Human soluble (S) and membrane-bound (MB) catechol O-methyltransferase (COMT, EC 2.1.1.6) enzymes have been expressed at sufficiently high levels in Escherichia coli and in baculovirus-infected insect cells to allow kinetic characterization of the enzyme forms. The use of tight-binding inhibitors such as entacapone enabled the estimation of actual enzyme concentrations and, thereby, comparison of velocity parameters, substrate selectivity, and regioselectivity of the methylation of both enzyme forms. Kinetics of the methylation reaction of dopamine, (-)-noradrenaline, L-dopa, and 3,4-dihydroxybenzoic acid was studied in detail. Here, the catalytic number (Vmax) of S-COMT was somewhat higher than that of MB-COMT for all four substrates. The Km values varied considerably, depending on both substrate and enzyme form. S-COMT showed about 15 times higher Km values for catecholamines than MB-COMT. The distinctive difference between the enzyme forms was also the higher affinity of MB-COMT for the coenzyme S-adenosyl-L-methionine (AdoMet). The average dissociation constants Ks were 3.4 and 20.2 microM for MB-COMT and S-COMT, respectively. Comparison between the kinetic results and the atomic structure of S-COMT is presented, and a revised mechanism for the reaction cycle is discussed. Two recently published human COMT cDNA sequences differed in the position of S-COMT amino acid 108, the residue being either Val-108 [Lundström et al. (1991) DNA Cell. Biol. 10, 181-189] or Met-108 [Bertocci et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1416-1420].(ABSTRACT TRUNCATED AT 250 WORDS)

NF-B transcription factors are retained in the cytoplasm in an inactive form until they are activated and rapidly imported into the nucleus. We identified importin ␣3 and importin ␣4 as the main importin ␣ isoforms mediating TNF-␣-stimulated NF-B p50/p65 heterodimer translocation into the nucleus. Importin ␣3 and ␣4 are close relatives in the human importin ␣ family. We show that importin ␣3 isoform also mediates nuclear import of NF-B p50 homodimer in nonstimulated cells. Importin ␣3 is shown to directly bind to previously characterized nuclear localization signals (NLSs) of NF-B p50 and p65 proteins. Importin ␣ molecules are known to have armadillo repeats that constitute the N-terminal and C-terminal NLS binding sites. We demonstrate by site-directed mutagenesis that NF-B p50 binds to the N-terminal and p65 to the Cterminal NLS binding site of importin ␣3. In vitro competition experiments and analysis of cellular NF-B suggest that NF-B binds to importin ␣ only when it is free of IB␣. The present study demonstrates that the nuclear import of NF-B is a highly regulated process mediated by a subset of importin ␣ molecules.

Influenza A virus nonstructural protein 1 (NS1A protein) is a virulence factor which is targeted into the nucleus. It is a multifunctional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. We show that the NS1A protein can interact with all six human importin ␣ isoforms, indicating that the nuclear translocation of NS1A protein is mediated by the classical importin ␣/␤ pathway. The NS1A protein of the H1N1 (WSN/33) virus has only one N-terminal arginine-or lysine-rich nuclear localization signal (NLS1), whereas the NS1A protein of the H3N2 subtype (Udorn/72) virus also has a second C-terminal NLS (NLS2). NLS1 is mapped to residues 35 to 41, which also function in the double-stranded RNA-binding activity of the NS1A protein. NLS2 was created by a 7-amino-acid C-terminal extension (residues 231 to 237) that became prevalent among human influenza A virus types isolated between the years 1950 to 1987. NLS2 includes basic amino acids at positions 219, 220, 224, 229, 231, and 232. Surprisingly, NLS2 also forms a functional nucleolar localization signal NoLS, a function that was retained in H3N2 type virus NS1A proteins even without the C-terminal extension. It is likely that the evolutionarily well-conserved nucleolar targeting function of NS1A protein plays a role in the pathogenesis of influenza A virus.The influenza A virus genome consisting of eight separate RNA segments encodes 11 viral structural and nonstructural proteins. In addition to the viral hemagglutinin, nonstructural protein 1 (NS1A) is one of the major viral virulence factors. The evolution of NS1A genes appears to be species specific, and the evolution of the present human NS1A genes began in 1918 when H1N1 type viruses emerged and became pandemic (20).The NS1A protein is a multifunctional protein that participates in both protein-RNA (7, 16, 28, 57) and protein-protein (23, 25, 38) interactions. The NS1A protein contains an Nterminal double-stranded RNA (dsRNA)-binding domain and a C-terminal effector domain (45). The three-dimensional structures of the dsRNA-binding and effector domains of NS1A have been determined (3,6,27). The NS1A protein exists as a dimer, and the structure of its RNA-binding domain differs markedly from all other known RNA-binding proteins. The effector domain binds two cellular proteins that are essential for the 3Ј end processing of cellular pre-mRNAs (5, 26, 38). As a result, the processing of cellular pre-mRNAs, including beta interferon (IFN-␤) pre-mRNA and the pre-mRNAs of other antiviral proteins, is inhibited, thereby suppressing the amount of mature IFN-␤ mRNA that is produced in infected cells (38,39,49,55). The role of the dsRNA-binding activity is controversial and may be virus strain specific. The role of the dsRNA-binding activity of the NS1A protein of the human H3N2 influenza A/Udorn/72 virus was determined using a recombinant virus expressing a NS1 protein lacking dsRNAbinding activity. Analysis of the defect in virus replication demonstrated that the primary ...

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Interferon stimulation results in tyrosine phosphorylation, dimerization, and nuclear import of STATs (signal transducers and activators of transcription). Proteins to be targeted into the nucleus usually contain nuclear localization signals (NLSs), which interact with importin ␣. Importin ␣ binds to importin ␤, which docks the protein complex to nuclear pores, and the complex translocates into the nucleus. Here we show that baculovirus-produced and -activated STAT1 homodimers and STAT1-STAT2 heterodimers directly interacted with importin ␣5 (NPI-1). This interaction was very stable and was dependent on lysines 410 and 413 of STAT1. Only STAT dimers that had two intact NLS elements, one in each monomer, were able to bind to importin ␣5. heterodimers. In the nucleus STATs interact with IRF-9/p48 protein to form ISGF3 complexes, which bind to well conserved interferon-stimulated response elements in the promoter regions of IFN-␣/␤-responsive genes and activate transcription (5-8). Binding of type II IFN (IFN-␥) to its receptor leads to the activation of JAK1 and JAK2 and tyrosine phosphorylation of STAT1 (also at Tyr-701). Activated STAT1 forms homodimers, which translocate into the nucleus and bind to GAS elements and activate transcription of IFN-␥-inducible genes (1, 2). Although the structure-function relationships of STATs have been carefully analyzed, the mechanisms of nuclear import of this important group of transcription factors have remained less well characterized. Recently we and others have shown that STAT1 and STAT2 have an arginine/lysine-rich nuclear localization signal (NLS) that mediates their nuclear translocation in dimeric complexes (9, 10). STAT-importinActive nuclear transport of large macromolecules occurs via the nuclear pore complex (11). Proteins to be imported into the nucleus usually contain a mono-or bipartite basic-type NLS, which binds to a specific NLS receptor, importin ␣ (12-14). The N-terminal importin ␤ binding (IBB) domain of importin ␣ interacts with importin ␤ (15), which mediates the docking of NLS-containing cargo-importin ␣/␤ complex to the cytoplasmic side of the nuclear pore, and the complex translocates into the nucleus (16,17). Inside the nucleus RanGTPase is involved in the disassembly of the cargo-importin complex (14,18,19). IFN-␥-induced nuclear import of STAT1 has been suggested to be dependent on one importin ␣ subtype, importin ␣5 (20), and the RanGTPase (21). However, the elements that regulate STAT-importin ␣5 interactions have remained elusive.In the present work we show, by using a baculovirus-reconstituted STAT activation system, that homodimeric STAT1 or heterodimeric STAT1-STAT2 complexes directly interact with importin ␣5. The interaction of STAT dimers with importin ␣ is very stable and is dependent on NLS situated in the DNA binding domain of STATs. The STAT-importin ␣5 complex consists of two importin ␣ and two STAT molecules. STAT-binding GAS oligonucleotides efficiently prevent the binding of dimeric STATs with importin ␣. We also demonstrate by co...