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Biology and Enzyme Regulation of Inositol Signal Transduction Pathways
Summary: John York studies the biology and enzyme regulation of inositol cellular signal transduction pathways, and the mechanisms of lithium action as it pertains to treatment of bipolar disorder (manic depression).
My laboratory is interested in cellular communication networks and the mechanisms by which defects in these pathways contribute to the pathophysiology of human disease. Extracellular signals are transduced across the membrane through receptors and are converted to intracellular "second" messengers, which amplify and propagate signals. We study a widely utilized communication network, the inositol signal transduction pathway. In recent years the complexity of the inositol metabolic pathway has become evident. These pathways have emerged as a multifaceted ensemble of cellular switches that regulate a number of processes well beyond inositol 1,4,5-trisphosphate (IP3)-mediated calcium release, including membrane trafficking, channel activity, and nuclear function. More than 30 inositol messengers are found in eukaryotic cells that may be generally grouped into two classes: (1) inositol lipid or phosphoinositide (PIP) and (2) water-soluble inositol polyphosphate (IP). Insights into the roles of these messengers have come through the characterization of numerous gene products—more than 80 in humans and 26 in budding yeast—that control the metabolism of PIPs and IPs.
Our research effort focuses on the enzyme regulation of PIP and IP messengers and the mechanistic understanding of cellular targets and processes influenced by these messengers. We use a multidisciplinary approach that includes pharmacology, biochemistry, genetics, biophysics, and cell molecular biology. We have identified novel roles for inositol messengers in the regulation of membrane trafficking, cytoskeletal organization, gene expression, and mRNA export. Through crystallographic studies of an IP phosphatase, we have uncovered a novel family of lithium targets with relevance to bipolar disorder (manic-depressive disease). Overall, we seek to understand a fundamental problem in biology—how diverse stimuli utilize IP signaling pathways to achieve specific cellular responses.
Nuclear Inositol Signaling
A recurring theme in intracellular signaling is the spatial restriction of pathways to selective intracellular compartments. Over the past decade, we have examined nuclear inositol signaling pathways. More recently our studies in the budding yeast Saccharomyces cerevisiae have identified nuclear processes regulated by IP messengers. Our recent work has reinforced that these pathways and their components are evolutionarily conserved in plants, flies, and mice.
The budding yeast genome contains a single phosphoinositide-specific phospholipase C gene (PLC1). Examination of IP metabolism in a variety of yeast strains reveals that Plc1 activation results in the production of IP3, which is then sequentially phosphorylated by two inositol polyphosphate kinases (IPKs) to inositol hexakisphosphate (IP6). In collaboration with Susan Wente's laboratory (Vanderbilt University), we discovered that these two kinase activities and their IP products, most likely IP6, are important for proper mRNA export from the nucleus. We now have generated plants that lack the ability to generate IP6 in their tissue and seeds.
In cloning the yeast IP3 kinase, designated IPK2, we found it to be identical to ARG82, a previously characterized regulator of gene expression through the ArgR-Mcm1 transcription complex. Ipk2 is a 6-/3-/5-kinase that sequentially converts IP3 to IP5, is localized within the nucleus, and is required to assemble protein complexes on DNA-promoter elements. Both Plc1 activity and Ipk2-mediated IP4/IP5 production are required for ArgR-Mcm1 transcriptional activation. Our results indicate Ipk2 influences transcriptional responses through a two-step mechanism. First, Ipk2 protein but not IP synthesis is needed to enable formation of ArgR-Mcm1 complexes on DNA promoter elements. Second, production of IP4 and possibly IP5 through both phospholipase C and Ipk2 kinase activity is required to execute transcriptional control properly. Whole-genome array analysis has revealed several new clusters of genes regulated by this phospholipase C–dependent IP signaling pathway. Furthermore, we determined that Ipk1 is not required for complex formation or transcription control, demonstrating that two independent signals arise from this pathway, IP4/IP5 as a regulator of transcription and IP6 as a regulator of mRNA export. Ipk2 has recently been identified by Erin O'Shea's lab (HHMI, University of California, San Francisco) as a regulator of chromatin-remodeling complexes in yeast. We have cloned and characterized Ipk2 orthologs from plants, flies, and mammals and find that Ipk2 pathways are important for production of numerous Ips, indicating that these IP signaling pathways are functionally conserved in higher eukaryotes. Additionally, we have found that a novel IP kinase is involved in the regulation of telomere length.
Phosphatase Regulation of Inositol Lipid Messengers
While inositol lipids are classically viewed as precursors to signaling molecules, the discovery of PI 3-kinase signaling demonstrates that PIPs also function as messengers in their own right. Through studies of inositol lipid phosphatases, we have helped to uncover processes influenced by lipid messengers. In light of several recent studies demonstrating that defects in inositol lipid phosphatases are observed in human diseases such as Lowe syndrome, cancer, and myotubular myopathy, these processes are significant. Our biochemical and genetic studies of four yeast inositol lipid 5-phosphatases (Inp51, Inp52, Inp53, and Inp54) have demonstrated that they play an essential role in survival, membrane trafficking, and actin cytoskeleton.
Yeast Inp52 and Inp53, also known as Sjl2 and Sjl3 (synaptojanin-like), share a highly similar domain structure with the mammalian synaptojanin proteins (discovered as regulators of synaptic vesicle recycling by the lab of Pietro De Camilli [HHMI, Yale University]). These proteins have a central 5-phosphatase catalytic domain that is flanked by an amino-terminal SAC1-like domain and a variable proline-rich carboxyl-terminal region. We found that SAC1-like domains encode a novel inositol lipid phosphatase (PPIPase). This discovery provides a biochemical mechanism explaining genetic studies of SAC1, which linked it to regulating actin cytoskeleton, secretion from the Golgi, and microsomal ATP transport. Significantly, we have identified the synaptojanin class of inositol regulatory enzymes as those that harbor two distinct catalytic active sites on a single polypeptide chain. We are currently exploring the roles for each phosphatase activity in cells. Suppressor analysis has revealed that PIP receptors include a number of known trafficking machinery.
Lithium Pharmacology
Bipolar or manic-depressive disease has been effectively treated with lithium for more than 40 years. Despite the enduring pharmacologic 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 characterization of two enzymes in the IP signaling pathway, inositol monophosphatase and inositol polyphosphate 1-phosphatase, each potently inhibited by therapeutic doses of lithium. We have determined the three-dimensional structure of 1-phosphatase, which has led to the identification of a family of structurally conserved lithium-inhibited phosphatases that function in diverse cellular pathways. Our hypothesis is that members of this family are the toxic and therapeutic targets of lithium.
We have characterized BPNT1, a new member of the family, and have found that it possesses lithium-inhibited bisphosphate nucleotidase activity. BPNT1 is highly expressed in regions of the distal nephron, and our data indicate that chronic lithium use in humans may induce nephrogenic diabetes insipidus through BPNT1 inhibition. Recently we have defined a pharmacological "antidote" to lithium toxicity. Characterization of the cellular function of individual enzymes in the family should elucidate lithium's mechanism of action. Ultimately this will enable the development of improved therapies for the treatment of bipolar disease.
This work was supported by grants from the Burroughs Wellcome Fund and currently receives support from the National Institutes of Health.
Last updated July 17, 2003
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