Deletion of the gene Pip4k2c, a novel phosphatidylinositol kinase, results in hyperactivation of the immune system Hyeseok Shima,b,c,1, Chuan Wud, Shivan Ramsamooja,b, Kaitlyn N. Boscha,b, Zuojia Chend, Brooke M. Emerlinga,b, Jihye Yuna,b, Hui Liue, Rayman Choo-Winga,b, Zhiwei Yanga,b, Gerburg M. Wulfe, Vijay Kumar Kuchrood, and Lewis C. Cantleya,b,2 aMeyer Cancer Center, Weill Cornell Medical College, New York, NY 10065; bDepartment of Medicine, Weill Cornell Medical College, New York, NY 10065; cBiological and Biomedical Sciences Graduate Program, Harvard Medical School, Boston, MA 02115; dEvergrande Center for Immunologic Diseases, Harvard Medical School and Brigham and Women’s Hospital, Boston, MA 02115; and eDivision of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA 02115 Contributed by Lewis C. Cantley, May 6, 2016 (sent for review January 28, 2016; reviewed by Richard A. Flavell and Robin F. Irvine) − − Type 2 phosphatidylinositol-5-phosphate 4-kinase (PI5P4K) converts with Trp53 / mice results in early embryonic lethality for the − − − − phosphatidylinositol-5-phosphate to phosphatidylinositol-4,5-bisphos- subset of embryos that are Pip4k2b / , Trp53 / , indicating a phate. Mammals have three enzymes PI5P4Kα,PI5P4Kβ, and PI5P4Kγ, synthetic lethality relationship between these genes. In contrast, − − and these enzymes have been implicated in metabolic control, Pip4k2a / mice do not exhibit any of the phenotypes observed in − − growth control, and a variety of stress responses. Here, we show the Pip4k2b / mice (5). They are not protected from obesity or that mice with germline deletion of type 2 phosphatidylinositol-5- insulin resistance, they do not exhibit a synthetic lethality re- phosphate 4-kinase gamma (Pip4k2c), the gene encoding PI5P4Kγ, lationship with Trp53, and in all ways examined, they resemble appear normal in regard to growth and viability but have increased wild-type mice (5). However, germline deletion of one allele of − − inflammation and T-cell activation as they age. Immune cell infiltrates Pip4k2a in the context of germline Pip4k2b / causes enhance- −/− − − increased in Pip4k2c mouse tissues. Also, there was an increase in ment of the phenotypes of the Pip4k2b / mice. Deletion of both γ proinflammatory cytokines, including IFN , interleukin 12, and inter- alleles of Pip4k2a and Pip4k2b did not have any observable effect Pip4k2c−/− Pip4k2c−/− leukin 2 in plasma of mice. mice had an in- on embryonic development up until the time of birth, but resulted crease in T-helper-cell populations and a decrease in regulatory T-cell in perinatal lethality of all pups. These results indicate that these populations with increased proliferation of T cells. Interestingly, genes do not play a major role in embryonic development and mammalian target of rapamycin complex 1 (mTORC1) signaling that Pip4k2a provides a backup for Pip4k2b that becomes critical was hyperactivated in several tissues from Pip4k2c−/− mice and − − at the time of birth. treating Pip4k2c / mice with rapamycin reduced the inflamma- tory phenotype, resulting in a decrease in mTORC1 signaling in tissues and a decrease in proinflammatory cytokines in plasma. Significance These results indicate that PI5P4Kγ plays a role in the regulation of the immune system via mTORC1 signaling. The mammalian target of rapamycin complex 1 (mTORC1) sig- naling pathway is an important facet of the immune system, PIP4K2C | mTORC1 | autoimmunity | PI5P4K | inflammation including that it regulates T-cell differentiation and activation. Here, we report that type 2 phosphatidylinositol-5-phosphate 4-kinase gamma (protein, PI5P4Kγ; gene, PIP4K2C) plays a role ype 2 phosphatidylinositol-5-phosphate 4-kinase (PI5P4K) con- in the regulation of the immune system by manipulating verts phosphatidylinositol-5-phosphate to phosphatidylinositol- T mTORC1 signaling. These results suggest that the SNP at the 4,5-bisphosphate. Mammals have three genes, PIP4K2A, PIP4K2B, PIP4K2C locus (rs1678542) in human patients with autoimmunity and PIP4K2C that encode the enzymes PI5P4Kα, PI5P4Kβ, and might cause a decrease in PI5P4Kγ expression and thereby an PI5P4Kγ, respectively. increase in mTORC1 signaling. In addition, these results imply All three isoforms are highly expressed in brain, whereas their that inhibition of mTORC1 would be beneficial to these patients. relative expressions in other tissues vary. PI5P4Kα is highly These studies also suggest that agents that inhibit PIP4K2C expressed in spleen and the peripheral blood. PI5P4Kβ is highly function could be useful to enhance cancer immunotherapy. expressed in muscle, whereas PI5P4Kγ is highly expressed in γ kidney (1). In kidney, PI5P4K is mostly detected in the cortex Author contributions: H.S., C.W., and L.C.C. designed research; H.S., C.W., S.R., K.N.B., Z.C., and the outer medulla (1). All tissues examined appear to express J.Y., H.L., R.C.-W., and Z.Y. performed research; B.M.E., G.M.W., and V.K.K. contributed at least one isoform of PI5P4K. At the cellular level, PI5P4Ks are new reagents/analytic tools; H.S. and C.W. analyzed data; and H.S. and L.C.C. wrote found in several organelles, including plasma membrane, cytosol, the paper. nucleus, and vesicular compartments (2, 3). It is not simple to Reviewers: R.A.F., Yale School of Medicine, Howard Hughes Medical Institute; and R.F.I., define unique compartmentalization of the individual enzymes Cambridge University. because all three isoforms can homodimerize or heterodimerize Conflict of interest statement: L.C.C. owns equity in, receives compensation from, and α β serves on the board of directors and scientific advisory board of Agios Pharmaceuticals. with each other. At the sequence level, PI5P4K and PI5P4K Agios Pharmaceuticals is identifying metabolic pathways of cancer cells and developing are more homologous to each other than either is to PI5P4Kγ. drugs to inhibit such enzymes to disrupt tumor cell growth and survival. L.C.C. owns Also, PI5P4Kγ is only about 1% as active as the other isoforms, equity in, receives compensation from, and serves on the scientific advisory board of Petra Pharmaceuticals, a company that develops targeted therapies for cancer treatment. In raising the possibility that its major role may be to localize or addition, Petra Pharmaceuticals will be providing funds to support research in the L.C.C. regulate the activities of PI5P4Kα and PI5P4Kβ. laboratory, although the research described in this paper predated the existence of this Germline deletion of both alleles of Pip4k2a or Pip4k2b in collaborative agreement. mice results in healthy mice that live a normal life span. The 1Present address: Petra Pharmaceuticals, New York, NY 10016. −/− Pip4k2b mice have increased insulin sensitivity and are pro- 2To whom correspondence should be addressed. Email: [email protected]. tected from obesity, insulin resistance, and type 2 diabetes when This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. −/− placed on a high-fat diet (4). Also, crossing the Pip4k2b mice 1073/pnas.1600934113/-/DCSupplemental. 7596–7601 | PNAS | July 5, 2016 | vol. 113 | no. 27 www.pnas.org/cgi/doi/10.1073/pnas.1600934113 Downloaded by guest on September 29, 2021 Whereas less is known about PI5P4Kγ, it has been linked to In addition, they exhibit hyperactivation of mTORC1 signaling in the mammalian target of rapamycin signaling complex (mTORC) multiple tissues, suggesting that Pip4k2c negatively regulates and to cellular immunity. Mackey et al. argued that PI5P4Kγ was mTORC1. These results, along with a recent observation that the negatively regulated by mTORC1 through direct phosphorylation enzyme encoded by this gene is a substrate of mTORC1 (6), at serine 324 and serine 328 (6). On the other hand, it was suggest a close relationship between mTORC1 and Pip4k2c. − − reported that knocking out the only PI5P4K isoform in Drosophila Moreover, the hyperimmune phenotype of the Pip4k2c / mice resulted in lower body weight and that this correlated with de- could be partially ameliorated by treatment with the mTORC1 creased mTORC1 signaling (7). Of particular interest, multiple inhibitor, rapamycin. Importantly, a SNP (rs1678542) located near studies have shown a link between a SNP (rs1678542) in the the PIP4K2C locus has been correlated with familial autoim- human PIP4K2C locus and familial autoimmunity (8, 9). The munity (8) and the results presented here suggest that loss of immune system is a complex network that evolved to protect PI5P4Kγ function could explain this disease. organisms from invasion of various microbes. Whereas an active immune system protects from microbes, overactivation of the Results immune system can result in autoimmunity. An understanding of Generation of Pip4k2c−/− Mice. To investigate the role of PI5P4Kγ − − the molecular underpinnings of an overactive or underactive in mammals, PI5P4Kγ knockout (Pip4k2c / ) mice were gener- immune system is important for developing therapies for immune- ated using Pip4k2c-targeted ES cell clones obtained from the related diseases from immunodeficiency to autoimmunity and knockout mouse project (KOMP) repository (www.komp.org). even cancers. mTORC1, a central regulator of cell survival, In the targeting construct, exons 3 and 4 of Pip4k2c were growth, and metabolism, also plays a critical role in regulation of bracketed by loxP sequences, so deletion of these exons could immune cells. mTORC1 responds to intra- and extracellular remove much of the kinase domain and