Identification of novel in vivo phosphorylation sites of the human proapoptotic protein BAD: pore-forming activity of BAD is regulated by phosphorylation.
BAD is a proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Although much attention has been devoted to the identification of phosphorylation sites in murine BAD, little data are available with respect to phosphorylation of human BAD protein. Using mass spectrometry, we identified here besides the established phosphorylation sites at serines 75, 99, and 118 several novel in vivo phosphorylation sites within human BAD (serines 25, 32/34, 97, and 124). Furthermore, we investigated the quantitative contribution of BAD targeting kinases in phosphorylating serine residues 75, 99, and 118. Our results indicate that RAF kinases represent, besides protein kinase A, PAK, and Akt/protein kinase B, in vivo BAD-phosphorylating kinases. RAF-induced phosphorylation of BAD was reduced to control levels using the RAF inhibitor BAY 43-9006. This phosphorylation was not prevented by MEK inhibitors. Consistently, expression of constitutively active RAF suppressed apoptosis induced by BAD and the inhibition of colony formation caused by BAD could be prevented by RAF. In addition, using the surface plasmon resonance technique, we analyzed the direct consequences of BAD phosphorylation by RAF with respect to association with 14-3-3 and Bcl-2/Bcl-X(L) proteins. Phosphorylation of BAD by active RAF promotes 14-3-3 protein association, in which the phosphoserine 99 represented the major binding site. Finally, we show here that BAD forms channels in planar bilayer membranes in vitro. This pore-forming capacity was dependent on phosphorylation status and interaction with 14-3-3 proteins. Collectively, our findings provide new insights into the regulation of BAD function by phosphorylation.
Polzien
Lisa
L
Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg.
Baljuls
Angela
A
Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg.
Rennefahrt
Ulrike E E
UEE
Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg.
Fischer
Andreas
A
Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg.
Schmitz
Werner
W
Institute of Physiological Chemistry, University of Wuerzburg, 97078 Wuerzburg.
Zahedi
Rene P
RP
Institute for Analytical Sciences, Department of Bioanalytics, 44139 Dortmund.
Sickmann
Albert
A
Institute for Analytical Sciences, Department of Bioanalytics, 44139 Dortmund; Medical Proteome Center, Ruhr University of Bochum, 44801 Bochum, Germany.
Metz
Renate
R
Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg.
Albert
Stefan
S
Julius-von-Sachs Institute for Biosciences, University of Wuerzburg, 97078 Wuerzburg.
Benz
Roland
R
Institute of Biotechnology, University of Wuerzburg, 97078 Wuerzburg.
Hekman
Mirko
M
Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg.
Rapp
Ulf R
UR
Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg. Electronic address: rappur@mail.uni-wuerzburg.de.
D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006801:Humans; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D051025:bcl-Associated Death Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism; D054462:p21-Activated Kinases / Q000378:metabolism; D048490:raf Kinases / Q000235:genetics / Q000378:metabolism
D048948:14-3-3 Proteins; C494819:BAD protein, human; C494820:Bad protein, mouse; D007473:Ion Channels; D008051:Lipid Bilayers; D010455:Peptides; D019253:Proto-Oncogene Proteins c-bcl-2; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein; D051057:Proto-Oncogene Proteins c-akt; D054462:p21-Activated Kinases; D048490:raf Kinases; D017868:Cyclic AMP-Dependent Protein Kinases