NPHP4^304–950 is sufficient to control Pyk2-induced tyrosine phosphorylation of NPHP1 by interfering with the NPHP1/Pyk2 interaction. A, NPHP4^304–950 controls tyrosine phosphorylation of NPHP1 and interferes with the NPHP1/Pyk2 interaction. F.NPHP1 was precipitated, and tyrosine phosphorylation of precipitated proteins was detected with an anti-phosphotyrosine antibody in the presence of either a control protein, WT NPHP4, or different NPHP4 truncations. Precipitates were also stained for co-precipitating Pyk2. WT NPHP4 and NPHP4^304–950 decrease the tyrosine phosphorylation of NPHP1 (upper panel) and abolish the NPHP1/Pyk2 interaction (lower panel). B, NPHP1 co-precipitates with NPHP4^304–950. FLAG-tagged proteins were precipitated, and co-precipitating V5.NPHP1 was evaluated. V5.NPHP1 specifically co-precipitated with both WT NPHP4 and the NPHP4^304–950 truncation. The immunoprecipitation experiments were performed four times.

Tyrosine phosphorylation of NPHP1 facilitates interaction with PACS-1. A, FLAG-tagged proteins were precipitated (IP), and co-precipitating HA.PACS-1 was analyzed (upper panels). IB, immunoblotting. The expression of WT Pyk2 enhances the amount of co-precipitating PACS-1 with WT NPHP1 compared with the presence of the kinase-dead (kd) variant of Pyk2. B, this effect was lost after mutation of the three phosphorylated tyrosines to phenylalanines, showing the importance of NPHP1 phosphorylation. C, the presence of NPHP4 significantly weakened the interaction of NPHP1 with PACS-1. D, this was also the case for the NPHP1 Y46F/Y349F/Y721F triple mutant. All experiments were performed at least three times.

NPHP4 controls Pyk2-induced tyrosine phosphorylation of NPHP1. A, Pyk2 induces tyrosine phosphorylation of NPHP1. F.NPHP1 was precipitated (IP), and tyrosine phosphorylation of precipitated proteins was detected with an anti-phosphotyrosine antibody (pY99, upper panel). Strong tyrosine phosphorylation of precipitated NPHP1 and co-precipitating Pyk2 was dependent on Pyk2 kinase activity (kd, kinase-dead mutant). IB, immunoblotting. B, phosphoamino acid analysis of precipitated NPHP1 after in vivo labeling of HEK293T cells with ³²P. Phosphorylated amino acids were identified by co-migration with standard nonradioactive phosphoamino acids, ninhydrin staining (indicated by circles), and autoradiography. C, wild-type NPHP4 (WT NPHP4) but not various patient mutations abolishes Pyk2-induced tyrosine phosphorylation of NPHP1. F.NPHP1 was precipitated, and tyrosine phosphorylation was evaluated with an anti-phosphotyrosine antibody. All NPHP4 constructs reduced tyrosine phosphorylation of NPHP1 compared with the empty vector control, but not to the degree of WT NPHP4. NPHP3^517–841 served as control protein. All experiments were performed at least three times with identical results.

Pyk2 induces NPHP1 phosphorylation of tyrosines 46, 349, and 721. A, transiently transfected HEK293T cells were labeled with ³²P and lysed, and F.NPHP1 was immunoprecipitated. Precipitated NPHP1 was separated by SDS-PAGE, transferred to nitrocellulose membranes, and digested in situ with trypsin, and the peptides were separated on TLC plates by high voltage electrophoresis and ascending chromatography. + and − indicate voltage during electrophoresis. Separated phosphopeptides were visualized by autoradiography. Two Pyk2-dependent phosphopeptide fragments could be identified as phosphotyrosine-containing peptides (N1 and N2, marked with circles). B, phosphopeptides N1 and N2 were analyzed by Edman degradation which identified the position of the phosphorylated tyrosine residue in the peptide. C, tyrosine residues 46, 349, and 721 in NPHP1 were mutated to phenylalanine residues, and FLAG-tagged versions of these single mutants were co-expressed with empty vector (lanes 1, 4, 7, and 10), HA-tagged Pyk2 (lanes 2, 5, 8, and 11) or Src^Y527F, a constitutively active mutant of c-Src (lanes 3, 6, 9, and 12). NPHP1 was precipitated and analyzed for tyrosine phosphorylation (upper panel). D, combined mutation of tyrosine residues 46 and 349 does not abrogate Pyk2-induced phosphorylation. Double mutant NPHP1 Y46F/Y349F was precipitated (IP), and tyrosine phosphorylation was monitored in presence of wild-type Pyk2 or a kinase-dead mutant. IB, immunoblotting. E, wild-type (WT) c-Src induces phosphorylation of the NPHP1 Y46F/Y349F double mutant, but not of the NPHP1 Y46F/Y349F/Y721F triple mutant. NPHP1 proteins were precipitated, and tyrosine phosphorylation was monitored. F, tyrosine residue 721 is endogenously phosphorylated. HEK293T cells were transfected and incubated for 15 min with peroxovanadate to inhibit tyrosine dephosphorylation. After cell lysis, NPHP1 proteins were precipitated and stained for phosphotyrosine. Mutation of Tyr-721 attenuated tyrosine phosphorylation of NPHP1. G, combined mutation of the mapped tyrosine residues abrogates tyrosine phosphorylation of NPHP1. NPHP1 proteins were precipitated, and tyrosine phosphorylation was monitored, revealing tyrosine phosphorylation of WT NPHP1 but not of the triple mutant. All immunoprecipitation experiments were performed at least three times.

NPHP4 controls the subcellular localization of NPHP1 in human ciliated retinal pigment epithelial cells. A, characterization of a novel murine monoclonal antibody detecting the N terminus of NPHP1. FLAG-tagged NPHP1 and NPHP1^237–670 or NPHP4^304–950 were expressed in HEK293T cells. Immunoblotting (IB) with an anti-FLAG antibody revealed expression of the indicated proteins. The novel NPHP1 mAb detected full-length NPHP1 but neither NPHP4^304–950 nor NPHP1^237–670. B, characterization of NPHP4 siRNA. Plasmids for F.NPHP4 and F.Eps^1–225 (epidermal growth factor receptor pathway substrate 15-like 1) and the indicated siRNAs were co-transfected in HEK293T cells. NPHP4 siRNAs 2, 3, and 4 specifically reduced NPHP4 expression. C, NPHP4 knockdown leads to an accumulation of NPHP1 in the trans-Golgi network. hTERT-RPE1 were transfected with either control siRNA or siRNA directed against NPHP4. After methanol fixation immunofluorescence staining was performed with the indicated antibodies in the absence of Triton X-100. Transfection of NPHP4 siRNA led to an increased NPHP1 signal co-localizing with a staining of the trans-Golgi marker Golgin-97. All RNAi experiments were performed three times.