You are here: Home > Balla Lab

Phosphoinositide Messengers in Cellular Signaling and Trafficking

• Tamás Balla, MD, PhD, Head, Section on Molecular Signal Transduction
• Alejandro Alvarez-Prats, PhD, Staff Scientist
• Yeun Ju Kim, PhD, Staff Scientist
• Gergo Gulyás, MD, PhD, Postdoctoral Fellow
• Amrita Mandal, PhD, Postdoctoral Fellow
• Yang Niu, PhD, Postdoctoral Fellow
• Joshua Pemberton, PhD, Postdoctoral Fellow
• Vijay Joshi, BS, Postbaccalaureate Intramural Research Training Award Fellow
• Marek Korzeniowski, PhD, Special Volunteer
• Ljubiša Vitković, PhD, Special Volunteer

Every biochemical process in a eukaryotic cell relies upon a molecular information flow that leads from receptors that inform the cell about its environment all the way to the molecular effectors that determine the appropriate cellular response. Proper information transmission requires a high degree of organization, where the molecular players are organized into different cellular compartments so that the specificity of the cellular response can be properly maintained. Breakdown of this organization is the ultimate cause of all human diseases, even if the affected molecular pathways differ according to the kind of disease, such as cancer, diabetes, or neuro-degenerative diseases, just to name a few.

Our research focuses on the question as to how cells organize their internal membranes to provide a structural framework on which molecular signaling complexes assemble to ensure proper information processing. The lipid composition of cellular membranes is a major determinant of their biophysical properties and is unique to the different cellular organelles. How cells achieve and maintain the proper lipid composition of their membranes is poorly understood. Cellular processes that affect the membrane lipid composition of organelles are often targeted by cellular pathogens, such as viruses, to force the cells to produce the pathogen instead of performing the cells' normal functions. Better understanding of these processes can not only provide new strategies to fight various human diseases but also to intercept the life cycle of cellular pathogens, offering an alternative to antimicrobial drugs.

Lipid transport and Ca²⁺ signaling are closely interrelated in plasma membrane (PM)–endoplasmic reticulum (ER) contact sites.

Membrane contacts sites (MCS) between various organelles are emerging as key structural elements, where important communication between organelles takes place. MCS have been primarily featured in non-vesicular lipid transfer and Ca²⁺ signal propagation, but their importance is likely to reach beyond these two processes. An important class of molecules that function at MCSs are the ORPs (oxysterol binding-protein–related proteins), which are the mammalian orthologs of the yeast Osh (oxysterol-binding homolog) proteins and mediate the transport of specific lipids between cellular membranes. One of the salient features of Osh/ORP proteins is that they use a phosphatidylinositol 4-phosphate (PI4P) gradient as a driving force as they counter-transport PI4P in exchange for the specific lipids they move between membranes. Therefore, lipid transport by Osh/ORP proteins is linked to the activity of PI 4-kinases.

We investigated the impact of changing PM PI4P levels on the Ca²⁺ entry process mediated by the STIM1–Orai1 molecular complex, which underlies the refilling of the ER luminal Ca²⁺ stores during receptor stimulation. We found that changing PM PI4P levels through inhibition of the lipid kinase (PI4KA), which produces PI4P in the PM, potently inhibits Ca²⁺ influx by interrupting the association of the calcium sensor STIM1 (stromal interaction molecule 1) with the PM. Similarly, manipulation of PM PI4P levels through the expression of ORP5 and ORP8 proteins had a major impact on Ca²⁺ influx. Our studies revealed a tight connection between Ca²⁺ entry mediated by the STIM1–Orai1 complex and the PI4P–driven lipid transport process at PM–ER contact sites.

The critical role of specific phosphoinositide lipids in the late stage of cell division

Separation of the two daughter cells at the last stage of mitosis, called cytokinetic membrane abscission, is a spatially and temporally regulated process that requires membrane remodeling at the midbody, a subcellular organelle that defines the cleavage site. The process is mediated by a multi-protein molecular complex, called ESCRT (endosomal sorting complex required for transport), and its defective function can lead to cataracts. It is not known how ESCRT defects can lead to cataracts and whether they are related to cytokinesis defects. In a collaborative study led by the Hirsch laboratory, it was found that a lens-specific cytokinetic process required the lipid kinase PI3K-C2 (phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2) and its lipid product PI(3,4)P2 (phosphatidylinositol 3,4-bisphosphate). These studies showed that the ESCRT-II subunit VPS36 (vacuolar protein-sorting-associated protein 36) requires PI(3,4)P2 binding, and loss of each of the ESCRT-II components led to impaired cytokinesis, triggering premature senescence in the lens of fish, mice, and humans. Importantly, the PI4P substrate for the PI3K-C2 enzyme to support this process was provided by the PI4KA enzyme. This evolutionarily conserved pathway underlies the cell type–specific control of cytokinesis, which helps to prevent early-onset cataract by protecting from senescence.

A specific calcineurin splice form targets the multi-protein complex of PI4KA.

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca²⁺ signaling. In a collaborative study, led by the Cyert laboratory, that focused on the understudied calcineurin isoform CNA1, it was discovered that, unlike canonical cytosolic calcineurin, CNA1 localizes to the plasma membrane and Golgi as a result of reversible palmitoylation of its divergent C-terminal tail. Palmitoylation targets CNA1 to a distinct set of membrane-associated interactors, including the multi-protein PI4KA complex containing EFR3B (palmitoylated plasma-membrane protein that controls responsiveness to G-protein–coupled receptors [GPCR]), PI4KA, TTC7B (tetratricopeptide repeat protein, a component of a complex required to localize PI4K to the plasma membrane), and FAM126A (hyacin; regulates PI4K synthesis at the plasma membrane). Hydrogen-deuterium exchange studies, performed in the Burke laboratory, found multiple contacts in the calcineurin–PI4KA complex, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which was established as a calcineurin substrate. In cellular studies, calcineurin inhibitors reduced PI4P production during Gq–coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. The work thus revealed a calcineurin-regulated signaling pathway and identified the PI4KA complex as a regulatory target. It also showed that dynamic palmitoylation provides the CNA1 enzyme with a unique localization to increase its access its substrates, lending the protein unique specificity and regulation.

PI4KA variants in human cause neurological, intestinal, and immunological disease.

The lipid kinase PI4KA generates PI4P in the PM, playing critical roles in the physiology of many cell types. PI4KA requires its assembly into a heterotetrameric complex with EFR3, TTC7 and FAM126. Sequence alterations in two of these molecular partners, TTC7 (encoded by TTC7A or TCC7B) and FAM126, in humans have been associated with a heterogeneous group of either neurological (FAM126A) or intestinal and immunological (TTC7A) conditions. In this multi-center collaborative study led by Andrew Crosby and Emma Baple, biallelic PI4KA sequence alterations in humans were shown to be associated with neurological disease, in particular hypomyelinating leukodystrophy. In addition, some affected individuals may also present with inflammatory bowel disease, multiple intestinal atresia, and combined immunodeficiency. Biochemical and structural modelling studies indicated that PI4KA–associated phenotypical outcomes probably stem from impairment of PI4KIIIα-TTC7-FAM126's organ-specific functions as the result of defective catalytic activity or altered intra-complex functional interactions. Together, these data define PI4KA gene alteration as a cause of a variable phenotypical spectrum and provide fundamental new insights into the combinatorial biology of the PI4KIIIα-FAM126-TTC7-EFR3 molecular complex.

Publications

1. Prinz WA, Toulmay A, Balla T. The functional universe of membrane contact sites. Nat Rev Mol Cell Biol 2020 1:7–24.
2. Gulyas G, Korzeniowski MK, Eugenio CEB, Vaca L, Kim YJ, Balla T. LIPID transfer proteins regulate store-operated calcium entry via control of plasma membrane phosphoinositides. Cell Calcium 2022 106:102631.
3. Gulluni F, Prever L, Li H, Krafcikova P, Corrado I, Lo WT, Margaria JP, Chen A, De Santis MC, Cnudde SJ, Fogerty J, Yuan A, Massarotti A, Sarijalo NT, Vadas O, Williams RL, Thelen M, Powell DR, Schueler M, Wiesener MS, Balla T, Baris HN, Tiosano D, McDermott BM Jr, Perkins BD, Ghigo A, Martini M, Haucke V, Boura E, Merlo GR, Buchner DA, Hirsch E. PI(3,4)P2-mediated cytokinetic abscission prevents early senescence and cataract formation. Science 2021 374:abk0410.
4. Ulengin-Talkish I, Parson MAH, Jenkins ML, Roy J, Shih AZL, St-Denis N, Gulyas G, Balla T, Gingras AC, Várnai P, Conibear E, Burke JE, Cyert MS. Palmitoylation targets the calcineurin phosphatase to the phosphatidylinositol 4-kinase complex at the plasma membrane. Nat Commun 2021 12:6064.
5. Salter CG, Cai Y, Lo B, Helman G, Taylor H, McCartney A, Leslie JS, Accogli A, Zara F, Traverso M, Fasham J, Lees JA, Ferla MP, Chioza BA, Wenger O, Scott E, Cross HE, Crawford J, Warshawsky I, Keisling M, Agamanolis D, Ward Melver C, Cox H, Elawad M, Marton T, Wakeling MN, Holzinger D, Tippelt S, Munteanu M, Valcheva D, Deal C, Van Meerbeke S, Walsh Vockley C, Butte MJ, Acar U, van der Knaap MS, Korenke GC, Kotzaeridou U, Balla T, Simons C, Uhlig HH, Crosby AH, De Camilli P, Wolf NI, Baple EL. Biallelic PI4KA variants cause neurological, intestinal and immunological disease. Brain 2021 144:3597–3610.

Collaborators

• Emma Baple, MBBS, MRCPCH, PhD, University of Exeter Medical School, Exeter, UK
• Evžen Boura, PhD, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic
• John Burke, PhD, University of Victoria, Victoria, Canada
• Andrew Crosby, PhD, University of Exeter Medical School, Exeter, UK
• Martha Cyert, PhD, Stanford University, Stanford, CA
• Emilio Hirsch, PhD, Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy
• Juan Marugan, PhD, Division of Pre-Clinical Innovation, NCATS, Bethesda, MD
• Péter Várnai, MD, PhD, Semmelweis University, Faculty of Medicine, Budapest, Hungary

Contact

For more information, email ballat@mail.nih.gov or visit https://ballalab.nichd.nih.gov.

Top of Page