Nuclear localisation of the G-actin sequestering peptide thymosin β4

Abstract

Thymosin β4 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 β4 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 β4 has a diffuse cytoplasmic and a pronounced nuclear staining. Enzymatic cleavage of fluorescently labelled thymosin β4 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 β4 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 β4 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.

A Novel Role of the Actin-nucleating Arp2/3 Complex in the Regulation of RNA Polymerase II-dependent Transcription

“…For example, it has been reported that phosphoinositide, which is known to stimulate polymerization, is found in the nucleus, and phosphoinositide signaling is initiated by a nucleus-specific isoform of phospholipase C (53,54). Several other actin-binding proteins, including cofilin, thymosin b4, gelsolin, and CapG, have also been observed in the nucleus (55)(56)(57)(58)(59)(60). These proteins function to decrease actin polymerization and/or regulate the size of actin polymers through their severing and capping activities (41,(55)(56)(57)(58)(59)(60), thus antagonizing the stimulatory functions of N-WASP and the Arp2/3 complex.…”

“…Several other actin-binding proteins, including cofilin, thymosin b4, gelsolin, and CapG, have also been observed in the nucleus (55)(56)(57)(58)(59)(60). These proteins function to decrease actin polymerization and/or regulate the size of actin polymers through their severing and capping activities (41,(55)(56)(57)(58)(59)(60), thus antagonizing the stimulatory functions of N-WASP and the Arp2/3 complex. Therefore, the regulation of nuclear actin is likely to involve both mechanisms that stimulate polymerization and depolymerization as well as those that control the size of actin filaments.…”

Proteomic characterization of Her2/neu‐overexpressing breast cancer cells

“…The protein profile obtained aftermanual curation of the data (Supplementary Figure 1), and in particular Table 1, explains the ability of H6O5 cells to form solid tumors when grafted onto mice (Figure 1d). In brief, H6O5 cells were found to overexpress Thymosin beta 4, an actin-binding protein previously described as having a role in angiogenesis [34,35], while having low protein levels of thrombospondin 1, an angiogenesis inhibitor, that binds to proteins on the cell surface, thereby modulating cell motility and cell adhesion events [36,37].…”

MRTF-A controls vessel growth and maturation by increasing the expression of CCN1 and CCN2

“…SRF is co-activated by myocardin 15 or MRTFs A and B 16 . These SRF coactivators are interacting with G-actin and translocate to the nucleus 17 , when G-actin levels decrease, for example, due to sequestration by the G-actinbinding peptide thymosin 4 (T 4) [18][19][20] . Since T 4 is a potent pro-angiogenic factor 21 , an involvement of the T 4-MRTF-SRF axis in vascular growth would require transcriptional activation beyond well-known myogenic proteins 13,14 .…”