Nucleolar Localization Signals of LIM Kinase 2 Function as a Cell- Penetrating Peptide
Abstract:LIM Kinase 2 (LIMK2) is a LIM domain-containing protein kinase which regulates actin polymerization thorough phosphorylation of the actin depolymerizing factor cofilin. It is also known to function as a shuttle between the cytoplasm and nucleus in endothelial cells. A basic amino acid-rich motif in LIMK2 was previously identified to be responsible for this shuttling function, as a nucleolar localization signal (NoLS). Here it is shown that this nucleolar localization signal sequence also has the characteristic function of a cell-penetrating peptide (CPP). We synthesized LIMK2 NoLSconjugated peptides and a protein and analyzed their cell-penetrating abilities in various types of cells. The BC-box motif of the Von Hippel-Lindau (VHL) protein was used for the peptide. This motif previously has been reported to be involved in the neural differentiation of bone marrow stromal cells and skin-derived precursor cells. Green fluorescence protein (GFP) was used as a large biologically active biomolecule for the protein. The LIMK2 NoLS-conjugated peptides and protein translocated across the cell membranes of fibroblast cells, neural stem cells, and even iPS cells. These results suggest that LIMK2 NoLS acts as a cell-penetrating peptide and its cell-penetrating ability is not restricted by cell type. Moreover, from an in vivo assay using a mouse brain, it was confirmed that LIMK2 NoLS has potential for transporting biomolecules across the blood-brain barrier.
Keywords: 2-mercaptoethanol; 9-fluorenylmethoxycarbonyl solid phase synthesis; Blood-brain barrier (BBB); Cell Culture; Cell-Penetrating Peptide; Cell-penetrating peptides (CPPs); DMEM/F12; Drosophila melanogaster transcription factor; Drug delivery system (DDS); Dulbecco's modified Eagle's medium/F-12; Eagle's minimum essential; FITC-Labeled Peptides; Green fluorescence protein; HisTrap; Human iPS cells; LIM Kinase 2; LIM Kinase 2 (LIMK2); LIM Kinase 2 Function; LIM domain-containing protein kinase; LIMK2 NoLS-Conjugated Peptide; Nucleolar localization signal (NoLS); Peptide Synthesis; Protein transduction domain (PTD); Rink amide MBHA resin (Novabiochem); Von Hippel-Lindau; amphipathic peptides; baculovirus-silkworm expression system; blood-brain barrier; cell adhesion; cytoarchitecture; depolymerizing factor cofilin; endocytosis; fetal bovine serum; fibroblasts; gene expression; imidazole; induced pluripotent stem; medium (EMEM); nucleolar localization signal; nucleolar translocation signal; pM15; phosphorylation; proteoglycans; reverse-phase high performance liquid chromatography (HPLC; streptomycin; transactivating protein; β-karyopherin (kap) family
Document Type: Research Article
Publication date: 2010-12-01
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