engleski

Subcellular localization of NDPKs: How far is a sponge from a human?

Title: Subcellular localization of NDPKs: How far is a sponge from a human? Authors: Maja Herak Bosnar, Jean de Gunzburg, Ružica Bago, Igor Weber, Jasminka Pavelić, Dragutin Perina, Andreja Mikoč, Helena Ćetković Aims: The aim of this work was to determine the specific, and potentially distinct subcellular localizations of human Nm23-H1/NDPK A and Nm23-H2/NDPK B, and the Nm23-H1/NDPK A carrying the mutation in the active site of the enzyme. This basic goal was broadened by defining its localization in three points of the cell cycle (late G1, S and G2/M phase), as well as trying to determine the subcellular localization of the recently discovered NDPK A homologue from marine sponge Suberites domuncula – Nm23-SD1. Hypothesis: Based on the multifunctional nature of the Nm23/NDPKs, we expected that the Nm23-H1/NDPK A and Nm23-H2/NDPK B occupy several locations in the cell. We have also predicted that if NDPK A and B have some distinct functions in the cell they should, at least partly, occupy distinct localizations or cellular compartments. Since the spongian Nm23-SD1 exhibits 71% amino acid homology with the human NDPK A we predict their localization to be at least partly identical. Methods: The issue was addressed by subcloning nm23-H1, nm23-H2, nm23-H1H118N (carrying a point mutation in the kinase domain), and the spongian nm23-SD1 full-length cDNA fragments into pEGFP and pDsRed vectors generating in frame fusion proteins. The constructs were lipofected into HEp-2 cells (squamous cell carcinoma of the larynx). The colocalization experiments with different cellular compartments were addressed by immunofluorescent techniques followed by epifluorescent and confocal microscopy. Cell-cycle dependent distribution of Nm23/NDPK proteins was revealed by treating transfected cells with dihydroxyurea and nocodazole and by BrdU staining. Results: Our experiments revealed that GFP-fused Nm23-H1 and Nm23-H2 proteins are found in the cytosol and colocalize with the endoplasmic reticulum. Some of the transfected cells display a various number of spherical “ granum-like” structures, typically positioned adjacent to the nuclear wall. The “ granum – like” structures were absent in cells transfected with the mutated form of nm23-H1. A part of the cells exhibit nuclear staining especially in late G1 phase. The experiments on spongian Nm23-SD1 are still under investigation but preliminary results reveal that its colocalization might be similar to the human homologue. Conclusions: The GFP-Nm23-H1 and H2 proteins display the same localization in transfected cells. They are principally found in the cytosol, and colocalize with the endoplasmic reticulum. Moreover, some cells exhibit nuclear staining, which appears to be cell cycle-dependent. References: Bosnar, MH, et al.(2004) Subcellular localization of A and B Nm23/NDPK subunits. Exp Cell Res 298, 275-284. Hercet M, et al. (2005) Identification and analysis of cDNAs encoding two nucleoside diphosphate kinases (NDPK/Nm23) from the marine sponge Suberites domuncula. Croat Chem Acta 78, 343-348.

nm23/NDPK; GFP; subcellular localization; spongia

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