A critical period requiring Rho proteins for cell cycle progression uncovered by reversible protein transduction in endothelial cells

Abstract

The time-specific requirement of Rho proteins for the S phase progression of vascular endothelial cells was determined by reversibly introducing inhibitor proteins with a cell-penetrating peptide. We found evidence of the reversibility of protein transduction. The removal of extracellular protein caused the transduced protein to decay in a manner sensitive to low temperatures. The time required for a 50% decay correlated with the protein size. The time-specific transduction of the inhibitor proteins uncovered a critical period requiring Rho proteins in the G 1 -S transition phase. Reversible protein transduction may thus be a powerful tool to investigate the time-specific role of signaling proteins.

Distinct Ca2+ Requirement for NO Production between Proteinase-Activated Receptor 1 and 4 (PAR1 and PAR4) in Vascular Endothelial Cells

“…The transduction of TATHA-Akt and TATHA-RB into BAEC was confirmed by an immunoblot analysis as described previously (Hirano et al, 2004b;Koga et al, 2004;Yufu et al, 2005). In brief, the cells on 100-mm dishes were harvested and resuspended in phosphatebuffered saline (PBS) at room temperature.…”

“…M63167), whereas (His) 6 -Akt lacks a cell-penetrating peptide and an HA epitope (Koga et al, 2004). TATHA-RB consisted of a hexahistidine tag, a cell-penetrating peptide, an HA epitope, and the RhoA-binding region of Rho kinase (Hirano et al, 2004b;Shiga et al, 2005). The recombinant proteins were affinity-purified through Ni 2ϩ -loaded HiTrap chelating column on Akta Prime (GE Healthcare, Tokyo, Japan).…”

“…BAEC, PAEC, and A7r5 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum as described previously (Hirano et al, 2001(Hirano et al, , 2004bEto et al, 2003). HUVEC were cultured in MCDB104 supplemented with 5% fetal bovine serum and endothelial cell growth supplements (Nissui Pharmaceutical, Tokyo, Japan), as described previously (Nakayama et al, 2004).…”

“…A dominant negative mutant of Akt with (TATHA-Akt) and without [(His) 6 -Akt] a cell-penetrating peptide, as well as a RhoA-inhibitory protein with a cell-penetrating peptide (TATHA-RB), was expressed in bacteria and prepared as described previously (Hirano et al, 2004b;Koga et al, 2004;Shiga et al, 2005;Yufu et al, 2005). TATHA-Akt consisted of a hexahistidine tag, a cell-penetrating peptide of Tat, a hemagglutinin (HA) epitope, and the N-terminal 147 amino acids of Akt1 (Accession no.…”

Regulation of myosin phosphorylation and myofilament Ca²⁺ sensitivity in vascular smooth muscle

“…This protein transduction technique is thus anticipated to be applicable to various types of cells. We and others have successfully applied this protein transduction technique to the vascular biology (Sauzeau et al, 2001;Hirano et al, 2004a;Hirano et al, 2004b;Koga et al, 2004). The specific peptide inhibitor can be introduced using cell-penetrating peptides as previously proposed (Niv et al, 2004).…”