Distinct LIM domains of Hic-5/ARA55 are required for nuclear matrix targeting and glucocorticoid receptor binding and coactivation

Hydrogen peroxide-inducible clone-5 (Hic-5), belongs to the group III LIM domain protein family and contains four carboxyl-terminal LIM domains (LIM1-LIM4). In addition to its role in focal adhesion signaling, Hic-5 acts in the nucleus as a coactivator for some steroid hormone receptors such as the glucocorticoid receptor (GR) and androgen receptor (AR). Based upon its effect on AR transactivation, Hic-5 has also been designated as ARA55. Here, we report mapping studies of Hic-5/ARA55 functional domains and establish that LIM3 and LIM4 are necessary for maximal effects on GR transactivation. However, results from yeast two-hybrid assays demonstrated that these two LIM domains together, while necessary, are not sufficient to interact with the tau2 transactivation domain of GR. LIM4 also functions as a nuclear matrix targeting sequence (NMTS) for Hic-5/ARA55, as it is both necessary and sufficient to target a heterologous protein to the nuclear matrix. Thus, as suggested from previous analysis of LIM domain-containing proteins, separate but highly related LIM domains serve distinct functions.     

The N domain of Smad7 is essential for specific inhibition of transforming growth factor-beta signaling.

Inhibitory Smads (I-Smads) repress signaling by cytokines of the transforming growth factor-beta (TGF-beta) superfamily. I-Smads have conserved carboxy-terminal Mad homology 2 (MH2) domains, whereas the amino acid sequences of their amino-terminal regions (N domains) are highly divergent from those of other Smads. Of the two different I-Smads in mammals, Smad7 inhibited signaling by both TGF-beta and bone morphogenetic proteins (BMPs), whereas Smad6 was less effective in inhibiting TGF-beta signaling. Analyses using deletion mutants and chimeras of Smad6 and Smad7 revealed that the MH2 domains were responsible for the inhibition of both TGF-beta and BMP signaling by I-Smads, but the isolated MH2 domains of Smad6 and Smad7 were less potent than the full-length Smad7 in inhibiting TGF-beta signaling. The N domains of I-Smads determined the subcellular localization of these molecules. Chimeras containing the N domain of Smad7 interacted with the TGF-beta type I receptor (TbetaR-I) more efficiently, and were more potent in repressing TGF-beta signaling, than those containing the N domain of Smad6. The isolated N domain of Smad7 physically interacted with the MH2 domain of Smad7, and enhanced the inhibitory activity of the latter through facilitating interaction with TGF-beta receptors. The N domain of Smad7 thus plays an important role in the specific inhibition of TGF-beta signaling.     

Disorder in a target for the smad2 mad homology 2 domain and its implications for binding and specificity

The Smad2 Mad homology 2 (MH2) domain binds to a diverse group of proteins which do not share a common sequence motif. We have used NMR to investigate the structure of one of these interacting proteins, the Smad binding domain (SBD) of Smad anchor for receptor activation (SARA). Our results indicate that the unbound SBD is highly disordered and forms no stable secondary or tertiary structures. Additionally we have used fluorescence binding studies to study the interaction between the MH2 domain and SBD and find that no region of the SBD dominates the interaction between the MH2 and the SBD. Our results are consistent with a series of hydrophobic patches on the MH2 that are able to recognize disordered regions of proteins. These findings elucidate a mechanism by which a single domain (MH2) can specifically recognize a diverse set of proteins which are unrelated by sequence, lead to a clearer picture of how MH2 domains function in the transforming growth factor-beta-signaling pathway and suggest possible mechanisms for controlling interactions with MH2 domains.     

The PDZ Domain of the LIM Protein Enigma Binds to β-Tropomyosin

PDZ and LIM domains are modular protein interaction motifs present in proteins with diverse functions. Enigma is representative of a family of proteins composed of a series of conserved PDZ and LIM domains. The LIM domains of Enigma and its most related family member, Enigma homology protein, bind to protein kinases, whereas the PDZ domains of Enigma and family member actin-associated LIM protein bind to actin filaments. Enigma localizes to actin filaments in fibroblasts via its PDZ domain, and actin-associated LIM protein binds to and colocalizes with the actin-binding protein alpha-actinin-2 at Z lines in skeletal muscle. We show that Enigma is present at the Z line in skeletal muscle and that the PDZ domain of Enigma binds to a skeletal muscle target, the actin-binding protein tropomyosin (skeletal beta-TM). The interaction between Enigma and skeletal beta-TM was specific for the PDZ domain of Enigma, was abolished by mutations in the PDZ domain, and required the PDZ-binding consensus sequence (Thr-Ser-Leu) at the extreme carboxyl terminus of skeletal beta-TM. Enigma interacted with isoforms of tropomyosin expressed in C2C12 myotubes and formed an immunoprecipitable complex with skeletal beta-TM in transfected cells. The association of Enigma with skeletal beta-TM suggests a role for Enigma as an adapter protein that directs LIM-binding proteins to actin filaments of muscle cells.     

Roles for the tubulin- and PTP-PEST-binding paxillin LIM domains in cell adhesion and motility

Cell dynamics mediated through cell-extracellular matrix contacts, such as adhesion and motility involve the precise regulation of large complexes of structural and signaling molecules called focal adhesions (FAs). Paxillin is a multi-domain FA adaptor protein containing five amino-terminal paxillin leucine-aspartate repeat (LD) motifs and four carboxyl-terminal Lin-11 Isl-1 and Mec-3 (LIM) domains. The LD motifs support paxillin binding to actopaxin, integrin linked kinase (ILK), FA kinase (FAK), paxillin kinase linker (PKL) and vinculin. Of the LIM domains, LIM2 and 3 comprise the paxillin FA-targeting motif, with phosphorylation of these domains modulating paxillin targeting and cell adhesion to fibronectin (Fn). The identity of the paxillin FA targeting partner remains to be determined; however, the LIM domains mediate interactions with tubulin and the protein-tyrosine phosphatase (PTP)-PEST. PTP-PEST binding requires both LIM3 and 4, whereas, the precise LIM target of tubulin binding is not known. In this report, we demonstrate that the individual paxillin LIM2 and 3 domains support specific binding to tubulin and suggest a potential role for this interaction in the regulation of paxillin sub-cellular compartmentalization. In addition, expression of paxillin molecules with mutations in the tubulin- and PTP-PEST-binding LIM domains differentially impaired Chinese hamster ovary K1 (CHO.K1) cell adhesion and migration to Fn. Perturbation of LIM3 or 4 inhibited adhesion while mutation of LIM2 or 4 decreased cell motility. Interestingly, expression of tandem LIM2-3 inhibited cell adhesion and spreading while LIM3-4 stimulated a well-spread polarized phenotype. These data offer further support for a critical role for paxillin in cell adhesion and motility.     

Distinct domain utilization by Smad3 and Smad4 for nucleoporin interaction and nuclear import

Smad proteins undergo rapid nuclear translocation upon stimulation by transforming growth factor-beta (TGFbeta) and in so doing transduce the signal into the nucleus. In this report we unraveled nuclear import mechanisms of Smad3 and Smad4 that are dependent on their interaction with FG-repeat-containing nucleoporins such as CAN/Nup214, without the involvement of importin molecules that are responsible for most of the known nuclear import events. A surface hydrophobic corridor within the MH2 domain of Smad3 is critical for association with CAN/Nup214 and nuclear import, whereas Smad4 interaction with CAN/Nup214, and nuclear import requires structural elements present only in the full-length Smad4. As exemplified by the different susceptibility to inhibition of import by cytoplasmic retention factor SARA (Smad anchor for receptor activation), such utilization of distinct domains for nuclear import of Smad3 and Smad4 suggests that nuclear transport of Smad3 and Smad4 is subject to control by different retention factors.