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: Cell-penetrating peptides (CPPs), Nucleolar localization signal (NoLS), Blood-brain barrier (BBB), LIM Kinase 2 (LIMK2), Protein transduction domain (PTD), Drug delivery system (DDS), LIM Kinase 2 Function, Cell-Penetrating Peptide, LIM domain-containing protein kinase, phosphorylation, depolymerizing factor cofilin, Von Hippel-Lindau, Green fluorescence protein, LIM Kinase 2, nucleolar translocation signal, transactivating protein, Drosophila melanogaster transcription factor, amphipathic peptides, proteoglycans, endocytosis, gene expression, cytoarchitecture, cell adhesion, induced pluripotent stem, blood-brain barrier, Peptide Synthesis, 9-fluorenylmethoxycarbonyl solid phase synthesis, Rink amide MBHA resin (Novabiochem), reverse-phase high performance liquid chromatography (HPLC, baculovirus-silkworm expression system, pM15, HisTrap, imidazole, Cell Culture, fibroblasts, Eagle's minimum essential, medium (EMEM), fetal bovine serum, Dulbecco's modified Eagle's medium/F-12, DMEM/F12, streptomycin, Human iPS cells, 2-mercaptoethanol, FITC-Labeled Peptides, LIMK2 NoLS-Conjugated Peptide, nucleolar localization signal, β-karyopherin (kap) family
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