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Functional analysis of MODY2 mutations in the nuclear export signal of glucokinase

AutorNavas, María-Angeles; Gutiérrez-Nogués, Ángel; García-Herrero, Carmen-María; Vincent, Olivier
Fecha de publicación2013
Citación49th EASD Annual Meeting (2013)
Resumen[Background and aims]: Glucokinase (GK) is a key enzyme in glucose homeostasis. GK acts as a glucose sensor in the pancreatic beta cell regulating insulin secretion and also plays a key role in glucose metabolism in the liver. Mutations in the GCK gene cause different monogenic glycaemic disorders, being the most common the familial, mild fasting hyperglycaemia or MODY2, caused by heterozygous inactivating mutations. The specific function of GK is based on the particular kinetic characteristics of this enzyme in addition to other regulatory mechanisms including covalent modifications, protein-protein interactions and subcellular localization of the enzyme. The aim of this work is to study the nuclear export mechanism of GK from the analysis of MODY2 mutations located in the nuclear export signal (NES) of the enzyme. [Materials and methods]: MODY2 mutations located in the NES sequence of GK (residues 300-310) have been introduced by directed mutagenesis into the human beta cell isoform of GCK. The biochemical effects of these mutations on the catalytic activity and thermostability of the enzyme were measured with bacterially expressed GST-GK fusion proteins. Physical interaction of GK with its regulatory protein GKRP was analyzed by enzymatic assays and/or the yeast two hybrid system. Glucokinase NES function was analyzed with a GFP fusion to rat GK residues 299-359 (pEGFP-N2-GK(299-359)). Confocal microscopy was used to study the nucleo-cytoplasmic localization of GFP constructs in transiently transfected Cos7 and Hek293T cells. [Results]: Mutations V302E, L304P and L306R impaired GK enzymatic activity, reducing the activity index up to 3% of wild type level in the case of L306R. In addition, mutations V302E and L304P caused protein instability and mutations V302E, L306R and L309P altered the interaction of GK with GKRP. When transfected into Cos7 or Hek293T cells, the wild type GK(residues 299-359)-GFP fusion protein was homogenously localized in the nucleus and cytoplasm in more than 80% of transfected cells. This percentage decreased up to 65% in cells expressing GFP fusion proteins containing mutations L309P or L309H and up to 20% in cells expressing mutations V302E, R303W, L304P and R308W, where the fluorescence distribution is nuclear-enriched in more than 50% of transfected cells. Treatment with leptomycin b resulted in nuclear enrichment of fluorescence in more than 80% of cells transfected with the wild type fusion protein and did not affect the predominant nuclear distribution of mutants V302E, R303W, L304P and R308W. Moreover, overexpression of exportin-1 by cotransfection of cells with pCMV-HA-CRM1 and wild type GFP-fusion constructs resulted in an increase of cells with cytoplasmic enrichment of fluorescence, whilst cotransfection with the R308W mutant still resulted in a relative high percentage of cells with nuclear enrichment of fluorescence. [Conclusion]: Our results indicate that GK nuclear export directed by the NES (residues 300-310) is mediated by the exportin-1 complex and MODY2 mutations in the NES sequence can affect this process, thus identifying an additional mechanism contributing to the disease.
DescripciónResumen del póster presentado al 49th European Association for the Study of Diabetes Annual Meeting, celebrado en Barcelona (España) del 23 al 27 de septiembre de 2013.-- et al.
URIhttp://hdl.handle.net/10261/125417
Aparece en las colecciones: (IIBM) Comunicaciones congresos
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