The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium.
Bottom Line: Mutations in RPGRIP1L result in severe human diseases called ciliopathies.Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l.We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l.
Affiliation: Institute for Animal Developmental and Molecular Biology, Heinrich-Heine University Düsseldorf, 40225 Düsseldorf, Germany Christoph.Gerhardt@hhu.de.Show MeSH
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Mentions: The observations of significant elongation of cilia in our in vitro and in vivo model systems led us to the question if ciliary signaling is affected in these systems. Previously, we found an elevated Gli3-190/Gli3-83 ratio in Rpgrip1l−/− mouse embryos as a result of a reduced proteolytic processing of Gli3 (Vierkotten et al., 2007). Consistent with this suspicion, the Gli3-190/Gli3-83 ratios are also elevated in Rpgrip1l−/− MEFs and isolated limbs (Fig. 3, A and B). Because previous studies showed that the deficiency of other TZ proteins affects events upstream of Gli3 in the Shh signal cascade (Garcia-Gonzalo et al., 2011) and since Smo regulates Gli3 modification via the Evc–Evc2 complex (Dorn et al., 2012), we analyzed the amount of Smo and Evc in cilia of SAG (Shh agonist)-treated Rpgrip1l−/− MEFs. We could not detect any alteration in the amount of either protein in cilia of Rpgrip1l−/− MEFs (Fig. S1, A and B) making it likely that Rpgrip1l affects Shh signaling at the level of Gli3. In search of the cause for the increased Gli3-190/Gli3-83 ratio, we investigated proteasomal activity. However, changes in overall cellular proteasomal activity were not detected in Rpgrip1l−/− MEFs (Fig. 3 C). Because Gli3-processing is dependent on cilia (Haycraft et al., 2005; Besse et al., 2011), we could not exclude that proteasomal activity is reduced exclusively at cilia. Potentially, an alteration of proteasomal degradation specific at cilia is undetectable in measurements of general proteasomal substrates. To test this hypothesis, we measured the amount of phospho-(S33/37/T41)-β-Catenin, a well-known proteasomal substrate (Aberle et al., 1997; Hart et al., 1999; Kitagawa et al., 1999; Latres et al., 1999; Liu et al., 1999; Winston et al., 1999). A previous study showed a localization of phospho-(S33/37/T41)-β-Catenin at the ciliary base (Corbit et al., 2008), suggesting that this is the location of its degradation. In total lysates of serum-starved Rpgrip1l−/− MEFs (on average, 82% of all cells had cilia), the amount of phospho-(S33/37/T41)-β-Catenin is significantly increased in comparison to serum-starved WT MEFs (on average, 88.6% of all cells produced cilia; Fig. 4 A), whereas non–phospho-(S33/37/T41)-β-Catenin is unaltered (Fig. 4 B). In total lysates of non–serum-starved Rpgrip1l−/− MEFs (on average, 4% of all cells possessed cilia), the amount of phospho-(S33/37/T41)-β-Catenin is not changed in comparison to non–serum-starved WT MEFs (on average, 6.67% of all cells had cilia; Fig. 4 C), demonstrating that the degradation of phospho-(S33/37/T41)-β-Catenin is cilia dependent. The amount of phospho-(S33/37/T41)-β-Catenin is significantly increased at the ciliary base of Rpgrip1l−/− MEFs (Fig. 4 D). To confirm these data, which were collected by using standard resolution microscopy, we also measured the amount of phospho-(S33/37/T41)-β-Catenin using super-resolution microscopy. 3D structured illumination microscopy (SIM; Gustafsson et al., 2008; Schermelleh et al., 2008) analyses confirmed the finding of significantly elevated levels of phospho-(S33/37/T41)-β-Catenin at the ciliary TZ in Rpgrip1l−/− MEFs (Fig. 4 E).
Affiliation: Institute for Animal Developmental and Molecular Biology, Heinrich-Heine University Düsseldorf, 40225 Düsseldorf, Germany Christoph.Gerhardt@hhu.de.