An expanded view of inositol signaling

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Introduction

Over 40 years ago, Mabel and Lowell Hokin published their seminal work demonstrating that phosphoinositides (PI) are rapidly metabolized by cells in response to stimuli, thereby founding the field of inositol signaling (1). In more recent years the tremendous complexity of the inositol metabolic pathway has become evident 2., 3., 4., 5., 6., 7.. Diverse stimuli such as growth factors, hormones, neurotransmitters, cell differentiation and developmental signals, cytokines and light activate molecular programs that lead to the production of numerous inositol polyphosphate (IP) messenger molecules. Compartmentalization of IP pathways, including those in the nucleus, indicates that spatial restrictions further increase the complexity. In all, over 30 lipid- and water-soluble IP molecules have been identified in eukaryotic cells, many of which have not been assigned a function in cells and hence have been designated as “orphan” IP molecules.

Conventional wisdom predicts that the major role of IP signaling activation is to mobilize calcium through IP3 and to stimulate protein kinase C through production of 1,2-diacylglycerol 5., 6., 8.. While these roles are widely accepted, the field has been slow to accept the notion that the other 29 “orphan” IP messengers may have equally important signaling roles. Our research efforts have focused on expanding the view of the pathway by seeking to identify the cellular targets and processes influenced by these “orphan” IP messengers. We have utilized a multidisciplinary approach, including genetic studies in budding yeast, to characterize the function of over 10 gene products that regulate the synthesis and breakdown of these molecules. This work has helped identify new roles for “orphan” IP messengers in regulating membrane trafficking cytoskeletal organization, gene expression, and mRNA export, and has uncovered a family of novel lithium targets with potential relevance to manic depressive disease 9., 10., 11., 12., 13., 14., 15., 16., 17., 18., 19., 20., 21., 22.. Here we integrate our knowledge of several of these genes and the cellular processes that they control.

Section snippets

Budding yeast—a genetically tractable model eukaryotic cell

One method for identifying potential roles for orphan inositol messengers is to create mutant cells that either fail to produce or overproduce a given signaling molecule. Subsequent phenotypic characterization may yield specific insights into the processes regulated by that given messenger. To accomplish this, it is necessary to first identify the genes encoding the cellular activities that regulate the synthesis and breakdown of the orphan molecule, i.e. the lipases, kinases and phosphatases.

A dual-functional inositol lipid phosphatase family

Classically, inositol lipids, such as phosphatidylinositol 4,5-bisphosphate [PI4., 5.P2], were thought to serve as precursors to signaling molecules. Upon agonist stimulation, receptor-mediated activation of PI-specific phospholipase C results in the hydrolysis of PI4., 5.P2 to liberate inositol 1,4,5-trisphosphate (IP3) into the cytoplasm where it facilitates the release of calcium from intracellular stores through binding to its protein receptor 5., 8.. Such signals are terminated by further

A phospholipase C dependent IP3 to IP6 pathway

Our studies of the Inp5 s were a starting point for probing the function of other IPs in budding yeast. Since the Inp5 s did not seem to be involved in termination of IP3 signaling, we began to think about what happens to IP3 upon formation. Yeast has a single PI-specific phospholipase C, Plc1 that cleaves PI4., 5.P2 to produce diacylglycerol and IP3 55., 56., 57.. Gain and loss of Plc1 mutants exhibit a pleiotropic range of defects indicative of its essential role in many cellular processes.

A role for IP6 production in the regulation of mRNA export

The ensemble of IP kinases responsible for generating a phospholipase C-dependent pool of IP6 had not been fully characterized at a molecular level in eukaryotic cells. Analysis of IP metabolism and inositol kinase activities from a variety of eukaryotes has indicated that anywhere from two to four distinct kinases are necessary to synthesize IP6 (7). In plants and animals, two out of four kinase have been cloned, an I 1., 4., 5. P3 3-kinase and an I 1., 3., 4. P3 5/6-kinase 58., 59., 60., 61..

A role for nuclear IP4/IP5 production in regulating gene expression

Our studies of gsl3 mutants indicated that it participated in the phospholipase C-dependent IP signaling pathway as an IP3 kinase or regulator thereof (20). In order to identify GSL3, we speculated that an evolutionary relationship might exist among IP3 kinases (21). A family of metazoan IP3 3-kinases had been identified by other groups, which upon multi-sequence alignment identified seven conserved elements. These were then used to pattern search the yeast genome database, and revealed Arg82p

Lithium pharmacology: a family of structurally conserved lithium targets

Bipolar or manic depressive disease has been effectively treated with lithium for over forty years. Despite the enduring pharmacological impact of this drug, the molecular basis for its therapeutic effect has remained elusive, thus hindering the search for more effective drugs that have fewer harmful side effects. Insight into lithium's mode of action has come from the cloning and characterization of two enzymes in the IP signaling pathway, inositol monophosphatase and 1-ptase 9., 10., 11., 12.

Summary

The long-range goal of our work is to elucidate how diverse extracellular stimuli elicit selective cellular responses through the activation of inositol polyphosphate (IP) signaling pathways. Defects in IP signaling pathways result in disease states such as Lowe syndrome, myotubular myopathy and cancer—through defects in the tumor suppressor PTEN. There are over 30 IP molecules, the majority of which have not been studied as messengers. It is our hypothesis that such IPs, designated as

Acknowledgements

We wish to thank Dr. Susan R. Wente (Washington University, St. Louis, MO), without whom much of this work would have not been possible. Our collaboration has been a truly uplifting experience that we hope continues well into the new millennium. This work is supported by a Burroughs Wellcome Fund Career Award in the Biomedical Sciences, the Howard Hughes Medical Institute, and the National Institutes of Health (HL-055672).

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