates the accessibility of its PDZ domains for cognate ligands and the formation of EBP50 oligomers, both of which are required for EBP50 to act as a scaffold protein.…”

The reno-protective effect of a phosphoinositide 3-kinase inhibitor wortmannin on streptozotocin-induced proteinuric renal disease rats

“…The appropriate regulation of GTPase is very important for maintaining differentiation of podocytes (Gundersen et al, 1980). The other GTPases, RhoA, has already been extensively studied in this field (Schmieder et al, 2004;Asanuma et al, 2006;Shibata et al, 2006;Hidaka et al, 2008). Although the role of RhoA in diabetic renal diseases has been evaluated, changes in expression and activity of Rac1/Cdc42 in diabetic kidneys have not been reported.…”

Control of cell adhesion and migration by podocalyxin. Implication of Rac1 and Cdc42

“…1). Since ezrin may interact with PODXL not involving the DTHL domain [31], perhaps it should also be considered as a potential factor involved in controlling the PODXL action.…”