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Phosphoinositide Messengers in Cellular Signaling and Trafficking

• Tamás Balla, MD, PhD, Head, Section on Molecular Signal Transduction
• Yeun Ju Kim, PhD, Staff Scientist
• Alejandro Alvarez-Prats, PhD, Postdoctoral Fellow
• Takashi Baba, PhD, Postdoctoral Fellow
• Joshua Pemberton, PhD, Postdoctoral Fellow
• Nivedita Sengupta, PhD, Postdoctoral Fellow
• Mira Sohn, PhD, Postdoctoral Fellow
• Dániel Tóth, MD, PhD, Postdoctoral Fellow
• Ljubisa Vitkovic, PhD, Special Volunteer
• Elisa Arthofer, MS, Graduate Student

We investigate signal transduction pathways that mediate the actions of hormones, growth factors, and neurotransmitters in mammalian cells, with special emphasis on the role of phosphoinositide-derived messengers. Phosphoinositides constitute a small fraction of the cellular phospholipids but play critical roles in the regulation of many signaling protein complexes, which assemble on the surface of cellular membranes and are intracellular lipid messengers controlling a variety of cellular functions. Phosphoinositides regulate protein kinases and GTP–binding proteins as well as membrane transporters, including ion channels, thereby controlling many cellular processes such as proliferation, apoptosis, metabolism, cell migration, and differentiation. We focus on the phosphatidylinositol 4 (PtdIns4)–kinases (PI4Ks), a family of enzymes that catalyze the first committed step in polyphosphoinositide synthesis. Current work aims to: (1) understand the function and regulation of several PI4Ks involved in the control of cellular signaling and trafficking pathways; (2) find specific inhibitors for the individual PI4Ks; (3) define the molecular basis of phosphatidylinositol 4-phosphate (PtdIns4P)–regulated pathways through identification of PtdIns4P–interacting molecules; (4) develop tools to analyze inositol lipid dynamics in live cells; and (5) determine the importance of the lipid-protein interactions in the activation of cellular responses by G protein–coupled receptors and receptor tyrosine kinases.

Control of phosphatidylserine metabolism by phosphatidylinositol 4-phosphate cycling

We discovered that phosphatidylinositol 4-kinase alpha (PI4KA), one of the lipid kinases that phosphorylate the important lipid phosphatidylinositol (PI) at the plasma membrane (PM), is essential for the transport and synthesis of a critical structural lipid, phosphatidylserine (PS). Prolonged (24–36 hours) treatment of cells with a PI4KA–specific inhibitor caused a 50% reduction in cellular PS content, and the inhibitor acutely blocked PS synthesis. We demonstrated that the inhibition was not attributable to a direct effect on the PS–synthesizing enzymes but was the result of a transport defect impeding transport of PS out of its site of synthesis in the endoplasmic reticulum (ER) to the PM. Given that PS synthesis is under very strong feed-back inhibition, PS transport defects out of the ER lead to inhibition of PS synthesis. A recent study identified mutations in the PS–synthetic enzyme PSS1, which rendered the enzyme insensitive to PS–mediated feed-back inhibition. The PSS1 mutations were found to be the cause of Lenz-Majewski syndrome (LMS), a rare human disease. We found that mutant PSS1 enzymes not only raised PS to a very high level but also reduced PtdIns4P levels in several membrane compartments, including the Golgi and PM. This was owing to the activation of the PtdIns4P phosphatase enzyme in the ER by accumulating PS. We postulated the existence of a mechanism that transports PS from the ER to the PM using the energy of a PtdIns4P gradient generated between the PM and the ER. The PtdIns4P gradient is maintained by PI4KA in the PM and the Sac1 phosphatase in the ER. While our studies were in progress, a paper was published showing that oxysterol binding protein–related proteins 5 and 8 (ORP5 and ORP8) are able to transport PS from the ER to the PM using the PtdIns4P gradient set up between the PM and the ER. Indeed, we showed that ORP8 bridges the ER and the PM in contact sites and that its PM binding requires PM PtdIns4P. These discoveries have important implications given that PI4KA is an essential host protein that supports hepatitis C virus (HCV) replication in the liver. It is likely that the important role of PI4KA in PS metabolism is critical for the HCV lifecycle. The studies also contributed to our understanding of how single mutations in the PSS1 enzyme cause the pleiotropic developmental defects described in patients of Lenz-Majewski syndrome.

Importance of the phospholipid exchange between the ER and plasma membrane and its impairment in some forms of Lou-Gehrig’s disease

In this set of studies, we investigated the impact of mutations in the ER–localized VAP-B protein (vesicle-associated membrane protein-associated protein B) identified in familial forms of amyotrophic lateral sclerosis (ALS or Lou-Gehrig’s disease) on the distribution and function of phospholipid transport between the ER and PM. Phospholipase C (PLC)–mediated hydrolysis of the limited pool of PM phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] requires replenishment from a larger pool of phosphatidylinositol (PI) via sequential phosphorylation by PI 4-kinases and PIP 5-kinases. Given that PI is synthesized in the ER and PtdIns(4,5)P₂ is generated in the PM, PI transfer proteins (PITPs) are believed to mediate this lipid transfer function. Recent studies identified the large PITP protein Nir2 as important for PtdIns transfer from the ER to the PM. In a previous study, we found that Nir2 was also required for the transfer of phosphatidic acid (PA) from the PM to the ER. In Nir2–depleted cells, activation of PLC leads to PA accumulation in the PM, and PI synthesis becomes severely impaired. In quiescent cells, Nir2 is localized to the ER via an interaction through its FFAT domain [two phenylalanines (FF) in an acidic tract] with ER–bound VAP-A and VAP-B proteins. After PLC activation, Nir2 also binds to the PM via interaction of its C-terminal domains with diacylglycerol and PA. Through these interactions, Nir2 functions in ER–PM contact zones. We found that mutations in VAP-B, which were identified in familial forms of ALS (Lou-Gehrig’s disease), caused aggregation of the VAP-B protein, which then impaired its binding to several proteins, including Nir2. However, the two mutations P56S and T46I did not cause a defect of similar severity. While the P56S mutation completely prevented association of VAP-B with Nir2, the T46I mutation was less severe so that Nir2 association was still supported, as were Nir2 translocation responses after PLC activation, although to a significantly lesser extent than was the case with the wild-type protein. The findings shed new light on the importance of non-vesicular lipid transfer of PI and PA in ER–PM contact zones and suggest that mutations in the VAP-B protein negatively affect this important homeostatic mechanism, possibly contributing to the progression of the devastating human disease ALS.

Determination of the structural basis of PI4KB and ACBD3 protein interaction important for viral replication

In collaboration with the group of Evžen Boura, we investigated the structural features of the interaction between PI4KB (one of the four PI4-kinase enzymes that can phosphorylate PI) and the ACBD3 (acyl-CoA binding domain–containing 3) protein, a protein involved in the maintenance of Golgi structure and function. PI4KB and ACBD3 proteins were both found to be important host factors for the replication of several enteroviruses in mammalian cells. It appears that the interaction of the two proteins is essential for the viral life cycle because the virus uses ACBD3 to recruit the lipid kinase to the replication organelle. Boura’s group identified the minimum interaction domains between the two proteins and solved the structure of this complex. The studies identified key residues on both proteins essential for the interaction. Our group used mutant ACBD3 proteins in intact cells to demonstrate that the residues are indeed essential for the ACDB3 protein to recruit the kinase to specific membrane compartments. The structural studies could help identify pharmacological means to interrupt this association as a way of blocking enteroviral replication in mammalian cells.

New tools for the detection of inositide-based messengers in cell populations

This year we also collaborated with our long-standing collaborator, Péter Várnai, to develop a method by which the level of inositol 1,4,5-trisphosphate (InsP3) can be followed in cell populations or in single living cells. For this we used the InsP3–binding domain of the type-I InsP3 receptor and introduced mutations to fine-tune its affinity for optimal on-off kinetics of InsP3 binding. These optimal sensors were then engineered as molecular sensors based on fluorescent resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) principles for single-cell or cell-population measurements, respectively. In addition to these sensors for measuring the soluble messenger InsP3, we also worked on methods, using BRET analysis, to follow inositol lipid changes in various intracellular domains. First, the Várnai group created BRET sensors for detecting PtdIns4P, PtdIns4,5P2 (phosphatidylinositol 4,5-bisphosphate), and PtdIns3,4,5P3 (phosphatidylinositol 3,4,5-trisphosphate) changes exclusively in the PM. The sensors were based on our previously characterized lipid-binding domains fused to Renilla luciferase and a PM–targeted yellow fluorescent protein called Venus expressed from a single plasmid, using the T2 viral sequence for separate expression of the two fusion proteins. We also generated tools based on the same principles for following phosphatidylserine (PS), phosphatidic acid (PA), diacylglycerol (DG), and cholesterol. We also made a tool-set for the detection of PtdIns4P in endosomal membranes. The new tools will be of great value in studies in which InsP3 and lipid changes need to be assessed in living cells, and they can be easily adapted for high-throughput screening applications.

VEGF signaling in astrocytes

The spatial organization of vascular endothelial growth factor (VEGF) signaling is a key determinant of vascular patterning during development and tissue repair. How VEGF signaling becomes spatially restricted and the role of VEGF secreting astrocytes in this process remains poorly understood. We developed a green fluorescent protein (GFP)–fused VEGF that is biologically active and is capable of indicating where and how VEGF is secreted from the cells. In collaboration with Jozef Kiss, we used the VEGF–GFP fusion protein to observe the intracellular routing, secretion, and immobilization of VEGF in scratch-activated living astrocytes, using confocal time-lapse microscopy. We found that VEGF is directly transported to cell-extracellular matrix attachment sites, where it is incorporated into fibronectin fibrils. VEGF accumulated at β1 integrin–containing fibrillar adhesions and was translocated along the cell surface prior to internalization and degradation. We also found that only the astrocyte-derived, matrix-bound, and thus not soluble VEGF lowered β1 integrin turnover in fibrillar adhesions. The results suggest that polarized VEGF release and extracellular matrix (ECM) remodeling by VEGF–secreting cells is key to the control of the local concentration and signaling of VEGF, and they also highlight the importance of astrocytes in directing VEGF functions, identifying these mechanisms as promising targets for anti-angiogenic approaches.

Funding

• NSERC Postdoctoral Fellowship supporting Dr. Joshua Pemberton
• JSPS-NIH Fellowship supporting Dr. Takashi Baba

Publications

1. Sohn M, Ivanova P, Brown HA, Toth DJ, Varnai P, Kim YJ, Balla T. Lenz-Majewski mutations in PTDSS1 affect phosphatidylinositol 4-phosphate metabolism at ER-PM and ER-Golgi junctions. Proc Natl Acad Sci USA 2016;113:4314-4319.
2. Klima M, Tóth DJ, Hexnerova R, Baumlova A, Chalupska D, Tykvart J, Rezabkova L, Sengupta N, Man P, Dubankova A, Humpolickova J, Nencka R, Veverka V, Balla T, Boura E. Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein. Sci Rep 2016;6:23641.
3. Tóth JT, Gulyás G, Tóth DJ, Balla A, Hammond GR, Hunyady L, Balla T, Várnai P. BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation. Biochim Biophys Acta 2016;1861:177-187.
4. Kim YJ, Guzman-Hernandez ML, Wisniewski E, Echeverria N, Balla T. Phosphatidylinositol and phosphatidic acid transport between the ER and plasma membrane during PLC activation requires the Nir2 protein. Biochem Soc Trans 2016;44:197-201.
5. Egervari K, Potter G, Guzman-Hernandez ML, Salmon P, Soto-Ribeiro M, Kastberger B, Balla T, Wehrle-Haller B, Kiss JZ. Astrocytes spatially restrict VEGF signaling by polarized secretion and incorporation of VEGF into the actively assembling extracellular matrix. Glia 2016;64:440-456.

Collaborators

• Evžen Boura, PhD, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic
• Alex H. Brown, PhD, Vanderbilt-Ingram Cancer Center, The Vanderbilt Institute of Chemical Biology, Vanderbilt University Medical School, Nashville, TN
• Jozsef Z. Kiss, MD, University of Geneva, Geneva, Switzerland
• Péter Várnai, MD, PhD, Semmelweis University, Faculty of Medicine, Budapest, Hungary

Contact

For more information, email ballat@mail.nih.gov or visit https://irp.nih.gov/pi/tamas-balla.

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