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.

Regulation of RAF activity by 14-3-3 proteins: RAF kinases associate functionally with both homo- and heterodimeric forms of 14-3-3 proteins.

Mammalian 14-3-3 proteins play a crucial role in the activation process of RAF kinases. However, little is known about the selectivity of the mammalian 14-3-3 isoforms with respect to RAF association and activation. Using mass spectrometry, we analyzed the composition of the 14-3-3 isoforms attached to RAF kinases and found that B-RAF associates in vivo with 14-3-3 at much higher diversity than A- and C-RAF. We also examined in vitro binding of purified mammalian 14-3-3 proteins to RAF kinases using surface plasmon resonance techniques. While B- and C-RAF exhibited binding to all seven 14-3-3 isoforms, A-RAF bound with considerably lower affinities to epsilon, tau, and sigma 14-3-3. These findings indicate that 14-3-3 proteins associate with RAF isoforms in a pronounced isoform-specific manner. Because 14-3-3 proteins appear in dimeric forms, we addressed the question of whether both homo- and heterodimeric forms of 14-3-3 proteins participate in RAF signaling. For that purpose, the budding yeast Saccharomyces cerevisiae, possessing only two 14-3-3 isoforms (BMH1 and BMH2), served as testing system. By deletion of the single BMH2 gene, we found that both homo- and heterodimeric forms of 14-3-3 can participate in RAF activation. Furthermore, we show that A-, B-, and C-RAF activity is differentially regulated by its C-terminal and internal 14-3-3 binding domain. Finally, prohibitin, a scaffold protein that affects C-RAF activation in a stimulatory manner, proved to interfere with the internal 14-3-3 binding site in C-RAF. Together, our results shed more light on the complex mechanism of RAF activation, particularly with respect to activation steps that are mediated by 14-3-3 proteins and prohibitin.

Dynamic changes in C-Raf phosphorylation and 14-3-3 protein binding in response to growth factor stimulation: differential roles of 14-3-3 protein binding sites.

Phosphorylation events play a crucial role in Raf activation. Phosphorylation of serines 259 and 621 in C-Raf and serines 364 and 728 in B-Raf has been suggested to be critical for association with 14-3-3 proteins. To study the functional consequences of Raf phosphorylations at these positions, we developed and characterized phosphospecific antibodies directed against 14-3-3 binding epitopes: a monoclonal phosphospecific antibody (6B4) directed against pS621 and a polyclonal antibody specific for B-Raf-pS364 epitope. Although 6B4 detected both C- and B-Raf in Western blots, it specifically recognizes the native form of C-Raf but not B-Raf. Contrary to B-Raf, a kinase-dead mutant of C-Raf was found to be only poorly phosphorylated in the Ser-621 position. Moreover, serine 259 to alanine mutation prevented the Ser-621 phosphorylation suggesting an interdependence between these two 14-3-3 binding domains. Direct C-Raf.14-3-3 binding studies with purified proteins combined with competition assays revealed that the 14-3-3 binding domain surrounding pS621 represents the high affinity binding site, whereas the pS259 epitope mediates lower affinity binding. Raf isozymes differ in their 14-3-3 association rates. The time course of endogenous C-Raf activation in mammalian cells by nerve growth factor (NGF) has been examined using both phosphospecific antibodies directed against 14-3-3 binding sites (6B4 and anti-pS259) as well as phosphospecific antibodies directed against the activation domain (anti-pS338 and anti-pY340/pY341). Time course of Ser-621 phosphorylation, in contrast to Ser-259 phosphorylation, exhibited unexpected pattern reaching maximal phosphorylation within 30 s of NGF stimulation. Phosphorylation of tyrosine 340/341 reached maximal levels subsequent to Ser-621 phosphorylation and was coincident with emergence of kinase activity. Taken together, we found substantial differences between C-Raf.14-3-3 binding epitopes pS259 and pS621 and visualized for the first time the sequence of the essential C-Raf phosphorylation events in mammalian cells in response to growth factor stimulation.