Interaction of cyclooxygenases with an apoptosis- and autoimmunity-associated protein.
Cyclooxygenases (COXs) 1 and 2 are 72-kDa, intralumenal residents of the endoplasmic reticulum (ER) and nuclear envelope, where they catalyze the rate-limiting steps in the conversion of arachidonate to the physiologically dynamic prostanoids. Recent studies, including the generation of knockout mice, show COX-1 and COX-2 to have biologically distinct roles within cells and organisms. Also apparent is that arachidonate substrate is selectably metabolized by COX-2 after mitogen stimulation in many cells that contain both isoforms. Because COX-1 and COX-2 are highly conserved in all residues needed for catalysis and in their purified forms have almost identical kinetic properties, we have searched for COX-interacting ER proteins that might mediate these unique isoenzymic properties. Using COXs as bait in the yeast two-hybrid system, we identified autoimmunity- and apoptosis-associated nucleobindin (Nuc) as a protein that specifically interacts with both isoenzymes. COX-Nuc binding was substantiated by immunoprecipitation experiments, which showed that COX-1 and, to a lesser extent, COX-2 form complexes with Nuc in vitro. When overexpressed in COS-1 cells, Nuc was found to be extracellularly released. However, when Nuc was co-overexpressed with COX-1 or COX-2, its release was reduced by >80%. This finding suggests that COX isoenzymes participate in the retention of Nuc within the lumen of the ER, where COX may regulate the release of Nuc from the cell. It also identifies Nuc as a potential regulator of COXs through this interaction.
Ballif
B A
BA
Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USA.
Mincek
N V
NV
Barratt
J T
JT
Wilson
M L
ML
Simmons
D L
DL
D000818:Animals; D017209:Apoptosis; D001327:Autoimmune Diseases; D001665:Binding Sites; D002135:Calcium-Binding Proteins; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004926:Escherichia coli; D006133:Growth Substances / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008861:Microsomes / Q000201:enzymology; D008958:Models, Molecular; D009419:Nerve Tissue Proteins; D009685:Nuclear Envelope / Q000201:enzymology; D000080866:Nucleobindins; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis
D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D006133:Growth Substances; D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D011994:Recombinant Proteins; D011451:Prostaglandin-Endoperoxide Synthases; D001616:beta-Galactosidase