The tumor necrosis factor-inducible zinc finger protein A20 interacts with TRAF1/TRAF2 and inhibits NF-kappaB activation.

TRAF1 and TRAF2 form an oligomeric complex that associates with the cytoplasmic domains of various members of the tumor necrosis factor (TNF) receptor superfamily. TRAF2 action is required for activation of the transcription factor NF-kappaB triggered by TNF and the CD40 ligand. Here we show that TRAF1 and TRAF2 interact with A20, a zinc finger protein, whose expression is induced by agents that activate NF-kappaB. Mutational analysis revealed that the N-terminal half of A20 interacts with the conserved C-terminal TRAF domain of TRAF1 and TRAF2. In cotransfection experiments, A20 blocked TRAF2-mediated NF-kappaB activation. A20 also inhibited TNF and IL-1-induced NF-kappaB activation, suggesting that it may inhibit NF-kappaB activation signaled by diverse stimuli. The ability of A20 to block NF-kappaB activation was mapped to its C-terminal zinc finger domain. Thus, A20 is composed of two functionally distinct domains, an N-terminal TRAF binding domain that recruits A20 to the TRAF2-TRAF1 complex and a C-terminal domain that mediates inhibition of NF-kappaB activation. Our findings suggest a possible molecular mechanism that could explain A20's ability to negatively regulate its own TNF-inducible expression.

Novel unconventional binding site in the variable region of immunoglobulins.

The variable immunoglobulin (Ig) domains contain hypervariable regions that are involved in the formation of the antigen binding site. Besides the canonical antigen binding site, so-called unconventional sites also reside in the variable region that bind bacterial and viral proteins. Docking to these unconventional sites does not typically interfere with antigen binding, which suggests that these sites may be a part of the biological functions of Igs. Herein, a novel unconventional binding site is described. The site is detected with 8-azidopurine nucleotide photoaffinity probes that label antibodies efficiently and under mild conditions. Tryptic peptides were isolated from photolabeled monoclonal antibodies and aligned with the variable antibody domains of heavy and light chains. The structure of a variable Ig fragment was used to model the binding of the purine nucleotide to invariant residues in a hydrophobic pocket of the Ig molecule at a location distant from the antigen binding site. Monoclonal and polyclonal antibodies were biotinylated with the photoaffinity linker and used in fluorescence-activated cell sorter and ELISA analyses. The data support the utility of this site for tethering diagnostic and therapeutic agents to the variable Ig fragment region without impairing the structural and functional integrity of antibodies.

The multidomain protein Trio binds the LAR transmembrane tyrosine phosphatase, contains a protein kinase domain, and has separate rac-specific and rho-specific guanine nucleotide exchange factor domains.

rho-like GTP binding proteins play an essential role in regulating cell growth and actin polymerization. These molecular switches are positively regulated by guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP. Using the interaction-trap assay to identify candidate proteins that bind the cytoplasmic region of the LAR transmembrane protein tyrosine phosphatase (PT-Pase), we isolated a cDNA encoding a 2861-amino acid protein termed Trio that contains three enzyme domains: two functional GEF domains and a protein serine/threonine kinase (PSK) domain. One of the Trio GEF domains (Trio GEF-D1) has rac-specific GEF activity, while the other Trio GEF domain (Trio GEF-D2) has rho-specific activity. The C-terminal PSK domain is adjacent to an Ig-like domain and is most similar to calcium/calmodulin-dependent kinases, such as smooth muscle myosin light chain kinase which similarly contains associated Ig-like domains. Near the N terminus, Trio has four spectrin-like repeats that may play a role in intracellular targeting. Northern blot analysis indicates that Trio has a broad tissue distribution. Trio appears to be phosphorylated only on serine residues, suggesting that Trio is not a LAR substrate, but rather that it forms a complex with LAR. As the LAR PTPase localizes to the ends of focal adhesions, we propose that LAR and the Trio GEF/PSK may orchestrate cell-matrix and cytoskeletal rearrangements necessary for cell migration.

Identification of functional domains and evolution of Tc1-like transposable elements.

Tc1-like transposable elements from teleost fish have been phylogenetically examined to determine the mechanisms involved in their evolution and conserved domains of function. We identified two new functional domains in these elements. The first is a bipartite nuclear localization signal, indicating that transposons can take advantage of the transport machinery of host cells for nuclear uptake of their transposases. The second is a novel combination of a paired domain-related protein motif juxtaposed to a leucine zipper-like domain located in the putative DNA-binding regions of the transposases. This domain coexists with a special inverted repeat structure in certain transposons in such phylogenetically distant hosts as fish and insects. Our data indicate that reassortment of functional domains and horizontal transmission between species are involved in the formation and spread of new types of transposable elements.

In its active form, the GTP-binding protein rab8 interacts with a stress-activated protein kinase.

Rab8 is a small GTP-binding protein that plays a role in vesicular transport from the trans-Golgi network to the basolateral plasma membrane in polarized epithelial cells (MDCK), and to the dendritic surface in hippocampal neurons. As is the case for most other rab proteins, the precise molecular interactions by which rab8 carries out its function remain to be elucidated. Here we report the identification and the complete cDNA-derived amino acid sequence of a murine rab8-interacting protein (rab8ip) that specifically interacts with rab8 in a GTP-dependent manner. Rab8ip displays 93% identity with the GC kinase, a serine/threonine protein kinase recently identified in human lymphoid tissue that is activated in the stress response. Like the GC kinase, rab8ip has protein kinase activity manifested by autophosphorylation and phosphorylation of the classical serine/threonine protein kinase substrates, myelin basic protein and casein. When coexpressed in transfected 293T cells, rab8 and the rab8ip/GC kinase formed a complex that could be recovered by immunoprecipitation with antibodies to rab8. Cell fractionation and immunofluorescence analyses indicate that in MDCK cells endogenous rab8ip is present both in the cytosol and as a peripheral membrane protein concentrated in the Golgi region and basolateral plasma membrane domains, sites where rab8 itself is also located. In light of recent evidence that rab proteins may act by promoting the stabilization of SNARE complexes, the specific GTP-dependent association of rab8 with the rab8ip/GC kinase raises the possibility that rab-regulated protein phosphorylation is important for vesicle targeting or fusion. Moreover, the rab8ip/GC kinase may serve to modulate secretion in response to stress stimuli.

Homophilic adhesion mediated by the neural cell adhesion molecule involves multiple immunoglobulin domains.

The neural cell adhesion molecule (N-CAM) mediates homophilic binding between a variety of cell types including neurons, neurons and glia, and neurons and muscle cells. The mechanism by which N-CAM on one cell interacts with N-CAM on another, however, is unknown. Attempts to identify which of the five immunoglobulin-like domains (Ig I-V) and the two fibronectin type III repeats (FnIII 1-2) in the extracellular region of N-CAM are involved in this process have led to ambiguous results. We have generated soluble recombinant proteins corresponding to each of the individual immunoglobulin domains and the combined FnIII 1-2 and prepared polyclonal antibodies specific for each. The purified proteins and antibodies were used in aggregation experiments with fluorescent microspheres and chicken embryo brain cells to determine possible contributions of each domain to homophilic adhesion. The recombinant domains were tested for their ability to bind to purified native N-CAM, to bind to each other, and to inhibit the aggregation of N-CAM on microspheres and the aggregation of neuronal cells. Each of the immunoglobulin domains bound to N-CAM, and in solution all of the immunoglobulin domains inhibited the aggregation of N-CAM-coated microspheres. Soluble Ig II, Ig III, and Ig IV inhibited neuronal aggregation; antibodies against whole N-CAM, the Ig III domain, and the Ig I domain all strongly inhibited neuronal aggregation, as well as the aggregation of N-CAM-coated microspheres. Of all the domains, the third immunoglobulin domain alone demonstrated the ability to self-aggregate, whereas Ig I bound to Ig V and Ig II bound to Ig IV. The combined FnIII 1-2 exhibited a slight ability to self-aggregate but did not bind to any of the immunoglobulin-like domains. These results suggest that N-CAM-N-CAM binding involves all five immunoglobulin domains and prompt the hypothesis that in homophilic cell-cell binding mediated by N-CAM these domains may interact pairwise in an antiparallel orientation.

Human semaphorins A(V) and IV reside in the 3p21.3 small cell lung cancer deletion region and demonstrate distinct expression patterns.

Semaphorins and collapsins make up a family of conserved genes that encode nerve growth cone guidance signals. We have identified two additional members of the human semaphorin family [human semaphorin A(V) and human semaphorin IV] in chromosome region 3p21.3, where several small cell lung cancer (SCLC) cell lines exhibit homozygous deletions indicative of a tumor suppressor gene. Human semaphorin A(V) has 86% amino acid homology with murine semaphorin A, whereas semaphorin IV is most closely related to murine semaphorin E, with 50% homology. These semaphorin genes are approximately 70 kb apart flanking two GTP-binding protein genes, GNAI-2 and GNAT-1. In contrast, other human semaphorin gene sequences (human semaphorin III and homologues of murine semaphorins B and C) are not located on chromosome 3. Human semaphorin A(V) is translated in vitro into a 90-kDa protein, which accumulates at the endoplasmic reticulum. The human semaphorin A(V) (3.4-kb mRNA) and IV (3.9- and 2.9-kb mRNAs) genes are expressed abundantly but differentially in a variety of human neural and nonneural tissues. Human semaphorin A(V) was expressed in only 1 out of 23 SCLCs and 7 out of 16 non-SCLCs, whereas semaphorin IV was expressed in 19 out of 23 SCLCs and 13 out of 16 non-SCLCs. Mutational analysis in semaphorin A(V) revealed mutations (germ line in one case) in 3 of 40 lung cancers. Our data suggest the need to determine the function of human semaphorins A(V) and IV in nonneural tissues and their role in the pathogenesis of lung cancer.

Modulation of promoter occupancy by cooperative DNA binding and activation-domain function is a major determinant of transcriptional regulation by activators in vivo.

Binding of transcriptional activators to a promoter is a prerequisite process in transcriptional activation. It is well established that the efficiency of activator binding to a promoter is determined by the affinity of direct interactions between the DNA-binding domain of an activator and its specific target sequences. However, I describe here that activator binding to a promoter is augmented in vivo by the effects of two other determinants that have not been generally appreciated: (i) the number of activator binding sites present in a promoter and (ii) the potency of activation domains of activators. Multiple sites within a promoter can cooperatively recruit cognate factors regardless of whether they contain an effective activation domain. This cooperativity can result in the synergistic activation of transcription. The second effect is the enhancement of activator binding to a promoter by the presence of activation domains. In this case, activation domains are not simply tethered to the promoter by the DNA-binding domain but instead assist the DNA-binding domain being tethered onto the promoter. This effect of activation domains on DNA binding is instrumental in determining how potent activators can induce steep transcriptional increases at low concentrations.

Evidence for distinct signaling mechanisms in two mammalian olfactory sense organs.

In mammals, olfactory stimuli are detected by sensory neurons at two distinct sites: the olfactory epithelium (OE) of the nasal cavity and the neuroepithelium of the vomeronasal organ (VNO). While the OE can detect volatile chemicals released from numerous sources, the VNO appears to be specialized to detect pheromones that are emitted by other animals and that convey information of behavioral or physiological importance. The mechanisms underlying sensory transduction in the OE have been well studied and a number of components of the transduction cascade have been cloned. Here, we investigated sensory transduction in the VNO by asking whether VNO neurons express molecules that have been implicated in sensory transduction in the OE. Using in situ hybridization and Northern blot analyses, we found that most of the olfactory transduction components examined, including the guanine nucleotide binding protein alpha subunit (G-alpha-olf), adenylyl cyclase type III, and an olfactory cyclic nucleotide-gated (CNG) channel subunit (oCNC1), are not expressed by VNO sensory neurons. In contrast, VNO neurons do express a second olfactory CNG channel subunit (oCNC2). These results indicate that VNO sensory transduction is distinct from that in the OE but raise the possibility that, like OE sensory transduction, sensory transduction in the VNO might involve cyclic nucleotide-gated ion channels.

A role for the Msx-1 homeodomain in transcriptional regulation: residues in the N-terminal arm mediate TATA binding protein interaction and transcriptional repression.

In a previous study we showed that the murine homeodomain protein Msx-1 is a potent transcriptional repressor and that this activity is independent of its DNA binding function. The implication of these findings is that repression by Msx-1 is mediated through its association with certain protein factors rather than through its interaction with DNA recognition sites, which prompted investigation of the relevant protein factors. Here we show that Msx-1 interacts directly with the TATA binding protein (TBP) but not with several other general transcription factors. This interaction is mediated by the Msx-1 homeodomain, specifically through residues in the N-terminal arm. These same N-terminal arm residues are required for repression by Msx-1, suggesting a functional relationship between TBP association and transcriptional repression. This is further supported by the observation that addition of excess TBP blocks the repressor action of Msx-1 in in vitro transcription assays. Finally, DNA binding activity is separable from both TBP interaction and repression, which further shows that these other activities of the Msx-1 homeodomain are distinct. Therefore, these findings define a role for the Msx-1 homeodomain, particularly the N-terminal arm residues in protein-protein interaction and transcriptional repression, and implicate a more complex role overall for homeodomains in transcriptional regulation.

Detection of sub-8-nm movements of kinesin by high-resolution optical-trap microscopy.

Kinesin is a molecular motor that transports organelles along microtubules. This enzyme has two identical 7-nm-long motor domains, which it uses to move between consecutive tubulin binding sites spaced 8 nm apart along a microtubular protofilament. The molecular mechanism of this movement, which remains to be elucidated, may be common to all families of motor proteins. In this study, a high-resolution optical-trap microscope was used to measure directly the magnitude of abrupt displacements produced by a single kinesin molecule transporting a microscopic bead. The distribution of magnitudes reveals that kinesin not only undergoes discrete 8-nm movements, in agreement with previous work [Svoboda, K., Schmidt, C. F., Schnapp, B. J. & Block, S.M. (1993) Nature (London) 365, 721-727], but also frequently exhibits smaller movements of about 5 nm. A possible explanation for these unexpected smaller movements is that kinesin's movement from one dimer to the next along a protofilament involves at least two distinct events in the mechanical cycle.

ARD1, a 64-kDa bifunctional protein containing an 18-kDa GTP-binding ADP-ribosylation factor domain and a 46-kDa GTPase-activating domain.

The alpha subunits of the heterotrimeric guanine nucleotide-binding proteins (G proteins) hydrolyze GTP at a rate significantly higher than do most members of the Ras family of approximatelly 20-kDa GTP-binding proteins, which depend on a GTPase-activating protein (GAP) for acceleration of GTP hydrolysis. It has been demonstrated that an inserted domain in the G-protein alpha subunit, not present in the much smaller Ras-like proteins, is responsible for this difference [Markby, D. W., Onrust, R. & Bourne, H. R. (1993) Science 262, 1895-1900]. We report here that ARD1, a 64-kDa protein with an 18-kDa carboxyl-terminal ADP-ribosylation factor (ARF) domain, exhibited significant GTPase activity, whereas the ARF domain, expressed as a recombinant protein in Escherichia coli, did not. Addition of the 46-kDa amino-terminal extension (similarly synthesized in E. coli) to the GTP-binding ARF-domain of ARD1 enhanced GTPase activity and inhibited GDP dissociation. The kinetic properties of mixtures of the ARF and non-ARF domains were similar to those of an intact recombinant ARD1. Physical association of the two proteins was demonstrated directly by gel filtration and by using the immobilized non-ARF domain. Thus, like the alpha subunits of heterotrimeric G proteins, ARD1 appears to consist of two domains that interact to regulate the biological activity of the protein.

Cloning, expression, and properties of the microtubule-stabilizing protein STOP.

Nerve cells contain abundant subpopulations of cold-stable microtubules. We have previously isolated a calmodulin-regulated brain protein, STOP (stable tubule-only polypeptide), which reconstitutes microtubule cold stability when added to cold-labile microtubules in vitro. We have now cloned cDNA encoding STOP. We find that STOP is a 100.5-kDa protein with no homology to known proteins. The primary structure of STOP includes two distinct domains of repeated motifs. The central region of STOP contains 5 tandem repeats of 46 amino acids, 4 with 98% homology to the consensus sequence. The STOP C terminus contains 28 imperfect repeats of an 11-amino acid motif. STOP also contains a putative SH3-binding motif close to its N terminus. In vitro translated STOP binds to both microtubules and Ca2+-calmodulin. When STOP cDNA is expressed in cells that lack cold-stable microtubules, STOP associates with microtubules at 37 degrees C, and stabilizes microtubule networks, inducing cold stability, nocodazole resistance, and tubulin detyrosination on microtubules in transfected cells. We conclude that STOP must play an important role in the generation of microtubule cold stability and in the control of microtubule dynamics in brain.

Factor XII-induced mitogenesis is mediated via a distinct signal transduction pathway that activates a mitogen-activated protein kinase.

Clotting factor XII (Hageman factor) contains epidermal growth factor (EGF)-homologous domains and is reported to be a potent mitogen for human hepatoma (HepG2) cells. In this study, we tested whether factor XII exhibits growth factor activity on several other EGF-sensitive target cells, including fetal hepatocytes, endothelial cells, alveolar type II cells, and aortic smooth muscle cells. We found that factor XII significantly enhanced [3H]thymidine incorporation in aortic smooth muscle cells (SMCs) and all other cells tested. Tyrphostin, a growth factor receptor/tyrosine kinase antagonist, inhibited both EGF- and factor XII-induced responses. However, differences in the levels of magnitude of DNA synthesis, the observed synergism between EGF and factor XII, and the differential sensitivity to tyrphostin suggest that the EGF receptor and the factor XII receptor may be nonidentical. The factor XII-induced mitogenic response was achieved at concentrations that were 1/10th the physiologic range for the circulating factor and was reduced by popcorn inhibitor, a specific factor XII protease inhibitor. Treatment of aortic SMCs with factor XII, as well as activated factor XII, resulted in a rapid and transient activation of a mitogen-activated/extracellular signal-regulated protein kinase with peak activity/tyrosine phosphorylation observed at 5 to 10 min of exposure. Taken together, these data (i) confirm that clotting factor XII functions as a mitogenic growth factor and (ii) demonstrate that factor XII activates a signal transduction pathway, which includes a mitogen-activated protein kinase.

Differential functions of the two Src homology 2 domains in protein tyrosine phosphatase SH-PTP1.

SH-PTP1 (also known as PTP1C, HCP, and SHP) is a non-transmembrane protein tyrosine phosphatase (PTPase) containing two tandem Src homology 2 (SH2) domains. We show here that the two SH2 (N-SH2 and C-SH2) domains in SH-PTP1 have different functions in regulation of the PTPase domain and thereby signal transduction. While the N-terminal SH2 domain is both necessary and sufficient for autoinhibition through an intramolecular association with the PTPase domain, truncation of the C-SH2 domain [SH-PTP1 (delta CSH2) construct] has little effect on SH-PTP1 activity. A synthetic phosphotyrosine residue (pY) peptide derived from the erythropoietin receptor (EpoR pY429) binds to the N-SH2 domain and activates both wild-type SH-PTP1 and SH-PTP1 (delta CSH2) 60- to 80-fold. Another pY peptide corresponding to a phosphorylation site on the IgG Fc receptor (Fc gamma RIIB1 pY309) associates with both the C-SH2 domain (Kd = 2.8 microM and the N-SH2 domain (Kd = 15.0 microM) and also activates SH-PTP1 12-fold. By analysis of the effect of the Fc gamma RIIB1 pY309 peptide on SH-PTP1 (delta CSH2), SH-PTP1 (R30K/R33E), SH-PTP1 (R30K/R136K), and SH-PTP1 (R136K) mutants in which the function of either the N- or C-SH2 domain has been impaired, we have determined that both synthetic pY peptides stimulate SH-PTP1 by binding to its N-SH2 domain; binding of pY ligand to the C-SH2 domain has no effect on SH-PTP1 activity. We propose that the N-terminal SH2 domain serves both as a regulatory domain and as a recruiting unit, whereas the C-terminal SH2 domain acts merely as a recruiting unit.