Rho GTPases regulate the transcription factor SRF via their ability to induce actin polymerization. SRF activity responds to G actin, but the mechanism of this has remained unclear. We show that Rho-actin signaling regulates the subcellular localization of the myocardin-related SRF coactivator MAL, rearranged in t(1;22)(p13;q13) AML. The MAL-SRF interaction displays the predicted properties of a Rho-regulated SRF cofactor. MAL is predominantly cytoplasmic in serum-starved cells, but accumulates in the nucleus following serum stimulation. Activation of the Rho-actin signaling pathway is necessary and sufficient to promote MAL nuclear accumulation. MAL N-terminal sequences, including two RPEL motifs, are required for the response to signaling, while other regions mediate its nuclear export (or cytoplasmic retention) and nuclear import. MAL associates with unpolymerized actin through its RPEL motifs. Constitutively cytoplasmic MAL derivatives interfere with MAL redistribution and Rho-actin signaling to SRF. MAL associates with several SRF target promoters regulated via the Rho-actin pathway.

Myocardin (MC) family proteins are transcriptional coactivators for serum response factor (SRF). Each family member possesses a conserved N-terminal region containing three RPEL motifs (the "RPEL domain"). MAL/ MKL1/myocardin-related transcription factor A is cytoplasmic, accumulating in the nucleus upon activation of RhoGTPase signaling, which alters interactions between G-actin and the RPEL domain. We demonstrate that MC, which is nuclear, does not shuttle through the cytoplasm and that the contrasting nucleocytoplasmic shuttling properties of MAL and MC are defined by their RPEL domains. We show that the MAL RPEL domain binds actin more avidly than that of MC and that the RPEL motif itself is an actin-binding element. RPEL1 and RPEL2 of MC bind actin weakly compared with those of MAL, while RPEL3 is of comparable and low affinity in the two proteins. Actin binding by all three motifs is required for MAL regulation. The differing behaviors of MAL and MC are specified by the RPEL1-RPEL2 unit, while RPEL3 can be exchanged between them. We propose that differential actin occupancy of multiple RPEL motifs regulates nucleocytoplasmic transport and activity of MAL.The myocardin (MC) family of transcriptional coactivators regulates the activity of the transcription factor serum response factor (SRF) through association with its DNA-binding domain (2,14,17,21,24,27). Two of the proteins, MAL/MKL1/ myocardin-related transcription factor A (MRTF-A) and MAL16/MKL2/MRTF-B, are ubiquitously expressed, while the expression of MC, the founding family member, is restricted to smooth and cardiac muscle. In contrast to MC, which appears constitutively nuclear (24), the other MC family members redistribute from the cytoplasm to the nucleus upon activation of Rho signaling in many other cell lines (5,14).In fibroblasts, the regulation of MAL localization and activity is controlled largely by Rho-dependent changes in the dynamics of actin turnover between its monomeric (G-actin) and filamentous (F-actin) states, and blockade of Rho-induced actin polymerization prevents MAL-mediated activation of SRF target genes (11,13,14,23). MAL constantly circulates between nucleus and cytoplasm in serum-starved cells. Its cytoplasmic steady-state localization is maintained by very efficient CRM1-dependent nuclear export, which also requires its interaction with actin in the nucleus (23). MAL senses the cellular G-actin concentration by direct interaction (Fig. 1A), and reduction of this interaction, whether it results from Rhoinduced depletion of the G-actin pool or from direct disruption by actin-binding drugs, such as cytochalasin D (CD), leads to MAL nuclear accumulation (Fig. 1A) (14, 23).MC family proteins possess a conserved N-terminal region containing three RPEL motifs (Pfam no. 02755) (6), termed the RPEL domain, and form one of two families of RPELcontaining proteins in metazoans (Fig. 1B). The MAL RPEL domain forms a stable complex with three molecules of actin in solution (18,23). Alanine substitution at the conserved R or P residu...

Nuclear accumulation of the serum response factor coactivator MAL/MKL1 is controlled by its interaction with G-actin, which results in its retention in the cytoplasm in cells with low Rho activity. We previously identified actin mutants whose expression promotes MAL nuclear accumulation via an unknown mechanism. Here, we show that actin interacts directly with MAL in vitro with high affinity. We identify a further activating mutation, G15S, which stabilises F-actin, as do the activating actins S14C and V159N. The three mutants share several biochemical properties, but can be distinguished by their ability to bind cofilin, ATP and MAL. MAL interaction with actin S14C is essentially undetectable, and that with actin V159N is weakened. In contrast, actin G15S interacts more strongly with MAL than the wild-type protein. Strikingly, the nuclear accumulation of MAL induced by overexpression of actin S14C is substantially dependent on Rho activity and actin treadmilling, while that induced by actin G15S expression is not. We propose a model in which actin G15S acts directly to promote MAL nuclear entry.

Multisite phosphorylation regulates many transcription factors, including the Serum Response Factor partner Elk-1. Phosphorylation of the transcriptional activation domain (TAD) of Elk-1 by the protein kinase ERK at multiple sites potentiates recruitment of the Mediator transcriptional coactivator complex and transcriptional activation, but the roles of individual phosphorylation events remained unclear. Using time-resolved nuclear magnetic resonance spectroscopy, we found that ERK2 phosphorylation proceeds at markedly different rates at eight TAD sites in vitro, which we classified as fast, intermediate and slow. Mutagenesis experiments showed that phosphorylation of fast and intermediate sites promoted Mediator interaction and transcriptional activation, whereas modification of slow sites counteracted both functions, thereby limiting Elk-1 output. Progressive Elk-1 phosphorylation thus ensures a self-limiting response to ERK activation, which occurs independently of antagonizing phosphatase activity. Results and DiscussionMultisite protein phosphorylation increases the complexity of functional signaling outputs that can be generated from single protein kinase inputs. It can set thresholds for activity, or transform graded signals into switch-like responses (1-4). Many transcription factors and their interacting regulatory proteins are subject to multisite phosphorylation, which allows distinct aspects of protein function, including protein turnover, nuclear import and export, and specific protein interactions, to be controlled independently (5). However, in general, the † The ternary complex factor (TCF) subfamily of Ets-domain transcription factors, consisting of Elk-1, SAP-1, and Net, provides an example of multisite phosphorylation in transcriptional activation. TCFs, together with their partner protein SRF, function in many biological processes by coupling SRF target genes to mitogen-activated protein kinase (MAP kinase) signaling (5). Mitogenic and stress stimuli induce phosphorylation of TCF Cterminal transcriptional activation domains (TADs) at multiple S/T-P (Ser-or Thr-Pro) phosphorylation sequences, of which eight are conserved across the family ( Fig. 1A; fig. S1) (6-11). Two MAP kinase-docking sites, the D-box and the Phe-Gln-Phe-Pro (FQFP) motif, control phosphorylation of these sites (12-15). Multisite phosphorylation triggers transcriptional activation by TCFs, facilitating their interaction with the Mediator transcriptional co-activator complex (16-19) but the kinetics with which the different sites are phosphorylated, and whether they serve distinct functions, remain unclear.To obtain atomic-resolution insights into phosphorylation of the Elk-1 TAD, we used nuclear magnetic resonance (NMR) spectroscopy (20) Fig. 2A). In the fast site mutant Elk-1F, phosphorylation rates of intermediate and slow sites increased, whereas those of the fast and slow sites increased in the intermediate-site mutant Elk-1I; in both cases the altered kinetics fit well with those predicted by the model (Fig 2B,...