In Vivo, Pikfyve Generates PI(3,5)P2, Which Serves As Both a Signaling Lipid and the Major Precursor for PI5P

In vivo, Pikfyve generates PI(3,5)P2, which serves as both a signaling lipid and the major precursor for PI5P

Sergey N. Zolova,b, Dave Bridgesb, Yanling Zhanga,b, Wei-Wei Leeb, Ellen Riehleb, Rakesh Vermab, Guy M. Lenkc, Kimber Converso-Barand, Thomas Weidee, Roger L. Albinf,g, Alan R. Saltielb,h,i, Miriam H. Meislerc,f, Mark W. Russelld, and Lois S. Weismana,b,1

Departments of aCell and Developmental Biology, cHuman Genetics, fNeurology, hInternal Medicine, and iMolecular and Integrative Physiology, dDivision of Pediatric Cardiology, Department of Pediatrics and Communicable Diseases, and bLife Sciences Institute, University of Michigan, Ann Arbor, MI 48109; eDivision of Molecular Nephrology, Department of Internal Medicine D, University Hospital Münster, 48149 Münster, Germany; and gGeriatric Research, Education and Clinical Center, Veterans Administration Ann Arbor Healthcare System, Ann Arbor, MI 48105

Edited by Pietro De Camilli, Yale University and Howard Hughes Medical Institute, New Haven, CT, and approved September 19, 2012 (received for review February 21, 2012)

Mutations that cause defects in levels of the signaling lipid (10), vacuole membrane ﬁssion (6), and vacuole acidiﬁcation (3).

phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] lead to profound Similarly, in mammalian cells, PIKfyve colocalizes with late endo- neurodegeneration in mice. Moreover, mutations in human FIG4 somes and lysosomes (16, 17) and with early endosomes (17–19).

predicted to lower PI(3,5)P2 levels underlie Charcot–Marie–Tooth Defects observed when Pikfyve function is compromised include type 4J neuropathy and are present in selected cases of amyotro- the formation of large vacuoles that contain LAMP1 and LAMP2 fi phic lateral sclerosis. In yeast and mammals, PI(3,5)P2 is generated (19-25), defects in retrograde traf c from early endosomes to by a protein complex that includes the lipid kinase Fab1/Pikfyve, the trans-Golgi network (19), defects in EGF receptor degrada- the scaffolding protein Vac14, and the lipid phosphatase Fig4. tion (22), defects in autophagy (22, 26, 27), and dysregulation − − − − Fibroblasts cultured from Vac14 / and Fig4 / mouse mutants have of TRPML1 (28). Mouse models with mutations in Vac14 and Fig4 genes exhibit a 50% reduction in the levels of PI(3,5)P2, suggesting that there may be PIKfyve-independent pathways that generate this lipid. Here, we profound neurodegeneration (5, 21, 29). Moreover, Pikfyve plays β β characterize a Pikfyve gene-trap mouse (Pikfyve -geo/ -geo), a hypo- critical roles in early development. A whole-body knockout of Pikfyve causes preimplantation lethality (30). morph with ∼10% of the normal level of Pikfyve protein. shRNA β β Here we characterize Pikfyve -geo/ -geo, a gene-trap mouse silencing of the residual Pikfyve transcript in fibroblasts demon- β β mutant. Pikfyve -geo/ -geo, a hypomorph, generates about 10% of strated that Pikfyve is required to generate all of the PI(3,5)P 2 the wild-type levels of Pikfyve protein. In fibroblasts, shRNA pool. Surprisingly, Pikfyve also is responsible for nearly all of silencing of the residual Pikfyve transcript revealed that the en- the phosphatidylinositol-5-phosphate (PI5P) pool. We show that tire PI(3,5)P2 pool is generated by Pikfyve. Moreover, most of PI5P is generated directly from PI(3,5)P2, likely via 3′-phosphatase β β the PI5P pool depends on Pikfyve. Furthermore, characteriza- Pikfyve -geo/ -geo β β activity. Analysis of tissues from the mouse tion of the Pikfyve -geo/ -geo mouse revealed additional pheno- mutants reveals that Pikfyve is critical in neural tissues, heart, types caused by defects in Pikfyve: In addition to neurode- β β lung, kidney, thymus, and spleen.Thus,PI(3,5)P2 and PI5P have generation, the Pikfyve -geo/ -geo mice exhibit profound defects in major roles in multiple organs. Understanding the regulation of the heart, lung, kidney, spleen, and thymus. these lipids may provide insights into therapies for multiple diseases. Results Characterization of a Pikfyveβ-geo/β-geo Hypomorphic Mouse. Mice phosphoinositide | spongiform were generated from embryonic stem cells with a gene-trap insertion in Pikfyve. The insertion site in intron 17 is upstream of hosphorylated phosphatidylinositol lipids (PPI) function as the kinase domain and within the CCT domain (Fig. 1A). The Psignaling molecules. Mammalian cells phosphorylate PI on predicted chimeric protein contains the first 765 amino acids of three available hydroxyl groups, in all combinations, giving rise Pikfyve fused to β-galactosidase-neomycin phosphotransferase II to seven lipids. Each PPI regulates multiple pathways through (β-Geo) and is the same size as wild-type Pikfyve. specific downstream effectors. The localization of lipid kinases Offspring from intercrosses of heterozygous mice produced and phosphatases dictate the organelles where each lipid species pups at the expected Mendelian ratio of 1:2:1 (Fig. S1A). At is generated. birth, homozygous mutants were about 50% smaller than wild- In the yeast Saccharomyces cerevisiae, only four of the seven PPIs type and heterozygote littermates (Fig. S1 B and C). Most died β β are made, and the initial elucidation of some of the pathways re- within a few days of birth. A few Pikfyve -geo/ -geo mutants sur- quired to synthesize and catabolize these lipids was determined in vived to 2 wk of age (Fig. S1A). yeast. The yeast PI3P 5-kinase, Fab1, converts PI3P to phospha- To test the efficacy of the gene trap, Pikfyve mRNA levels were tidylinositol 3,5-bisphosphate [PI(3,5)P2] (1, 2). Generation of determined in fibroblasts from postnatal day 0 (P0) pups (Fig. 1A PI(3,5)P2 requires a protein complex composed of Fab1, the- and Fig. S1D). Quantitative real-time PCR of exons 2 and 3 did scaffolding protein Vac14 (3-5), the PI(3,5)P2 5-phosphatase not detect significant differences in transcript levels of Pikfyve in Fig4 (6, 7), a Fab1 activator, Vac7 (8, 9), and a negative regulator, Atg18 (10, 11). Although Fig4 is a lipid phosphatase, its presence also is required for Fab1 activity (6, 12). Thus, Author contributions: S.N.Z. and L.S.W. designed research; S.N.Z., D.B., Y.Z., W.-W.L., E.R., the absence of Vac14 or Fig4 results in a similar defect, and K.C.-B. performed research; R.V., G.M.L., K.C.-B., M.H.M., and M.W.R. contributed reduced PI(3,5)P2. new reagents/analytic tools; S.N.Z., D.B., Y.Z., W.-W.L., G.M.L., K.C.-B., T.W., R.L.A., A.R.S., All homologs of Fab1 [Pikfyve in mammals (13)], contain M.H.M., M.W.R., and L.S.W. analyzed data; and S.N.Z., D.B., Y.Z., T.W., R.L.A., A.R.S., M.H.M., a FYVE domain predicted to bind PI3P (14). Mammalian Pik- M.W.R., and L.S.W. wrote the paper. fyve, Vac14, and Fig4 form a complex (5, 15). The authors declare no conflict of interest. Yeast Fab1 and its regulators are localized on the vacuole This article is a PNAS Direct Submission. (lysosome) and on late endosomes (1–6, 8, 9, 12), localizations 1To whom correspondence should be addressed. E-mail: [email protected]. that are consistent with the phenotypes associated with the loss This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of Fab1, including defects in retrograde traffic from vacuoles 1073/pnas.1203106109/-/DCSupplemental.

17472–17477 | PNAS | October 23, 2012 | vol. 109 | no. 43 www.pnas.org/cgi/doi/10.1073/pnas.1203106109 Downloaded by guest on September 28, 2021 β β protein (Fig. S1G). Thus, the Pikfyve -geo/ -geo chimera does not integrate into the Vac14 complex. Furthermore, depletion of Pikfyve does not interfere with the ability of Vac14 and Fig4 to form a complex.

Pikfyve Protein Is Not Depleted Uniformly in Tissues of the Pikfyveβ-geo/β-geo Mouse. We determined relative levels of Pikfyve, Vac14, and Fig4 in multiple tissues from wild-type P0 pups. The levels of Pikfyve, Vac14, and Fig4 were highest in the brain, β β twofold higher than in other tissues (Fig. S2A). In Pikfyve -geo/ -geo P0 pups, Pikfyve was reduced dramatically in all tissues, by 65– 90%, although the extent of Pikfyve reduction was not uniform (Fig. S2 B and C). There was much less change in the levels of β β Vac14 or Fig4 in the tissues of Pikfyve -geo/ -geo mice.

Neonatal Pikfyveβ-geo/β-geo Mice Display Modest Defects in the Nervous β β System. Pikfyve -geo/ -geo P0 mice died perinatally; however the general cytoarchitecture of the brains was unexpectedly normal. There was no obvious vacuolation in most brain regions. One exception was the caudal cortical subventricular neuroepithelium, a region of rapidly dividing cells, where there was modest vacuolation (Fig. S3A). This spongiform appearance was present in all β β four Pikfyve -geo/ -geo brains examined but in none of the four + + Pikfyve / brains. There were vacuoles in the dorsal root ganglia β β of the four Pikfyve -geo/ -geo pups; the surrounding tissue appeared normal (Fig. S3B). To test the potential of neurons from the brain to form vacuoles, we generated primary neuronal cultures from the β β hippocampus of Pikfyve -geo/ -geo and wild-type P0 mice. Many of β-geo/β-geo β-geo/β-geo the neurons cultured from Pikfyve mice formed vacuoles, Fig. 1. The Pikfyve mutant has low levels of Pikfyve protein. (A) but neurons from wild-type mice did not (Fig. S3C). These results β β β Schematic of the Pikfyve coding region indicating position of the -Geo in- demonstrate that neuronal tissues from the Pikfyve -geo/ -geo sert, the 5′ and 3′ regions with primers spanning exons 2 and 3 and exons 41 and 42 used for quantitative RT-PCR (green arrows), the shRNA target site mouse have the potential to become vacuolated when cultured (red arrow), and peptide (asterisk) used to produce the mouse monoclonal ex vivo. antibody. (B) Western blots show that Pikfyveβ-geo/β-geo fibroblasts have re- Pikfyveβ-geo/β-geo Mutants that Survive Beyond 2 Wk Develop Extensive duced Pikfyve protein but no difference in Vac14 and Fig4 protein levels. β β Neurodegeneration. A small number of Pikfyve -geo/ -geo mutant mice survived for 16–19 d (Fig. S1A). In studies of the survivors, + β wild-type, heterozygous, and homozygous fibroblasts, indicating Pikfyve / -geo heterozygous littermates served as controls; there are no differences in appearance or behavior of wild-type and that the chimeric and wild-type transcripts are equally stable. + β Pikfyve / -geo heterozygous mice. Differences were observed for exons 41 and 42, which are β β present in the wild-type allele only. In heterozygous mice, the At P16 mutant Pikfyve -geo/ -geo pups had half the body weight ∼ (Fig. S1A) and 30% the brain weight of heterozygous littermates levels of transcript containing exons 41 and 42 was 50% of wild- β β type. Notably, ∼15% of full-length Pikfyve transcripts were still (Fig. S4 B and C). The three homozygous Pikfyve -geo/ -geo mice β-geo/β-geo fi that survived for 18–19 d also displayed severely impaired mo- present in homozygous Pikfyve broblasts. β β To distinguish the gene-trap chimeric protein from wild-type bility (Movie S1). At 16 d, the Pikfyve -geo/ -geo pups had prom- Pikfyve, we generated a monoclonal antibody to residues 1793– inent vacuolation in the deep cerebellar nuclei and in the 1995 within the kinase domain (Fig. 1A, asterisk). The level of brainstem, particularly the midbrain and pons (Fig. S4D). There β β Pikfyve in Pikfyve -geo/ geo fibroblasts was reduced, but Vac14 also was vacuolation in the spinal cord (Fig. S4E). Brain and and Fig4 were present at normal levels (Fig. 1B). This result spinal cord from heterozygous littermates showed no evidence of − − β β contrasts with that in Vac14 / fibroblasts, which have no Vac14 degeneration. Most Pikfyve -geo/ -geo mice died around P0, but in and have reduced Fig4 (Fig. 1B and ref. 31). Western blots of a the surviving mutants, defects in the nervous system become

β β CELL BIOLOGY dilution series of cell lysates indicated that the Pikfyve -geo/ -geo more prominent later, raising the likelihood of serious defects in β + and Pikfyve -geo/ mutant fibroblasts expressed about 10% (Fig. other organs. S1E), and 50% (Fig. S1F), of the wild-type levels of Pikfyve, β β respectively. Neonatal Pikfyve -geo/ -geo Mice Exhibit Defects in Several Organs. β + β β Heterozygous Pikfyve -geo/ mice have no apparent defects, The hearts of Pikfyve -geo/ -geo mice were severely affected. β β β suggesting that the Pikfyve -geo allele does not have a dominant Compared with wild-type littermates, the Pikfyve -geo/ -geo hearts negative effect in vivo. Moreover, this allele does not interact with had a reduced density of atrial myocytes, consistent with reduced Vac14. In wild-type lysates, immunoprecipitation of Pikfyve, using myofibrillar content. In addition, large translucent vacuoles were β β an amino-terminal Pikfyve antibody, coprecipitates Vac14 and present (Fig. 2). The lungs of the Pikfyve -geo/ -geo mutants also Fig4. Conversely, immunoprecipitation of Vac14 coprecipitates were abnormal, with an increase in the ratio of cellularity in the β β Pikfyve and Fig4 (Fig. S1G). In lysates from Pikfyve -geo/ -geo alveolar region and decreased airspace in the lung (Fig. 2 and fibroblasts, immunoprecipitation with the amino terminal Pikfyve Fig. S5A). β β − − antibody immunoprecipitated both the Pikfyve -geo/ -geo chimera We analyzed four P0 Vac14 / (Fig. S5B)andthreeE18.5 − − − − − − and residual full-length Pikfyve; however, little Vac14 or Fig4 Fig4 / mice (Fig. S5C). The hearts from the Vac14 / and Fig4 / coprecipitated. This small amount is likely caused by a pull-down mutants had significant degeneration of the atria marked by the of residual full-length Pikfyve. Consistent with this postulate, im- presence of large, transparent vacuoles. These data strongly sug- β-geo/β-geo munoprecipitation of Vac14 from Pikfyve fibroblasts gest that defects in the levels of PI(3,5)P2 and PI5P lead to de- coprecipitated Fig4 and full-length Pikfyve but not the chimeric generation of the heart, particularly the atria.

Zolov et al. PNAS | October 23, 2012 | vol. 109 | no. 43 | 17473 Downloaded by guest on September 28, 2021 contained numerous small vacuoles (Fig. S5 D and H and Fig. β β S6A). Kidneys from Pikfyve -geo/ -geo mutants exhibited defects at P0, and the defects were worse in older animals. At P0 the kidneys contained multiple small vacuoles in renal tubules, but the overall structure of the glomeruli was unaffected (Fig. S6B). At P19 there was a progression in the kidney degeneration in β β Pikfyve -geo/ -geo homozygotes (Fig. S6C), with some tubular cells ﬁlled with vacuoles and some apparent loss of tubules.

Pikfyve Generates All of the Cellular PI(3,5)P . The dramatic re- β 2 β duction of Pikfyve protein in the Pikfyve -geo/ -geo mutant mice (Figs. S1E and S2 B and C) suggested that the mutants would be largely depleted of PI(3,5)P2. We examined steady-state levels of β-geo/β-geo PI(3,5)P2 in heterozygous and homozygous Pikfyve fibroblasts, where the levels of Pikfyve are about 50% and 10% of wild-type levels, respectively (Fig. S1 F and E). Heterozygous + β Pikfyve / -geo fibroblasts had wild-type levels of PI(3,5)P (Fig. β β 2 S7A). Pikfyve -geo/ -geo fibroblasts had a 50% reduction in PI(3,5) P2, with a corresponding 40% rise in the precursor, PI3P. These findings indicate that Pikfyve has a role in the generation of PI (3,5)P2 from PI3P. Note that these data provide information about PI(3,5)P2 levels only in fibroblasts. To measure the effects of these mutations on PPI levels in specific tissues, new techni- ques will need to be developed. Notably, although PI(3,5)P levels were normal in heterozy- + β 2 gous Pikfyve / -geo fibroblasts, there was a 30% reduction of PI5P β β (Fig. S7A). Moreover, in homozygous Pikfyve -geo/ -geo fibroblasts there was an additional reduction of PI5P to 50% of wild-type levels, which is comparable to the reduction of PI(3,5)P2 in the β β same cells. These observations suggest that Pikfyve also plays Fig. 2. Spongiform degeneration of the neonatal Pikfyve -geo/ -geo mouse β β a major role in the generation of PI5P. There were no detectable heart. Sagittal sections of neonatal wild-type (WT) and Pikfyve -geo/ -geo mice stained with H&E. The atria (At), ventricles (V), and lung (L) of wild-type differences in the levels of PI4P or PI(4,5)P2 (Fig. S7A). littermates exhibit normal morphology. At higher magnification, atrial and To test further the requirement for Pikfyve in the generation of PI(3,5)P , we depleted the remaining Pikfyve protein in wild-type ventricular myocytes strongly stain with H&E. There is no apparent vacuo- 2 β β β β -geo/ -geo fi lation. Pikfyve -geo/ -geo mice have reduced H&E staining of atrial myocytes and Pikfyve broblasts by culture for 4 d in the presence β β consistent with reduced myofibrillar content. In the Pikfyve -geo/ -geo of shRNA (Fig. S7B). This depletion resulted in the formation of β-geo/β-geo mutants, both atria and ventricles have vacuoles (arrows), with a high level vacuoles (Fig. S7C). In the Pikfyve fibroblasts, vacuoles of vacuolation in the atria. Smaller vacuoles (arrowheads) give the myocytes were present before shRNA silencing. After silencing, the num- [identified by heavily stained myofibrils (white arrow)] a foamy appearance. ber and size of the vacuoles increased. shRNA knockdown in wild-type fibroblasts resulted in a 85% reduction in Pikfyve pro- β β tein, whereas shRNA knockdown in Pikfyve -geo/ -geo fibroblasts β β The hearts from 2-wk-old Pikfyve -geo/ -geo mutants were smaller caused a complete loss of detectable protein (Fig. 3A). In β β than those of their heterozygous littermates (Fig. S5D). The right Pikfyve -geo/ -geo fibroblasts, there also was a loss of the β-gal gene- atrium and superior vena cava were enlarged, but cardiac structure trap chimeric protein. There was no detectable reduction in was normal otherwise. There was extensive vacuolation of the Vac14 or Fig4. β β atrial myocytes. Smaller vacuoles also were noted in ventricular Depletion of Pikfyve by shRNA in Pikfyve -geo/ -geo cells myocytes (Fig. S5E). resulted in a fivefold elevation of PI3P and total loss of PI(3,5)P2 To determine if these histologic abnormalities would result in (Fig. 3B); no peak was detected at the position of PI(3,5)P2 (Fig. myocardial dysfunction, echocardiograms were performed on S8A). Summation of the 2-min elution region that includes these fi four pups that survived beyond 2 wk. The hearts were signi - products demonstrated that PI(3,5)P2 in wild-type cells com- cantly smaller, resulting in a decreased calculated left ventricular prised 0.040 ± 0.006% of total phosphoinositide lipids, whereas (LV) mass (Fig. S5F). Because of the marked difference between in the mutant cells PI(3,5)P was not detectable (0.001 ± β β + β 2 the size of the Pikfyve -geo/ -geo mutants and their Pikfyve / -geo 0.002%). These findings show that the total cellular pool of PI littermates, the calculated LV mass was corrected for calculated (3,5)P2 is generated via the Pikfyve pathway and that Pikfyve is body surface area. After correction, no detectable difference in the source of PI(3,5)P2 in mammalian cells. LV size or LV mass was noted (Fig. S5F). However, there were differences in right ventricular (RV) size and function. When Most of the PI5P Pool Is Generated from PI(3,5)P . shRNA treatment β β 2 corrected for differences in body surface area, the RV end di- of Pikfyve -geo/ -geo fibroblasts resulted in a loss of 85% of PI5P β β astolic dimension was significantly larger in the Pikfyve -geo/ -geo (Fig. 3B and Fig. S8B). These findings provide strong support for + β mutants than in their Pikfyve / -geo littermates (Fig. S5F), as is the hypothesis that Pikfyve is required to generate most of the consistent with RV dilatation. PI5P pool. Moreover, Vac14- and Fig4-deficient cells also have These defects suggested a primary pulmonary pathology. In- a 50% (21) and 55% (Fig. S8C) reduction in PI5P, respectively. deed, the lungs had marked abnormalities that might contribute The small amount of PI5P detectable after shRNA treatment β β to the early demise of the Pikfyve -geo/ -geo mutants (Fig. S5G). leaves open the possibility that a minor portion of the PI5P pool There was increased cellularity with reduced alveolar cross-sec- is generated via Pikfyve-independent pathways. tional area and apparent pulmonary vascular congestion. In ad- In addition to effects on PI5P, there was an ∼30% reduction in β-geo/β-geo dition, there were areas lacking alveolar development. These PI(4,5)P2 after shRNA treatment of Pikfyve fibroblasts findings suggest a primary role for Pikfyve in lung development. (Fig. S7D). This finding raises the possibility that the Pikfyve- Analysis of other organs revealed that the thymus and spleens dependent PI5P pool contributes to the generation of PI(4,5)P . β β 2 from the P16 Pikfyve -geo/ -geo mutants were smaller and had less Note that nearly complete reduction of Pikfyve protein is cellular density than the organs from wild-type littermates and necessary for substantial changes in PI(3,5)P2 levels (Fig. S7E).

17474 | www.pnas.org/cgi/doi/10.1073/pnas.1203106109 Zolov et al. Downloaded by guest on September 28, 2021 Fig. 3. Generation of PI(3,5)P2 and most of the PI5P requires Pikfyve. Lentiviral expression of Pikfyve shRNA for 4 d. Scrambled sequence was used as a negative control. (A) After shRNA, Pikfyve protein levels in the wild-type and Pikfyveβ-geo/β-geo mouse fibroblasts were decreased or were below detectable levels, respectively. (B) Depletion of Pikfyve in β β Pikfyve -geo/ -geo fibroblasts resulted in a fivefold elevation of PI3P, a loss of 85% of PI5P, and nearly

complete loss of PI(3,5)P2.

Because Pikfyve is an enzyme, only a catalytic amount of Pikfyve Discussion is required to act on a large pool of substrate. For example, These studies revealed that Pikfyve is responsible for all of the shRNA knockdown of Pikfyve to 17% of its normal levels in intracellular PI(3,5)P2 pool. While this hypothesis was previously −/− −/− wild-type cells reduces the level of PI(3,5)P2 by only 40%. Thus, assumed, observations that Vac14 and Fig4 fibroblasts have fi unless Pikfyve is lowered to nondetectable levels, signi cant half the normal levels of PI(3,5)P2 raised the possibility that amounts of PI(3,5)P2 and PI5P will remain. other pathways contribute to PI(3,5)P pools. However, after 2 β β The requirement for Pikfyve for normal steady-state levels of knockdown of residual Pikfyve protein in Pikfyve -geo/ -geo fibro- PI5P levels could be a direct effect of Pikfyve catalyzing the blasts, no PI(3,5)P2 was detected. generation of PI5P from phosphatidylinositol or could occur Pikfyve is indirectly responsible for most of the PI5P pool. In − − β β − − indirectly through enzymes with 3′-phosphatase activity acting on Vac14 / , Pikfyve -geo/ -geo,orFig4 / fibroblasts, PI5P levels are PI(3,5)P2, or through a combination of the two. We designed reduced ∼50% (Figs. S7A and S8C) (21). When Pikfyve is fur- β β three approaches to distinguish between these models. ther reduced by shRNA knockdown in Pikfyve -geo/ -geo cells, only Pikfyve was inhibited acutely in wild-type fibroblasts using the 15% of wild-type PI5P levels remain (Fig. 3B and Fig. S8B). catalytic Pikfyve inhibitor, YM201636 (23). After treatment, PI Furthermore, acute treatment of wild-type fibroblasts with the fi (3,5)P2 levels were reduced fully at the rst time point assessed, Pikfyve inhibitor YM201636 resulted in a rapid loss of PI5P (Fig. 2.5 min. There was a slower, but still rapid, reduction in PI5P, 4A and ref. 34). Thus, loss of Vac14, Fig4, or Pikfyve activity with a half-life of ∼4.5 min (Fig. 4A). This reduction was ac- results in significant loss of PI5P. companied by elevation of PI3P during the 30-min treatment. No We used three tests to determine whether Pikfyve has a direct fi signi cant changes were detected for PI4P or PI(4,5)P2 (Fig. 4A). role in the generation of PI5P from phosphatidylinositol or, al- Starvation, followed by nutrient refeeding, stimulates Pikfyve ternatively, whether PI5P is generated from PI(3,5)P2 via 3′- activity as measured by an increase in PI(3,5)P2 (32). We tested phosphatases. That PI5P levels are 10-fold higher than PI(3,5)P2 the effects of nutrient refeeding on the pools of PI5P and PI(3,5) levels initially raised doubt that PI(3,5)P2 would be a major P2. Fibroblasts were starved by 1-h incubation in HBSS buffer, precursor for the generation of PI5P. However, our data suggest causing an acute decrease in all phosphoinositide lipids; PI(3,5) that most of the PI5P is derived from PI(3,5)P2. P2 and PI5P were reduced ∼50% (Fig. 4B). Upon readdition of The strongest support for this hypothesis is that human PIK- complete medium, PI(3,5)P2 plateaued at prestarvation levels FYVE expressed in yeast is highly active and produced a fivefold within 2.5 min. In contrast, PI5P returned to prestarvation levels elevation in PI(3,5)P2 (Fig. 4C and Fig. S8D). However, the at 5 min and continued to rise for another 5 min. This time lag is small additional amount of PI5P produced was sixfold lower than consistent with a precursor/product relationship between PI(3,5) PI(3,5)P2. Notably, if all of the PI5P generated by PIKFYVE was P2 and PI5P. PI3P, PI4P, and PI(4,5)P2 returned to prestarvation derived indirectly from PIKFYVE via PI(3,5)P2, then the sum fi levels during the rst 2.5 min of refeeding (Fig. 4B). total of PI5P plus PI(3,5)P2 plus PI3P, should be equal in wild- To test further whether most of the PI5P pool in fibroblasts is type yeast and yeast expressing PIKFYVE—as, indeed, was ob-

derived via Pikfyve generation of PI5P from phosphatidylinositol served (Fig. 4D). These observations strongly support the hy- CELL BIOLOGY (PI), we tested whether human PIKFYVE can synthesize PI5P pothesis that the PIKFYVE protein in vivo directly catalyzes the directly in S. cerevisiae, which makes little to no PI5P (1, 33). In conversion of PI3P to PI(3,5)P2 but does not generate PI5P wild-type yeast there was a small peak in the region where PI5P from PI. should elute, which was reduced 1.7-fold in a fab1Δ mutant (Fig. The experiments performed in fibroblasts also fit this hy- 4C and Fig. S8E). Overexpression human PIKFYVE cDNA in pothesis. Upon acute inhibition of Pikfyve, PI(3,5)P2 was fully wild-type yeast caused a fivefold elevation in the levels of PI(3,5) depleted at the first measurable time point, 2.5 min. In contrast, P2, demonstrating enzymatic activity of human PIKFYVE (Fig. PI5P declined more slowly; at 7.5 min, about half of the PI5P 4C and Fig. S8D). However, there was only a small increase in remained. Similarly, using 1-h starvation followed by nutrient PI5P (Fig. 4C and Fig. S8E). The PIKFYVE-dependent pool of refeeding to stimulate Pikfyve, PI(3,5)P2 fully increased to its PI5P was sixfold less than the PIKFYVE-dependent PI(3,5)P2 new steady-state levels at 2.5 min following refeeding, whereas pool. There was a corresponding reduction in PI3P levels (Fig. the rate of increase of PI5P levels apparently was slower and was 4C). Moreover, the sum of new PI5P plus PI(3,5)P2 was equal to completed at 10 min. The more rapid disappearance of PI(3,5)P2 the total decrease in PI3P (Fig. 4D). There were no alterations in after inhibition of Pikfyve, compared with the disappearance of the levels of PI4P or PI(4,5)P2 (Fig. S8F). Together, these studies PI5P, and the more rapid appearance of PI(3,5)P2, after stimu- fit best with the hypothesis that PI5P pools in fibroblasts are lation of Pikfyve fit the hypothesis that PI(3,5)P2 and PI5P are regulated indirectly by Pikfyve via the conversion of PI(3,5)P2 generated by different enzymes and are consistent with a pre- to PI5P. cursor/product relationship between PI(3,5)P2 and PI5P.

Zolov et al. PNAS | October 23, 2012 | vol. 109 | no. 43 | 17475 Downloaded by guest on September 28, 2021 generate PI5P from PI in vitro (35), another in vitro study showed that Pikfyve directly generates PI5P (13). However, note that in the latter study Pikfyve was pulled down from cell types that express many lipid 3′-phosphatases, including several myotubularins. A tight association of Pikfyve with myotubularins in vivo and in the immunoprecipitation protocol would explain the differences between in vitro studies of Pikfyve immunoprecipitated from COS cells and of Pikfyve expressed heterologously in yeast. The most likely candidate 3′-phosphatases for the generation of PI5P from PI(3,5)P2 are myotubularins. Several myotubularin 3′-lipid phosphatases, including Mtm1, Mtmr2, Mtmr3, and Mtmr6, can convert PI(3,5)P2 to PI5P in vitro and, when expressed heterologously, in S. cerevisiae (33, 36–38). Thus, myotubularins may convert PI(3,5)P2 to PI5P in vivo. However, a direct test of this role is not feasible, because there are at least eight active myotubularins (39, 40). Some PI5P generated via Pikfyve may contribute to PI(4,5)P2 pools (Fig. 5). When PI5P is depleted, PI(4,5)P2 levels are reduced (Fig. S7D). PI5P is converted to PI(4,5)P2 by type II PI-5- P 4-kinases (PIP4K IIs) (41, 42). The discovery that Pikfyve is critical for the generation of most of the cellular PI5P pool provides a caveat to the interpretation of phenotypes caused by perturbation of Pikfyve activity. Meth- ods will need to be developed that can distinguish between phenotypes caused by loss of PI5P and those caused by loss of PI (3,5)P2. Loss of PI(3,5)P2 rather than PI5P is likely responsible for the vacuoles observed during inhibition of Pikfyve activity (Fig. S7C and refs. 5, 19–25, and 29). Elevation of PI5P affects early endosomes without affecting late endosomes (43), and large vacuoles seen in metazoans likely are related to the large vacuoles in yeast, an organism that does not make PI5P. Large vacuoles are a hallmark of partial to full inhibition of Pikfyve activity, but vacuoles may not be the primary cause of organ damage in animals. Neurons become vacuolated ex vivo, suggesting that vacuole formation may not be the most proximal

Fig. 4. Perturbation of Pikfyve activity suggests that the PI5P derives from

PI(3,5)P2. (A) Inhibition of Pikfyve in mouse primary fibroblasts by 1.6 μM YM201636 for the times specified results in a rapid depletion of PI(3,5)P2 and PI5P (n = 4). (B) Stimulation of mouse fibroblasts by refeeding with normal growth medium, insulin, and transferrin following 1-h starvation in HBSS + + with Mg2 and Ca2 reveals that within 2.5 min all lipids, with the exception of PI5P, increased and plateaued at a new steady-state level. PI5P plateaued at its new steady-state level at 10 min (n = 3). (C) Expression of human

PIKFYVE in wild-type yeast resulted in a large elevation in PI(3,5)P2, a small increase in PI5P, and a corresponding reduction in PI3P. (D) The sum of PI3P,

PI(3,5)P2, and PI5P was the same in wild-type yeast with PIKFYVE overexpression and with vector control (n = 3). This result suggests that PI5P

levels are dependent on PI3P via dephosphorylation of PI(3,5)P2.

An alternative interpretation for the studies in fibroblasts is that the rate of turnover of PI5P is slower than the rate of turnover of PI(3,5)P2. However, collective interpretation of the fibroblast and yeast studies strongly supports the hypothesis that most of the Pikfyve-dependent PI5P pool in fibroblasts is generated via the action of 3′-lipid phosphatases on PI(3,5)P2 (Fig. 5). Fig. 5. Model in which most of the PI5P pool derives from PI(3,5)P2 that is Two observations support the model that Pikfyve makes PI5P generated via Pikfyve. PI is converted to PI3P by Vps34 and possibly other + β PIKfyve / -geo fi PI3Ks (A) (48). Some PI3P is converted to PI(3,5)P2 by Pikfyve (Fab1) (B) (1, 2, directly in vivo. First, in heterozygous broblasts ′ (Fig. S7A), PI5P is reduced by 30%, whereas PI(3,5)P levels are 13). Fig4 is both a lipid 3 -phosphatase and an activator of Fab1 (6, 7). Pikfyve 2 generates all of the PI(3,5)P pool (Fig. 3). Most of the PI5P pool indirectly unaffected. One explanation is that mechanisms that coordinate 2 fi requires Pikfyve (C). The Pikfyve-dependent pool of PI(3,5)P2 is dephos- both synthesis and turnover ensure speci c levels of PI(3,5)P2. phorylated to produce PI5P. The most likely 3′-phosphatases are myotubu- Perhaps minor perturbations in PI(3,5)P2 synthesis in the het- larins (MTMRs) (33, 36–38). Blue arrows denote the indirect generation of

erozygous mutant are compensated by less turnover of PI(3,5)P2 PI5P from PI3P through dephosphorylation of PI(3,5)P2. PI5P also may serve to PI5P. Second, although one study found that Pikfyve cannot as a precursor for some of the PI(4,5)P2 pool (ref. 42 and Fig. S8D)(D).

17476 | www.pnas.org/cgi/doi/10.1073/pnas.1203106109 Zolov et al. Downloaded by guest on September 28, 2021 or serious defect caused by loss of Pikfyve activity. PI(3,5)P2 and the sole source of PI(3,5)P2, and the resultant PI(3,5)P2 serves as PI5P likely regulate multiple additional pathways. It is of interest the precursor for most of the cellular pools of PI5P. that several aspects of the Pikfyve depletion phenotype closely resemble those observed in mice deficient for Fig4 and Vac14, Materials and Methods consistent with the coordinated role of the three proteins in All experiments with animals were performed in compliance with guidelines regulation of phosphoinositide levels. of the University Committee on Use and Care of Animals of the University of Francois–Neetens mouchetée fleck corneal dystrophy (44) is Michigan and National Institutes of Health. Animals were housed and cared associated with mutations in the PIKFYVE gene. Mutations in for in accordance with National Institutes of Health guidelines. See SI the PIKFYVE regulator FIG4 are responsible for the Charcot– Materials and Methods for details of experimental procedures. Marie–Tooth disorder CMT4J (29, 31, 45, 46) and for some cases of amyotrophic lateral sclerosis and primary lateral scle- ACKNOWLEDGMENTS. We thank Dr. Shenghui He for help with quantitative RT-PCR and Judy Poore and the Microscopy and Image Analysis rosis (47). It is likely that additional neurological diseases may be Laboratory at the University of Michigan for H&E sample preparation. This linked to PIKFYVE and its regulators. Moreover, additional work used the Vector and Hybridoma Cores of the Michigan Diabetes Re- pathological consequences of mutations in PIKFYVE and its search and Training Center, funded by DK020572 from the National Insti- β β regulators are likely. Most striking in the Pikfyve -geo/ -geo mutant tute of Diabetes and Digestive and Kidney Disease, and the University of are the defects in the heart and lung. Michigan Echocardiography and Transgenic Animal Model Cores. This work fi was supported by National Institutes of Health Research Grants R01 NS064015 Together, these nding demonstrate that defects in the levels (to L.S.W.), R01 GM24872 (to M.H.M.), and R01 DK061618 (to A.R.S.); by a Vet- of PI(3,5)P2 and PI5P cause premature lethality and degeneration erans Administration Merit Review Grant (to R.L.A.), and by a grant from the of multiple organs. Furthermore, in mammalian tissues, Pikfyve is Fritz Thyssen Foundation (to T.W.).

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