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My research interests center around an inositol kinase which can also function as a protein kinase and as an isomerase. This enzyme (5/6-kinase) converts inositol 1,3,4-trisphosphate to one of two inositol tetrakisphosphate isomers, either inositol 1,3,4,6-tetrakisphosphate or inositol 1,3,4,5-tetrakisphosphate. As an isomerase, it can interconvert these two IP4 isomers. This enzyme acts at a branch point in inositol metabolism, and is the rate-limiting step for the synthesis of higher phosphorylated forms of inositol, including IP6. IP6 has been implicated in a variety of cellular processes, including mRNA export from the nucleus, binding to the clathrin assembly proteins AP2 and AP180, inhibition of clathrin cage assembly in vitro, inhibition of protein kinases, regulation of nonhomologous DNA end joining of double strand breaks, endocytosis, adenosine deaminase function, and nonvisual arrestin oligomerization.

Unlike protein kinases and other small molecule kinases (e.g. sugar kinases), there are no canonical kinase domains, including a recognizable ATP-binding domain, in 5/6-kinase. The enzyme is highly conserved throughout evolution, and we have exploited this by mutating residues which are absolutely conserved. Each mutant has been characterized with regard to its enzymatic activity. Together with the recently solved crystal structure of the Entamoeba histolytica 5/6-kinase, we have been able to define the active site of this enzyme.

We have analyzed the expression pattern of 5/6-kinase during mouse development, using in situ hybridization and Xgal staining of transgenic mice harboring a β-galactosidase gene trap cassette in the 5/6-kinase gene. The enzyme is strongly expressed in the neural tube early in development and throughout the central and peripheral nervous systems at later developmental stages, as well as in arterial smooth muscle cells and in adipose tissue, particularly brown fat. In addition, in vitro differentiation of 3T3L1 cells into adipocytes was associated with upregulation of 5/6-kinase expression.

We have most recently shown that mice expressing reduced levels of 5/6-kinase (5/6-kinase hypomorphs) develop neural tube defects (NTDs) with a penetrance of 12%. These are exhibited as either exencephaly, spina bifida, or both. We are currently modifying the diets of these animals to both increase and decrease the level of inositol in the diet, to see whether these dietary manipulations alter the prevalence of NTDs. We are also testing whether folic acid supplementation will reduce the number of embryos with an NTD, as 30% of NTDs in humans are folate resistant. We postulate that the NTDs seen in the 5/6-kinase hypomorphs are likely to fall into this category and will therefore be unresponsive to folic acid supplementation.

Top: Whole-mount Xgal staining of a mouse embryo for 5/6-kinase Bottom: Scanning electron micrographs of four littermates with reduced levels of 5/6-kinase. Neural tube defects are indicated by arrows From: Wilson MP, Hugge C, Bielinska M, Nicholas P, Majerus PW, Wilson DB Neural tube defects in mice with reduced levels of inositol 1,3,4-trisphosphate 5/6-kinase. Proc Natl Acad Sci U S A 2009 May 29; [Epub ahead of print]

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• Updated: June 15, 2009
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