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.     

SPARC interacts with AMPK and regulates GLUT4 expression

AMP-activated protein kinase (AMPK) is a critical regulator of glucose metabolism. To elucidate the biochemical mechanisms by which AMPK regulates glucose and fat metabolism, we conducted a yeast two-hybrid screen to identify its interacting partners. A yeast two-hybrid system was used to screen a mouse embryo cDNA library for proteins able to bind mouse AMPKα1. We also demonstrated an endogenous interaction between AMPKα1 and its interacting partner by co-immunoprecipitation of the endogenous proteins using specific antibodies in HepG2 cells, and in rat kidney, liver, skeletal muscle, and fat tissue. We show that secreted protein acidic and rich in cysteine (SPARC) is an AMPK-interacting protein, and the two proteins enhance each other. AMPK activation increases SPARC expression, and knockdown of AMPK to inhibit endogenous AMPK expression reduces SPARC protein levels. On the other hand, SPARC siRNA reduces AICAR-stimulated AMPK phosphorylation. SPARC affects AMPK-mediated glucose metabolism through regulation of Glut4 expression in L6 myocytes. Our findings suggest that SPARC may be involved in regulating glucose metabolism via AMPK activation. These results provide a starting point for efforts to clarify the relationship between AMPK and SPARC, and deepen our understanding of their roles in fat and glucose metabolism.     

Identification of two proteins, S14 and UIP1, that interact with UCH37

By the use of the yeast two-hybrid screen we have identified two proteins that interacted with UCH37: S14, which is a subunit of PA700 and a novel protein, UIP1 (UCH37 interacting protein 1). The interaction of UCH37 with S14 or UIP1 was confirmed by in vitro binding assay and in vivo co-immunoprecipitation analysis. The C-terminal extension of UCH37 is essential for interaction with S14 or UIP1 as shown by the yeast two-hybrid assay and the in vitro binding assay. Furthermore, UIP1 blocked the interaction between UCH37 and S14 in vitro.     

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.     

Identification and Characterization of MT-1X as a Novel FHL3-Binding Partner

Four and a half LIM domain protein 3 (FHL3) is a member of the FHL protein family that plays roles in the regulation of cell survival, cell adhesion and signal transduction. However, the mechanism of action for FHL3 is not yet clear. The aim of present study was to identify novel binding partner of FHL3 and to explore the underlying mechanism. With the use of yeast two-hybrid screening system, FHL3 was used as the bait to screen human fetal hepatic cDNA library for interacting proteins. Methionine-1X was identified as a novel FHL3 binding partner. The interaction between FHL3 and the full length MT-1X was further confirmed by yeast two-hybrid assay, co-immunoprecipitation and GST pull-down assays. Furthermore,the result demonstrated that MT-1X knockdown promoted the FHL3-induced inhibitory effect on HepG2 cells by regulating FHL3-mediated Smad signaling and involving in the modulation the expression of G2/M phase-related proteins through interaction with FHL3. These findings suggest that functional interactions between FHL3 and MT-1X may provide some clues to the mechanisms of FHL3-regulated cell proliferation.