N-terminus-mediated dimerization of ROCK-I is required for RhoE binding and actin reorganization

ROCK-I (Rho-associated kinase 1) is a serine/threonine kinase that can be activated by RhoA and inhibited by RhoE. ROCK-I has an N-terminal kinase domain, a central coiled-coil region and a RhoA-binding domain near the C-terminus. We have previously shown that RhoE binds to the N-terminal 420 amino acids of ROCK-I, which includes the kinase domain as well as N-terminal and C-terminal extensions. In the present study, we show that N-terminus-mediated dimerization of ROCK-I is required for RhoE binding. The central coiled-coil domain can also dimerize ROCK-I in cells, but this is insufficient in the absence of the N-terminus to allow RhoE binding. The kinase activity of ROCK-I(1-420) is required for dimerization and RhoE binding; however, inclusion of part of the coiled-coil domain compensates for lack of kinase activity, allowing RhoE to bind. N-terminus-mediated dimerization is also required for ROCK-I to induce the formation of stellate actin stress fibres in cells. These results indicate that dimerization via the N-terminus is critical for ROCK-I function in cells and for its regulation by RhoE.     

A large family of anti‐activators accompanying XylS/AraC family regulatory proteins

AraC Negative Regulators (ANR) suppress virulence genes by directly down‐regulating AraC/XylS members in Gram‐negative bacteria. In this study, we sought to investigate the distribution and molecular mechanisms of regulatory function for ANRs among different bacterial pathogens. We identified more than 200 ANRs distributed in diverse clinically important gram negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC) and enteroaggregative E. coli (EAEC), and members of the Pasteurellaceae. By employing a bacterial two hybrid system, pull down assays and surface plasmon resonance (SPR) analysis, we demonstrate that Aar (AggR‐activated regulator), a prototype member of the ANR family in EAEC, binds with high affinity to the central linker domain of AraC‐like member AggR. ANR‐AggR binding disrupted AggR dimerization and prevented AggR‐DNA binding. ANR homologs of Vibrio cholerae, Citrobacter rodentium, Salmonella enterica and ETEC were capable of complementing Aar activity by repressing aggR expression in EAEC strain 042. ANR homologs of ETEC and Vibrio cholerae bound to AggR as well as to other members of the AraC family, including Rns and ToxT. The predicted proteins of all ANR members exhibit three highly conserved predicted α‐helices. Site‐directed mutagenesis studies suggest that at least predicted α‐helices 2 and 3 are required for Aar activity. In sum, our data strongly suggest that members of the novel ANR family act by directly binding to their cognate AraC partners.     

Residues near the amino terminus of Rns are essential for positive autoregulation and DNA binding

Most members of the AraC/XylS family contain a conserved carboxy-terminal DNA binding domain and a less conserved amino-terminal domain involved in binding small-molecule effectors and dimerization. However, there is no evidence that Rns, a regulator of enterotoxigenic Escherichia coli virulence genes, responds to an effector ligand, and in this study we found that the amino-terminal domain of Rns does not form homodimers in vivo. Exposure of Rns to the chemical cross-linker glutaraldehyde revealed that the full-length protein is also a monomer in vitro. Nevertheless, deletion analysis of Rns demonstrated that the first 60 amino acids of the protein are essential for the activation and repression of Rns-regulated promoters in vivo. Amino-terminal truncation of Rns abolished DNA binding in vitro, and two randomly generated mutations, I14T and N16D, that independently abolished Rns autoregulation were isolated. Further analysis of these mutations revealed that they have disparate effects at other Rns-regulated promoters and suggest that they may be involved in an interaction with the carboxy-terminal domain of Rns. Thus, evolution may have preserved the amino terminus of Rns because it is essential for the regulator's activity even though it apparently lacks the two functions, dimerization and ligand binding, usually associated with the amino-terminal domains of AraC/XylS family members.     

Mechanism of cullin3 e3 ubiquitin ligase dimerization

Cullin E3 ligases are the largest family of ubiquitin ligases with diverse cellular functions. One of seven cullin proteins serves as a scaffold protein for the assembly of the multisubunit ubiquitin ligase complex. Cullin binds the RING domain protein Rbx1/Rbx2 via its C-terminus and a cullin-specific substrate adaptor protein via its N-terminus. In the Cul3 ubiquitin ligase complex, Cul3 substrate receptors contain a BTB/POZ domain. Several studies have established that Cul3-based E3 ubiquitin ligases exist in a dimeric state which is required for binding of a number of substrates and has been suggested to promote ubiquitin transfer. In two different models, Cul3 has been proposed to dimerize either via BTB/POZ domain dependent substrate receptor homodimerization or via direct interaction between two Cul3 proteins that is mediated by Nedd8 modification of one of the dimerization partners. In this study, we show that the majority of the Cul3 proteins in cells exist as dimers or multimers and that Cul3 self-association is mediated via the Cul3 N-terminus while the Cul3 C-terminus is not required. Furthermore, we show that Cul3 self-association is independent of its modification with Nedd8. Our results provide evidence for BTB substrate receptor dependent Cul3 dimerization which is likely to play an important role in promoting substrate ubiquitination.