The ubiquitin-activating enzyme E1 is phosphorylated and localized to the nucleus in a cell cycle-dependent manner.
The ubiquitin-activating enzyme E1 exists as two isoforms, E1a (117 kDa) and E1b (110 kDa). E1a is phosphorylated, whereas E1b is not. In the present study we have demonstrated the cell cycle dependence of E1a phosphorylation: a 2-fold increase in the specific phosphorylation of E1a in G2 compared with the basal level of phosphorylation in the other stages of the cell cycle. Two-dimensional gel electrophoresis resolved E1 into the two isoforms E1a and E1b; E1a resolved further as three phosphorylated forms and one nonphosphorylated form, while E1b resolved as one nonphosphorylated form. E1a is found predominantly in the phosphorylated forms. However, the distribution of E1a among these different phosphorylated forms was not cell cycle-dependent. We next evaluated the enzymatic activity of E1 as well as its subcellular localization throughout the cell cycle. 32P-Pyrophosphate exchange activity of E1 did not vary along the cell cycle; however, the amount of ubiquitin-protein conjugates decreased by 50% in G2. Nuclear and cytosolic fractionation of cells revealed the nuclear to cytosolic ratio of phosphorylated E1a was 3-fold greater in G2 compared with the other stages of the cell cycle. Finally, purified nuclear extracts supported E1-dependent ubiquitin conjugation of exogenous substrates as did purified cytosol. However, in nuclear extracts but not in cytosol the amount of E1 activity was rate-limiting. Thus we establish nuclear E1-dependent protein ubiquitination and propose that an increase in phosphorylation of E1a in G2 functions to increase the import and/or retention of E1a in the nucleus and may modulate nuclear protein ubiquitination.
Stephen
A G
AG
Edward Mallincrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Trausch-Azar
J S
JS
Ciechanover
A
A
Schwartz
A L
AL
D002451:Cell Compartmentation; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D006367:HeLa Cells; D006801:Humans; D008025:Ligases / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism
D010750:Phosphoproteins; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes