NEMO specifically recognizes K63-linked poly-ubiquitin chains through a new bipartite ubiquitin-binding domain.

An important property of NEMO, the core element of the IKK complex involved in NF-kappaB activation, resides in its ability to specifically recognize poly-ubiquitin chains. A small domain called NOA/UBAN has been suggested to be responsible for this property. We recently demonstrated that the C-terminal Zinc Finger (ZF) of NEMO is also able to bind ubiquitin. We show here by ZF swapping and mutagenesis that this represents its only function. While neither NOA nor ZF shows any preference for K63-linked chains, we demonstrate that together they form a bipartite high-affinity K63-specific ubiquitin-binding domain. A similar domain can be found in two other proteins, Optineurin and ABIN2, and can be freely exchanged with that of NEMO without interfering with its activity. This suggests that the main function of the C-terminal half of NEMO is to specifically bind K63-linked poly-ubiquitin chains. We also demonstrate that the recently described binding of NEMO to linear poly-ubiquitin chains is dependent on the NOA alone and does not require the presence of the ZF.

Identification of novel interaction partners for the conserved membrane proximal region of alpha-integrin cytoplasmic domains.

The alpha3Abeta1 integrin is a laminin receptor with a broad specificity for different laminin isoforms. Furthermore, it regulates the function of other integrins, like alpha2beta1, alpha5beta1 and alpha6Abeta1. In a yeast two hybrid screen of a human placenta cDNA library, we identified cDNAs coding for four different proteins that strongly interact with the conserved region of the cytoplasmic domain of the alpha3A integrin subunit. In addition to the cDNA for nucleotide exchange factor Mss4 and the putative tumour suppressor protein BIN1, two novel cDNAs were identified. Association analysis with different integrin subunits revealed them as cDNAs that encode binding proteins which react with a broad spectrum of alpha subunits. The conserved membrane proximal region of the alpha3A chain was identified as the binding site for all four proteins. They, therefore, may be involved in the regulation of general functions of integrins.

Interaction between the cytodomains of the alpha 3 and beta 1 integrin subunits regulates remodelling of adhesion complexes on laminin.

The first step of laminin 1-induced signal transduction is initiated by the formation of alpha 6 beta 1 integrin-specific adhesion complexes. In contrast, on other laminin isoforms the adhesion complexes are alpha 3 beta 1 integrin-specific due to a transdominant regulation of the alpha 6 beta 1 integrin by the alpha 3 beta 1 integrin. To determine the mechanism of this regulation, peptides representing the cytoplasmic domain of the alpha 3 or alpha 6 integrin subunits were microinjected together with recombinant enhanced green fluorescence protein into live fibroblasts. Microinjection of the alpha 3 integrin peptide to laminin 1-adherent cells displaying alpha 6 beta 1 integrin-specific adhesion complexes resulted in the disengagement of the alpha 6 beta 1 integrin, while microinjection of green fluorescence protein alone or in combination with the alpha 6 integrin cytodomain had no effect. Further surface plasmon resonance studies revealed that the cytodomain of the beta 1 integrin subunit interacts with low affinity with the cytoplasmic tail of the alpha 3 integrin subunit, but not with that of several other alpha subunits including alpha 6. These results imply that the cytoplasmic tails of the integrin alpha subunits play a critical role in the regulation of integrin-induced signal transduction. In particular, the intracellular tail of the alpha 3 integrin subunit controls the formation of adhesion complexes in cells adhering to laminins.

The LIM-only protein DRAL/FHL2 binds to the cytoplasmic domain of several alpha and beta integrin chains and is recruited to adhesion complexes.

LIM proteins contain one or more double zinc finger structures (LIM domains) mediating specific contacts between proteins that participate in the formation of multiprotein complexes. We report that the LIM-only protein DRAL/FHL2, with four and a half LIM domains, can associate with alpha(3A), alpha(3B), alpha(7A), and several beta integrin subunits as shown in yeast two-hybrid assays as well as after overexpression in human cells. The amino acid sequence immediately following the conserved membrane-proximal region in the integrin alpha subunits or the C-terminal region with the conserved NXXY motif of the integrin beta subunits are critical for binding DRAL/FHL2. Furthermore, the DRAL/FHL2 associates with itself and with other molecules that bind to the cytoplasmic domain of integrin alpha subunits. Deletion analysis of DRAL/FHL2 revealed that particular LIM domains or LIM domain combinations bind the different proteins. These results, together with the fact that full-length DRAL/FHL2 is found in cell adhesion complexes, suggest that it is an adaptor/docking protein involved in integrin signaling pathways.