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Volume 272, Number 16, Issue of April 18, 1997 pp. 10895-10903
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Identification of a Region within the Ubiquitin-activating Enzyme Required for Nuclear Targeting and Phosphorylation

(Received for publication, January 10, 1997)

Andrew G. Stephen , Julie S. Trausch-Azar , Patricia M. Handley-Gearhart , Aaron Ciechanover ^¶ and Alan L. Schwartz

From the  Edward Mallinckrodt Departments of Pediatrics and Molecular Biology and Pharmacology, Washington University School of Medicine and Division of Pediatric Hematology-Oncology, Children's Hospital, St. Louis, Missouri 63110 and the ^¶ Unit of Biochemistry and Rappaport Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel

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.

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