Intramolecular masking of the nuclear location signal and dimerization domain in the precursor for the p50 NF-kappa B subunit.

We show that the non-DNA-binding precursor for the p50 subunit (p110), like NF-kappa B, is subject to control of nuclear uptake. In contrast to p50, p110 was excluded from nuclei and unable to associate detectably with p50 or p65 NF-kappa B subunits. The nuclear location signal in the N-terminal half of p110 was not accessible for monospecific antibodies. Removal of only 191 amino acids from the C-terminus of p110 restored antibody accessibility as well as nuclear uptake. The C-terminal half of p110, which is linked to the p50 portion via a glycine-rich hinge, could also noncovalently bind to p50. This helps to explain why p50, after cleavage of the precursor in intact cells, was still retained in an inactive form in the cytoplasm. Our study describes a novel mechanism of nuclear uptake control by masking of a nuclear location signal through a remote domain within a precursor molecule.     

Human NCU-G1 can function as a transcription factor and as a nuclear receptor co-activator

Background  

Novel, uncharacterised proteins represent a challenge in biochemistry and molecular biology. In this report we present an initial functional characterization of human kidney predominant protein, NCU-G1.     

The NF-kappa B precursor p105 and the proto-oncogene product Bcl-3 are I kappa B molecules and control nuclear translocation of NF-kappa B.

We have examined the interaction of the NF-kappa B precursor p105 with NF-kappa B subunits. Similar to an I kappa B molecule, p105 associates in the cytoplasm with p50 or p65. Through this assembly, p105 efficiently blocks nuclear transfer of either subunit. Moreover, the p105 protein inhibits DNA binding of dimeric NF-kappa B subunits in a similar, but not identical, manner to its isolated C-terminal domain, which contains an ankyrin-like repeat domain (ARD). The proto-oncogene product Bcl-3 also controls nuclear translocation of p50, but not of p65. Hence, p50 can be retained in the cytoplasm via at least three distinct interactions: through direct interactions either with its own precursor, with Bcl-3 or indirectly through I kappa B alpha or -beta when attached to p65. We discuss a function of p105 as a cytoplasmic assembly unit for homo- and heteromeric NF-kappa B complexes and of Bcl-3 as an I kappa B with novel subunit specificity.     

Human RNA 5'-kinase (hClp1) can function as a tRNA splicing enzyme in vivo

Yeast and human Clp1 proteins are homologous components of the mRNA 3′-cleavage-polyadenylation machinery. Recent studies highlighting an association of human Clp1 (hClp1) with tRNA splicing endonuclease and an intrinsic RNA-specific 5′-OH polynucleotide kinase activity of hClp1 have prompted speculation that Clp1 might play a catalytic role in tRNA splicing in animal cells. Here, we show that expression of hClp1 in budding yeast can complement conditional and lethal mutations in the essential 5′-OH RNA kinase module of yeast or plant tRNA ligases. The tRNA splicing activity of hClp1 in yeast is abolished by mutations in the kinase active site. In contrast, overexpression of yeast Clp1 (yClp1) cannot rescue kinase-defective tRNA ligase mutants, and, unlike hClp1, the purified recombinant yClp1 protein has no detectable RNA kinase activity in vitro. Mutations of the yClp1 ATP-binding site do not affect yeast viability. These findings, and the fact that hClp1 cannot complement growth of a yeast clp1Δ strain, indicate that yeast and human Clp1 proteins are not functional orthologs, despite their structural similarity. Although hClp1 can perform the 5′-end-healing step of a yeast-type tRNA splicing pathway in vivo, it is uncertain whether its kinase activity is necessary for tRNA splicing in human cells, given that other mammalian counterparts of yeast-type tRNA repair enzymes are nonessential in vivo.     

DNA binding alters the protease susceptibility of the p50 subunit of NF-kappa B.

The subdomain structure of the p50 subunit of NF-kappa B (amino acids 35-381) was investigated by partial proteolysis of the native protein. Trypsin cleaves p50 at a limited number of sites with an initial cleavage at low trypsin concentration occurring after R362 and a second cleavage taking place at higher trypsin concentration after K77. The cleavage after R362 does not alter the DNA binding characteristics of p50 but removes the nuclear localisation signal indicating that this region occupies a highly exposed position on the surface of the protein. The second cleavage after K77 generates a protein that although dimeric is incapable of binding DNA, thus emphasising the importance of residues 35-77 in DNA recognition. However p50 dimers containing one molecule cleaved after K77 and one molecule with this region intact are capable of binding DNA. When very high concentrations of trypsin are employed p50 is completely degraded. However if p50 is bound tightly to DNA containing its specific recognition site prior to trypsin addition the cleavage after K77 is almost completely blocked and the protein becomes highly resistant to proteolysis. These data suggest that bound DNA may mask critical trypsin cleavage sites or that DNA binding is accompanied by a conformational change in protein structure that renders the protein resistant to proteolysis.     

BCL3 encodes a nuclear protein which can alter the subcellular location of NF-kappa B proteins.

BCL3 is a candidate proto-oncogene involved in the recurring translocation t(14;19) found in some patients with chronic lymphocytic leukemia. BCL3 protein acts as an I kappa B in that it can specifically inhibit the DNA binding of NF-kappa B factors. Here, we demonstrate that BCL3 is predominantly a nuclear protein and provide evidence that its N terminus is necessary to direct the protein into the nucleus. In contrast to I kappa B alpha (MAD3), BCL3 does not cause NF-kappa B p50 to be retained in the cytoplasm; instead, in cotransfection assays, it alters the subnuclear localization of p50. The two proteins colocalize, suggesting that they interact in vivo. Further immunofluorescence experiments showed that a mutant p50, lacking a nuclear localization signal and restricted to the cytoplasm, is brought into the nucleus in the presence of BCL3. Correspondingly, a wild-type p50 directs into the nucleus a truncated BCL3, which, when transfected alone, is found in the cytoplasm. We tested whether BCL3 could overcome the cytoplasmic retention of p50 by I kappa B alpha. Results from triple cotransfection experiments with BCL3, I kappa B alpha, and p50 implied that BCL3 can successfully compete with I kappa B alpha and bring p50 into the nucleus; thus, localization of NF-kappa B factors may be affected by differential expression of I kappa B proteins. These novel properties of BCL3 protein further establish BCL3 as a distinctive member of the I kappa B family.     

The bZIP transactivator of Epstein-Barr virus, BZLF1, functionally and physically interacts with the p65 subunit of NF-kappa B.

The Epstein-Barr virus (EBV) BZLF1 (Z) immediate-early transactivator initiates the switch between latent and productive infection in B cells. The Z protein, which has homology to the basic leucine zipper protein c-Fos, transactivates the promoters of several replicative cycle proteins. Transactivation efficiency of the EBV BMRF1 promoter by Z is cell type dependent. In B cells, in which EBV typically exists in a latent form, Z activates the BMRF1 promoter inefficiently. We have discovered that the p65 component of the cellular factor NF-kappa B inhibits transactivation of several EBV promoters by Z. Furthermore, the inhibitor of NF-kappa B, I kappa B alpha, can augment Z-induced transactivation in the B-cell line Raji. Using glutathione S-transferase fusion proteins and coimmunoprecipitation studies, we demonstrate a direct interaction between Z and p65. This physical interaction, which requires the dimerization domain of Z and the Rel homology domain of p65, can be demonstrated both in vitro and in vivo. Inhibition of Z transactivation function by NF-kappa B p65, or possibly by other Rel family proteins, may contribute to the inefficiency of Z transactivator function in B cells and may be a mechanism of maintaining B-cell-specific viral latency.