Identification of nucleolin as an AU-rich element binding protein involved in bcl-2 mRNA stabilization.
bcl-2 mRNA contains an AU-rich element (ARE) that functions in regulating bcl-2 stability. Our earlier studies indicated that taxol- or okadaic acid-induced bcl-2 mRNA destabilization in HL-60 cells is associated with decreased binding of trans-acting factors to the ARE. To identify factors that play a role in the regulation of bcl-2 mRNA stability, bcl-2 ARE-binding proteins were purified from HL-60 cells. Three polypeptides of 100, 70, and 32 kDa were isolated from a bcl-2 ARE affinity matrix. Matrix-assisted laser desorption ionization mass spectroscopy analysis identified these proteins as full-length nucleolin and proteolytic fragments of nucleolin. RNA gel shifts assays indicated that recombinant nucleolin (residues 284-707) binds specifically to bcl-2 ARE RNA. In addition, recombinant nucleolin decreases the rate of decay of mRNA in HL-60 cell extracts in an ARE-dependent manner. Taxol or okadaic acid treatment of HL-60 cells results in proteolysis of nucleolin in a similar time frame as drug-induced bcl-2 mRNA down-regulation. These findings suggest that nucleolin functions as a bcl-2-stabilizing factor and that taxol and okadaic acid treatment induces apoptosis in HL-60 cells through a process that involves down-regulation of nucleolin and destabilization of bcl-2 mRNA.
Sengupta
Tapas K
TK
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Bandyopadhyay
Sumita
S
Fernandes
Daniel J
DJ
Spicer
Eleanor K
EK
D017209:Apoptosis / Q000187:drug effects; D019254:Genes, bcl-2; D018922:HL-60 Cells; D006801:Humans; D019319:Okadaic Acid / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D011485:Protein Binding; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000097796:Nucleolin
D019319:Okadaic Acid; D017239:Paclitaxel; D010750:Phosphoproteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000097796:Nucleolin