No abstract

The Rel-associated protein pp40 is functionally related to I kappa B, an inhibitor of the transcription factor NF-kappa B. Purified pp40 inhibits the DNA binding activity of the NF-kappa B protein complex (p50:p65 heterodimers), p50:c-Rel heteromers, and c-Rel homodimers. The sequence of the complementary DNA encoding pp40 revealed similarity to the gene encoding MAD-3, a protein with mammalian I kappa B-like activity. Protein sequencing of I kappa B purified from rabbit lung confirmed that MAD-3 encodes a protein similar to I kappa B. The sequence similarity between MAD-3 and pp40 includes a casein kinase II and consensus tyrosine phosphorylation site, as well as five repeats of a sequence found in the human erythrocyte protein ankyrin. These results suggest that rel-related transcription factors, which are capable of cytosolic to nuclear translocation, may be held in the cytosol by interaction with related cytoplasmic anchor molecules.

To understand the mechanism by which pp4O/IKcBl inhibits DNA binding activity of the rel/NF-jcB family of transcription factors, we have investigated the role of ankyrin repeats on the biological function of pp4O by deleting or mutating conserved residues. We show that (s) ankyrin repeats alone are not sufficient to manifest biological activity but require the C-terminal region of the pp4O protein; (ii) four out of the five ankyrin repeats are essential for inhibiting the DNA binding activity; (Mi) pp4O mutants that do not inhibit DNA binding of rel protein also do not associate with rel; (iv) although pp4O can associate with the p65 and p50 subunits of NF-cB, pp4O inhibits the DNA binding activity of only the p50-p65 heterodimer and the p65 homodimer; and (v) pp4Oinhibits the transcription of genes linked to KB site; however, mutants that do not affect DNA binding have no effect. We propose that the ankyrin repeats and the C-terminal region of pp4O form a structure that associates with the rel homology domain to inhibit DNA binding activity.

The structural genes coding for both subunits of adenosylcobalamin-dependent methylmalonyl-CoA mutase from the Gram-positive bacterium Propionibacterium shermanii have been cloned, with the use of synthetic oligonucleotides as primary hybridization probes. The genes are closely linked and are transcribed in the same direction. Nucleotide sequence analysis of 4.5 kb of DNA encompassing both genes allowed us to infer the complete amino acid sequence of the two subunits: the beta-subunit is the product of the upstream gene, and consists of 638 amino acid residues (Mr 69465) and the alpha-subunit consists of 728 amino acid residues (Mr 80,147). There is a very close structural homology between the two subunits, reflecting the probable duplication of a common ancestral gene. A sequence present only in the alpha-subunit is significantly homologous to a portion of the sequence of the methylmalonyl-CoA-binding subunit of transcarboxylase from P. shermanii [Samols, Thornton, Murtif, Kumar, Haase & Wood (1988) J. Biol. Chem. 263, 6461-6464], and this homologous region may form part of the CoA ester-binding site in both enzymes.

Synemin is an intermediate filament protein present in glioblastomas (GBMs) but not in normal brain. In GBM cells synemin interacts with and antagonizes PP2A, which is the phosphatase dephosphorylating Akt. This maintains the phosphorylation status of Akt sites that are substrates for PDPK1 and mTORc2, thereby fostering proliferation.

5'-Deoxyadenosylcobalamin-dependent methylmalonyl-CoA mutase was purified to homogeneity from Propionibacterium shermanii by a simplified procedure. The native enzyme has an apparent Mr of 165,000, similar to the enzyme from other sources but larger than previously reported. It consists of two non-identical subunits, of Mr 79,000 and 67,000 respectively. The smaller subunit is apparently not a proteolytic fragment of the larger one. The final preparation usually contained some inactive mutase, bearing a tenaciously bound cobalamin species. This protein proved to be readily separable from apoenzyme by fast protein liquid chromatography on anion-exchange columns.

~ ~~ ~~~~Two independent mutants defective in glutathione reductase (EC 1 .6.4.2) were isolated in an Escherichia coli K12 strain lysogenized with bacteriophage Mu. The prophage was lost (and the ability to reduce glutathione regained) by 32% of the xylose-positive transductants when T4GT7 was used as the vector, but the markers were not cotransduced by P1. Similarly, the prophage site and ma2A were cotransduced by T4GT7 but not by P 1. The gor gene maps between min 77 and 78 on the E. coli genome, and the mutation causes no growth defect.

The v-rel oncogene of avian reticuloendotheliosis virus type T (REV-T) encodes a 59-kilodalton (kDa) phosphoprotein located principally in the cytosol of transformed lymphoid cells. All of the detectable pp59v-rel was present in high-molecular-weight complexes containing at least five cellular proteins (p124, p115, p75c-rel p70hsC, and pp4O). Antiserum was developed against the 40-kDa protein, the most abundant cellular protein associated with the complex. The 40-kDa phosphoprotein was complexed with pp59v-rel in REV-T-transformed lymphoid cell lines arrested at different stages of B-cell development as well as in lymphoid tumor cells and in fibrosarcomas. The half-life (8 h) of pp4O in REV-T-transformed lymphoid cells was the same as that of pp59v-rel. Antiserum against pp4O permitted the identification of two pp59v-rel complexes. The most abundant cytoplasmic complex contained approximately 75% of the pp59v-rel and all of the detectable pp4O in REV-T-transformed lymphoid cells. Twenty-five percent of the pp59v-rel was present in a minor complex that contained the majority of p75crel along with p115 and p124. In nuclear extracts of REV-T-transformed lymphoid cells, pp59v-re was complexed with pp4O. The two high-molecular-weight proteins (p115 and p124) and p75c're were not detected in the nuclear complex. In the cytosolic complexes, pp4O was heavily phosphorylated, whereas the nuclear form was much less extensively phosphorylated.