Overexpression of the Bcl-2 protein increases the half-life of p21Bax.
Bcl-2 and Bax are homologous proteins which can heterodimerize with each other. These proteins have opposing effects on cell survival when overexpressed in cells, with Bcl-2 blocking and Bax promoting apoptosis. Here we demonstrate that gene transfer-mediated elevations in Bcl-2 protein levels result in a marked increase in the steady-state levels of endogenous p21Bax protein as determined by immunoblotting in the Jurkat T-cell and 697 pre-B-cell leukemia cell lines, but not in several other cell lines including CEM T-cell leukemia, 32D.3 myeloid progenitor, PC12 pheochromocytoma, and NIH-3T3 fibroblasts. Steady-state levels of p21Bax protein were also elevated in the lymph nodes of Bcl-2 transgenic mice in which a BCL-2 transgene is expressed at high levels in B-cells. Northern blot analysis of BCL-2-transfected and control-transfected Jurkat and 697 leukemia cells revealed no Bcl-2-induced alterations in the steady-state levels of BAX mRNAs. In contrast, L-[35S]methionine pulse-chase analysis indicated a marked increase in the half-life (t1/2) of the p21Bax protein in BCL-2-transfected 697 cells compared to control-transfected cells (t1/2 > 24 h versus approximately 4 h), whereas the rate of Bax degradation was unaltered in Bcl-2-transfected CEM cells. The results demonstrate that levels of the proapoptotic p21Bax protein can be post-translationally regulated by Bcl-2, probably in a tissue-specific fashion, and suggest the existence of a feedback mechanism that may help to maintain the ratio of Bcl-2 to Bax protein in physiologically appropriate ranges.
Miyashita
T
T
La Jolla Cancer Research Foundation, California 92037, USA.
Kitada
S
S
Krajewski
S
S
Horne
W A
WA
Delia
D
D
Reed
J C
JC
D016475:3T3 Cells; D000310:Adrenal Gland Neoplasms; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D005246:Feedback; D015870:Gene Expression; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D015458:Leukemia, T-Cell; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010673:Pheochromocytoma; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014162:Transfection; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; C494827:Bax protein, mouse; C494828:Bax protein, rat; D017931:DNA Primers; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D011994:Recombinant Proteins; D051028:bcl-2-Associated X Protein; D008715:Methionine; D011505:Protein-Tyrosine Kinases