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Mutations in INPP5E, encoding inositol polyphosphate-5-phosphatase E, link phosphatidyl inositol signaling to the ciliopathies.

Bielas SL, Silhavy JL, Brancati F, Kisseleva MV, Al-Gazali L, Sztriha L, Bayoumi RA, Zaki MS, Abdel-Aleem A, Rosti RO, Kayserili H, Swistun D, Scott LC, Bertini E, Boltshauser E, Fazzi E, Travaglini L, Field SJ, Gayral S, Jacoby M, Schurmans S, Dallapiccola B, Majerus PW, Valente EM, Gleeson JG - Nat. Genet. (2009)

Bottom Line: In individuals with Joubert disease genetically linked to JBTS1, we identified mutations in the INPP5E gene, encoding inositol polyphosphate-5-phosphatase E, which hydrolyzes the 5-phosphate of PtdIns(3,4,5)P3 and PtdIns(4,5)P2.Mutations clustered in the phosphatase domain and impaired 5-phosphatase activity, resulting in altered cellular PtdIns ratios.These data link PtdIns signaling to the primary cilium, a cellular structure that is becoming increasingly recognized for its role in mediating cell signals and neuronal function.

View Article: PubMed Central - PubMed

Affiliation: Neurogenetics Laboratory, Howard Hughes Medical Institute, Department of Neurosciences and Pediatrics, University of California, San Diego, La Jolla, USA.

ABSTRACT
Phosphotidylinositol (PtdIns) signaling is tightly regulated both spatially and temporally by subcellularly localized PtdIns kinases and phosphatases that dynamically alter downstream signaling events. Joubert syndrome is characterized by a specific midbrain-hindbrain malformation ('molar tooth sign'), variably associated retinal dystrophy, nephronophthisis, liver fibrosis and polydactyly and is included in the newly emerging group of 'ciliopathies'. In individuals with Joubert disease genetically linked to JBTS1, we identified mutations in the INPP5E gene, encoding inositol polyphosphate-5-phosphatase E, which hydrolyzes the 5-phosphate of PtdIns(3,4,5)P3 and PtdIns(4,5)P2. Mutations clustered in the phosphatase domain and impaired 5-phosphatase activity, resulting in altered cellular PtdIns ratios. INPP5E localized to cilia in major organs affected by Joubert syndrome, and mutations promoted premature destabilization of cilia in response to stimulation. These data link PtdIns signaling to the primary cilium, a cellular structure that is becoming increasingly recognized for its role in mediating cell signals and neuronal function.

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Impaired 5-phosphatase activity and altered ratio of PtdIns(4,5)P2 to PtdIns(4)P associated with JBTS1 INPP5E mutations. (a) Summary of PtdIns metabolism. P = phosphate. A block in INPP5E function is predicted to increase the PtdIns(4,5)P2:PtdIns(4)P ratio. (b-c) More severe reduction in 5-phosphatase activity of mutant INPP5E against PtdIns(3,4,5)P3 than PtdIns(4,5)P2 substrates. Note that activity was largely retained against PtdIns(4,5)2 for some mutations (mutants R435Q, R512W/R515W and K580E are severely defective, whereas R378C and R563H are only slightly diminished). D477N is known phosphatasedead, compared with each of the patient mutations. (N = 3 for each sample) (d) Elevated ratio of PtdIns(4,5)P2 to PtdIns(4)P in patient primary fibroblast lines MTI-610-V-2 and V-1, compared with control fibroblast. * represent p < 0.05 ANOVA two way corrected for multiple comparisons.
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Figure 2: Impaired 5-phosphatase activity and altered ratio of PtdIns(4,5)P2 to PtdIns(4)P associated with JBTS1 INPP5E mutations. (a) Summary of PtdIns metabolism. P = phosphate. A block in INPP5E function is predicted to increase the PtdIns(4,5)P2:PtdIns(4)P ratio. (b-c) More severe reduction in 5-phosphatase activity of mutant INPP5E against PtdIns(3,4,5)P3 than PtdIns(4,5)P2 substrates. Note that activity was largely retained against PtdIns(4,5)2 for some mutations (mutants R435Q, R512W/R515W and K580E are severely defective, whereas R378C and R563H are only slightly diminished). D477N is known phosphatasedead, compared with each of the patient mutations. (N = 3 for each sample) (d) Elevated ratio of PtdIns(4,5)P2 to PtdIns(4)P in patient primary fibroblast lines MTI-610-V-2 and V-1, compared with control fibroblast. * represent p < 0.05 ANOVA two way corrected for multiple comparisons.

Mentions: To test the effects of INPP5E mutations on PtdIns phosphatase activity, we immunoprecipated tagged wildtype and enzymatically (D477N 6), together with each mutation separately from mammalian cells (resulting in similar protein levels, Supplemental Fig. 3) and then analyzed phosphatase activity against PtdIns(3,4,5)P3 and PtdIns(4,5)P2, the presumed cellular INPP5E substrates (Fig. 2a). We found that each of the JBTS1 missense mutations severely disrupted phosphatase activity towards PtdIns(3,4,5)P3 (Fig. 2b, p < 0.05 activity for each mutant, N = 3 independent experiments). Of note, since the R512W/R515W variants were observed on a single haplotype, we reasoned that probably only one was driving the defect in enzymatic activity, and under separate analysis, the R515W mutation was shown to be the major contributor to the defective enzymatic activity (data not shown). Comparison with phosphatase activity using PtdIns(4,5)P2 as substrate showed similar although not as severely compromised results as compared with wildtype (Fig. 2c), suggesting that PtdIns(3,4,5)P3 may be the relevant substrate in JBTS1. Because overexpression of INPP5E was previously shown to block Akt phosphorylation in response to PDGF stimulation in cultured cells (presumably by depleting levels of PtdIns(3,4,5)P3 and PtdIns(4,5)P2) 7, we next tested the effect of overexpression of each of the patient mutations in this published assay. Not only did patient mutations fail to block Akt signaling, but elevated basal levels of pAkt were also apparent in unstimulated cells (Supplemental Fig. 4). We conclude that the JBTS1-associated missense mutations impair phosphatase activity towards putative PtdIns substrates, which can alter downstream signaling events.


Mutations in INPP5E, encoding inositol polyphosphate-5-phosphatase E, link phosphatidyl inositol signaling to the ciliopathies.

Bielas SL, Silhavy JL, Brancati F, Kisseleva MV, Al-Gazali L, Sztriha L, Bayoumi RA, Zaki MS, Abdel-Aleem A, Rosti RO, Kayserili H, Swistun D, Scott LC, Bertini E, Boltshauser E, Fazzi E, Travaglini L, Field SJ, Gayral S, Jacoby M, Schurmans S, Dallapiccola B, Majerus PW, Valente EM, Gleeson JG - Nat. Genet. (2009)

Impaired 5-phosphatase activity and altered ratio of PtdIns(4,5)P2 to PtdIns(4)P associated with JBTS1 INPP5E mutations. (a) Summary of PtdIns metabolism. P = phosphate. A block in INPP5E function is predicted to increase the PtdIns(4,5)P2:PtdIns(4)P ratio. (b-c) More severe reduction in 5-phosphatase activity of mutant INPP5E against PtdIns(3,4,5)P3 than PtdIns(4,5)P2 substrates. Note that activity was largely retained against PtdIns(4,5)2 for some mutations (mutants R435Q, R512W/R515W and K580E are severely defective, whereas R378C and R563H are only slightly diminished). D477N is known phosphatasedead, compared with each of the patient mutations. (N = 3 for each sample) (d) Elevated ratio of PtdIns(4,5)P2 to PtdIns(4)P in patient primary fibroblast lines MTI-610-V-2 and V-1, compared with control fibroblast. * represent p < 0.05 ANOVA two way corrected for multiple comparisons.
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Figure 2: Impaired 5-phosphatase activity and altered ratio of PtdIns(4,5)P2 to PtdIns(4)P associated with JBTS1 INPP5E mutations. (a) Summary of PtdIns metabolism. P = phosphate. A block in INPP5E function is predicted to increase the PtdIns(4,5)P2:PtdIns(4)P ratio. (b-c) More severe reduction in 5-phosphatase activity of mutant INPP5E against PtdIns(3,4,5)P3 than PtdIns(4,5)P2 substrates. Note that activity was largely retained against PtdIns(4,5)2 for some mutations (mutants R435Q, R512W/R515W and K580E are severely defective, whereas R378C and R563H are only slightly diminished). D477N is known phosphatasedead, compared with each of the patient mutations. (N = 3 for each sample) (d) Elevated ratio of PtdIns(4,5)P2 to PtdIns(4)P in patient primary fibroblast lines MTI-610-V-2 and V-1, compared with control fibroblast. * represent p < 0.05 ANOVA two way corrected for multiple comparisons.
Mentions: To test the effects of INPP5E mutations on PtdIns phosphatase activity, we immunoprecipated tagged wildtype and enzymatically (D477N 6), together with each mutation separately from mammalian cells (resulting in similar protein levels, Supplemental Fig. 3) and then analyzed phosphatase activity against PtdIns(3,4,5)P3 and PtdIns(4,5)P2, the presumed cellular INPP5E substrates (Fig. 2a). We found that each of the JBTS1 missense mutations severely disrupted phosphatase activity towards PtdIns(3,4,5)P3 (Fig. 2b, p < 0.05 activity for each mutant, N = 3 independent experiments). Of note, since the R512W/R515W variants were observed on a single haplotype, we reasoned that probably only one was driving the defect in enzymatic activity, and under separate analysis, the R515W mutation was shown to be the major contributor to the defective enzymatic activity (data not shown). Comparison with phosphatase activity using PtdIns(4,5)P2 as substrate showed similar although not as severely compromised results as compared with wildtype (Fig. 2c), suggesting that PtdIns(3,4,5)P3 may be the relevant substrate in JBTS1. Because overexpression of INPP5E was previously shown to block Akt phosphorylation in response to PDGF stimulation in cultured cells (presumably by depleting levels of PtdIns(3,4,5)P3 and PtdIns(4,5)P2) 7, we next tested the effect of overexpression of each of the patient mutations in this published assay. Not only did patient mutations fail to block Akt signaling, but elevated basal levels of pAkt were also apparent in unstimulated cells (Supplemental Fig. 4). We conclude that the JBTS1-associated missense mutations impair phosphatase activity towards putative PtdIns substrates, which can alter downstream signaling events.

Bottom Line: In individuals with Joubert disease genetically linked to JBTS1, we identified mutations in the INPP5E gene, encoding inositol polyphosphate-5-phosphatase E, which hydrolyzes the 5-phosphate of PtdIns(3,4,5)P3 and PtdIns(4,5)P2.Mutations clustered in the phosphatase domain and impaired 5-phosphatase activity, resulting in altered cellular PtdIns ratios.These data link PtdIns signaling to the primary cilium, a cellular structure that is becoming increasingly recognized for its role in mediating cell signals and neuronal function.

View Article: PubMed Central - PubMed

Affiliation: Neurogenetics Laboratory, Howard Hughes Medical Institute, Department of Neurosciences and Pediatrics, University of California, San Diego, La Jolla, USA.

ABSTRACT
Phosphotidylinositol (PtdIns) signaling is tightly regulated both spatially and temporally by subcellularly localized PtdIns kinases and phosphatases that dynamically alter downstream signaling events. Joubert syndrome is characterized by a specific midbrain-hindbrain malformation ('molar tooth sign'), variably associated retinal dystrophy, nephronophthisis, liver fibrosis and polydactyly and is included in the newly emerging group of 'ciliopathies'. In individuals with Joubert disease genetically linked to JBTS1, we identified mutations in the INPP5E gene, encoding inositol polyphosphate-5-phosphatase E, which hydrolyzes the 5-phosphate of PtdIns(3,4,5)P3 and PtdIns(4,5)P2. Mutations clustered in the phosphatase domain and impaired 5-phosphatase activity, resulting in altered cellular PtdIns ratios. INPP5E localized to cilia in major organs affected by Joubert syndrome, and mutations promoted premature destabilization of cilia in response to stimulation. These data link PtdIns signaling to the primary cilium, a cellular structure that is becoming increasingly recognized for its role in mediating cell signals and neuronal function.

Show MeSH
Related in: MedlinePlus