Regulation of RAF Activity by 14-3-3 Proteins

The Syk protein-tyrosine kinase is phosphorylated on multiple tyrosines after the aggregation of the B cell antigen receptor. However, metabolic labeling experiments indicate that Syk is inducibly phosphorylated to an even greater extent on serine after receptor ligation. A combination of phosphopeptide mapping and mass spectrometric analyses indicates that serine 291 is a major site of phosphorylation. Serine 291 lies within a 23-amino acid insert located within the linker B region that distinguishes Syk from SykB and Zap-70. The phosphorylation of serine-291 by protein kinase C enhances the ability of Syk to couple the antigen receptor to the activation of the transcription factors NFAT and Elk-1. Protein interaction studies indicate a role for the phosphorylated linker insert in promoting an interaction between Syk and the chaperone protein, prohibitin.The Syk protein-tyrosine kinase is an essential component of the protein machinery that couples receptors bearing immunoreceptor tyrosine-based activation motifs to intracellular signal transduction pathways in hematopoietic cells (for a recent review, see Ref. 1). Syk is recruited via its N-terminal pair of Src homology 2 domains to a pair of tyrosines located in the cytoplasmic immunoreceptor tyrosine-based activation motifs of immune recognition receptors that become phosphorylated after ligand-induced aggregation. For example, clustering of the B cell receptor for antigen (BCR) 4 leads to the phosphorylation of the accessory molecules CD79a and CD79b, resulting in the recruitment of Syk to the clustered BCR complex. Receptor binding leads to the activation of Syk and to its phosphorylation on tyrosines that regulate its activity and interactions with downstream effectors that bear Src homology 2 or other phosphotyrosine binding domains. This in turn couples the BCR to the activation of, among others, the phospholipase ␥/PKC, PI3K/Akt, and Ras/Raf/Erk signaling pathways. In the absence of Syk, few if any signals are sent after BCR clustering (2-4).Syk contains a stretch of 23 amino acids known as the "linker insert" that lies within the linker B region that separates the tandem pair of Src homology 2 domains from the C-terminal catalytic domain. The presence of this insert distinguishes Syk from SykB, a shorter alternatively spliced isoform, and from ZAP-70, the Syk paralog found in T cells and natural killer cells (5, 6). The role of the linker insert in modulating the properties of Syk is poorly understood, but two functions have been proposed. In one, the linker insert harbors a signal sequence that allows the kinase to transit into the nucleus (7). In another, the insert enhances the ability of the kinase to interact with immunoreceptor tyrosine-based activation motif-bearing receptors (8). Within the linker insert (TWSPGGIISRIKSYSFPKPGHKK) are several potential sites of phosphorylation. The tyrosine (Tyr-290 in the murine Syk sequence) is a site of autophosphorylation in vitro (9) but to our knowledge is not known to be phosphorylated in response to r...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Regulation of Syk by Phosphorylation on Serine in the Linker Insert

Paris

Galán

et al. 2010

“…Purification of Kinases, BAD, Bcl-X L , and 14-3-3 ProteinsExpression and purification of RAF kinases, PAK1, Akt/PKB, 14-3-3 proteins, and Bcl-X L from Sf9 insect cells (an insect cell line derived from pupal ovarian tissue of Spodoptera frugiperda) were performed as previously described (14,(51)(52)(53). For purification of phosphorylated His-hBAD, Sf9 cells were infected with baculoviruses at a multiplicity of infection of 5 and incubated for 48 h at 27°C.…”

Section: Methodsmentioning

confidence: 99%

Identification of Novel in Vivo Phosphorylation Sites of the Human Proapoptotic Protein BAD

Polzien

Baljuls

Rennefahrt

et al. 2009

Self Cite

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.Apoptosis is a genetically programmed, morphologically distinct form of cell death that can be triggered by a variety of physiological and pathological stimuli (1-3). This form of cellular suicide is widely observed in nature and is not only essential for embryogenesis, immune responses, and tissue homeostasis but is also involved in diseases such as tumor development and progression. Bcl-2 family proteins play a pivotal role in controlling programmed cell death. The major function of these proteins is to directly modulate outer mitochondrial membrane permeability and thereby regulate the release of apoptogenic factors from the intermembrane space into the cytoplasm (for a recent review, see Ref. 4). On the basis of various structural and functional characteristics, the Bcl-2 family of proteins is divided into three subfamilies, including proteins that either inhibit (e.g. Bcl-2, Bcl-X L , or Bcl-w) or promote programmed cell death (e.g. Bax, Bak, or Bok) (5, 6). A second subclass of proapoptotic Bcl-2 family members, the BH3 2 -only proteins, comprises BAD, Bik, Bmf, Hrk, Noxa, truncated Bid, Bim, and Puma (4). BH3-only proteins share sequence homology only at the BH3 domain. The amphipathic helix formed by the BH3 domain (and neighboring residues) associates with a hydrophobic groove of ...

“…Akt/ PKB plasmids were kindly provided by Jakob Troppmair (Innsbruck, Austria). Expression and purification of RAF kinases, PAK1, and Akt/PKB from Sf9 insect cells were performed as described previously (27,28). GST-tagged hBAD was expressed in Escherichia coli using pGEX-2T vector (GE Healthcare) and isolated by glutathione-Sepharose affinity chromatography.…”

Section: Dna Expression Plasmids and Purification Of Proteins-mentioning

confidence: 99%

BAD Contributes to RAF-mediated Proliferation and Cooperates with B-RAF-V600E in Cancer Signaling

Polzien

Baljuls

Albrecht

et al. 2011

Self Cite

BAD (Bcl-2 antagonist of cell death) belongs to the proapoptotic BH3-only subfamily of Bcl-2 proteins. Physiological activity of BAD is highly controlled by phosphorylation. To further analyze the regulation of BAD function, we investigated the role of recently identified phosphorylation sites on BAD-mediated apoptosis. We found that in contrast to the N-terminal phosphorylation sites, the serines 124 and 134 act in an antiapoptotic manner because the replacement by alanine led to enhanced cell death. Our results further indicate that RAF kinases represent, besides PAK1, BAD serine 134 phosphorylating kinases. Importantly, in the presence of wild type BAD, co-expression of survival kinases, such as RAF and PAK1, leads to a strongly increased proliferation, whereas substitution of serine 134 by alanine abolishes this process. Furthermore, we identified BAD serine 134 to be strongly involved in survival signaling of B-RAF-V600E-containing tumor cells and found that phosphorylation of BAD at this residue is critical for efficient proliferation in these cells. Collectively, our findings provide new insights into the regulation of BAD function by phosphorylation and its role in cancer signaling.Apoptosis is a programmed cell death mechanism that regulates the destruction of cells. This is crucial for multicellular organisms in processes such as development and tissue homeostasis (1-3). Mitochondria constitute a convergence point in the process of apoptosis (3, 4). Crucial regulators of apoptosis at the level of mitochondria are the proteins of the Bcl-2 family that are characterized by the presence of Bcl-2 homology domains (BH1-BH4).2 The Bcl-2 family of proteins is divided into three subfamilies, including proteins that either inhibit (e.g. Bcl-X L , Bcl-2, or Bcl-w) or promote (e.g. Bak, Bax, or Bok) programmed cell death (4 -6). A second subclass of proapoptotic Bcl-2 family members consists of the BH3-only proteins that share sequence homology only at the BH3 domain. This subclass comprises BAD, Bmf, Bik, Noxa, Hrk, Bim, Bid, and Puma (4). A complex interplay between anti-and proapoptotic proteins of this family mediates induction and execution of programmed cell death. Surprisingly, several proteins of the Bcl-2 family shaped up as regulators of various physiological processes other than apoptosis, such as autophagy, mitochondrial respiration, and the glucose metabolism (7).BAD (Bcl-2-associated death promoter, Bcl-2 antagonist of cell death) is a BH3-only protein that promotes apoptosis by forming heterodimers with the prosurvival proteins Bcl-2 and Bcl-X L (8). Phosphorylation of specific serine residues, Ser-112 and Ser-136 of murine BAD (mBAD) or the corresponding phosphorylation sites Ser-75 and Ser-99 of human BAD (hBAD) leads to complex formation with 14-3-3 proteins and subsequent relocation of BAD (9, 10). Phosphorylation of mBAD at Ser-155 (Ser-118 of hBAD) disrupts the association with Bcl-X L or Bcl-2, provoking cell survival (11). Therefore, the phosphorylation status of BAD at specific serine resi...