Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two members of the STAT signal transducer and activator of transcription family, STAT1 and STAT2, are rapidly phosphorylated on tyrosine in response to alpha interferon (IFN-alpha). Previous work showed that in the mutant human cell line U6A, which lacks STAT2 and is completely defective in IFN-alpha signaling, the phosphorylation of STAT1 is very weak, revealing that activation of STAT1 depends on STAT2. We now find that STAT2 binds to the cytoplasmic domain of the IFNAR2c (also known as IFNAR2-2) subunit of the IFN-alpha receptor in extracts of untreated cells. STAT1 also binds but only when STAT2 is present. The activities of chimeric STAT2-STAT1 proteins were assayed in U6A cells to define regions required for IFN-alpha signaling. Previous work showed that a point mutation in the Src homology 2 (SH2) domain prevents STAT2 from binding to phosphotyrosine 466 of the IFNAR1 subunit of the activated receptor. However, we now find that the entire SH2 domain of STAT2 can be replaced by that of STAT1 without loss of function, revealing that other regions of STAT2 are required for its specific interaction with the receptor. A chimeric protein, in which the N-terminal third of STAT2 has replaced the corresponding region of STAT1, did preassociate with the IFNAR2c subunit of the receptor, became phosphorylated when IFN-alpha was added, and supported the phosphorylation of endogenous STAT1. These results are consistent with a model in which STAT2 and STAT1 are prebound to the IFNAR2c subunit of the resting receptor. Upon activation, the IFNAR1 subunit is phosphorylated on Tyr-466, allowing the SH2 domain of STAT2 to bind to it; this is followed by the sequential phosphorylation of STAT2 and STAT1.
Mol Cell Biol 1997 Apr
PMID:Functional subdomains of STAT2 required for preassociation with the alpha interferon receptor and for signaling. 912 53

The NPM-ALK fusion gene, formed by the t(2;5)(p23;q35) translocation in non-Hodgkin's lymphoma, encodes a 75-kDa hybrid protein that contains the amino-terminal 117 amino acid residues of the nucleolar phosphoprotein nucleophosmin (NPM) joined to the entire cytoplasmic portion of the receptor tyrosine kinase ALK (anaplastic lymphoma kinase). Here, we demonstrate the transforming ability of NPM-ALK and show that oncogenesis by the chimeric protein requires the activation of its kinase function as a result of oligomerization mediated by the NPM segment. Sedimentation gradient experiments revealed that NPM-ALK forms in vivo multimeric complexes of approximately 200 kDa or greater that also contain normal NPM. Cell fractionation studies of the t(2;5) translocation-containing lymphoma cell line SUP-M2 showed NPM-ALK to be localized within both the cytoplasmic and nuclear compartments. Immunostaining performed with both polyclonal and monoclonal anti-ALK antibodies confirmed the dual location of the oncoprotein and also indicated that NPM-ALK is abundant within both the nucleoplasm and the nucleolus. An intact NPM segment is absolutely required for NPM-ALK-mediated oncogenesis, as indicated by our observation that three different NPM-ALK mutant proteins lacking nonoverlapping portions of the NPM segment were each unable to form complexes, lacked kinase activity in vivo, and failed to transform cells. However, NPM could be functionally replaced in the fusion protein with the portion of the unrelated translocated promoter region (TPR) protein that activates the TPR-MET fusion kinase by mediating dimerization through its leucine zipper motif. This engineered TPR-ALK hybrid protein, which transformed cells almost as efficiently as NPM-ALK, was localized solely within the cytoplasm of cells. These data indicate that the nuclear and nucleolar localization of NPM-ALK, which probably occur because of transport via the shuttling activity of NPM, is not required for oncogenesis. Further, the activation of the truncated ALK protein by a completely heterologous oligomerization domain suggests that the functionally important role of the NPM segment of NPM-ALK in transformation is restricted to the formation of kinase-active oligomers and does not involve the alteration of normal NPM functions.
Mol Cell Biol 1997 Apr
PMID:Role of the nucleophosmin (NPM) portion of the non-Hodgkin's lymphoma-associated NPM-anaplastic lymphoma kinase fusion protein in oncogenesis. 912 81

Many thylakoid lumenal proteins are nuclear encoded, cytosolically synthesized, and reach their functional location after posttranslational targeting across two chloroplast envelope membranes and the thylakoid membrane via proteinaceous transport systems. To study whether these transmembrane transport machineries can translocate folded structures, we overexpressed the 17-kDa subunit of the oxygen-evolving complex of photosystem II (prOE17) that had been modified to contain a unique C-terminal cysteine. This allowed us to chemically link a terminal 6.5-kDa bovine pancreatic trypsin inhibitor (BPTI) moiety to prOE17 to create the chimeric protein prOE17-BPTI. Redox reagents and an irreversible sulfhydryl-specific cross-linker, bis-maleimidohexane, were used to manipulate the structure of BPTI. Import of prOE17-BPTI into isolated chloroplasts and thylakoids demonstrates that the small tightly folded BPTI domain is carried across both the chloroplast envelopes and the delta pH-dependent transmembrane transporter of the thylakoid membrane when linked to the correctly targeted OE17 precursor. Transport proceeded even when the BPTI moiety was internally cross-linked into a protease-resistant form. These data indicate that unfolding is not a ubiquitous requirement for protein translocation and that at least some domains of targeted proteins can maintain a nonlinear structure during their translocation into and within chloroplasts.
Mol Biol Cell 1997 May
PMID:A folded protein can be transported across the chloroplast envelope and thylakoid membranes. 916 75

Related outer membrane proteins, termed secretins, participate in the secretion of macromolecules across the outer membrane of many Gram-negative bacteria. In the pullulanase-secretion system, PulS, an outer membrane-associated lipoprotein, is required both for the integrity and the proper outer membrane localization of the PulD secretin. Here we show that the PulS-binding site is located within the C-terminal 65 residues of PulD. Addition of this domain to the filamentous phage secretin, pIV, or to the unrelated maltose-binding protein rendered both proteins dependent on PulS for stability. A chimeric protein composed of bacteriophage f1 pIV and the C-terminal domain of PuID required properly localized PulS to support phage assembly. An in vivo complex formed between the pIV-PulD65 chimera and PulS was detected by co-immunoprecipitation and by affinity chromatography.
Mol Microbiol 1997 May
PMID:The C-terminal domain of the secretin PulD contains the binding site for its cognate chaperone, PulS, and confers PulS dependence on pIVf1 function. 917 41

Members of the Src family of protein tyrosine kinases are localized to subspecialized regions of the plasma membrane. Herein we show that the N-terminal SH4 region of the Src family member p59fyn (Fyn) is both necessary and sufficient for targeting of Fyn and heterologous proteins to the plasma membrane and detergent-insoluble subdomains. Attachment of the first 16 amino acids of Fyn to a normally cytosolic protein, beta-galactosidase, resulted in distinct plasma membrane localization of the chimeric protein. Mutation of the palmitoylation site (cysteine-3) within Fyn16-beta-galactosidase or wild-type Fyn abrogated plasma membrane localization, resulting in redistribution of the mutant proteins into intracellular membranes. Substitution of the SH4 motif within Fyn with heterologous sequences from other palmitoylated proteins (G alpha o and GAP43) revealed that the presence of palmitate is sufficient to direct plasma membrane localization independent of surrounding amino acid sequences and myristate. Palmitoylated Fyn chimeras were also enriched in the Triton X-100-resistant matrix, whereas nonpalmitoylated forms of these proteins were detected in the detergent-soluble fraction. The palmitate moiety on Fyn exhibited a half-life of 1.5-2 h. In contrast, the half-life of the polypeptide backbone was 8 h, indicating that palmitoylation is a reversible modification. These studies establish that the palmitoylated SH4 sequence of Fyn can be used to specifically target proteins to the plasma membrane in a reversible manner.
Mol Biol Cell 1997 Jun
PMID:Palmitoylation of p59fyn is reversible and sufficient for plasma membrane association. 920 23

The myeloperoxidase (MPO) gene is transcribed specifically in immature myeloid cells and is regulated in part by a 414-bp proximal enhancer. Mutation of a core binding factor (CBF)-binding site at -288 decreased enhancer activity 30-fold in 32D cl3 myeloid cells cultured in granulocyte colony-stimulating factor (G-CSF). A novel functional analysis, linking the CBF-binding site to an enhancer deletion series, located at -147 an evolutionarily conserved c-Myb-binding site which was required for optimal enhancer activity and synergy with CBF in 32D cells. These sites cooperated in isolation and independent of a precise spacing. Deletional analysis carried out in the absence of the c-Myb-binding site at -147 located at -301 a second c-Myb-binding site which also synergized with CBF to activate the enhancer. A GA-rich region at -162 contributed to cooperation with CBF when the adjacent c-Myb-binding site was intact. Mutation of both c-Myb-binding sites in the context of the entire enhancer greatly impaired activation by endogenous CBF in 32D cells. Similarly, activation by c-Myb was impaired in constructs lacking the CBF-binding site. CBF and c-Myb were required for induction of MPO proximal enhancer activity when 32D cells differentiated in response to G-CSF. A fusion protein containing the Gal4 DNA-binding domain and the AML-1B activation domain, amino acids 216 to 480, activated transcription alone and cooperatively with c-Myb in nonmyeloid CV-1 cells. Determining how CBF and c-Myb synergize in myeloid cells might contribute to our understanding of leukemogenesis by the AML1-ETO, AML1-MDS1, CBFbeta-SMMHC, and v-Myb oncoproteins.
Mol Cell Biol 1997 Sep
PMID:Core binding factor cannot synergistically activate the myeloperoxidase proximal enhancer in immature myeloid cells without c-Myb. 927 90

We recently demonstrated that cyclic GMP (cGMP)-dependent protein kinase (G-kinase) activates the human fos promoter in a strictly cGMP-dependent manner (T. Gudi et al., J. Biol. Chem. 271:4597-4600, 1996). Here, we demonstrate that G-kinase translocates to the nucleus by an active transport mechanism which requires a nuclear localization signal (NLS) and is regulated by cGMP. Immunofluorescent staining of G-kinase was predominantly cytoplasmic in untreated cells, but intense nuclear staining appeared in 8-bromo (Br)-cGMP-treated cells. We identified a putative NLS in the G-kinase ATP binding domain which resembles the NLS of the interleukin-1alpha precursor. Fusion of the G-kinase NLS to the N terminus of beta-galactosidase produced a chimeric protein which localized to the nucleus. Mutation of a single amino acid residue (K407-->E) within the G-kinase NLS produced an enzyme with normal cGMP-dependent activity in vitro which did not translocate to the nucleus and did not transactivate the fos promoter in the presence of 8-Br-cGMP in vivo. In contrast, N-terminally truncated versions of G-kinase with constitutive, cGMP-independent activity in vitro localized to the nucleus and transactivated the fos promoter in the absence of 8-Br-cGMP. These results indicate that nuclear localization of G-kinase is required for transcriptional activation of the fos promoter and suggest that a conformational change of the kinase, induced by cGMP binding or by removal of the N-terminal autoinhibitory domain, functionally activates an otherwise cryptic NLS.
Mol Cell Biol 1997 Sep
PMID:Regulation of gene expression by cyclic GMP-dependent protein kinase requires nuclear translocation of the kinase: identification of a nuclear localization signal. 927 2

Listeria monocytogenes is an intracellular bacterial pathogen that expresses several surface proteins critical for the infectious process. Such proteins include InlA (internalin) and InlB, involved in bacterial entry into the host cell, and ActA, required for bacterially induced actin-based motility. Although the molecular mechanisms of attachment of InlA and ActA have been characterized, essentially nothing is known about how InlB is anchored to the bacterial surface. Using a genetic approach, we demonstrate that the last 232 amino acids of InlB are both necessary and sufficient for anchoring this protein to the bacterial surface. An InlB mutant protein deleted for the last 232 amino acids was secreted and not detected at the cell surface. A 'domain-swapping' strategy in which these 232 amino acids were used to replace the normal cell wall-anchoring domain of InlA resulted in a chimeric protein that was anchored to the cell surface and able to confer entry. Interestingly, surface association of InlB also occurred when InlB was added externally to bacteria, suggesting that association may be able to occur after secretion. This association was productive for invasion, as it conferred bacterial entry into host cells. The C-terminal anchoring region in InlB contains 80-amino-acid repeats beginning with the sequence GW that is also present in a newly identified surface-associated bacteriolysin of L. monocytogenes, called Ami. Addition of GW repeats to the C-terminal of InlB improves anchoring of the protein to the cell surface. These and other data suggest that such 'GW' repeats may constitute a novel motif for cell-surface anchoring in Listeria and other Gram-positive bacteria. This motif may have important consequences for the release of surface proteins involved in interactions with eukaryotic cells.
Mol Microbiol 1997 Jul
PMID:InlB: an invasion protein of Listeria monocytogenes with a novel type of surface association. 928 40

The calcium-binding basement membrane protein fibulin-1C was shown to bind nidogen in a calcium-dependent fashion. Fibulin-1C consists of small N (domain 1) and C-terminal (domain III) globular structures connected by a central rod (domain II) composed of nine epidermal growth factor (EG) modules, eight of which possess a consensus sequence for calcium binding. Several point and deletion mutants and chimeric protein constructs were used to define the nidogen binding epitope of fibulin-1C by surface plasmon resonance and solid phase assays. All recombinant products were obtained from transfected kidney cells in a folded form as shown by CD spectroscopy, electron microscopy and proteolysis. They were used to demonstrate that calcium-binding is essentially due to the EG modules possessing the consensus binding sequence. Deletion of domain III caused a 30-fold reduction in nidogen binding, whereas deletion of domain I had no effect, yet domain III alone was also inactive. Successive deletions of two to seven EG modules of domain II also caused partial of complete inactivation of binding depending on how many were deleted or their position relative to domain III. Site-directed mutagenesis within the calcium binding consensus sequences demonstrated a similar dependence. Replacement of seven of the calcium-binding modules by a similar tandem array from a related protein showed a distinct (fibulin-2) to almost complete loss of binding (fibrillin-1). This indicates a complex epitope structure involving domains II and III, which each may provide binding epitopes or stabilize each other.
J Mol Biol 1997 Sep 19
PMID:Binding of fibulin-1 to nidogen depends on its C-terminal globular domain and a specific array of calcium-binding epidermal growth factor-like (EG) modules. 929 50

A gene-trap vector, pWH14, has been developed to tag genes expressed in embryonic stem (ES) cells of the mouse. The approach relies on the ability of the endogenous promoter to drive promoterless neo-IRES-lacZ construct producing a dicistronic mRNA consisting of the neomycin-resistance (neo) gene and the beta-galactosidase gene sequence. The neo gene produces a chimeric protein with the truncated product of the tagged gene and serves as a selectable marker for an insertion into an expressed gene. The internal ribosome entry site (IRES) sequence from murine encephalomyocarditis virus allows the translation of the second cistron, lacZ, to produce beta-galactosidase that can be used as a reporter for the expression of the tagged gene. The pWH14 vector was introduced into ES cells by electroporation, and the cells were selected for G418-resistance. About 50% of the G418-resistant colonies were stained positive for the beta-galactosidase activity. Southern analysis showed that each clone had one or more vector sequences integrated. Northern blot analysis of the clones positive for beta-galactosidase indicated that the fused RNAs containing the neo and the beta-gal genes were derived from the endogenous promoters of the tagged genes. Seven clones were chosen and injected into blastocysts, and chimeras were obtained. Two of the gene-trap insertions (wh14.1 and wh14.3) were transmitted through germ-line. In these two lines, the pattern of lacZ expression was restricted to early stages of embryos. This gene-trap vector may provide a means for tagging and studying the active genes in vivo in early embryogenesis.
Mol Cells 1997 Aug 31
PMID:Dicistronic tagging of genes active in embryonic stem cells of mice. 933 94


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