Overexpressed full-length human BCL2 extends the survival of baculovirus-infected Sf9 insect cells.
Full-length and truncated human BCL2 lacking the entire C-terminal hydrophobic domain have been overexpressed in Spodoptera frugiperda insect cells with the baculovirus expression system. Immunoblot analysis with BCL2-specific antibodies revealed that both full-length and truncated BCL2 are expressed as multiple immunoreactive species, suggesting posttranslational modifications. The expression of the full-length but not the truncated BCL2 extended the survival of baculovirus-infected cells by preventing virus-induced DNA cleavage. This result is consistent with the reported protective effect of BCL2 against apoptosis in mammalian lymphocytes and suggests a conserved function in evolution. Subcellular fractionation and indirect immunofluorescence studies in intact cells demonstrated that the recombinant full-length and truncated BCL2 proteins were expressed predominantly as nuclear membrane-associated proteins. These results imply that BCL2 must utilize hydrophobic domains other than the deleted domain for its association with the subcellular membranes. Metabolic labeling of insect cells expressing the full-length and the truncated form of BCL2 with 32P(i) demonstrated that BCL2 is a phosphoprotein.
Alnemri
E S
ES
Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107.
Robertson
N M
NM
Fernandes
T F
TF
Croce
C M
CM
Litwack
G
G
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D016923:Cell Death; D002470:Cell Survival / Q000235:genetics*; D019938:Cyclin D1; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009036:Moths; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011519:Proto-Oncogenes; D015183:Restriction Mapping; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D019938:Cyclin D1; D011505:Protein-Tyrosine Kinases