Bcl-2 undergoes phosphorylation by c-Jun N-terminal kinase/stress-activated protein kinases in the presence of the constitutively active GTP-binding protein Rac1.
We have studied the phosphorylation of the Bcl-2 family of proteins by different mitogen-activated protein (MAP) kinases. Purified Bcl-2 was found to be phosphorylated by the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) p54-SAPKbeta, and this is specific insofar as the extracellular signal-regulated kinase 1 (ERK1) and p38/RK/CSBP (p38) catalyzed only weak modification. Bcl-2 undergoes similar phosphorylation in COS-7 when coexpressed together with p54-SAPKbeta and the constitutive Rac1 mutant G12V. This is seen by both 32PO4 labeling and the appearance of five discrete Bcl-2 bands with reduced gel mobility. As anticipated, both intracellular p54-SAPKbeta activation and Bcl-2 phosphorylation are blocked by co-transfection with the MAP kinase specific phosphatase MKP3/PYST1. MAP kinase specificity is also seen in COS-7 cells as Bcl-2 undergoes only weak phosphorylation when co-expressed with enzymatically activated ERK1 or p38. Four critical residues undergoing phosphorylation in COS-7 cells were identified by expression of the quadruple Bcl-2 point mutant T56A,S70A,T74A, S87A. Sequencing phosphopeptides derived from tryptic digests of Bcl-2 indicates that purified GST-p54-SAPKbeta phosphorylates identical sites in vitro. This is the first report of Bcl-2 phosphorylation by the JNK/SAPK class of MAP kinases and could indicate a key modification allowing control of Bcl-2 function by cell surface receptors, Rho family GTPases, and/or cellular stresses.
Maundrell
K
K
Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development S.A., CH-1228 Plan-les-Ouates, Geneva, Switzerland.
Antonsson
B
B
Magnenat
E
E
Camps
M
M
Muda
M
M
Chabert
C
C
Gillieron
C
C
Boschert
U
U
Vial-Knecht
E
E
Martinou
J C
JC
Arkinstall
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D017931:DNA Primers; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases; D048057:Mitogen-Activated Protein Kinase 10; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D020744:rac GTP-Binding Proteins
D017931:DNA Primers; D010446:Peptide Fragments; D010748:Phosphopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D048057:Mitogen-Activated Protein Kinase 10; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins