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Nucleolar Localization Signals of LIM Kinase 2 Function as a Cell- Penetrating Peptide

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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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  • Protein & Peptide Letters publishes short papers in all important aspects of protein and peptide research, including structural studies, recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, drug design etc. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallisation, and preliminary structure determinations of biologically important proteins are acceptable. Purely theoretical papers are also acceptable provided they provide new insight into the principles of protein/peptide structure and function.
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