A novel PTB-PDZ domain interaction mediates isoform-specific ubiquitylation of mammalian Numb

LNX was originally cloned as a Numb PTB-binding molecule, and it was subsequently found to act as a RING finger-type E3 ubiquitin ligase for the ubiquitylation and degradation of mNumb. Numb is a PTB domain-containing protein that functions as an intrinsic determinant of cell fate in asymmetric cell division. In mammals, four protein isoforms arise from alternative mRNA splicing. Here we report that while all four protein isoforms bind to LNX, only p72 and p66 Numb isoforms are ubiquitylated and degraded. The p72 and p66 Numb proteins differ from the other two isoforms by the presence of an 11-amino acid sequence insert in the PTB domain (PTBi). We demonstrate that the isoform-specific ubiquitylation of mNumb is due to a novel interaction between the first PDZ domain (PDZ1) of LNX and the PTBi variant. Deletion of LNX PDZ1 domain resulted in loss of ubiquitylation and subsequent degradation of the PTBi form of Numb. Interestingly efficient PTBi ubiquitylation not only depends on association with the LNX PDZ1 domain but also requires binding to the canonical PTB-binding motif NPAY in LNX. Thus two distinct modes of PTBi-mediated interaction with LNX work in concert to allow the effective and specific degradation of the p72 and p66 isoforms of mNumb.     

The basic region of Fos mediates specific DNA binding.

The DNA-binding domains of the members of the Fos and Jun families of proteins consist of a basic region followed by a dimerizing segment with heptad repeats of leucine. Fos-Jun heterodimers and Jun alone, but not Fos alone, bind to the symmetrical sequences TGACTCA (AP-1 site) or TGACGTCA (cAMP response element or CRE). We set out to test the hypothesis that in the Fos-Jun heterodimer the basic region of Fos confers specific DNA-binding properties equivalent to the contribution of the basic region of Jun. Fos-Jun chimeric proteins were prepared consisting of the basic region of one protein joined to the leucine repeat of the other. Heterodimers with mixed Fos and Jun leucine repeat segments showed high affinity binding to the AP-1 site or CRE whether they contained two basic regions from Jun, two basic regions from Fos, or one from each source. Heterodimers with two Fos basic regions showed somewhat greater affinity for the CRE and AP-1 site than the heterodimer with two Jun basic regions. The DNA sequence specificity and the purine and phosphate DNA contact sites for each heterodimer were similar. We conclude that in the Fos-Jun heterodimer the basic region of Fos contributes specific DNA-binding properties equivalent to those of Jun. Our results support a model in which the Fos and Jun basic regions of the Fos-Jun heterodimer each interact with symmetrical DNA half sites.     

Architecture of basic building blocks in protein and domain structural interaction networks

MOTIVATION: The structural interaction of proteins and their domains in networks is one of the most basic molecular mechanisms for biological cells. Topological analysis of such networks can provide an understanding of and solutions for predicting properties of proteins and their evolution in terms of domains. A single paradigm for the analysis of interactions at different layers, such as domain and protein layers, is needed. RESULTS: Applying a colored vertex graph model, we integrated two basic interaction layers under a unified model: (1) structural domains and (2) their protein/complex networks. We identified four basic and distinct elements in the model that explains protein interactions at the domain level. We searched for motifs in the networks to detect their topological characteristics using a pruning strategy and a hash table for rapid detection. We obtained the following results: first, compared with a random distribution, a substantial part of the protein interactions could be explained by domain-level structural interaction information. Second, there were distinct kinds of protein interaction patterns classified by specific and distinguishable numbers of domains. The intermolecular domain interaction was the most dominant protein interaction pattern. Third, despite the coverage of the protein interaction information differing among species, the similarity of their networks indicated shared architectures of protein interaction network in living organisms. Remarkably, there were only a few basic architectures in the model (>10 for a 4-node network topology), and we propose that most biological combinations of domains into proteins and complexes can be explained by a small number of key topological motifs. CONTACT: doheon@kaist.ac.kr.     

The beta 4 subunit cytoplasmic domain mediates the interaction of alpha 6 beta 4 integrin with the cytoskeleton of hemidesmosomes.

The alpha 6 beta 4 integrin is structurally distinct from all the other known integrins because the cytoplasmic domain of beta 4 is unusually large and contains four type III fibronectin-like modules toward its C-terminus. To examine the function of the beta 4 cytoplasmic tail, we have expressed full-length and truncated human beta 4 cDNAs in rat bladder epithelial 804G cells, which form hemidesmosome-like adhesions in vitro. The cDNA encoded wild-type beta 4 subunit associated with endogenous alpha 6 and was recruited at the cell surface within hemidesmosome-like adhesions. A recombinant form of beta 4, lacking almost the entire cytoplasmic domain associated with alpha 6, reached the cell surface but remained diffusely distributed. A beta 4 molecule lacking almost the entire extracellular portion did not associate with alpha 6 but was correctly targeted to the hemidesmosome-like adhesions. Thus, the cytoplasmic portion of beta 4 contains sequences that are required and may be sufficient for the assembly of the alpha 6 beta 4 integrin into hemidesmosomes. To localize these sequences we examined the properties of additional mutant forms of beta 4. A truncated beta 4 subunit, lacking the most C-terminal pair of type III fibronectin homology domains, was incorporated into hemidesmosome-like adhesions, but another recombinant beta 4 molecule, lacking both pairs of type III fibronectin repeats, was not. Finally a recombinant beta 4 molecule, which was created by adjoining the region of the cytoplasmic domain including all type III repeats to the transmembrane segment, was efficiently recruited in hemidesmosome-like adhesions. Taken together these results suggest that the assembly of the alpha 6 beta 4 integrin into hemidesmosomes is mediated by a 303-amino acid region of beta 4 tail that comprises the first pair of type III fibronectin repeats and the segment between the second and third repeats. These data imply a function of a specific segment of the beta 4 cytoplasmic domain in interaction with cytoskeletal components of hemidesmosomes.     

A negatively charged domain of LAT mediates its interaction with the active form of Lck

Abstract

We have shown previously that in T cells, LAT co-immunoprecipitates with the active but not the inactive-‘closed’ form of Lck, and that this interaction impacts negatively on Lck activity. Here we confirm that activation of T cells induced a transient LAT/Lck association within 4 min after stimulation, returning to basal levels by 30 min. Interestingly, autoimmune T cells isolated from patients with systemic lupus erythematosus, which contain a larger pool of active Lck and LAT, exhibited increased LAT/Lck association compared to healthy controls. To identify the domain of LAT responsible for its interaction with active Lck, a series of LAT truncation mutants were constructed and tested in co-immunoprecipitation experiments. We found that the segment comprising residues 112–126 of human LAT is required for its interaction with Lck. This domain is rich in negatively charged amino acids and is conserved among different species. Therefore, in addition to the conserved tyrosines, the 112–126 domain identified here could be important for certain functions of LAT in T cells.     

CD98hc (SLC3A2) interaction with the integrin beta subunit cytoplasmic domain mediates adhesive signaling

In mammals, beta1 integrin adhesion receptors generate signals that mediate cell spreading, migration, proliferation, and survival. CD98, a heterodimeric transmembrane protein, physically associates with certain integrin beta subunit cytoplasmic domains (tails) via its heavy chain, CD98hc (SLC3A2), and loss of CD98hc impairs integrin signaling. Here we have used the lack of CD98hc interaction with the Drosophila integrin betaPS tail for a homology scanning analysis that implicated the C-terminal 8 residues of beta3 (Thr(755)-Thr(802)) in CD98hc binding. We then identified point mutations in the beta3 C terminus (T755K and T758M) that abolish CD98hc association and a double mutation in the corresponding residues in the betaPS tail (K839T,M842T), which resulted in gain of CD98hc interaction. Furthermore, the loss of function beta3(T755K) mutation or the gain of function beta3/betaPS(K839T,M842T) led to a loss or gain of integrin-mediated cell spreading, respectively. Thus, we have identified critical integrin residues required for CD98hc interaction and in doing so have shown that CD98c interaction with the integrin beta tail is required for its ability to mediate integrin signaling. These studies also provide new insights into how CD98hc may cooperate with other cytoplasmic domain binding proteins to modulate integrin functions and into the evolution of integrin signaling.     

A negatively charged domain of LAT mediates its interaction with the active form of Lck

We have shown previously that in T cells, LAT co-immunoprecipitates with the active but not the inactive-'closed' form of Lck, and that this interaction impacts negatively on Lck activity. Here we confirm that activation of T cells induced a transient LAT/Lck association within 4 min after stimulation, returning to basal levels by 30 min. Interestingly, autoimmune T cells isolated from patients with systemic lupus erythematosus, which contain a larger pool of active Lck and LAT, exhibited increased LAT/Lck association compared to healthy controls. To identify the domain of LAT responsible for its interaction with active Lck, a series of LAT truncation mutants were constructed and tested in co-immunoprecipitation experiments. We found that the segment comprising residues 112-126 of human LAT is required for its interaction with Lck. This domain is rich in negatively charged amino acids and is conserved among different species. Therefore, in addition to the conserved tyrosines, the 112-126 domain identified here could be important for certain functions of LAT in T cells.     

OB-fold domain of KREPA4 mediates high-affinity interaction with guide RNA and possesses annealing activity

KREPA4, also called MP24, is an essential mitochondrial guide RNA (gRNA)-binding protein with a preference for the 3′ oligo(U) tail in trypanosomes. Structural prediction and compositional analysis of KREPA4 have identified a conserved OB (oligonucleotide/oligosaccharide-binding)-fold at the C-terminal end and two low compositional complexity regions (LCRs) at its N terminus. Concurrent with these predictions, one or both of these regions in KREPA4 protein may be involved in gRNA binding. To test this possibility, deletion mutants of KREPA4 were made and the effects on the gRNA-binding affinities were measured by quantitative electrophoretic mobility shift assays. The gRNA-binding specificities of these mutants were evaluated by competition experiments using gRNAs with U-tail deletions or stem–loop modifications and uridylated nonguide RNAs or heterologous RNA. Our results identified the predicted OB-fold as the functional domain of KREPA4 that mediates a high-affinity interaction with the gRNA oligo(U) tail. An additional contribution toward RNA-binding function was localized to LCRs that further stabilize the binding through sequence-specific interactions with the guide secondary structure. In this study we also found that the predicted OB-fold has an RNA annealing activity, representing the first report of such activity for a core component of the RNA editing complex.     

A conserved domain of the arabidopsis GNOM protein mediates subunit interaction and cyclophilin 5 binding

The Arabidopsis GNOM protein, a guanine nucleotide exchange factor (GEF) that acts on ADP ribosylation factor (ARF)-type G proteins, is required for coordination of cell polarity along the apical-basal embryo axis. Interallelic complementation of gnom mutants suggested that dimerization is involved in GNOM function. Here, direct interaction between GNOM molecules is demonstrated in vitro and by using a yeast two-hybrid system. Interaction was confined to an N-terminal domain conserved within a subgroup of large ARF GEFs. The same domain mediated in vitro binding to cyclophilin 5 (Cyp5), which was identified as a GNOM interactor in two-hybrid screening. Cyp5 displayed peptidylprolyl cis/trans-isomerase and protein refolding activities that were sensitive to cyclosporin A. Cyp5 protein accumulated in several plant organs and, like GNOM, was partitioned between cytosolic and membrane fractions. Cyp5 protein was also expressed in the developing embryo. Our results suggest that Cyp5 may regulate the ARF GEF function of the GNOM protein during embryogenesis.     

A novel cysteine-rich domain of Sep15 mediates the interaction with UDP-glucose:glycoprotein glucosyltransferase

Selenium is an essential trace element with potent cancer prevention activity in mammals. The 15-kDa selenoprotein (Sep15) has been implicated in the chemopreventive effect of dietary selenium. Although the precise function of Sep15 remains elusive, Sep15 co-purifies with UDP-glucose:glycoprotein glucosyltransferase (GT), an essential regulator of quality control mechanisms within the endoplasmic reticulum. Recent studies identified two GT and two Sep15 homologues in mammals. We characterize interactions between these protein families in this report. Sep15 and GT form a tight 1:1 complex, and these interactions are conserved between mammals and fruit flies. In mammalian cells, Sep15 co-immunoprecipitates with both GT isozymes. In contrast, a Sep15 homologue, designated selenoprotein M (SelM), does not form a complex with GT. Sequence analysis of members of the Sep15 family identified a novel N-terminal cysteine-rich domain in Sep15 that is absent in SelM. This domain contains six conserved cysteine residues that form two CxxC motifs that do not coordinate metal ions. If this domain is deleted or the cysteines are mutated, Sep15 no longer forms a complex with GT. Conversely, if the cysteine-rich domain of Sep15 is fused to the N-terminus of SelM, the resulting chimera is capable of binding GT. These data indicate that the cysteine-rich domain of Sep15 exclusively mediates protein-protein interactions with GT.     

Aromatic and basic residues within the EVH1 domain of VASP specify its interaction with proline-rich ligands.

Short contiguous peptides harboring proline-rich motifs are frequently involved in protein-protein interactions, such as associations with Src homology 3 (SH3) and WW domains. Although patches of aromatic residues present in either domain interact with polyprolines, their overall structures are distinct, suggesting that additional protein families exist that use stacked aromatic amino acids (AA domains) to bind polyproline motifs [1] [2] [3]. A polyproline motif (E/DFPPPPTD/E in the single-letter amino-acid code), present in the ActA protein of the intracellular bacterial pathogen Listeria monocytogenes, serves as a ligand for the Ena/VASP protein family --the vasodilator-stimulated phosphoprotein (VASP), the murine protein Mena, Drosophila Enabled (Ena) and the Ena/VASP-like protein Evl [4] [5] [6] [7]. These share a similar overall structure characterized by the two highly conserved Ena/VASP homology domains (EVH1 and EVH2) [5]. Here, using three independent assays, we have delineated the minimal EVH1 domain. Mutations of aromatic and basic residues within two conserved hydrophilic regions of the EVH1 domain abolished binding to ActA. Binding of an EVH1 mutant with reversed charges could partially be rescued by introducing complementary mutations within the ligand. Like SH3 domains, aromatic residues within the EVH1 domain interacted with polyprolines, whereas the ligand specificity of either domain was determined by reciprocally charged residues. The EVH1 domain is therefore a new addition to the AA domain superfamily, which includes SH3 and WW domains.     

The DELLA domain of GA INSENSITIVE mediates the interaction with the GA INSENSITIVE DWARF1A gibberellin receptor of Arabidopsis

Gibberellic acid (GA) promotes seed germination, elongation growth, and flowering time in plants. GA responses are repressed by DELLA proteins, which contain an N-terminal DELLA domain essential for GA-dependent proteasomal degradation of DELLA repressors. Mutations of or within the DELLA domain of DELLA repressors have been described for species including Arabidopsis thaliana, wheat (Triticum aestivum), maize (Zea mays), and barley (Hordeum vulgare), and we show that these mutations confer GA insensitivity when introduced into the Arabidopsis GA INSENSITIVE (GAI) DELLA repressor. We also demonstrate that Arabidopsis mutants lacking the three GA INSENSITIVE DWARF1 (GID1) GA receptor genes are GA insensitive with respect to GA-promoted growth responses, GA-promoted DELLA repressor degradation, and GA-regulated gene expression. Our genetic interaction studies indicate that GAI and its close homolog REPRESSOR OF ga1-3 are the major growth repressors in a GA receptor mutant background. We further demonstrate that the GA insensitivity of the GAI DELLA domain mutants is explained in all cases by the inability of the mutant proteins to interact with the GID1A GA receptor. Since we found that the GAI DELLA domain alone can mediate GA-dependent GID1A interactions, we propose that the DELLA domain functions as a receiver domain for activated GA receptors.