Nuclear localisation of the G-actin sequestering peptide thymosin beta4.
Thymosin beta4 is regarded as the main G-actin sequestering peptide in the cytoplasm of mammalian cells. It is also thought to be involved in cellular events like cancerogenesis, apoptosis, angiogenesis, blood coagulation and wound healing. Thymosin beta4 has been previously reported to localise intracellularly to the cytoplasm as detected by immunofluorescence. It can be selectively labelled at two of its glutamine-residues with fluorescent Oregon Green cadaverine using transglutaminase; however, this labelling does not interfere with its interaction with G-actin. Here we show that after microinjection into intact cells, fluorescently labelled thymosin beta4 has a diffuse cytoplasmic and a pronounced nuclear staining. Enzymatic cleavage of fluorescently labelled thymosin beta4 with AsnC-endoproteinase yielded two mono-labelled fragments of the peptide. After microinjection of these fragments, only the larger N-terminal fragment, containing the proposed actin-binding sequence exhibited nuclear localisation, whereas the smaller C-terminal fragment remained confined to the cytoplasm. We further showed that in digitonin permeabilised and extracted cells, fluorescent thymosin beta4 was solely localised within the cytoplasm, whereas it was found concentrated within the cell nuclei after an additional Triton X100 extraction. Therefore, we conclude that thymosin beta4 is specifically translocated into the cell nucleus by an active transport mechanism, requiring an unidentified soluble cytoplasmic factor. Our data furthermore suggest that this peptide may also serve as a G-actin sequestering peptide in the nucleus, although additional nuclear functions cannot be excluded.
Huff
Thomas
T
Institut für Biochemie, Medizinische Fakultät, Universität Erlangen-Nürnberg, Fahrstr. 17, 91054 Erlangen, Germany. t.huff@biochem.uni-erlangen.de
Rosorius
Olaf
O
Otto
Angela M
AM
Müller
Christian S G
CS
Ballweber
Edda
E
Hannappel
Ewald
E
Mannherz
Hans Georg
HG
D000199:Actins / Q000737:chemistry / Q000378:metabolism; D021581:Active Transport, Cell Nucleus; D000818:Animals; D001665:Binding Sites; D002103:Cadaverine / Q000494:pharmacology; D002264:Carboxylic Acids / Q000494:pharmacology; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D002851:Chromatography, High Pressure Liquid; D003593:Cytoplasm / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D009389:Neovascularization, Pathologic; D017830:Octoxynol / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013947:Thymosin / Q000737:chemistry*; D011503:Transglutaminases / Q000737:chemistry; D014709:Vero Cells; D014945:Wound Healing; D000099201:Thymosin beta(4)
D000199:Actins; D002103:Cadaverine; D002264:Carboxylic Acids; D003902:Detergents; D005456:Fluorescent Dyes; D017830:Octoxynol; D010455:Peptides; D013947:Thymosin; D011503:Transglutaminases; D000099201:Thymosin beta(4); C436864:Oregon Green 488 carboxylic acid