Megakaryoblastic leukemia 1 (MKL1) is a myocardin-related coactivator of the serum response factor (SRF) transcription factor, which has an integral role in differentiation, migration, and proliferation. Serum induces RhoA-dependent translocation of MKL1 from the cytoplasm to the nucleus and also causes a rapid increase in MKL1 phosphorylation. We have mapped a serum-inducible phosphorylation site and found, surprisingly, that its mutation causes constitutive localization to the nucleus, suggesting that phosphorylation of MKL1 inhibits its serum-induced nuclear localization. The key site, serine 454, resembles a mitogen-activated protein kinase phosphorylation site, and its modification was blocked by the MEK1 inhibitor U0126, implying that extracellular signal-regulated kinase 1/2 (ERK1/2) is the serum-inducible kinase that phosphorylates MKL1. Previous results indicated that G-actin binding to MKL1 promotes its nuclear export, and we found that MKL1 phosphorylation is required for its binding to actin, explaining its effect on localization. We propose a model in which serum induction initially stimulates MKL1 nuclear localization due to a decrease in G-actin levels, but MKL1 is then downregulated by nuclear export due to ERK1/2 phosphorylation.Megakaryoblastic leukemia 1/2 proteins (MKL1/2, MRTF-A/B, MAL, BSAC), along with the related protein myocardin, are transcriptional coactivators of serum response factor (SRF) (7). SRF is a transcription factor that belongs to the MADS box family and binds to serum response elements (SRE) in the promoters of various immediate-early and muscle-specific genes (32,33,38). The core sequence of the SRE has the consensus sequence CC(AT) 6 GG and is called a CArG box (13,17). Serum and growth factors stimulate SRF activity via two seemingly independent pathways, one that is dependent on the phosphorylation of ternary complex factors (TCFs) by a mitogen-activated protein kinase (MAPK) cascade and the other that is dependent on Rho signaling and actin dynamics (12, 40). The TCF proteins Elk-1, SAP-1, and Net make sequence-specific DNA contacts with Ets motifs adjoining the CArG boxes of some immediate-early genes, and phosphorylation of their transcriptional activation domains potentiates their ability to activate transcription (29). The second pathway that activates SRF involves the small GTPase RhoA, since the inhibition of RhoA blocks serum induction of TCF-independent SRE reporter genes and some SRF target genes, while activated RhoA can stimulate SRE reporter genes (12). RhoA activation causes stress fiber formation and the reduction of monomeric G-actin. The use of actin mutants and drugs that interfere with actin treadmilling suggests that SRE activation is controlled by the G-actin pool (21, 31).Myocardin was originally identified as a strong coactivator for SRF in heart and smooth muscle cells (35). We and others have identified two myocardin-related SRF-specific coactivators, MKL1 and MKL2, that are expressed in a wide range of embryonic and adult tissues and that stron...

The melastatin-like transient-receptor-potential-7 protein (TRPM7), harbouring a cation channel and a serine/threonine kinase, has been implicated in thymopoiesis and cytokine expression. Here we show, by analysing TRPM7 kinase-dead mutant (Trpm7 R/R) mice, that the enzymatic activity of the receptor is not essential for thymopoiesis, but is required for CD103 transcription and gut-homing of intra-epithelial lymphocytes. Defective T cell gut colonization reduces MHCII expression in intestinal epithelial cells. Mechanistically, TRPM7 kinase activity controls TGF-β-induced CD103 expression and pro-inflammatory T helper 17, but not regulatory T, cell differentiation by modulating SMAD2. Notably, we find that the TRPM7 kinase activity promotes gut colonization by alloreactive T cells in acute graft-versus-host disease. Thus, our results unravel a function of TRPM7 kinase in T cell activity and suggest a therapeutic potential of kinase inhibitors in averting acute graft-versus-host disease.

Expression of connective tissue growth factor (CTGF) in endothelial cells is modulated by shear stress affecting the organization of the cytoskeleton. The molecular connection between alterations of actin and CTGF expression was investigated in human umbilical vein endothelial cells (HUVEC) and a microvascular endothelial cell line. Overexpression of nonpolymerizable monomeric actin R62D interfered with stress fiber formation in HUVEC and concomitantly reduced immunoreactive CTGF. In microvascular endothelial cells, flow-dependent upregulation of CTGF was prevented by this actin mutant. In contrast, overexpression of actin S14C strengthened filamentous actin and increased CTGF expression. These data indicated an inverse relationship between CTGF expression and monomeric actin. Coexpression of the mutant actins and different CTGF promoter constructs revealed an actin-sensitive site between 3 and 4.5 kb of the CTGF promoter. A CArG-like box at -3791 bp was responsible for actin-dependent CTGF induction as shown by mutagenesis. Overexpression of actin S14C activated the nonmutated promoter significantly more strongly than the mutated promoter. Actin polymerization is regulated by the small GTPase RhoA and activation of serum response factor (SRF). Overexpression of constitutively active RhoA or SRF significantly increased CTGF protein synthesis. The 4.5-kb promoter construct, but not the construct with a mutation in the CArG box, was activated by SRF or RhoA, providing evidence for a functional role of this site in CTGF induction. These findings provide novel evidence that monomeric actin is the connecting link between alterations in the cytoskeleton and CTGF gene expression and demonstrate the importance of SRF in regulating CTGF transcription.

F-actin mediates motility both at the leading edge of a cell and in the nucleus.

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