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View; B, Tubule) BASIC RESEARCH www.jasn.org KIBRA Modulates Directional Migration of Podocytes Kerstin Duning,* Eva-Maria Schurek,*† Marc Schlu¨ter,* Michael Bayer,* Hans-Christian Reinhardt,* Albrecht Schwab,‡ Liliana Schaefer,§ Thomas Benzing,† ʈ Bernhard Schermer,† Moin A. Saleem, Tobias B. Huber,¶ Sebastian Bachmann,** Joachim Kremerskothen,* Thomas Weide,* and Hermann Pavensta¨dt* *Medizinische Klinik und Poliklinik D and ‡Institut fu¨r Physiologie II, Universita¨tsklinikum Mu¨nster, Mu¨nster, §Universita¨tsklinikum Frankfurt, Pharmazentrum, Frankfurt/Main, †Universita¨tsklinikum Ko¨ln, Innere Medizin IV, Nephrologie und Allgemeine Innere Medizin, Ko¨ln, ¶Medizinische Universita¨tsklinik, Abteilung Innere Medizin IV, Freiburg, and **Charite´–Universita¨tsmedizin Berlin, Institut fu¨r Vegetative Anatomie, Berlin, Germany; and ʈ Academic and Children’s Renal Unit, University of Bristol, Bristol, United Kingdom ABSTRACT Asymmetric delivery and distribution of macromolecules are essential for cell polarity and for cellular functions such as differentiation, division, and signaling. Injury of podocytes, which are polarized epithelial cells, changes the dynamics of the actin meshwork, resulting in foot process retraction and proteinuria. Although the spatiotemporal control of specific protein–protein interactions is crucial for the establishment of cell polarity, the mechanisms controlling polarity-dependent differentiation and division are incompletely understood. In this study, yeast two-hybrid screens were performed using a podocyte cDNA library and the polarity protein PATJ as bait. The protein KIBRA was identified as an interaction partner of PATJ and was localized to podocytes, tubular structures, and collecting ducts. The last four amino acids of KIBRA mediated binding to the eighth PDZ domain of PATJ. In addition, KIBRA directly bound to synaptopodin, an essential organizer of the podocyte cytoskeleton. Stable knockdown of KIBRA in immortalized podocytes impaired directed cell migration, suggesting that KIBRA modulates the motility of podocytes by linking polarity proteins and cytoskeleton-associated protein complexes. J Am Soc Nephrol 19: 1891–1903, 2008. doi: 10.1681/ASN.2007080916 Cell polarity regulates important processes, such as the actin cytoskeleton, resulting in foot process re- asymmetric cell division, cellular morphology, in- traction (foot process effacement) and proteinuria. tracellular signaling, and cell migration. So far, it is Obviously, a regulated cell polarity is essential for known that specific protein–protein interactions podocyte function. Nevertheless, the expression, are important for these processes, but the detailed molecular mechanisms that control cell polarity are Received August 20, 2007. Accepted April 11, 2008. poorly understood. Podocytes are highly polarized Published online ahead of print. Publication date available at epithelial cells that play a key role in the mainte- www.jasn.org. nance of the size-selective filtration barrier of the 1 K.D. and E.-M.S. contributed equally to this work, and T.W. and kidney. They consist of a cell body with primary H.P. contributed equally to this work. and highly branched secondary foot processes, Correspondence: Dr. Thomas Weide, UKM, Medizinische Klinik leading to a complex “neuron-like” cell architec- und Poliklinik D, Abteilung: Molekulare Nephrologie, Domagk- ture. The interdigitating secondary foot processes strasse 3a, D-48149 Mu¨nster, Germany. Phone: ϩ49-251-83- mediate the adhesion to the glomerular basement 57939; Fax: ϩ49-251-83-57943; E-mail: [email protected]; or Prof. Hermann Pavensta¨dt, UKM, Medizinische Klinik und membrane and form the slit diaphragm, unique Poliklinik D, Albert-Schweizer Strasse 33, D-48149 Mu¨nster, cell–cell contacts that serve as a final filtration bar- Germany. Phone: ϩ49-251-83-47516; Fax: ϩ49-251-83-46979; rier.1 E-mail: [email protected] Injury of podocytes leads to dynamic changes of Copyright ᮊ 2008 by the American Society of Nephrology J Am Soc Nephrol 19: 1891–1903, 2008 ISSN : 1046-6673/1910-1891 1891 BASIC RESEARCH www.jasn.org function, and cross-talk of polarity regulators and the molec- RESULTS ular links between polarity proteins and downstream effects such as signaling, differentiation, and directional migration of KIBRA Directly Interacts with Cell Polarity Protein podocytes are unknown. PATJ During the past decade, two polarity complexes have been During the past decade, it was shown that the Pals1-PATJ- described, the aPKC-PAR3-PAR6 (aPKC for atypical protein Crb3 and aPKC-PAR3-PAR6 cell polarity complexes are espe- kinase C; PAR for partitioning defective) and the Pals1-PATJ- cially important in diverse epithelia of various tissues; there- Crb complex (Pals1 for protein-associated with Lin7–1; PATJ fore, we first tested whether these core proteins of the apical for Pals1-associated tight junction protein; and Crb3 for polarity complexes are expressed in immortalized cultured Crumbs3).2–4 It has been shown that these complexes are part podocytes.22 We found mRNA expression of PATJ; Pals1; both of an evolutionarily conserved system that regulates apicobasal Crumbs3 isoforms (Crb3a/b); and aPKC␨, PAR3, and three polarity, tight junction formation, signaling, and directional PAR6 isoforms in podocyte cDNA library PCR reactions (Fig- migration of eukaryotic cells. All core components of the com- ure 1A). This expression pattern might be a first hint that the plexes carry multiple protein–protein interaction modules, cell polarity of podocytes could be regulated in a similar man- suggesting that they are part of multiprotein complexes.2–4 ner as already shown for many other cell types. Interestingly, PATJ (also called INADL or CIPP) was first iden- For further examinations, we focused on the multi-PDZ tified as a highly abundant protein in brain and kidney.5–7 In ad- protein PATJ and performed a Y2H screen, using a podocyte dition to the N-terminal L27 domain, PATJ contains ten PDZ (for cDNA library and PATJ as bait. One of the isolated yeast clones PSD95/discs large/zonula occludens 1 [ZO-1]) domains, suggest- encoded an N-terminal deletion of KIBRA.23 Mapping studies ing that PATJ acts as a scaffolding protein that is able to bind to using a set of PATJ deletion mutants in the Y2H system (Figure many cellular partners through these domains.3,6 Previously, it 1, B and C) and GST pull-down assays revealed that PDZ8 was shown that PATJ associates with neuronal proteins and chan- mediates the PATJ–KIBRA interaction (Figure 1D). nels (e.g., neurexin, neuroligin, members of the Kir-family, The identified KIBRA clone (clone 29) of the Y2H screen ASIC3, 5HT2A), indicating that it recruits receptors and struc- lacks the two N-terminal WW domains (amino acids [aa] 1 tural proteins at synaptic sites.8,9 A recent study reported that through 39 and 54 through 86) but contains a putative cal- PATJ interacts with the tuberous sclerosis complex protein 1 and cium-sensitive C2-like domain (aa 726 through 787), a gluta- 2 (TSC1/2) and thereby links the mammalian target of rapamycin mate-rich region (aa 845 through 868), and the aPKC␨-bind- (mTOR) signaling pathway to the Pals1-PATJ-Crb3 cell polarity ing domain (aa 953 through 996; Figure 1B, bottom). The complex, supporting the idea that cell polarity proteins are also C-terminal four aa of KIBRA (ADDV, single-letter code) con- involved in signaling pathways.10 tain a putative class III PDZ-binding site.24 To find out In addition, PATJ directly binds to the tight junction pro- whether the KIBRA-PATJ interaction is mediated by this mo- teins ZO-3 and claudin 1 via the sixth and eighth PDZ do- tif, we performed co-immunoprecipitation (Co-IP) assays mains, emphasizing that PATJ plays an important role in the with lysates from HEK293T cells expressing FLAG-tagged full- maintenance of cell polarity and tight junction establishment length KIBRA with and without the ADDV motif and EYFP- of epithelial cells.11–14 Recently, it was shown that PATJ is part tagged full-length PATJ, respectively. Only KIBRA containing of a huge Rich/Amot complex by interacting with members of this motif was able to precipitate EYFP-tagged PATJ in these the Amot protein family.12,15 This family plays a role in the assays (Figure 1E). In addition, lysates from HEK293T express- regulation of cell–cell junctions and cell motility.16 In this con- ing the GFP-tagged C-terminus of KIBRA with and without text, it is interesting that the knockdown of PATJ in epithelial the ADDV motif (aa 1045 through 1113/1109) were incubated cells results in an impaired migration of MDCK II cells, sug- with recombinant GST PDZ7–10 fusion proteins. Again, only gesting that PATJ regulates not only cell polarity and tight KIBRA deletion mutants that contained the ADDV motif in- junction establishment but also the directional migration of teracted with PATJ (Figure 1F). epithelial cells. In this study, we performed a yeast two-hybrid (Y2H) KIBRA Is Expressed in Various Renal Tissues screen, using a cDNA library from immortalized podocytes KIBRA and PATJ both are expressed in immortalized podo- and PATJ as bait. We found KIBRA (for kidney brain), a pro- cytes (Figure 2A). Furthermore, immunofluorescence analysis tein with high expression in the brain and kidney that probably revealed that KIBRA and PATJ co-localize throughout the cy- plays a central role in human memory, as interaction partner of toplasm in a predominant perinuclear pattern. Minor frac- PATJ.17–21 tions of both proteins were also found at the lamellipodia lead- Our investigations revealed that in the kidney, KIBRA is ing edge, similar to the recently described PATJ distribution in expressed in glomerular podocytes, in some tubules, and in the migrating MDCK II cells (Figure 2B).25 In addition, we found collecting duct. In addition to the PATJ interaction, we found that KIBRA partially co-localizes with the actin and tubulin that KIBRA binds to synaptopodin, an essential protein of podo- cytoskeleton at these leading edges (Figure 2C, a through f). cytes. Furthermore, a knockdown of the KIBRA expression re- By contrast, vimentin, a marker for intermediate filaments, sulted in an impairment of podocyte directional migration.
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