Extracellular Bad fused to toxin transport domains induces apoptosis.
Bad, a proapoptotic member of the Bcl-2 family, is inactivated by phosphorylation, and this loss of activity may contribute to the malignancy of certain types of tumors such as glioblastoma and prostate cancer. To determine whether extracellular Bad can be delivered into cells via cell surface receptor binding and induce apoptosis, we genetically fused the mouse Bad gene to the gene for the translocation and receptor-binding domains of diphtheria toxin (DTTR). The purified Bad (wild-type)-DTTR protein showed cytotoxicity to human glioma cells in a dose-dependent manner. Bad phosphorylation sites at codons 112 and 136 were mutated from serine to alanine to prevent Bad inactivation by kinases and to increase the toxicity of Bad. The Bad (S112A S136A)-DTTR protein was at least 5 times more toxic than Bad (wild-type)-DTTR with an IC(50) of 5 x 10(-8) M. The Bad (S112A S136A)-DTTR protein altered the subcellular distribution of Bcl-X(L), indicating that it enters the cell cytoplasm and binds Bcl-X(L). Bad (S112D S136A)-DTTR, mutated to mimic phosphorylation of Bad, showed lower toxicity than either Bad (wild-type)-DTTR or Bad (S112A S136A)-DTTR, additionally indicating that Bad-DTTR must bind Bcl-X(L) to stimulate apoptosis. We conclude that extracellular Bad can be delivered into cells via the transport domain of a bacterial toxin and may be used to induce apoptosis.
Ichinose
Makoto
M
Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
Liu
Xiu-Huai
XH
Hagihara
Naoshi
N
Youle
Richard J
RJ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001692:Biological Transport; D002352:Carrier Proteins / Q000494:pharmacology*; D004167:Diphtheria Toxin / Q000494:pharmacology*; D006801:Humans; D051059:PTEN Phosphohydrolase; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics; D010766:Phosphorylation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000235:genetics; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein
C494819:BAD protein, human; C494811:BCL2L1 protein, human; D002352:Carrier Proteins; D004167:Diphtheria Toxin; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein; D010744:Phosphoric Monoester Hydrolases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human