JLP associates with kinesin light chain 1 through a novel leucine zipper-like domain

Scaffolding proteins exist in eukaryotes to properly assemble signaling proteins into specific multimeric functional complexes. JLP is a novel leucine zipper protein belonging to a family of scaffolding proteins that assemble JNK signaling modules. JLP is a proline-rich protein that contains two leucine zipper domains and a highly conserved C-terminal domain. We have identified kinesin light chain 1 (KLC1) as a binding partner for the second leucine zipper domain of JLP using yeast two-hybrid screening. The interaction domain of KLC1 was mapped to its tetratripeptide repeat, which contains a novel leucine zipper-like domain that is crucial for the interaction with JLP. Mutations of Leu-280, Leu-287, Val-294, and Leu-301 within this domain of KLC1 disrupted its ability to associate with JLP. Immunofluorescence studies showed that JLP and KLC1 co-localized in the cytoplasm and that the localization of JLP was dependent on its second leucine zipper. Ectopic expression of a dominant negative form of KLC1 resulted in the mislocalization of endogenous JLP. Moreover, the association between JLP and KLC1 occurred in vivo and was important in the formation of ternary complex with JNK1. These results identify a novel protein-protein interaction between KLC1 and JLP that involves leucine zipper-like domains and support the role of motor proteins in the spatial regulation of signaling modules.     

Dimerization of cGMP-dependent protein kinase 1alpha and the myosin-binding subunit of myosin phosphatase: role of leucine zipper domains

Nitric oxide (NO) and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase (cGK)-mediated activation of myosin phosphatase, which dephosphorylates myosin light chains. We recently found that cGMP-dependent protein kinase 1alpha binds directly to the myosin-binding subunit (MBS) of myosin phosphatase via the leucine/isoleucine zipper of cGK. We have now studied the role of the leucine zipper domain of MBS in dimerization with cGK and the leucine/isoleucine zipper and leucine zipper domains of both proteins in homodimerization. Mutagenesis of the MBS leucine zipper domain disrupts cGKIalpha-MBS dimerization. Mutagenesis of the MBS leucine zipper eliminates MBS homodimerization, while similar disruption of the cGKIalpha leucine/isoleucine zipper does not prevent formation of cGK dimers. The MBS leucine zipper domain is phosphorylated by cGK, but this does not have any apparent effect on heterodimer formation between the two proteins. MBS LZ mutants that are unable to bind cGK were poor substrates for cGK. These data support the theory that the MBS leucine zipper domain is necessary and sufficient to mediate both MBS homodimerization and binding of the protein to cGK. In contrast, the leucine/isoleucine zipper of cGK is required for binding to MBS, but not for cGK homodimerization. These data support that the MBS and cGK leucine zipper domains mediate the interaction between these two proteins. The contribution of these domains to both homodimerization and their specific interaction with each other suggest that additional regulatory mechanisms involving these domains may exist.     

Identification of structural and functional domains in mixed lineage kinase dual leucine zipper-bearing kinase required for complex formation and stress-activated protein kinase activation

Accumulating evidence suggests that mitogen-activated protein kinase signaling pathways form modular signaling complexes. Because the mixed lineage kinase dual leucine zipper-bearing kinase (DLK) is a large modular protein, structure-function analysis was undertaken to examine the role of DLK domains in macromolecular complex formation. DLK mutants were used to demonstrate that a DLK leucine zipper-leucine zipper interaction is necessary for DLK dimerization and to show that DLK dimerization mediated by the leucine zipper domain is prerequisite for DLK activity and subsequent activation of stress-activated protein kinase (SAPK). Heterologous mixed lineage kinase family members can be co-immunoprecipitated. However, the DLK leucine zipper domain interacted specifically only with the DLK leucine zipper domain; in contrast, DLK NH(2)-terminal region was sufficient to co-immunoprecipitate leucine zipper kinase and DLK. DLK has been shown to associate with the putative scaffold protein JIP1. This association occurred through the DLK NH(2)-terminal region and occurred independently of DLK catalytic activity. Although the DLK NH(2)-terminal region associated directly with JIP-1, this region did not interact directly with either DLK or leucine zipper kinase. Therefore, DLK may interact with heterologous mixed lineage kinase proteins via intermediary proteins. The NH(2)-terminal region of overexpressed DLK was required for activation of SAPK. These results provide evidence that protein complex formation is required for signal transduction from DLK to SAPK.     

Human cytomegalovirus UL84 oligomerization and heterodimerization domains act as transdominant inhibitors of oriLyt-dependent DNA replication: evidence that IE2-UL84 and UL84-UL84 interactions are required for lytic DNA replication

Human cytomegalovirus (HCMV) UL84 encodes a 75-kDa protein required for oriLyt-dependent DNA replication and interacts with IE2 in infected and transfected cells. UL84 localizes to the nucleus of transfected and infected cells and is found in viral replication compartments. In transient assays it was shown that UL84 can interfere with the IE2-mediated transactivation of the UL112/113 promoter of HCMV. To determine whether UL84 protein-protein interactions are necessary for lytic DNA synthesis, we purified UL84 and used this protein to generate a monoclonal antibody. Using this antibody, we now show that UL84 forms a stable interaction with itself in vivo. The point of self-interaction maps to a region of the protein between amino acids 151 and 200, a domain that contains a series of highly charged amino acid residues. Coimmunoprecipitation assays determined that UL84 interacts with a protein domain present within the first 215 amino acids of IE2. We also show that an intact leucine zipper domain of UL84 is required for a stable interaction with IE2 and UL84 leucine zipper mutants fail to complement oriLyt-dependent DNA replication. UL84 leucine zipper mutants no longer interfere with IE2-mediated transactivation of the UL112/113 promoter, confirming that the leucine zipper is essential for a functional interaction with IE2. In addition, we demonstrate that both the leucine zipper and oligomerization domains of UL84 can act as transdominant-negative inhibitors of lytic replication in the transient assay, strongly suggesting that both an IE2-UL84 and a UL84-UL84 interaction are required for DNA synthesis.     

Molecular cloning,expression, overproduction and characterization of human TRAIP Leucine zipper protein

The TRAIP interacting protein is known as a negative regulator of TNF-induced-nuclear factor, kappa-light-chain-enhancer of activated B cell (NF-κB) by direct interaction with the adaptor protein TRAF2, which inhibits the function of TRAF2 via the RINGCC domain protein. The TRAIP protein is composed of 469 amino acids with an N-terminal RING motif that is followed by a coiled coil (CC) and leucine zipper domain. TRAIP proteins are critical in programmed cell death, cell proliferation and differentiation, and embryonic development. The critical functions of TRAIP together with the molecular inhibitory mechanism effect of TRAIP have been reported by two different studies and have opened up new research into the field of TRAF biology. In this study, we designed different constructs of the Leucine zipper domain to find the over –expressed construct for further studies. We successfully cloned the C-terminal TRAIP containing the leucine zipper domain. In addition, we have over-expressed and purified the TRAIP LZ for their biochemical characterization.     

Interferon-induced Mx proteins form oligomers and contain a putative leucine zipper.

Interferons induce a number of different proteins that mediate the antiproliferative, antiviral, and immunomodulatory functions of interferons. At least three different proteins mediate the antiviral response, and one of them, Mx protein, specifically inhibits the replication of influenza virus and (vesicular stomatitis virus). Mouse and rat Mx1 proteins are nuclear, whereas other presently known Mx proteins are cytoplasmic. The cellular functions of Mx proteins are unknown, but all of them contain a consensus GTP binding site. Very little information is available on the structure and characteristics of the mouse Mx1 protein itself. For biochemical characterization, we expressed mouse Mx1 protein in a baculovirus system and purified it to homogeneity. The purified protein as well as the authentic murine cellular Mx1 protein exists in dimers and trimers in the presence of dissociating solvents, whereas in physiological buffers they form aggregates. Cross-linking experiments done on Mx-expressing cells from various species revealed that mouse, rat, and human Mx proteins exist predominantly in trimers. Amino acid sequence analysis shows that all known Mx proteins have conserved leucine repeats typical for a leucine zipper at their COOH-terminal end. In vitro translation of chimeric catechol O-methyltransferase-Mx1 gene constructs revealed that the leucine zipper domain of Mx1 protein is responsible for the oligomerization. The COOH terminus also functions as a nuclear localization signal. Microinjection of purified oligomers into the cell cytoplasm resulted in a fast accumulation of the protein in the resulted in a fast accumulation of the protein in the nucleus. Immunoelectron microscopy revealed that nuclear murine Mx1 protein exists in distinct, electron-dense structures separate from nuclear membrane, and chromatin, or nucleolus. These observations reveal that a COOH-terminal leucine zipper domain is an important structural element of all Mx proteins. Its relevance to the biology and functions of Mx proteins is presently not known.     

IS911 transposition is regulated by protein-protein interactions via a leucine zipper motif

Efficient intermolecular transposition of bacterial insertion sequence IS911 involves the activities of two element-encoded proteins: the transposase, OrfAB, and a regulatory factor, OrfA. OrfA shares the majority of its amino acid sequence with the N-terminal part of OrfAB. This includes a putative helix-turn-helix and three of four heptads of a leucine zipper motif. OrfA strongly stimulates OrfAB-mediated intermolecular transposition both in vivo and in vitro. The present results support the notion that this is accomplished by direct interaction between these two proteins via the leucine zipper. We used both a genetic approach, based on gene fusions with phage lambda repressor, and a physical approach, involving co-immunoprecipitation, to show that OrfA not only undergoes oligomerisation but is capable of engaging with OrfAB to form heteromultimers, and that the leucine zipper is necessary for both types of interaction. Furthermore, mutation of the leucine zipper in OrfA inactivated its regulatory function. Previous observations demonstrated that the integrity of the leucine zipper motif was also important for OrfAB binding to the IS911 terminal inverted repeats. Here, we show, in gel shift experiments, using a derivative of OrfAB deleted for the C-terminal catalytic domain, OrfAB[1-149], that the protein is capable of pairing two inverted repeats to generate a species resembling a "synaptic complex". Preincubation of OrfAB[1-149] with OrfA dramatically reduced formation of this complex and favored formation of an alternative complex devoid of OrfA. Together these results suggest that OrfA exerts its regulatory effect by interacting transiently with OrfAB via the leucine zipper and modifying OrfAB binding to the inverted repeats.     

Dimerization of the Epstein-Barr virus ZEBRA protein in the yeast two-hybrid system. Comparison Of a ZEBRA variant with the B95-8 form

Epstein-Barr virus (EBV) is a herpes virus associated with several human tumors. The EBV protein, ZEBRA, is a transactivator of the basic leucine zipper family (bZip). It binds to specific sequences on DNA and is able to interact with cellular proteins such as p53. The interaction of the ZEBRA protein with its cognate DNA sequences is stable as long as the dimerization domain is functional. Recent work from this laboratory identified a ZEBRA variant (Z206) with a single amino acid change at residue 206. An alanine is substituted for a serine, and this replacement is present in 72% of nasopharyngeal carcinoma from Europe and North Africa. As amino acid 206 lies within the dimerization domain it could be instrumental in interactions with other proteins. The yeast two-hybrid system was used to study ZEBRA-protein interactions. As ZEBRA by itself is a transactivator in yeast, it cannot be used directly in this assay. This paper describes modifications in ZEBRA amino acid sequences, rendering it usable in the yeast two-hybrid assay. We compared the dimerization capacity of the Z206 variant to that of ZEBRA from B95-8 (Z95) and observed that reporter gene activity with Z206 was consistently lower than that of Z95 (P < 0.05). Furthermore, no interaction was found to occur between either form of ZEBRA (Z206 or Z95) and the tumor suppressor, p53 in the yeast two-hybrid system.     

Leucine zipper-mediated homodimerization of the p21-activated kinase-interacting factor, beta Pix. Implication for a role in cytoskeletal reorganization

Pix, a p21-activated kinase-interacting exchange factor, is known to be involved in the regulation of Cdc42/Rac GTPases. The 85-kDa betaPix-a protein contains an Src homology 3 domain, the tandem Dbl homology and Pleckstrin homology domains, a proline-rich region, and a GIT1-binding domain. In addition to those domains, betaPix-a also contains a putative leucine zipper domain at the C-terminal end. In this study, we demonstrate that the previously identified putative leucine zipper domain mediates the formation of betaPix-a homodimers. Using in vitro and in vivo methodologies, we show that deletion of the leucine zipper domain is sufficient to abolish betaPix-a homodimerization. In NIH3T3 fibroblast cells, expression of wild type betaPix-a induces the formation of membrane ruffles. However, cells expressing the leucine zipper domain deletion mutant could not form membrane ruffle structures. Moreover, platelet-derived growth factor-mediated cytoskeletal changes were completely blocked by the leucine zipper domain deletion mutant. The results suggest that the leucine zipper domain enables betaPix-a to homodimerize, and homodimerization is essential for betaPix-a signaling functions leading to the cytoskeletal reorganization.     

Characterization of Solt, a novel SoxLZ/Sox6 binding protein expressed in adult mouse testis

SoxLZ/Sox6, a member of the Sox protein family, contains a leucine zipper motif in addition to an HMG box, which is its DNA binding domain. Here we have identified a novel SoxLZ/Sox6 binding protein, termed Solt, which we obtained independently using both a far-Western blot and a yeast two-hybrid screen. Like SoxLZ/Sox6 mRNA, Solt mRNA was exclusively expressed in the testis in mouse. Solt contains an unusual leucine zipper, which bound to the leucine zipper region of SoxLZ/Sox6 in vitro. In transient transfection assays in CHO cells with SoxLZ/Sox6 containing the transactivational region of herpes simplex virus VP16, expression of a reporter gene that carries a cis binding region for Sox proteins was significantly enhanced by the co-expression of Solt and Ca(2+)/calmodulin-dependent protein kinase IV.     

Membrane targeting by APPL1 and APPL2: dynamic scaffolds that oligomerize and bind phosphoinositides

Human adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1 (APPL1) and adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 2 (APPL2) are homologous effectors of the small guanosine triphosphatase RAB5 that interact with a diverse set of receptors and signaling proteins and are proposed to function in endosome-mediated signaling. Herein, we investigated the membrane-targeting properties of the APPL1 and APPL2 Bin/Amphiphysin/Rvs (BAR), pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains. Coimmunoprecipitation and yeast two-hybrid studies demonstrated that full-length APPL proteins formed homooligomers and heterooligomers and that the APPL minimal BAR domains were necessary and sufficient for mediating APPL-APPL interactions. When fused to a fluorescent protein and overexpressed, all three domains (minimal BAR, PH and PTB) were targeted to cell membranes. Furthermore, full-length APPL proteins bound to phosphoinositides, and the APPL isolated PH or PTB domains were sufficient for in vitro phosphoinositide binding. Live cell imaging showed that full-length APPL-yellow fluorescent protein (YFP) fusion proteins associated with cytosolic membrane structures that underwent movement, fusion and fission events. Overexpression of full-length APPL-YFP fusion proteins was sufficient to recruit endogenous RAB5 to enlarged APPL-associated membrane structures, although APPL1 was not necessary for RAB5 membrane targeting. Taken together, our findings suggest a role for APPL proteins as dynamic scaffolds that modulate RAB5-associated signaling endosomal membranes by their ability to undergo domain-mediated oligomerization, membrane targeting and phosphoinositide binding.