Neph1 cooperates with nephrin to transduce a signal that induces actin polymerization

Abstract

While the mechanisms that regulate actin dynamics in cellular motility are intensively studied, relatively little is known about signaling events that transmit outside-in signals and direct assembly and regulation of actin polymerization complexes at the cell membrane. The kidney podocyte provides a unique model for investigating these mechanisms since deletion of Nephrin or Neph1, two interacting components of the specialized podocyte intercellular junction, results in abnormal podocyte morphogenesis and junction formation. We provide evidence that extends the existing model by which the Nephrin-Neph1 complex transduces phosphorylation-mediated signals that assemble an actin polymerization complex at the podocyte intercellular junction. Upon engagement, Neph1 is phosphorylated on specific tyrosine residues by Fyn, which results in the recruitment of Grb2, an event that is necessary for Neph1-induced actin polymerization at the plasma membrane. Importantly, Neph1 and Nephrin directly interact and, by juxtaposing Grb2 and Nck1/2 at the membrane following complex activation, cooperate to augment the efficiency of actin polymerization. These data provide evidence for a mechanism reminiscent of that employed by vaccinia virus and other pathogens, by which a signaling complex transduces an outside-in signal that results in actin filament polymerization at the plasma membrane.

FIG. 1.

Neph1 interacts with Grb2 in a tyrosine phosphorylation-dependent manner. (A) Rat glomerular lysate was incubated with GST-Neph1 expressed in tyrosine kinase-expressing E. coli TKB1 or E. coli BL21. Following pull-down and resolution of GST-Neph1-associated proteins on SDS-PAGE, blots were immunoblotted with indicated antibodies. (B) Neph1 and Grb2 associate in vivo. Coimmunoprecipitation experiments performed on cells from rat glomerular lysate using antibodies against Neph1 and Grb2 show endogenous association between these two proteins. (C) Direct interaction between Neph1 and Grb2. Purified recombinant GST-Neph1 and GST-Grb2 were mixed, and immunoprecipitation and immunoblotting were performed with the indicated antibodies. Only phosphorylated Neph1 (lane 5) associated with Grb2. Input recombinant proteins were identified by immunoblotting. Tyrosine phosphorylation of GST-Neph1 prepared in TKB1 was confirmed (data not shown). (D) Grb2 interacts with tyrosine-phosphorylated Neph1 via its SH2 domain. Lysates from COS7 cells expressing Flag-tagged wild-type Neph1 treated where indicated with pervanadate (PV) were mixed with GST fusion proteins containing full-length Grb2 and a truncated Grb2 containing only its SH2 domain. The Grb2 SH2 domain was sufficient to pull down tyrosine-phosphorylated Neph1. The Coomassie blue-stained SDS-polyacrylamide gel shows the purified GST recombinant protein input. Input COS7 cell lysates employed are shown. (E) GST-Grb2 overlay. Arrayed Neph1 oligopeptides synthesized with tyrosine phosphorylation at Y604, Y716, Y719, Y637, and Y638 were incubated with GST-Grb2 and then probed with anti-Grb2 antibody. Grb2 associated with peptides containing tyrosine phosphorylation at the Y⁶³⁷YNV motif. Relative molecular masses are in kDa. WB, Western blot; IP, immunoprecipitation.

FIG. 2.

Fyn catalyzes Neph1 tyrosine phosphorylation and is necessary for Neph1-Grb2 interaction. (A) Coimmunoprecipitation experiments on cells from rat glomerular lysates showing interaction between endogenous Neph1 and Fyn (PI, preimmune serum). (B) In vitro kinase assay. GST-Neph1 was incubated with active recombinant His-tagged Fyn in the presence of [γ-³²P]ATP for 20 min. Radiolabeled Fyn is observed as anticipated in lanes 2 to 4. Phosphorylation of both Neph1 and Fyn is attenuated in the presence of PP2, a Src family kinase inhibitor. A Coomassie blue-stained gel indicates expression of recombinant GST proteins. (C) Neph1-Grb2 interaction in Fyn-null mice. Glomerular lysates from kidneys of wild-type mice or mice with deletion of Fyn were immunoprecipitated with Neph1 antibody and blotted for Grb2. Grb2 coimmunoprecipitated with Neph1 in wild-type mice. Tyrosine phosphorylation of Neph1 was significantly attenuated in Fyn-null mice compared to control.

FIG. 3.

Mapping of Neph1 tyrosine residues phosphorylated by Fyn. (A) Peptide array screen. Oligopeptides (11- to 18-mers) were synthesized covering all 19 tyrosine residues in the Neph1 cytoplasmic domain and were arrayed. WT, wild type. Peptides with replacements of tyrosine by phenylalanine are indicated. Equal quantities of oligopeptides were blotted onto a PVDF membrane, incubated with or without active recombinant Fyn and [γ-³²P]ATP, and evaluated by phosphorimager analysis. Four tyrosine residues, Y637, Y638, Y716, and Y719, were phosphorylated in the presence of active Fyn. (B) COS7 cells were transfected with plasmids encoding Fyn or kinase-dead Fyn (KD) and with wild-type Neph1 or its indicated tyrosine mutants. Neph1 was immunoprecipitated from cell lysates and immunoblotted for phosphotyrosine content (pY). The graph displays band intensity normalized to total immunoprecipitated Neph1; represented are means ± standard errors of the means. Results are representative of three independent experiments. (C) Both tyrosines Y637 and Y638 are essential for Neph1-Grb2 interaction. COS7 cells were transiently transfected with plasmids encoding full-length wild-type Neph1 (Neph1 WT) and its tyrosine mutants. Grb2-GFP, Fyn, and kinase-dead (Fyn KD) plasmids were cotransfected where indicated. Neph1-Grb2 association was abrogated when both Y637 and Y638 were replaced with phenylalanine simultaneously.

FIG. 4.

Neph1 is tyrosine phosphorylated and recruits Grb2 following PAN-induced podocyte injury in vivo. (A) Time course of urine albumin/creatinine ratios of control or PAN-treated female rats (PAN-treated rats had increased proteinuria compared to controls; P < 0.05, day 3 and later). (B) Electron micrographs of kidneys from vehicle control and PAN-treated rats showing loss of normal foot process architecture at 72 h postinjection (CL, capillary lumen; GBM, glomerular basement membrane; P, podocyte). Bar, 1 μm. (C) Neph1 complexes immunoprecipitated from rat glomerular lysates treated with PAN or vehicle alone were evaluated by immunoblotting. (D) In similar experiments, Nephrin was immunoprecipitated from rat glomerular lysates prepared as indicated (PI, preimmune serum).

FIG. 5.

Grb2 is recruited to the CD16/CD7/Neph1CD cluster at the plasma membrane. (A) NIH 3T3 cells expressing indicated CD16/CD7 chimeric proteins (red) and Grb2-GFP (green) were treated with anti-CD16 antibody (primary) and rhodamine-labeled anti-IgG antibody (secondary) or secondary antibody only (top row, as control) and then fixed and examined by confocal microscopy. The farthest-right panels are images reconstructed in the yz plane. Colocalization of Grb2-GFP and CD16/CD7/Neph1CD appears yellow on merged images. Indicated CD16/CD7/Neph1CD chimeric proteins mutated at Y637 and/or Y638 were expressed with Grb2-GFP in NIH 3T3 cells. Complexes were clustered as described and evaluated by confocal microscopy. Data are representative of multiple experiments. Magnification, ×600. (B) NIH 3T3 cells expressing CD16/CD7/Neph1CD were treated with clustering antibodies (primary and secondary antibodies) for the time periods shown; following Neph1 immunoprecipitation, immune complexes were immunoblotted using antiphosphotyrosine antibody.

FIG. 6.

Fyn is necessary for Neph1-Grb2 recruitment. SYF cells were transfected with CD16/CD7/Neph1CD and Grb2-GFP. Grb2 was recruited to Neph1 only in the presence of Fyn. The extreme right panels show the images reconstructed in the yz plane with recruitment at the apical surface of the cell.

FIG. 7.

Neph1 activation results in Grb2-dependent actin polymerization. CD16/CD7/Neph1CD or CD16/CD7/HA chimeric protein (red), Grb2-GFP, and actin-CFP were expressed in NIH 3T3 cells, clustered with anti-CD16 antibody (primary) and rhodamine-labeled anti-IgG antibody (secondary), and then fixed and examined by confocal microscopy. In the presence of the Y⁶³⁷/Y⁶³⁸ Neph1 mutant neither Grb2 recruitment nor actin polymerization was seen. The bottom panels show a schematic of actin pedestal and enlarged views of representative actin pedestals identified in cells coexpressing clustered CD16/CD7/Neph1CD, GFP-Grb2, and actin-CFP.

FIG. 8.

Clustering of Nephrin cytoplasmic domain with Neph1 induces simultaneous tyrosine phosphorylation of both Nephrin and Neph1. (A) CD16/CD7/NephrinCD chimeric protein (red) and full-length wild-type Neph1-GFP were expressed in NIH 3T3 cells and were clustered with anti-CD16 antibody (primary) and rhodamine-labeled anti-IgG antibody (secondary) and then fixed and examined by confocal microscopy. Far right panels are images reconstructed in the yz plane. (B) CD16/CD7/NephrinCD chimeric protein and full-length Flag-tagged Neph1 were expressed in NIH 3T3 cells and clustered (right panel) or not clustered (left panel). Lysates were resolved with SDS-PAGE and probed with the indicated antibody. (C) HEK293 cells stably expressing human Nephrin were transfected with Flag-tagged Neph1 and Fyn. 50A9 antibody against human Nephrin ectodomain was used to induce Nephrin clustering. Lysates were immunoprecipitated with Neph1 antibody, resolved with SDS-PAGE, and probed with antiphosphotyrosine antibody.

FIG. 9.

Engagement of either Nephrin or Neph1 when in association results in recruitment of Nck and Grb2. Indicated expression plasmids were combined, expressed in NIH 3T3 cells, clustered with anti-CD16 antibody (primary) and rhodamine-labeled anti-IgG antibody (secondary), and then fixed and examined by confocal microscopy. The farthest-right panels are images reconstructed in the yz plane. Insets in the bottom two panels represent fivefold enlargements of a portion of the micrograph demonstrating a key feature.

FIG. 10.

Nephrin and Neph1 cooperate to induce actin polymerization in a Nck- and Grb2-dependent fashion. Quantification of actin tail-forming efficiency. As described in the text, combinations of CD16/CD7/NephrinCD, full-length wild-type Neph1, CD16/CD7/NephrinCD(TM), or full-length wild-type Neph1(DM) were expressed with Nck and/or Grb2 in NIH 3T3 cells as indicated. The number of actin tails per individual cell was counted in 30 cells for each condition. Each experiment was repeated four times; the graph represents the mean number of tails per cell ± standard deviation. The table provides results of the single-tailed paired t test used to compare groups. Neph1(DM), Y-to-F mutation at Y637 and Y638; Nephrin(TM), Y-to-F mutation at Y1191, Y1208, and Y1232. NA, not applicable.