Phosphoinositide-calcium Signaling In Cell Regulation

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Every biochemical process that happens 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. A 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 neurodegenerative diseases just to name a few. Research described in this report has focused on the question of how cells organize their internal membranes to provide a structural framework on which molecular signaling complexes assemble to ensure proper information processing. 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 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 not only can 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) - endoplasmatic 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 ORP (oxysterol-binding protein-related protein) proteins that are the mammalian orthologues of the yeast Osh proteins and mediate the transport of specific lipids between cellular membranes. One of the salient features of Osh/ORP proteins is that they use 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. In this research period, we investigated the impact of changing PM PI4P levels on the Ca2+ entry process mediated by the STIM1-Orai1 molecular complex that 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) that produces PI4P in the PM potently inhibits Ca2+ influx through interruption of STIM1 association with the PM. Similarly, manipulation of PM PI4P levels through the expression of ORP5 and ORP8 proteins had 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. This process mediated by a multi-protein molecular complex, called ESCRT and its defective function can lead to cataract. It is not known how ESCRT defects can lead to cataract and whether it is related to cytokinesis defects. In a collaborative study led by the Hirsch laboratory in Italy, 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 that helps to prevent early onset cataract by protecting from senescence. 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 at Stanford University that focused on the understudied calcineurin isoform, CNA1 it was discovered that unlike canonical cytosolic calcineurin, CNA1 localizes to the plasma membrane and Golgi due to 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, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange studies performed in the Burke laboratory at the University of Victoria, Canada, found multiple contacts in the calcineurin-PI4KA complex, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which was establish as a calcineurin substrate. In cellular studies, Calcineurin inhibitors decreased PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. This work 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 a unique localization to increase its access its substrates providing unique specificity and regulation to the protein. 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 multiple 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 researchers of the University of Exeter Medical School in the UK, 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 phenotypic outcomes probably stem from impairment of PI4KIII-TTC7-FAM126's organ-specific functions, due to 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 insight into the combinatorial biology of the PI4KIII-FAM126-TTC7-EFR3 molecular complex.