C, Cellular localization of endogenous RKIP in MDCK cells at confluent cell density such that the cells are quiescent. B, Cellular localization of endogenous phospho-RKIP in MDCK cells at subconfluent cell density such that cells are still proliferating. A, Cellular localization of total RKIP (RKIP and phospho-RKIP) in MDCK canine kidney epithelial cells at subconfluent cell density so that the cells are still growing. 1 Endogenous RKIP localizes to the nucleus of epithelial cells. Results RKIP localization in the nucleusįig. The putative NLS in RKIP is important to RKIP’s function in mitotic checkpoint control during the cell cycle in MCF-7 human breast cancer cells. RKIP interacts with an importin α isoform(s) in both BT-20 and MDA-MB-231 human triple-negative breast cancer cells. Fusion of the putative NLS sequence to GFP led to a greater nuclear accumulation of GFP when compared to GFP alone. We also found that the mutation of certain basic residues (substitution of arginines and lysines with asparagines alone and in combination) within the sequence impairs the nuclear accumulation of RKIP, consistent with the functioning of the sequence as an NLS. The regulatory serine residue 153, whose phosphorylation state helps determine which binding partner RKIP associates with, is located within this putative NLS sequence. We confirmed that the putative NLS sequence mediates nuclear transport of RKIP by analyzing the localization of RKIP point mutants of the putative NLS and an RKIP deletion mutant fused to green fluorescence protein (GFP) or GFP along with glutathione-S-transferase (GST). We identified a putative classical bipartite nuclear localization signal (NLS) consisting of 146-RGKFKVASFRKK-157 toward the C-terminus of the protein by bioinformatics analysis. We confirmed by confocal microscopy that RKIP localizes inside the nucleus (nucleoplasm) in MDCK cells. We also found similar results in Cal-51 triple-negative human breast cancer cells, as well as in RKIP reexpression experiments in RKIP-silenced BT-20 human triple-negative breast cancer cells. Here we report that RKIP localizes primarily to the nucleus in Madin-Darby canine kidney (MDCK) epithelial cells, both in cultures that are subconfluent (with growing cells) and those that are confluent (with quiescent cells). We also found that RKIP interacts with nuclear transport factor importin α in BT-20 and MDA-MB-231 human breast cancer cells, suggesting classical importin-mediated active nuclear transport. Evaluating the biological function of nuclear localization of RKIP, we found that the presence of the putative NLS is important for RKIP's role in mitotic checkpoint regulation in MCF-7 human breast cancer cells. A fusion construct of RKIP’s putative NLS alone to a heterologous reporter protein leads to nuclear localization of the fusion protein, demonstrating that this sequence alone is sufficient for import into the nucleus. We found consistent results in reexpression experiments with wildtype or mutant RKIP in RKIP-silenced cells. Point mutation of all the basic residues in RKIP’s putative NLS particularly strongly reduces nuclear localization. Deletion of and point mutations in the putative NLS lead to decreased nuclear localization. Like classical NLS sequences, RKIP’s putative NLS is rich in arginine and lysine residues. We have identified a putative bipartite nuclear localization signal (NLS) in RKIP that maps to the surface of the protein surrounding a known regulatory region. Although most of its functions have been assumed to be cytoplasmic, we show here that RKIP’s localization is primarily nuclear in both growing and quiescent Madin-Darby canine kidney epithelial cells and in Cal-51 and BT-20 human breast cancer cells. Raf kinase inhibitor protein (RKIP) is a multifunctional modulator of intracellular signal transduction. Glycobiology and Extracellular Matrices.
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