The septin cortex at the yeast mother-bud neck.

A specialized cortical domain is organized by the septins at the necks of budding yeast cells. Recent findings suggest that this domain serves as a diffusion barrier and also as a local cell-shape sensor. We review these findings along with what is known about the organization of the septin cortex and its regulation during the cell cycle.     

Interaction of the RAGE cytoplasmic domain with diaphanous-1 is required for ligand-stimulated cellular migration through activation of Rac1 and Cdc42

Cellular migration is a fundamental process linked to diverse pathological states such as diabetes and its complications, atherosclerosis, inflammation, and cancer. The receptor for advanced glycation end products (RAGE) is a multiligand cell surface macromolecule which binds distinct ligands that accumulate in these settings. RAGE-ligand interaction evokes central changes in key biological properties of cells, including proliferation, generation of inflammatory mediators, and migration. Although RAGE-dependent signal transduction is critically dependent on its short cytoplasmic domain, to date the proximate mechanism by which this RAGE domain engages and stimulates cytoplasmic signaling pathways has yet to be identified. Here we show that the RAGE cytoplasmic domain interacts with Diaphanous-1 (Dia-1) both in vitro and in vivo. We employed the human RAGE cytoplasmic domain as "bait" in the yeast two-hybrid assay and identified the formin homology (FH1) domain of Dia-1 as a potential binding partner of this RAGE domain. Immunoprecipitation studies revealed that the RAGE cytoplasmic domain interacts with the FH1 domain of Dia-1. Down-regulation of Dia-1 expression by RNA interference blocks RAGE-mediated activation of Rac-1 and Cdc42 and, in parallel, RAGE ligand-stimulated cellular migration. Taken together, these findings indicate that the interaction of the RAGE cytoplasmic domain with Dia-1 is required to transduce extracellular environmental cues evoked by binding of RAGE ligands to their cell surface receptor, a chief consequence of which is Rac-1 and Cdc42 activation and cellular migration. Because RAGE and Dia-1 are implicated in the regulation of inflammatory, vascular, and transformed cell migration, these findings highlight this interaction as a novel target for therapeutic intervention in inflammation, atherosclerosis, diabetes, and cancer.     

The WW domain: linking cell signalling to the membrane cytoskeleton.

The WW domain is one of the smallest yet most versatile protein-protein interaction modules. The ability of this simple domain to interact with a number of proline-containing ligands has resulted in a great deal of functional diversity. Most recently it has been shown that WW domain interactions can also be differentially regulated by tyrosine phosphorylation. Here we briefly review WW domain ligands and structure in comparison to SH3 domain ligands and structure and discuss recent findings with regard to the regulation of WW domain interactions by phosphorylation. In particular we describe the potential for differential binding of the b-dystroglycan WW domain ligand by dystrophin or caveolin-3 in skeletal muscle and show how this could act as a switch to alter the relative affinity of the muscle dystroglycan complex for caveolin-3 or dystrophin and utrophin.     

The critical role of the stem region as a functional domain responsible for the oligomerization and Golgi localization of N-acetylglucosaminyltransferase V. The involvement of a domain homophilic interaction

We demonstrated that a region in the stem of N-acetylglucosaminyltransferase V (GnT-V), a Golgi resident protein, is not required for enzyme activity but serves as functional domain, responsible for intracellular localization. Deletion of the domain led to complete retention of the kinetic properties but resulted in the cell surface localization of the enzyme as well as its efficient secretion into the medium. The lack of this domain concomitantly abolished the disulfide-mediated oligomerization of GnT-V, which appears to confer the Golgi retention. When the domain was inserted into the stem region of a cell surface-localized type II membrane protein, the resulting chimeric protein was substantially oligomerized and predominantly localized in the intracellular organelle. Furthermore, it was found that the presence of this domain is exclusively responsible for homo-oligomer formation. This homophilic interaction appears to involve a hydrophobic cluster of residues in the alpha-helix of the domain, as indicated by secondary structure predictions. These findings suggest that the domain specifically participates in the Golgi retention of GnT-V, probably via inducing homo-oligomer formation, and would also provide a possible mechanism for the oligomerization, which is critical for localization in the Golgi.     

Interaction of the Hsp110 Molecular Chaperones from S. cerevisiae with Substrate Protein

Hsp110 proteins act as nucleotide exchange factors of the molecular chaperone Hsp70 in eukaryotes. In addition, they have been reported to stabilize unfolded proteins for subsequent refolding. Hsp110 proteins belong to the Hsp70 superfamily and, in analogy to Hsp70, the substrate-binding site was proposed to be located at the interface of the β-sandwich domain and the three-helix-bundle domain. Saccharomyces cerevisiae has two closely related cytosolic isoforms of Hsp110, Sse1p and Sse2p. Under normal growth conditions, Sse1p is the predominant form. Sse2p is induced under stress conditions, such as heat shock. Consistent with these findings, we find that Sse2p has increased temperature stability. Both Sse1p and Sse2p accelerate nucleotide exchange on the yeast Hsp70 Ssa1p. Furthermore, Sse1p and Sse2p effectively compete for binding of unfolded luciferase. In contrast to Sse1p, however, Sse2p fails to stabilize this model substrate under thermal stress for subsequent Hsp70-mediated refolding. Using a domain shuffling approach, we show that both the nucleotide-binding domain and the β-sandwich domain of Sse1p are required to preserve nonnative luciferase in a folding-competent state. Our findings suggest that Sse1p must undergo partial unfolding for efficient protection of luciferase, and that the β-sandwich domain of Sse1p acts as an intramolecular chaperone for refolding of the nucleotide-binding domain. Under extreme stress conditions, Sse2p appears to take over the nucleotide exchange factor function of Sse1p and might promote the controlled aggregation of stress-denatured proteins.     

Interaction partners of the PDZ domain of erbin

In order to identify proteins that bind to the PDZ domain of Erbin, we tested the C-termini of several proteins in a yeast two-hybrid assay. ErbB2, APC, beta-catenin, c-Rel and HTLV-1 Tax were identified as ligands of the PDZ domain of Erbin. The interactions were verified by co-immunoprecipitation experiments. These findings demonstrate the promiscuity of the PDZ domain of Erbin.     

Effect of E2 envelope glycoprotein cytoplasmic domain mutations on Sindbis virus pathogenesis.

The cytoplasmic domain of the E2 envelope glycoprotein is important in Sindbis virus assembly, but little is known about its role in the pathogenesis of Sindbis virus encephalitis. To investigate its role in viral pathogenesis, we constructed six recombinant viruses containing site mutations in the E2 cytoplasmic domain, using the neurovirulent background strain, TE12. Our findings demonstrate that the E2 cytoplasmic domain is a determinant of Sindbis virus growth and neurovirulence in suckling mice as well as persistent infection in weanling scid mice. They also suggest that the tyrosine, serine, or threonine residues are not essential for replication in mouse brain or anti-E2 monoclonal antibody-mediated restriction of Sindbis virus replication.     

AH/PH domain-mediated interaction between Akt molecules and its potential role in Akt regulation.

The cytoplasmic serine-threonine protein kinase coded for by the c-akt proto-oncogene features a protein kinase C-like catalytic domain and a unique NH2-terminal domain (AH domain). The AH domain is a member of a domain superfamily whose prototype was observed in pleckstrin (pleckstrin homology, or PH, domain). In this communication, we present evidence that the AH/PH domain is a domain of protein-protein interaction which mediates the formation of Akt protein complexes. The interaction between c-akt AH/PH domains is highly specific, as determined by the failure of this domain to bind AKT2. The AH/PH domain-mediated interactions depend on the integrity of the entire domain. Akt molecules with deletions of the NH2-terminal portion (amino acids 11 to 60) and AH/PH constructs with deletions of the C-terminal portion of this domain (amino acids 107 to 147) fail to interact with c-akt. To determine the significance of these findings, we carried out in vitro kinase assays using Akt immunoprecipitates from serum-starved and serum-starved, platelet-derived growth factor-stimulated NIH 3T3 cells. Addition of maltose-binding protein-AH/PH fusion recombinant protein, which is expected to bind Akt, to the immunoprecipitates from serum-starved cells induced the activation of the Akt kinase.     

Kinetics of Protein-Protein Interactions of Connexins: Use of Enzyme Linked Sorbent Assays

Determination of the protein-protein interactions of connexins has become a rapidly expanding field of research. While there are multiple methods of determining the identity of binding partners, determination of the strengths of interactions is not as simple. Here we describe the use of the in vitromethod Enzyme Linked Sorbent Assay (ELSA) to compare binding affinities of known protein partners for Connexin43. We used the binding of Cx43 Carboxyl Terminal domain to the PDZ-2 domain of Zonula Occludens-1 and to the SH3 domain of c-Src. In the ELSA assay we found that while the binding of the SH3 domain of c-Src is pH-dependent, the interaction of the PDZ domain of ZO-1 is not. These data confirm findings using Surface Plasmon Resonance (1) and indicate that ELSA can be a useful tool in determining the kinetics of protein-protein interactions.     

Characterization of a novel rice kinesin O12 with a calponin homology domain

Genomic analysis predicted that the rice (Oryza sativa var. japonica) genome encodes at least 41 kinesin-like proteins including the novel kinesin O12, which is classified as a kinesin-14 family member. O12 has a calponin homology (CH) domain that is known as an actin-binding domain. In this study, we expressed the functional domains of O12 in Escherichia coli and determined its enzymatic characteristics compared with other kinesins. The microtubule-dependent ATPase activity of recombinant O12 containing the motor and CH domains was significantly reduced in the presence of actin. Interestingly, microtubule-dependent ATPase activity of the motor domain was also affected by actin in the absence of the CH domain. Our findings suggest that the motor activity of the rice plant-specific kinesin O12 may be regulated by actin.     

Kinetics of Protein-Protein Interactions of Connexins: Use of Enzyme Linked Sorbent Assays

Determination of the protein-protein interactions of connexins has become a rapidly expanding field of research. While there are multiple methods of determining the identity of binding partners, determination of the strengths of interactions is not as simple. Here we describe the use of the in vitro method Enzyme Linked Sorbent Assay (ELSA) to compare binding affinities of known protein partners for Connexin43. We used the binding of Cx43 Carboxyl Terminal domain to the PDZ-2 domain of Zonula Occludens-1 and to the SH3 domain of c-Src. In the ELSA assay we found that while the binding of the SH3 domain of c-Src is pH-dependent, the interaction of the PDZ domain of ZO-1 is not. These data confirm findings using Surface Plasmon Resonance (1) and indicate that ELSA can be a useful tool in determining the kinetics of protein-protein interactions.     

CARD11 and CARD14 are novel caspase recruitment domain (CARD)/membrane-associated guanylate kinase (MAGUK) family members that interact with BCL10 and activate NF-kappa B

The caspase recruitment domain (CARD) is a protein-binding module that mediates the assembly of CARD-containing proteins into apoptosis and NF-kappaB signaling complexes. We report here that CARD protein 11 (CARD11) and CARD protein 14 (CARD14) are novel CARD-containing proteins that belong to the membrane-associated guanylate kinase (MAGUK) family, a class of proteins that functions as molecular scaffolds for the assembly of multiprotein complexes at specialized regions of the plasma membrane. CARD11 and CARD14 have homologous structures consisting of an N-terminal CARD domain, a central coiled-coil domain, and a C-terminal tripartite domain comprised of a PDZ domain, an Src homology 3 domain, and a GUK domain with homology to guanylate kinase. The CARD domains of both CARD11 and CARD14 associate specifically with the CARD domain of BCL10, a signaling protein that activates NF-kappaB through the IkappaB kinase complex in response to upstream stimuli. When expressed in cells, CARD11 and CARD14 activate NF-kappaB and induce the phosphorylation of BCL10. These findings suggest that CARD11 and CARD14 are novel MAGUK family members that function as upstream activators of BCL10 and NF-kappaB signaling.     

Oligomerization and ligand-binding properties of the ectodomain of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluRD.

The extracellular part of ionotropic glutamate receptor (iGluR) subunits can be divided into a conserved two-lobed ligand-binding domain ("S1S2") and an N-terminal approximately 400-residue segment of unknown function ("X domain") which shows high sequence variation among subunits. To investigate the structure and properties of the N-terminal domain, we have now produced affinity-tagged recombinant fragments which represent the X domain of the GluRD subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptors either alone or covalently linked to the ligand-binding domain ("XS1S2"). These fragments were expressed in insect cells as secreted soluble proteins and were recognized by a conformation-specific anti-GluRD monoclonal antibody. A hydrodynamic analysis of the purified fragments revealed them to be dimers, in contrast to the S1S2 ligand-binding domain which is monomeric. The X domain did not bind radiolabeled AMPA or glutamate nor did its presence affect the ligand binding properties of the S1S2 domain. Our findings demonstrate that the N-terminal domain of AMPA receptor can be expressed as a soluble polypeptide and suggest that subunit interactions in iGluR may involve the extracellular domains.     

Common themes and variations in the rhodanese superfamily

The rhodanese homology domain is a ubiquitous fold found in several phylogenetically related proteins encoded by eubacterial, archeal, and eukaryotic genomes. Although rhodanese-like proteins share evolutionary relationships, analysis of their sequences highlights that they are so heterogeneous to form the rhodanese superfamily. The variability occurs at different levels including sequence, active site loop length, presence of a critical catalytic Cys residue, and domain arrangement. Even within the same genome, multiple genes encode rhodanese-like proteins presenting with variably arranged rhodanese domain(s): as single or tandem domain(s), or combined with other protein domain(s). Given the highly variable organization of the rhodanese domain(s) and the context where it is found, here we review the structural organization and function of the rhodanese-like proteins. The overview of the most recent findings about rhodanese allow us to depict a superfamily of versatile proteins relying on persulfide chemistry to accomplish cellular functions spanning from resistance to environmental threats, such as cyanide, and key cellular reactions related to sulfur metabolism and progression of cell cycle.     

BCR sequences essential for transformation by the BCR-ABL oncogene bind to the ABL SH2 regulatory domain in a non-phosphotyrosine-dependent manner.

BCR-ABL is a chimeric oncogene implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias. BCR first exon sequences specifically activate the tyrosine kinase and transforming potential of BCR-ABL. We have tested the hypothesis that activation of BCR-ABL may involve direct interaction between BCR sequences and the tyrosine kinase regulatory domains of ABL. Full-length c-BCR as well as BCR sequences retained in BCR-ABL bind specifically to the SH2 domain of ABL. The binding domain has been localized within the first exon of BCR and consists of at least two SH2-binding sites. This domain is essential for BCR-ABL-mediated transformation. Phosphoserine/phosphothreonine but not phosphotyrosine residues on BCR are required for interaction with the ABL SH2 domain. These findings extend the range of potential SH2-protein interactions in growth control pathways and suggest a function for SH2 domains in the activation of the BCR-ABL oncogene as well as a role for BCR in cellular signaling pathways.