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.

When directed to the nucleus by TGF-b or BMP signals, Smad proteins undergo cyclin-dependent kinase 8/9 (CDK8/9) and glycogen synthase kinase-3 (GSK3) phosphorylations that mediate the binding of YAP and Pin1 for transcriptional action, and of ubiquitin ligases Smurf1 and Nedd4L for Smad destruction. Here we demonstrate that there is an order of events-Smad activation first and destruction later-and that it is controlled by a switch in the recognition of Smad phosphoserines by WW domains in their binding partners. In the BMP pathway, Smad1 phosphorylation by CDK8/9 creates binding sites for the WW domains of YAP, and subsequent phosphorylation by GSK3 switches off YAP binding and adds binding sites for Smurf1 WW domains. Similarly, in the TGF-b pathway, Smad3 phosphorylation by CDK8/9 creates binding sites for Pin1 and GSK3, then adds sites to enhance Nedd4L binding. Thus, a Smad phosphoserine code and a set of WW domain code readers provide an efficient solution to the problem of coupling TGF-b signal delivery to turnover of the Smad signal transducers.

The transcription factor serum response factor (SRF) interacts with its cofactor, MAL/MKL1, a member of the myocardin-related transcription factor (MRTF) family, through its DNA-binding domain. We define a seven-residue sequence within the conserved MAL B1 region essential and sufficient for complex formation. The neighboring Q-box sequence facilitates this interaction. The B1 and Q-box regions also have antagonistic effects on MAL nuclear import, but the residues involved are largely distinct. Both MAL and the ternary complex factor (TCF) family of SRF cofactors interact with a hydrophobic groove and pocket on the SRF DNA-binding domain. Unlike the TCFs, however, interaction of MAL with SRF is impaired by SRF ␣I-helix mutations that reduce DNA bending in the SRF-DNA complex. A clustered SRF ␣I-helix mutation strongly impairs MAL-SRF complex formation but does not affect DNA distortion in the MAL-SRF complex. MAL-SRF complex formation is facilitated by DNA binding. DNase I footprinting indicates that in the SRF-MAL complex MAL directly contacts DNA. These contacts, which flank the DNA sequences protected from DNase I by SRF, are required for effective MAL-SRF complex formation in gel mobility shift assays. We propose a model of MAL-SRF complex formation in which MAL interacts with SRF by the addition of a ␤-strand to the SRF DNA-binding domain ␤-sheet region, while SRF-induced DNA bending facilitates MAL-DNA contact.Serum response factor (SRF) is a prototype of the MADS (Mcm1, Agamous, Deficiens, SRF) family of eukaryotic transcription factors, which play important roles in the specification of cell identity during development and differentiation (22,31). SRF interacts directly with at least two families of signalregulated cofactors. The ternary complex factor (TCF) family of Ets domain proteins, Elk-1, SAP-1, and Net, which respond to mitogen-activated protein kinase signaling, bind to both SRF and adjacent DNA sequences (34). In contrast, members of the myocardin-related transcription factor (MRTF) coactivator family (MAL/MKL1/MRTFa, MAL16/MKL2/MRTFb, and myocardin) apparently bind SRF without making specific DNA contacts (17,21,27,36,37). MAL and MAL16 respond to Rho-actin signaling, while activation by myocardin is apparently constitutive (5,23,28,36).The best-characterized MADS box regulatory complexes are those formed by SRF and its Saccharomyces cerevisiae relative, Mcm1. In each case, the MADS protein forms a three-layered structure comprising the 56-residue MADS box motif and its C-terminal flanking sequences (8,26,32). A long coiled-coil at the core of the MADS box dimer straddles the DNA minor groove, making sequence-specific major groove contacts at each side, and inserting its N-terminal extension into the minor groove. Above lies a four-stranded antiparallel ␤-sheet platform derived from the C-terminal sequences of each MADS motif. The third layer is structurally variable, being formed from subfamily-specific sequences C terminal to the MADS box and is required for high-affinity dimerization. Inter...