Physical interactions and functional coupling between Daxx and sodium hydrogen exchanger 1 in ischemic cell death

Daxx, a death domain-associated protein, is implicated in ischemic cell death. To clarify the mechanism of cell death mediated by Daxx, a yeast two-hybrid assay was performed. Sodium hydrogen exchanger isoform 1 (NHE1) was identified as a Daxx-interacting protein. During ischemic stress, Daxx translocates from the nucleus to the cytoplasm, where it colocalizes with NHE1. Daxx binds to the ezrin/radixin/moesin-interacting domain of NHE1, in competition with ezrin. Consistent with this finding, transfection of the constitutively cytoplasmic mutant, Daxx(W621A), inhibited ezrin-mediated Akt-1 activation. Moreover, transfection of Daxx(W621A), but not the Daxx(S667A) mutant that is confined to the nucleus, accelerated pH(i) recovery from an acid load, indicating that the cytoplasmic protein activates NHE1. Based on the results, we propose that ischemic insult triggers the nucleocytoplasmic translocation of Daxx, following which cytoplasmic Daxx stimulates the NHE1 transporter activity and suppresses activation of the NHE1-ezrin-Akt-1 pathway. Our data support a novel molecular function of Daxx as an upstream regulator of NHE1 in ischemic cell death.     

Interaction of hCLIM1, an enigma family protein, with alpha-actinin 2

Enigma proteins are proteins that possess a PDZ domain at the amino terminal and one to three LIM domains at the carboxyl terminal. They are cytoplasmic proteins that are involved with the cytoskeleton and signal transduction pathway. By virtue of the two protein interacting domains, they are capable of protein-protein interactions. Here we report a study on a human Enigma protein hCLIM1, in particular. Our study describes the interaction of the human 36 kDa carboxyl terminal LIM domain protein (hCLIM1), the human homologue of CLP36 in rat, with alpha-actinin 2, the skeletal muscle isoform of alpha-actinin. hCLIM1 protein was shown to interact with alpha-actinin 2 by yeast two-hybrid screening and immunochemical analyses. Yeast two-hybrid analyses also demonstrated that the LIM domain of hCLIM1 binds to the EF-hand region of alpha-actinin 2, defining a new mode of LIM domain interactions. Immunofluorescent study demonstrates that hCLIM1 colocalizes with alpha-actinin at the Z-disks in human myocardium. Taken together, our experimental results suggest that hCLIM1is a novel cytoskeletal protein and may act as an adapter that brings other proteins to the cytoskeleton.     

The insulin-sensitive glucose transporter, GLUT4, interacts physically with Daxx. Two proteins with capacity to bind Ubc9 and conjugated to SUMO1

In this study we have used the yeast two-hybrid system to identify proteins that interact with the carboxyl-cytoplasmic domain (residues 464-509) of the insulin-sensitive glucose transporter GLUT4 (C-GLUT4). Using as bait C-GLUT4, we have isolated the carboxyl domain of Daxx (C-Daxx), the adaptor protein associated with the Fas and the type II TGF-beta (TbetaRII) receptors (1,2 ). The two-hybrid interaction between C-GLUT4 and C-Daxx is validated by the ability of in vitro translated C-GLUT4 to interact with in vitro translated full-length Daxx and C-Daxx. C-Daxx does not interact with the C-cytoplasmic domain of GLUT1, the ubiquitous glucose transporter homologous to GLUT4. Replacement of alanine and serine for the dileucine pair (Leu(489)-Leu(490)) critical for targeting GLUT4 from the trans-Golgi network to the perinuclear intracellular store as well as for its surface internalization by endocytosis inhibits 2-fold the interaction of C-GLUT4 with Daxx. Daxx is pulled down with GLUT4 immunoprecipitated from lysates of 3T3-L1 fibroblasts stably transfected with GLUT4 and 3T3-L1 adipocytes expressing physiological levels of the two proteins. Similarly, GLUT4 is recovered with anti-Daxx immunoprecipitates. Using an established cell fractionation procedure we present evidence for the existence of two distinct intracellular Daxx pools in the nucleus and low density microsomes. Confocal immunofluorescence microscopy studies localize Daxx to promyelocytic leukemia nuclear bodies and punctate cytoplasmic structures, often organized in strings and underneath the plasma membrane. Daxx and GLUT4 are SUMOlated as shown by their reaction with an anti-SUMO1 antibody and by the ability of this antibody to pull down Daxx and GLUT4.     

Golgi-localizing, gamma-adaptin ear homology domain, ADP-ribosylation factor-binding (GGA) proteins interact with acidic dileucine sequences within the cytoplasmic domains of sorting receptors through their Vps27p/Hrs/STAM (VHS) domains

GGA (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding) proteins are potential effectors of ADP-ribosylation factors, are associated with the trans-Golgi network (TGN), and are involved in protein transport from this compartment. By yeast two-hybrid screening and subsequent two-hybrid and pull-down analyses, we have shown that GGA proteins, through their VHS (Vps27p/Hrs/STAM) domains, interact with acidic dileucine sequences found in the cytoplasmic domains of TGN-localized sorting receptors such as sortilin and mannose 6-phosphate receptor. A mutational analysis has revealed that a leucine pair and a cluster of acidic residues adjacent to the pair are mainly responsible for the interaction. A chimeric receptor with the sortilin cytoplasmic domain localizes to the TGN, whereas the chimeric receptor with a mutation at the leucine pair or the acidic cluster is mislocalized to punctate structures reminiscent of early endosomes. These results indicate that GGA proteins regulate the localization to or exit from the TGN of the sorting receptors.     

HCC-associated protein HCAP1, a variant of GEMIN4, interacts with zinc-finger proteins

The gene HCAP1 (HCC-associated Protein 1), one variant of GEMIN4, has been mapped in a minimum LOH region on chromosome 17p13.3 and encodes a 1047-amino acid protein. Function predictions based on the amino acid sequence of protein HCAP1 revealed it to contain one helix-loop-helix motif and one leucine zipper domain. Using yeast two-hybrid screening, five zinc-finger proteins were identified as HCAP1-interacting proteins. Among them, NDP52 (nuclear dot protein 52) appeared most frequently in positive clones and was the most strongly interacting protein. Then, the interaction between HCAP1 and NDP52 was confirmed by GST pull-down assay and a coimmunoprecipitation experiment. Moreover, an immunofluorescent staining assay indicated that NDP52 colocalizes with HCAP1 in the cytoplasm. By deletion analysis, the leucine zipper domain of HCAP1 and the zinc finger domain of NDP52 were identified as important regions responsible for the interaction.     

Specificity of interactions of galectin-3 with Chrp,a cysteine- and histidine-rich cytoplasmic protein

Earlier work described the cloning of a gene from murine 3T3 cells encoding a cytoplasmic protein Chrp containing a cysteine- and histidine-rich motif characteristic of Zn-finger proteins. The interaction of Chrp with murine galectin-3 first became evident in a yeast two-hybrid screen, but it was also observed in co-precipitation experiments from 3T3 cell lysates. Here, the formation of equimolar complexes by murine Chrp and hamster galectin-3 is shown. Moreover, we found that Chrp binds to the carbohydrate-recognition domain (CRD) of hamster galectin-3 and not to the N-terminal domain carrying the proline- and glycine-rich repeats characteristic of galectin-3 and absent in other galectins. However, galectin-1 does not bind to Chrp, although its CRD is homologous to the galectin-3 CRD. Finally, we report that galectin-3, in a complex with Chrp, binds to laminin in surface plasmon resonance experiments with similar kinetics and affinity as it does in the free state. The formation of higher-order complexes containing these proteins and additional binding partners may be relevant to cytoplasmic functions involving galectin-3.     

Rapid characterization of the binding property of HtrA2/Omi PDZ domain by validation screening of PDZ ligand library

HtrA2/Omi is a mammalian mitochondrial serine protease, and was found to have dual roles in mammalian cells, not only acting as an apoptosis-inducing protein but also maintaining mitochondrial homeostasis. PDZ domain is one of the most important protein-protein interaction modules and is involved in a variety of important cellular functions, such as signal transduction, degradation of proteins, and formation of cytoskeleton. Recently, it was reported that the PDZ domain of HtrA2/Omi might regulate proteolytic activity through its interactions with ligand proteins. In this study, we rapidly characterized the binding properties of HtrA2/Omi PDZ domain by validation screening of the PDZ ligand library with yeast two-hybrid approach. Then, we predicted its novel ligand proteins in human proteome and reconfirmed them in the yeast two-hybrid system. Finally, we analyzed the smallest networks bordered by the shortest path length between the protein pairs of novel interactions to evaluate the confidence of the identified interactions. The results revealed some novel binding properties of HtrA2/Omi PDZ domain. Besides the reported Class II PDZ motif, it also binds to Class I and Class III motifs, and exhibits restricted variability at P⁻³, which means that the P⁻³ residue is selected according to the composition of the last three residues. Seven novel ligand proteins of HtrA2/Omi PDZ domain were discovered, providing significant clues for further clarifying the roles of HtrA2/Omi. Moreover, this study proves the high efficiency and practicability of the newly developed validation screening of candidate ligand library method for binding property characterization of peptide-binding domains.     

Identification and characterization of RPK118, a novel sphingosine kinase-1-binding protein

Sphingosine kinase (SPHK) is a key enzyme catalyzing the formation of sphingosine 1 phosphate (SPP), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through intracellular as well as extracellular mechanisms. However, the molecular mechanism of the intracellular actions of SPP remains unclear. Here we have cloned a novel sphingosine kinase-1 (SPHK1)-binding protein, RPK118, by yeast two-hybrid screening. RPK118 contains several functional domains whose sequences are homologous to other known proteins including the phox homology domain and pseudokinase 1 and 2 domains and is shown to be a member of an evolutionarily highly conserved gene family. The pseudokinase 2 domain of RPK118 is responsible for SPHK1 binding as judged by yeast two-hybrid screening and immunoprecipitation studies. RPK118 is also shown to co-localize with SPHK1 on early endosomes in COS7 cells expressing both recombinant proteins. Furthermore, RPK118 specifically binds to phosphatidylinositol 3-phosphate. These results strongly suggest that RPK118 is a novel SPHK1-binding protein that may be involved in transmitting SPP-mediated signaling into the cell.     

Novel protein interactors of urokinase-type plasminogen activator receptor

The urokinase-type plasminogen activator receptor (uPAR) has been implicated in tumor growth and metastasis. The crystal structure of uPAR revealed that the external surface is largely free to interact with a number of proteins. Additionally, due to absence of an intracellular cytoplasmic protein domain, many of the biological functions of uPAR necessitate interactions with other proteins. Here, we used yeast two-hybrid screening of breast cancer cDNA library to identify hSpry1 and HAX1 proteins as putative candidate proteins that interact with uPAR bait constructs. Interaction between these two candidates and uPAR was confirmed by GST-pull down, co-immunoprecipitation assays and confocal microscopy. These novel interactions that have been identified may also provide further evidence that uPAR can interact with a number of other proteins which may influence a range of biological functions.     

Interaction of Daxx, a Fas binding protein, with sentrin and Ubc9

Sentrin is a ubiquitin-like protein that can covalently modify cellular proteins, and is a Fas binding protein that protects cells against anti-Fas induced cell death. However, the mechanism by which sentrin exerts its effect upon Fas-mediated apoptosis is not well known. Thus, this study examined the interaction of sentrin with Daxx. Sentrin interacted with Daxx but not with FADD when analyzed by yeast two-hybrid assay. In vitro translated Daxx bound to GST-sentrin fusion protein. FLAG-sentrin fusion protein was also coimmunoprecipitated with Daxx in BOSC23 cells. Also, Daxx interacted with Ubc9, an essential protein as a key conjugating enzyme. Amino acids 625-740 of Daxx, known as Fas binding region, was also mapped as sentrin and Ubc9 binding region. Colocalization of Fas, sentrin, and Ubc9 binding regions suggests the importance of that region upon the regulation of Daxx. Our data also demonstrated that sentrin could homooligomerize by protein-protein interaction.