Alexia-Ileana Zaromytidou

When directed to the nucleus by TGF-β 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-β 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-β 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 alphaI-helix mutations that reduce DNA bending in the SRF-DNA complex. A clustered SRF alphaI-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 beta-strand to the SRF DNA-binding domain beta-sheet region, while SRF-induced DNA bending facilitates MAL-DNA contact.