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ARL13B, PDE6D, and CEP164 Form a Functional Network for INPP5E Ciliary Targeting

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ARL13B, PDE6D, and CEP164 Form a Functional Network for INPP5E Ciliary Targeting ARL13B, PDE6D, and CEP164 form a functional network for INPP5E ciliary targeting Melissa C. Humberta,b, Katie Weihbrechta,b, Charles C. Searbyb,c, Yalan Lid, Robert M. Poped, Val C. Sheffieldb,c, and Seongjin Seoa,1 aDepartment of Ophthalmology and Visual Sciences, bDepartment of Pediatrics, cHoward Hughes Medical Institute, and dProteomics Facility, University of Iowa, Iowa City, IA 52242 Edited by Kathryn V. Anderson, Sloan-Kettering Institute, New York, NY, and approved October 19, 2012 (received for review June 28, 2012) Mutations affecting ciliary components cause a series of related polydactyly, skeletal defects, cleft palate, and cerebral develop- genetic disorders in humans, including nephronophthisis (NPHP), mental defects (11). Inactivation of Inpp5e in adult mice results in Joubert syndrome (JBTS), Meckel-Gruber syndrome (MKS), and Bar- obesity and photoreceptor degeneration. Interestingly, many pro- det-Biedl syndrome (BBS), which are collectively termed “ciliopa- teins that localize to cilia, including INPP5E, RPGR, PDE6 α and thies.” Recent protein–protein interaction studies combined with β subunits, GRK1 (Rhodopsin kinase), and GNGT1 (Transducin γ genetic analyses revealed that ciliopathy-related proteins form sev- chain), are prenylated (either farnesylated or geranylgeranylated), eral functional networks/modules that build and maintain the pri- and mutations in these genes or genes involved in their prenylation mary cilium. However, the precise function of many ciliopathy- (e.g., AIPL1 and RCE1) lead to photoreceptor degeneration in related proteins and the mechanisms by which these proteins are vertebrates and humans (12–18). Understanding the molecular targeted to primary cilia are still not well understood. Here, we mechanismsbywhichprenylatedproteinsaretransportedtopri- fi fi describe a protein–protein interaction network of inositol polyphos- mary cilia would have signi cant rami cations in developing ther- phate-5-phosphatase E (INPP5E), a prenylated protein associated apeutic strategies to treat blindness and other ciliopathy phenotypes with JBTS, and its ciliary targeting mechanisms. INPP5E is targeted associated with these genes. to the primary cilium through a motif near the C terminus and In this study, we sought to determine the functional network of prenyl-binding protein phosphodiesterase 6D (PDE6D)-dependent INPP5E and the mechanisms by which INPP5E is targeted to mechanisms. Ciliary targeting of INPP5E is facilitated by another primary cilia. We determined the ciliary targeting sequence (CTS) JBTS protein, ADP-ribosylation factor-like 13B (ARL13B), but not by of INPP5E, the small GTPase responsible for its ciliary targeting, ARL2 or ARL3. ARL13B missense mutations that cause JBTS in and an interaction network that connects INPP5E to other known ciliopathy genes and new candidates. humans disrupt the ARL13B–INPP5E interaction. We further demon- strate interactions of INPP5E with several ciliary and centrosomal Results proteins, including a recently identified ciliopathy protein centroso- CTS of INPP5E. Previously, INPP5E was shown to localize to primary mal protein 164 (CEP164). These findings indicate that ARL13B, cilia (10, 11). We confirmed its ciliary localization in hTERT– INPP5E, PDE6D, and CEP164 form a distinct functional network that RPE1 and IMCD3 cells by detecting endogenous and transfected is involved in JBTS and NPHP but independent of the ones previ- A B INPP5E fi FLAG-tagged INPP5E (Fig. 1 and ). Ablation of ex- ously de ned by NPHP and MKS proteins. pression by siRNA-mediated gene knockdown verified the speci- ficity of the antibody staining. To map the CTS of INPP5E, we photoreceptor degeneration | retinitis pigmentosa | leber congenital generated a series of deletion mutants with N-terminal FLAG tags amaurosis | polydactyly | cystic kidney and evaluated their localization in IMCD3 cells (Fig. 1 B and C). The constructs with N-terminal 289 or 604 residues showed dif- rimary cilia are microtubule-based cell surface projections that fused localization throughout the cell including the nucleus. In Pemanate from the centrosome. This subcellular organelle func- contrast, constructs with the C-terminal portion of INPP5E (amino tions as an antenna, sensing and transducing extracellular signals acids 79–644 and 288–644) showed specific ciliary localization. into the cell, and plays an essential role in regulating multiple cel- Consistent with the previous observation (11), the N-terminal 626 lular processes including the cell cycle, embryonic development, and residues of INPP5E were sufficient to localize to cilia, suggesting tissue homeostasis (1–3). Mutations affecting ciliary and cen- that the CTS of INPP5E is between the phosphatase catalytic trosomal components underlie a group of related human disorders domain and the CaaX motif, which is a prenylation signal, and that such as Joubert syndrome (JBTS), Meckel-Gruber syndrome prenylation itself is not necessary for INPP5E ciliary targeting. (MKS), nephronophthisis (NPHP), and Bardet-Biedl syndrome The amino acid sequence in this region (amino acids 605–626) (BBS), collectively termed ciliopathies (1–3). Recent protein– is highly conserved in vertebrates (Fig. 1D). For fine mapping of protein interaction studies have identified several functional the CTS in this region, we generated small deletion mutants modules or networks involved in these ciliopathies (4). For ex- (three to five residue deletions from the full-length construct) ample, BBS proteins and intraflagellar transport (IFT) proteins spanning this region and evaluated their localization (Fig. 1 D and form multiprotein complexes, the BBSome and the IFT com- E). Immunofluorescence microscopy results indicate that a motif plexes, respectively, and these complexes are involved in trans- consisting of seven residues (FDRELYL; amino acids 609–615) is porting ciliary proteins. Likewise, NPHP and MKS proteins form critical for INPP5E ciliary targeting. Mutations in VRP residues a distinct modular complex at the transition zone of primary cilia (amino acids 595–597), which resemble the ciliary targeting VxPx and regulate ciliary membrane compositions (5–9). However, there are many ciliary and centrosomal proteins [e.g., inositol polyphosphate-5-phosphatase E (INPP5E) and ADP-ribosylation Author contributions: S.S. designed research; M.C.H., K.W., C.C.S., Y.L., R.M.P., and S.S. factor-like 13B (ARL13B)] that have not been linked to any of the performed research; V.C.S. contributed new reagents/analytic tools; M.C.H., R.M.P., and S.S. known functional networks and their precise functions remain to analyzed data; and M.C.H. and S.S. wrote the paper. be elucidated. The authors declare no conflict of interest. INPP5E encodes an enzyme that hydrolyzes the 5-phosphate of This article is a PNAS Direct Submission. PtdIns(3,4,5)P3 and PtdIns(4,5)P2 and localizes to primary cilia. 1To whom correspondence should be addressed. E-mail: [email protected]. Mutations in this gene cause JBTS in humans (10, 11). In mice, loss This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. CELL BIOLOGY of Inpp5e activity results in cystic kidney, bilateral anophthalmia, 1073/pnas.1210916109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1210916109 PNAS | November 27, 2012 | vol. 109 | no. 48 | 19691–19696 Downloaded by guest on September 29, 2021 acetyl Ciliary AC 640 INPP5E + -tub merged 242 297 599 localization 1 644 Full-length Yes PRD IPPc CaaX 1-289 No siCTRL 1-604 No 1-626 Yes 79-644 Yes 288-644 Yes siINPP5E CTS Fig. 1. Ciliary targeting sequence of INPP5E. (A) BD594 INPP5E localizes to the primary cilium in hTERT–RPE1 ARL13B H. sapiens cells. Antibodies against acetylated tubulin and γ-tu- + -tub FLAG merged M. musculus B. taurus bulin (red) were used to mark cilia and centrosomes, C. lupus familiaris G. gallus respectively. Endogenous INPP5E (green) was detected X. tropicalis D. rerio within the primary cilium. siRNA against INPP5E was fi Full-length 633 transfected to verify the speci city of the staining. H. sapiens Merged images are shown (Right) with DAPI staining M. musculus B. taurus for the nucleus (blue). (B) Localization of INPP5E de- C. lupus familiaris 1-289 G. gallus letion mutants. FLAG-tagged INPP5E deletion mutants X. tropicalis D. rerio were transfected into IMCD3 cells and their localiza- tion was probed with anti-FLAG antibody (green). Cilia E ARL13B ARL13B and centrosomes (red) were labeled with ARL13B and 1-604 + -tub FLAG merged + -tub FLAG merged γ-tubulin antibodies, respectively. Numbers indicate the amino acid residues transfected. (C) Summary of LYL INPP5E immunolocalization study. Proline-rich domain VRP 1-626 (PRD), inositol polyphosphate phosphatase catalytic (IPPc) domain, and CaaX motif were marked in blue, red, and purple boxes, respectively. The location of KRR RDNIP ciliary targeting sequence (CTS) is highlighted (Bot- 79-644 tom). (D) Conservation of amino acid sequences around the CTS in vertebrates. The numbers above the KEI sequence denote the amino acid position in human LAAG INPP5E. Gray box marks conserved (identical or similar) 288-644 amino acids. Black bars represent the mutated resi- dues in each construct. Red box highlights the CTS FDR determined in E.(E) Fine mapping of the CTS in INPP5E. Mutated amino acids are shown (Left). motif found in rhodopsin, polycystin-1, and polycystin-2 (19–21), ciliary targeting of INPP5E is mediated through a different did not affect INPP5E ciliary localization. Mutations in KRR mechanism.
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