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          Institute: MPI für Herz- und Lungenforschung (W. G. Kerckhoff Institut)     Collection: Publikationen des W. G. Kerckhoff-Instituts     Display Documents



ID: 474481.0, MPI für Herz- und Lungenforschung (W. G. Kerckhoff Institut) / Publikationen des W. G. Kerckhoff-Instituts
Identification of novel in vivo phosphorylation sites of the human proapoptotic protein BAD: pore-forming activity of BAD is regulated by phosphorylation
Authors:Polzien, L.; Baljuls, A.; Rennefahrt, U. E.; Fischer, A.; Schmitz, W.; Zahedi, R. P.; Sickmann, A.; Metz, R.; Albert, S.; Benz, R.; Hekman, M.; Rapp, U. R.
Date of Publication (YYYY-MM-DD):2009
Title of Journal:J Biol Chem
Volume:284
Issue / Number:41
Start Page:28004
End Page:20
Audience:Not Specified
Abstract / Description: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.
Free Keywords:14-3-3 Proteins/genetics/metabolism; Amino Acid Sequence; Animals; Cyclic AMP-Dependent Protein Kinases/metabolism; Humans; Ion Channels/*chemistry/genetics/*metabolism; Lipid Bilayers/metabolism; Mass Spectrometry; Mice; Molecular Sequence Data; NIH 3T3 Cells; Peptides/chemistry/genetics/metabolism; Phosphorylation; Protein Binding; Proto-Oncogene Proteins c-akt/metabolism; Proto-Oncogene Proteins c-bcl-2/genetics/metabolism; Sequence Alignment; bcl-Associated Death Protein/*chemistry/genetics/*metabolism; bcl-X Protein/genetics/metabolism; p21-Activated Kinases/metabolism; raf Kinases/genetics/metabolism
External Publication Status:published
Document Type:Article
Communicated by:N. N.
Affiliations:MPI für physiologische und klinische Forschung
External Affiliations:Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg, Germany.
Identifiers:ISSN:1083-351X (Electronic) 0021-9258 (Linking)
URL:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=..
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