Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-beta 4 (T beta 4) was involved in the regulation of subcellular localization and activity of MRTF-A. T beta 4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin-MRTF-A complex formation. T beta 4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF-SRF signaling. The activation rate of MRTF-A by the T beta 4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type T beta 4. In contrast, the beta-actin mutant 3DA, which has a lower affinity for T beta 4, more effectively suppressed MRTF-A activity than wild-type beta-actin. Furthermore, ectopic T beta 4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, T beta 4 is an important MRTF regulator that controls the G-actin-MRTFs interaction. (C) 2013 Elsevier Inc. All rights reserved.