IDENTIFICATION OF A REGION WITHIN THE UBIQUITIN-ACTIVATING ENZYME REQUIRED FOR NUCLEAR TARGETING AND PHOSPHORYLATION

The ubiquitin-activating enzyme exists as two isoforms: Ela, localized predominantly in the nucleus, and E1b, localized in the cytoplasm, Pr eviously we generated hemagglutinin (HA) epitope-tagged cDNA construct s, 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, 35171-33178), which represent the n ative isoforms, HA1-E1 is exclusively nuclear, whereas HA2-E1 is found predominantly in the cytoplasm, Using high resolution isoelectric foc using and SDS-polyacrylamide gel electrophoresis, we confirm that thes e epitope-tagged constructs HA1-E1 and HA2-E1 represent the two isofor ms Ela and E1b, HA1E1/E1a exists as one non-phosphorylated and four ph osphorylated forms, and HA2-E1/E1b exists as one predominant non-phosp horylated form and two minor phosphorylated forms, We demonstrate that the first 11 amino acids are essential for phosphorylation and exclus ive nuclear localization of HA1-E1, Within this region are four serine residues and a putative nuclear localization sequence (NLS; (PLSKKRR) -P-5), Removal of these four serine residues reduced phosphorylation l evels by 60% but had no effect on nuclear localization of HA1-E1, Each serine residue was independently mutated to an alanine and analyzed b y two-dimensional electrophoresis; only serine 4 was phosphorylated, D isruption of the basic amino acids within the NLS resulted in loss of exclusive nuclear localization and a 90-95% decrease in the phosphoryl ation of HA1-E1. This putative NLS was able to confer nuclear import o n a nonnuclear protein in digitonin-permeabilized cells in a temperatu re- and ATP-dependent manner, Thus the predominant requirement for eff icient phosphorylation of HA1-E1/E1a is a functional NLS, suggesting t hat Ela may be phosphorylated within the nucleus.