Identification of a region within the ubiquitin-activating enzyme required for nuclear targeting and phosphorylation.
The ubiquitin-activating enzyme exists as two isoforms: E1a, localized predominantly in the nucleus, and E1b, localized in the cytoplasm. Previously we generated hemagglutinin (HA) epitope-tagged cDNA constructs, HA1-E1 (epitope tag placed after the first methionine) and HA2-E1 (epitope tag placed after the second methionine) (Handley-Gearhart, P. M., Stephen, A. G., Trausch-Azar, J. S., Ciechanover, A., and Schwartz, A. L. (1994) J. Biol. Chem. 269, 33171-33178), which represent the native isoforms. HA1-E1 is exclusively nuclear, whereas HA2-E1 is found predominantly in the cytoplasm. Using high resolution isoelectric focusing and SDS-polyacrylamide gel electrophoresis, we confirm that these epitope-tagged constructs HA1-E1 and HA2-E1 represent the two isoforms E1a and E1b. HA1-E1/E1a exists as one non-phosphorylated and four phosphorylated forms, and HA2-E1/E1b exists as one predominant non-phosphorylated form and two minor phosphorylated forms. We demonstrate that the first 11 amino acids are essential for phosphorylation and exclusive nuclear localization of HA1-E1. Within this region are four serine residues and a putative nuclear localization sequence (NLS; 5PLSKKRR). Removal of these four serine residues reduced phosphorylation levels by 60% but had no effect on nuclear localization of HA1-E1. Each serine residue was independently mutated to an alanine and analyzed by two-dimensional electrophoresis; only serine 4 was phosphorylated. Disruption of the basic amino acids within the NLS resulted in loss of exclusive nuclear localization and a 90-95% decrease in the phosphorylation of HA1-E1. This putative NLS was able to confer nuclear import on a non-nuclear protein in digitonin-permeabilized cells in a temperature- and ATP-dependent manner. Thus the predominant requirement for efficient phosphorylation of HA1-E1/E1a is a functional NLS, suggesting that E1a may be phosphorylated within the nucleus.
Stephen
A G
AG
Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Trausch-Azar
J S
JS
Handley-Gearhart
P M
PM
Ciechanover
A
A
Schwartz
A L
AL
D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology; D018076:DNA, Complementary; D000939:Epitopes; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D016324:Sequence Tagged Sites; D012694:Serine; D014162:Transfection; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases
D018076:DNA, Complementary; D000939:Epitopes; D007527:Isoenzymes; D010710:Phosphates; D011994:Recombinant Proteins; D012694:Serine; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes