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. 2011 May 16;193(4):633–642. doi: 10.1083/jcb.201009069

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© 2011 Habbig et al.

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Figure 3. — NPHP4 derepresses the inhibition of TAZ by Lats1. (a) NPHP4 abrogates the interaction of Lats1 and TAZ. FLAG-tagged Lats1 (F.Lats1) was coexpressed with GFP-tagged TAZ (GFP.TAZ) in the presence or absence of V5-tagged NPHP4 (V5.NPHP4) in HEK293T cells. After immunoprecipitation (IP) with FLAG (M2) beads, Western blot (WB) analysis revealed that the interaction of Lats1 and TAZ was abolished in the presence of NPHP4. FLAG.Eps served as a control protein. Both FLAG.Lats1 and FLAG.Eps were stained in the precipitates to control the comparable efficiency of the immunoprecipitation. (b) Overexpression of NPHP4 reduces the phosphorylation of TAZ by Lats1. HEK293T cells were transfected with the indicated constructs. Cell lysates were analyzed by Western blotting using the pYAP–Ser-127/pTAZ–Ser-89 antibody. (c) NPHP4 derepresses the inhibitory effect of Lats1 on TAZ signaling. Plasmids encoding the indicated proteins or empty pcDNA6 vector were cotransfected into HEK293T cells together with the TEAD reporter plasmids as indicated in Material and methods. As expected, Lats1 reduced TEAD/TAZ activity. Additional coexpression of NPHP4 reversed this effect (n = 4; *, P < 0.05). Expression of the indicated proteins was confirmed by Western blotting. (d) The Lats1-resistant mutant of TAZ (TAZ 4SA) could not be activated by NPHP4, suggesting that NPHP4 regulates TAZ signaling through interference with Lats1-dependent phosphorylation of TAZ (n = 3). Error bars represent SEM. WCL, whole cell lysate.