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NEPH1 defines a novel family of podocin interacting proteins ¹

Department of Internal Medicine, Division of Nephrology, University Hospital Freiburg, Germany

³Correspondence: Renal Division, University Hospital Freiburg, Hugstetterstrasse 55, 79106 Freiburg, Germany. E-mail: walz{at}med1.ukl.uni-freiburg.de

SPECIFIC AIMS

Characterization of NEPH1 and description of its family members NEPH2 and NEPH3.

PRINCIPAL FINDINGS

1. NEPH1 is a member of three structurally related proteins

2. NEPH1, NEPH2, and NEPH3 are expressed in podocytes and interact with podocin

3. NEPH1 is a signaling molecule that needs the presence of TEC kinases to fully trans-activate AP-1

CONCLUSIONS AND SIGNIFICANCE

The congenital nephrotic syndrome of the Finnish type is caused by mutations in NPHS1, the gene encoding for nephrin, and is characterized by massive proteinuria in utero and nephrosis at birth. Nephrin, a member of the immunoglobulin superfamily, is located at opposing sites of the secondary foot processes formed by podocytes, a specialized epithelial cell that ensures size and charge selective ultrafiltration. However, the precise function of nephrin remains unknown. A second steroid-resistant nephrotic syndrome is caused by mutations of NPHS2, the gene encoding for podocin. Podocin is a member of the stomatin protein family and is predicted to form a membrane-associated hairpin-like structure with a cytosolic amino- and carboxyl-terminal domain typical of stomatin-like proteins and caveolins. We have recently demonstrated that nephrin is a signaling molecule that activates canonical protein kinase cascades. Podocin interacts with the carboxy-terminal, cytoplasmic domain of nephrin and greatly enhances nephrin-induced signaling. NEPH1, a recently identified molecule with five extracellular immunoglobulin-like domains, is structurally related to nephrin. NEPH1 is abundantly expressed in the kidney, and disruption of the NEPH1 gene in mice results in effacement of glomerular podocytes, heavy proteinuria, and early postnatal death.

We report now that NEPH1 belongs to a family of three closely related proteins that bind to the carboxy-terminal domain of podocin. All three proteins belong to the immunoglobulin superfamily and share a common domain architecture consisting of five extracellular immunoglobulin-like repeats, followed by a transmembrane domain and a cytoplasmic domain of 198–235 amino acids. A survey of EST database entries and published data revealed that podocin and the NEPH family members have a similar tissue distribution. Immunohistochemistry revealed that both podocin and NEPH1 colocalize in renal glomeruli and outline the glomerular basal membrane. Since all three NEPH proteins can be detected in human podocytes, we speculated that NEPH proteins are a novel family of podocin binding proteins. Coimmunoprecipitation studies revealed that the cytoplasmic domain of all three proteins of this newly identified gene family binds the carboxyl terminus of podocin; furthermore, podocin precipitates endogenous NEPH1 from podocytes. The cytoplasmic domain of all three NEPH proteins contains a short but highly conserved stretch of nine conserved amino acids (KDPTNGYYxV). Mutational analysis of NEPH1 and tyrosine-to-alanine replacement at position 7 of this motif demonstrated that the integrity of this motif is essential for binding of podocin to NEPH1. Since this NEPH1 mutation substantially inhibits NEPH1-podocin interaction, we speculated that the interaction between podocin and NEPH1 may be regulated by a protein kinase that recognizes the PxxxY motif and phosphorylates tyrosine 637. Since NEPH1 contains a binding site for the SH2 domain of Itk and the PxxxY motif can be found in substrates of the src kinase family, NEPH1 and podocin were coexpressed with Itk and v-src. Both protein kinases substantially reduced the interaction of the carboxy-terminal domain of NEPH1 with podocin, indicating that tyrosine phosphorylation of the podocin binding motif regulates the interaction between NEPH1 and podocin (Fig. 1 ).

View larger version (20K): [in this window] [in a new window]   Figure 1. NEPH1, NEPH2, and NEPH3 interact with podocin. A) NEPH1 interacts with podocin. HEK 293T cells were transiently transfected with Flag-tagged green fluorescent protein (gfp), podocin, and mouse NEPH1. Immunoprecipitation (IP) was performed with the Flag-specific M2 beads. Immobilized NEPH1 was detected, using a NEPH1-specific antiserum in combination with Western blot analysis (upper panel). Equal expression of NEPH1 in all three transfection was verified by Western blot analysis of the lysates (lower panel). B) Podocin precipitates NEPH1. Immobilization of endogenous protein with podocin antiserum coprecipitates NEPH1 in lysates out of mouse podocytes. C) NEPH1 interacts with the carboxyl-terminal domain of podocin. HEK 293T cells were transiently transfected with NEPH1 and sIg.7 containing the CD5 leader sequence, followed by the CH2 and CH3 domain of human IgG, and the transmembrane domain of CD7, fused to either the amino- or carboxyl-terminal domain of podocin. Immunoprecipitation (IP) was performed with protein G Sepharose. Immobilized NEPH1 was detected using a NEPH1-specific antiserum in combination with Western blot analysis (upper panel). Equal expression of NEPH1 in all 3 transfections was verified by Western blot analysis of the lysates (lower panel). D) The carboxyl-terminal domains of nephrin, NEPH1, NEPH2, and NEPH3 interact with podocin. HEK 293T cells were transiently transfected with Flag-tagged podocin and sIg.7 fused to the carboxyl-terminal domain of either nephrin, NEPH1, NEPH2, or NEPH3. Immunoprecipitation (IP) was per- formed with protein G Sepharose. Immobilized podocin was detected, using the Flag-specific M2 monoclonal antibody in combination with Western blot analysis (upper panel). Comparable expression of podocin in all three transfection was verified by Western blot analysis of the lysates (lower panel). E) The podocin binding motif is conserved in all three NEPH family members. Nephrin shares with the NEPHs the proline at position 633 and the tyrosine at position 637 of NEPH1. F) Tyrosine 637 of NEPH1 is important for the NEPH1-podocin interaction. HEK 293T cells were transfected with plasmids as indicated. The Ig fusion proteins containing the carboxyl-terminal domain of NEPH1 or mutant NEPH1 were precipitated using protein G. Flag-tagged podocin was detected by Western blot analysis using the Flag-specific M2 antibody. The lysates demonstrate that the amount of podocin was similar in all conditions. Mutation of the tyrosine to alanine at position 637 of NEPH1 significantly reduces the interaction with podocin, indicating this tyrosine is crucial for the interaction with podocin. In contrast, substitution of proline 633 with serine appears to enhance binding of podocin to the mutant NEPH1. G) The podocin binding motif is recognized by src and Tec family kinases leading to a tyrosine phosphorylation within the PxxxY motif. The binding of podocin to NEPH1 is reduced if tyrosine kinases (v-src and ITK) are coexpressed.

All three NEPH proteins share a conserved Grb2 SH2 binding site as well as several conserved amino acids flanking a PDZK1 binding site at the carboxyl terminus. Both Grb2 and PDZK1 coimmunopreciptated with NEPH1 in transiently transfected cells, suggesting that NEPH molecules can connect to canonical signaling cascades. Since nephrin is a signaling molecule that activates the transcription factor AP-1, we compared the trans-activation mediated by nephrin and NEPH1. Nephrin and NEPH1 both triggered a significant increase in AP-1 activation in transiently transfected HEK 293T cells. However, NEPH1-mediated trans-activation was less striking than the activation induced by nephrin, and podocin had only a modest effect on the NEPH1-mediated AP-1 activation. Since NEPH1 contains an Itk SH2 binding site and both Itk and Tec increased tyrosine phosphorylation of NEPH1, we tested whether Itk influenced the NEPH1-mediated AP-1 activation. Itk by itself had little effect on trans-activation of the AP-1 reporter construct, but in combination with NEPH1 Itk triggered a fourfold increase in NEPH1-mediated AP-1 activation, suggesting that Itk or other members of the Tec family are required for efficient downstream signaling of NEPH1. Tec kinases are widely expressed, contribute to multiprotein complexes, and are essential for activation of PLC . They participate in signal transduction of growth factor receptors, cytokine receptors, G-protein coupled receptors, antigen receptors, and integrins as well as cytoskeletal reorganization, cellular migration, and regulation of the barrier function of epithelial cells. At least two family members, Bmx and Tec, can be found in the kidney, and both podocin and Tec kinases are present in lipid rafts. Hence, it is conceivable that podocin recruits NEPH1 and Tec kinases to specialized microdomains, where phosphorylation of NEPH1 releases this molecule from its interaction with podocin (Fig. 2 ). Our findings establish a new family of podocin interacting signaling molecules and demonstrate a dynamic recruitment of NEPH1 to podocin-containing microdomains. The NEPH3 gene is located on chromosome 19q13.1 immediately adjacent to the NPHS1 gene. It will be interesting to examine whether some patients with a hereditary nephrotic syndrome and an apparent linkage with the NPHS1 gene may in fact suffer from mutations in the NEPH3 gene.

View larger version (36K): [in this window] [in a new window]   Figure 2. Schematic diagram. A) Podocin (red) clusters and NEPH1 (blue) in specialized microdomains that also contain TEC kinases (purple). The tyrosine 637 within NEPH1 (yellow Y637) is unphosphorylated and thereby the binding to podocin facilitated. B) In the presence of TEC which has been found to bind to nephrin NEPH1 is tyrosine phosphorylated and dissociates out of the interaction with podocin, which can be mimicked by a mutation of tyrosine to alanine at amino acid 637.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0242fje; to cite this article, use FASEB J. (November 1, 2002) 10.1096/fj.02-0242fje

² These authors contributed equally to this work.

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