contributed equally to this work TIA-1 and TIAR are related proteins that bind to an AU-rich element (ARE) in the 3¢ untranslated region of tumor necrosis factor alpha (TNF-a) transcripts. To determine the functional signi®cance of this interaction, we used homologous recombination to produce mutant mice lacking TIA-1. Although lipopolysaccharide (LPS)-stimulated macrophages derived from wild-type and TIA-1 ±/± mice express similar amounts of TNF-a transcripts, macrophages lacking TIA-1 produce signi®cantly more TNF-a protein than wild-type controls. The half-life of TNF-a transcripts is similar in wild-type and TIA-1 ±/± macrophages, indicating that TIA-1 does not regulate transcript stability. Rather, the absence of TIA-1 signi®cantly increases the proportion of TNF-a transcripts that associate with polysomes, suggesting that TIA-1 normally functions as a translational silencer. TIA-1 does not appear to regulate the production of interleukin 1b, granulocyte±macrophage colony-stimulating factor or interferon g, indicating that its effects are, at least partially, transcript speci®c. Mice lacking TIA-1 are hypersensitive to the toxic effects of LPS, indicating that this translational control pathway may regulate the organismal response to microbial stress.

The ras-like GTP binding proteins cdc42, rac, and rho regulate diverse cellular processes including cell growth and actin remodeling associated with changes in cell morphology, growth, adhesion, and motility (1-4). In fibroblasts, cdc42 regulates actin polymerization and focal complexes necessary for filopodia formation, rac mediates actin polymerization and focal complex assembly within lamellipodia and membrane ruffles, and rho induces actin stress fiber and focal adhesion (FA) complex formation (5). A hierarchical relationship exists among cdc42, rac, and rho, whereby cdc42 regulates rac activity and rac regulates rho activity, suggesting that these proteins may orchestrate the spatial and temporal changes in the actin cytoskeleton necessary for cell movement (5, 6). cdc42 and rac also regulate activation of the c-Jun N-terminal kinase/stressactivated kinase via a mitogen-activated protein (MAP) kinase pathway (7-9), and rac and rho are essential for ras transformation (10, 11). cdc42, rho, and rac all appear to stimulate c-fos transcription (12), as well as cell cycle progression through GI and subsequent DNA synthesis (9). The activation state of ras-like GTP binding proteins is positively regulated by guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP, and negatively by GTPase activating proteins (2). A number of putative GEFs for rho-like GTPases have been identified by sequence comparison (2), and several of these demonstrate GEF activity in vitro. The dbl and ost oncogene products have cdc42 and rho GEF activity (13-15); the lbc oncogene product has rho GEF activity (16); the invasion-inducing Tiaml gene product has cdc42, rho, and rac GEF activity (17); and the yeast CDC24 gene product has cdc42 GEF activity (18). LAR is a broadly expressed transmembrane protein tyrosine phosphatase (PTPase) comprised of a cell adhesion-like extracellular region and two intracellular PTPase domains (19)(20)(21)(22). A role for LAR in regulating cell-matrix interactions was proposed, as LAR colocalizes with a coiled-coil protein, termed LAR interacting protein 1 (LIP.1) at the ends of FAs (23), and LAR expression was observed at regions of association between cells and basement membrane in various tissues (19). To identify putative substrates and other proteins involved in LAR-mediated signal transduction, we screened for proteins that bind the LAR PTPase domains using the interaction-trap assay and coimmunoprecipitation studies. A protein thus isolated is a novel multidomain GEF we have named Trio because it contains three enzyme domains: two GEF domains, one of which has racl GEF activity and the other has rhoA GEF activity, and a serine/threonine kinase (PSK) domain. In addition, Trio contains four N-terminal spectrinlike domains, two pleckstrin-like domains, and an Ig-like domain. Because proteins with cdc42, rac, or rho GEF activity are generally involved in regulating cytoskeletal organization (1), it is likely that Trio in conjunction with LAR plays a key role in coordinating the ...

Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% of the particle mass. We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis. Analysis of the protein sequence revealed a region of ∼350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP). PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. The catalytic domain of p193 was expressed and purified from bacterial extracts. Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP. Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity. Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP. Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s). A portion of p193 is nuclear and localizes to the mitotic spindle.

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Protein tyrosine phosphatases (PTPases), together with protein tyrosine kinases, regulate the tyrosine phosphorylation that controls cell activities and proliferation. Previously, it has been recognized that both cytosolic PTPases and membrane associated, receptor‐like PTPases exist. In order to examine the structural diversity of receptor‐like PTPases, we isolated human cDNA clones that cross‐hybridized to a Drosophila PTPase cDNA clone, DPTP12, under non‐stringent hybridization conditions. The cDNA clones thus isolated included LCA and six other novel receptor‐like PTPases, named HPTP alpha, beta, gamma, delta, epsilon, and zeta. The cytoplasmic regions of HPTP alpha and epsilon are highly homologous, and are composed of two tandemly duplicated PTPase‐like domains. The extracellular regions of HPTP alpha and epsilon are, respectively, 123 amino acids and 27 amino acids, and do not have obvious similarity to any known protein. The cytoplasmic region of HPTP beta contains only one PTPase domain. The extracellular region of HPTP beta, which is 1599 amino acids, is composed of 16 fibronectin type‐III repeats. HPTP delta is very similar to leukocyte common antigen related molecule (LAR), in both the extracellular and cytoplasmic regions. Partial sequences of HPTP gamma and zeta indicate that they are highly homologous and contain two PTPase‐like domains. The PTPase‐like domains of HPTP alpha, beta and delta expressed in Escherichia coli had tyrosine phosphatase activities.