Bcl-2 Phosphorylation by p38 MAPK: identification of target sites and biologic consequences

The antiapoptotic role of Bcl-2 can be regulated by its phosphorylation in serine and threonine residues located in a nonstructured loop that links BH3 and BH4 domains. p38 MAPK has been identified as one of the kinases able to mediate such phosphorylation, through direct interaction with Bcl-2 protein in the mitochondrial compartment. In this study, we identify, by using mass spectrometry techniques and specific anti-phosphopeptide antibodies, Ser(87) and Thr(56) as the Bcl-2 residues phosphorylated by p38 MAPK and show that phosphorylation of these residues is always associated with a decrease in the antiapoptotic potential of Bcl-2 protein. Furthermore, we obtained evidence that p38 MAPK-induced Bcl-2 phosphorylation plays a key role in the early events following serum deprivation in embryonic fibroblasts. Both cytochrome c release and caspase activation triggered by p38 MAPK activation and Bcl-2 phosphorylation are absent in embryonic fibroblasts from p38alpha knock-out mice (p38alpha(-/-) MEF), whereas they occur within 12 h of serum withdrawal in p38alpha(+/+) MEF; moreover, they can be prevented by p38 MAPK inhibitors and are not associated with the synthesis of the proapoptotic proteins Bax and Fas. Thus, Bcl-2 phosphorylation by activated p38 MAPK is a key event in the early induction of apoptosis under conditions of cellular stress.     

The apoptotic v-cyclin-CDK6 complex phosphorylates and inactivates Bcl-2

v-cyclin encoded by Kaposi's sarcoma herpesvirus/human herpesvirus 8 (KSHV or HHV8) associates with cellular cyclin-dependent kinase 6 (CDK6) to form a kinase complex that promotes cell-cycle progression, but can also induce apoptosis in cells with high levels of CDK6. Here we show that whereas HHV8-encoded v-Bcl-2 protects against this apoptosis, cellular Bcl-2 has lost its anti-apoptotic potential as a result of an inactivating phosphorylation in its unstructured loop region. Moreover, we identify Bcl-2 as a new substrate for v-cyclin-CDK6 in vitro, and show that it is present in a complex with CDK6 in cell lysates. A Bcl-2 mutant with a S70A S87A double substitution in the loop region is not phosphorylated and provides resistance to apoptosis, indicating that inactivation of Bcl-2 by v-cyclin-CDK6 may be required for the observed apoptosis. Furthermore, the identification of phosphorylated Bcl-2 in HHV8-positive Kaposi's sarcoma indicates that HHV8-mediated interference with host apoptotic signalling pathways may encourage the development of Kaposi's sarcoma.     

Evidence that Ser87 of BimEL is phosphorylated by Akt and regulates BimEL apoptotic function

Bim, the Bcl-2 interacting mediator of cell death, is a member of the BH3-only family of pro-apoptotic proteins. Recent studies have demonstrated that the apoptotic activity of Bim can be regulated through a post-translational mechanism whereby ERK phosphorylation serves as a signal for Bim ubiquitination and proteasomal degradation. In this report, we investigated the signaling pathways leading to Bim phosphorylation in Ba/F3 cells, an interleukin-3 (IL-3)-dependent B-cell line. IL-3 stimulation induced phosphorylation of Bim(EL), one of the predominant isoforms of Bim expressed in cells, at multiple sites, as evidenced by the formation of at least three to four bands by Western blotting that were sensitive to phosphatase digestion. The appearance of multiple, phosphorylated species of Bim(EL) correlated with Akt, and not ERK, activation. The PI3K inhibitor, LY294002, blocked IL-3-stimulated Akt activity and partially blocked Bim(EL) phosphorylation. In vitro kinase assays showed that recombinant Akt could directly phosphorylate a GST-Bim(EL) fusion protein and identified the Akt phosphorylation site in the Bim(EL) domain as Ser(87). Further, we demonstrated that cytokine stimulation promotes Bim(EL) binding to 14-3-3 proteins. Finally, we show that mutation of Ser(87) dramatically increases the apoptotic potency of Bim(EL). We propose that Ser(87) of Bim(EL) is an important regulatory site that is targeted by Akt to attenuate the pro-apoptotic function of Bim(EL), thereby promoting cell survival.     

The retinoblastoma protein is phosphorylated on multiple sites by human cdc2.

The retinoblastoma gene product (pRB) is a nuclear phosphoprotein that is thought to play a key role in the negative regulation of cellular proliferation. pRB is phosphorylated in a cell cycle dependent manner, and studies in both actively dividing and differentiated cells suggest that this modification may be essential for cells to progress through the cell cycle. Using tryptic phosphopeptide mapping we have shown that pRB is phosphorylated on multiple serine and threonine residues in vivo and that many of these phosphorylation events can be mimicked in vitro using purified p34cdc2. Using synthetic peptides corresponding to potential cdc2 phosphorylation sites, we have developed a strategy which has allowed the identification of five sites. S249, T252, T373, S807 and S811 are phosphorylated in vivo, and in each case these sites correspond closely to the consensus sequence for phosphorylation by p34cdc2. This and the observation that pRB forms a specific complex with p34cdc2 in vivo suggests that p34cdc2 or a p34cdc2-related protein is a major pRB kinase.     

The Equilibrative Nucleoside Transporter (ENT1) can be phosphorylated at multiple sites by PKC and PKA

Equilibrative Nucleoside Transporters (SLC29) are a family of proteins that transport nucleosides, nucleobases and nucleoside analogue drugs across cellular membranes. ENT1 is expressed ubiquitously in mammalian tissues and responsible for a significant portion of nucleoside analog drug uptake in humans. Despite the important clinical role of ENT1, many aspects of the regulation of this protein remain unknown. A major outstanding question in this field is the whether ENT1 is phosphorylated directly. To answer this question, we overexpressed tagged human (h) and mouse (m) ENT1, affinity purified protein using the tag, conducted phosphoamino acid analysis and found that m/hENT1 is predominantly phosphorylated at serine residues. The large intracellular loop of ENT1, between transmembrane domains 6 and 7, has been suggested to be a site of regulation by phosphorylation, therefore we generated His/Ubiquitin tagged peptides of this region and used them for in vitro kinase assays to identify target serines. Our data support a role for PKA and PKC in the phosphorylation of ENT1 within the intracellular loop and show that PKA can phosphorylate multiple sites within this loop while PKC specifically targets serines 279 and 286 and threonine 274. These data demonstrate, for the first time, that ENT1 is a phosphoprotein that can be directly phosphorylated at several sites by more than one kinase. The presence of multiple kinase targets within the loop suggests that ENT1 phosphorylation is considerably more complex than previously thought and thus ENT1 may be subject to phosphorylation by multiple pathways.     

The flexible loop of Bcl-2 is required for molecular interaction with immunosuppressant FK-506 binding protein 38 (FKBP38)

Bcl-2 contains an unusually long loop between the first and the second helices. This loop has been shown to be highly flexible based on NMR and X-ray crystallographic analyses of this region. Bcl-2 is regulated at the posttranslational level through phosphorylation of specific residues within the flexible loop. The biological role and posttranslational modifications of the loop of Bcl-2 is currently unclear. FK-506 binding protein 38 (FKBP38) has been reported to interact with Bcl-2, suggesting that FKBP38 could act as a docking molecule to localize Bcl-2 at the mitochondrial membrane [Shirane, M. and Nakayama, K.I. (2003) Inherent calcineurin inhibitor FKBP38 targets Bcl-2 to mitochondria and inhibits apoptosis. Nat. Cell Biol. 5, 28-37]. Here, we investigated the molecular interaction between FKBP38 and Bcl-2, and demonstrated that Bcl-2 interacts with FKBP38 through the unstructured loop, and the interaction appears to regulate phosphorylation in the loop of Bcl-2.     

Molecular dynamics simulations of the Bcl-2 protein to predict the structure of its unordered flexible loop domain

B-cell lymphoma (Bcl-2) protein is an anti-apoptotic member of the Bcl-2 family. It is functionally demarcated into four Bcl-2 homology (BH) domains: BH1, BH2, BH3, BH4, one flexible loop domain (FLD), a transmembrane domain (TM), and an X domain. Bcl-2’s BH domains have clearly been elucidated from a structural perspective, whereas the conformation of FLD has not yet been predicted, despite its important role in regulating apoptosis through its interactions with JNK-1, PKC, PP2A phosphatase, caspase 3, MAP kinase, ubiquitin, PS1, and FKBP38. Many important residues that regulate Bcl-2 anti-apoptotic activity are present in this domain, for example Asp34, Thr56, Thr69, Ser70, Thr74, and Ser87. The structural elucidation of the FLD would likely help in attempts to accurately predict the effect of mutating these residues on the overall structure of the protein and the interactions of other proteins in this domain. Therefore, we have generated an increased quality model of the Bcl-2 protein including the FLD through modeling. Further, molecular dynamics (MD) simulations were used for FLD optimization, to predict the flexibility, and to determine the stability of the folded FLD. In addition, essential dynamics (ED) was used to predict the collective motions and the essential subspace relevant to Bcl-2 protein function. The predicted average structure and ensemble of MD-simulated structures were submitted to the Protein Model Database (PMDB), and the Bcl-2 structures obtained exhibited enhanced quality. This study should help to elucidate the structural basis for Bcl-2 anti-apoptotic activity regulation through its binding to other proteins via the FLD.     

The Equilibrative Nucleoside Transporter (ENT1) can be phosphorylated at multiple sites by PKC and PKA

Abstract

Equilibrative Nucleoside Transporters (SLC29) are a family of proteins that transport nucleosides, nucleobases and nucleoside analogue drugs across cellular membranes. ENT1 is expressed ubiquitously in mammalian tissues and responsible for a significant portion of nucleoside analog drug uptake in humans. Despite the important clinical role of ENT1, many aspects of the regulation of this protein remain unknown. A major outstanding question in this field is the whether ENT1 is phosphorylated directly. To answer this question, we overexpressed tagged human (h) and mouse (m) ENT1, affinity purified protein using the tag, conducted phosphoamino acid analysis and found that m/hENT1 is predominantly phosphorylated at serine residues. The large intracellular loop of ENT1, between transmembrane domains 6 and 7, has been suggested to be a site of regulation by phosphorylation, therefore we generated His/Ubiquitin tagged peptides of this region and used them for in vitro kinase assays to identify target serines. Our data support a role for PKA and PKC in the phosphorylation of ENT1 within the intracellular loop and show that PKA can phosphorylate multiple sites within this loop while PKC specifically targets serines 279 and 286 and threonine 274. These data demonstrate, for the first time, that ENT1 is a phosphoprotein that can be directly phosphorylated at several sites by more than one kinase. The presence of multiple kinase targets within the loop suggests that ENT1 phosphorylation is considerably more complex than previously thought and thus ENT1 may be subject to phosphorylation by multiple pathways.     

Phosphorylation of Bcl-2 protein by CDC2 kinase during G2/M phases and its role in cell cycle regulation

Although it has been reported that Bcl-2 phosphorylation is associated with certain types of apoptosis, there is much controversy over the functional significance of and the kinases responsible for the phosphorylation. In this study, we examined whether Bcl-2 is phosphorylated by CDC2 kinase, a master regulator of G(2)/M transition in the eukaryotic cell cycle. When CDC2 was activated by okadaic acid in HL-60 cells, Bcl-2 phosphorylation was readily induced. The phosphorylation was correlated with the accumulation of cells in G(2)/M phases, but was not proportional to the level of apoptosis. Furthermore, we found that Bcl-2 was phosphorylated during G(2)/M phases of normal cell cycle. The ability of CDC2 to phosphorylate Bcl-2 was confirmed by in vitro kinase assay with a highly purified CDC2-cyclin B complex. Using synthetic peptides and mutant cell lines, we identified threonine 56, one of two consensus sites for CDC2 within the Bcl-2 sequence, as a residue phosphorylated by CDC2. Mutation at threonine 56 abrogated the cell cycle inhibitory effect of Bcl-2 without affecting anti-apoptotic function. These results suggest that two distinct functions of Bcl-2 (anti-apoptosis and cell cycle inhibition) are differentially regulated by post-translational mechanisms such as phosphorylation. CDC2-mediated phosphorylation of Bcl-2 may play some physiological roles in the negative regulatory events during mitosis.     

Molecular basis of Bcl-X(L)-p53 interaction: insights from molecular dynamics simulations

Bcl-X(L), an antiapoptotic Bcl-2 family protein, plays a central role in the regulation of the apoptotic pathway. Heterodimerization of the antiapoptotic Bcl-2 family proteins with the proapoptotic family members such as Bad, Bak, Bim and Bid is a crucial step in the apoptotic regulation. In addition to these conventional binding partners, recent evidences reveal that the Bcl-2 family proteins also interact with noncanonical binding partners such as p53. Our previous NMR studies showed that Bcl-X(L): BH3 peptide and Bcl-X(L): SN15 peptide (a peptide derived from residues S15-N29 of p53) complex structures share similar modes of bindings. To further elucidate the molecular basis of the interactions, here we have employed molecular dynamics simulations coupled with MM/PBSA approach. Bcl-X(L) and other Bcl-2 family proteins have 4 hydrophobic pockets (p1-p4), which are occupied by four systematically spaced hydrophobic residues (h1-h4) of the proapoptotic Bad and Bak BH3 peptides. We observed that three conserved hydrophobic residues (F19, W23 and L26) of p53 (SN15) peptide anchor into three hydrophobic pockets (p2-p4) of Bcl-X(L) in a similar manner as BH3 peptide. Our results provide insights into the novel molecular recognition by Bcl-X(L) with p53.     

Hydrophobic motif phosphorylation is not required for activation loop phosphorylation of p70 ribosomal protein S6 kinase 1 (S6K1)

p70 ribosomal protein S6 kinase 1 (S6K1) is regulated by multiple phosphorylation events. Three of these sites are highly conserved among AGC kinases (cAMP dependent Protein Kinase, cGMP dependent Protein Kinase, and Protein Kinase C subfamily): the activation loop in the kinase domain, and two C-terminal sites, the turn motif and the hydrophobic motif. The common dogma has been that phosphorylation of the hydrophobic motif primes S6K1 for the phosphorylation at the activation loop by phosphoinositide-dependent protein kinase 1 (PDK1). Here, we show that the turn motif is, in fact, phosphorylated first, the activation loop second, and the hydrophobic motif is third. Specifically, biochemical analyses of a construct of S6K1 lacking the C-terminal autoinhibitory domain as well as full-length S6K1, reveals that S6K1 is constitutively phosphorylated at the turn motif when expressed in insect cells and becomes phosphorylated in vitro by purified PDK1 at the activation loop. Only the species phosphorylated at the activation loop by PDK1 gets phosphorylated at the hydrophobic motif by mammalian target of rapamycin (mTOR) in vitro. These data are consistent with a previous model in which constitutive phosphorylation of the turn motif provides the key priming step in the phosphorylation of S6K1. The data provide evidence for regulation of S6K1, where hydrophobic motif phosphorylation is not required for PDK1 to phosphorylate S6K1 at the activation loop, but instead activation loop phosphorylation of S6K1 is required for mTOR to phosphorylate the hydrophobic motif of S6K1.     

Posttranslational modification of Bcl-2 facilitates its proteasome-dependent degradation: molecular characterization of the involved signaling pathway

The ratio of proapoptotic versus antiapoptotic Bcl-2 members is a critical determinant that plays a significant role in altering susceptibility to apoptosis. Therefore, a reduction of antiapoptotic protein levels in response to proximal signal transduction events may switch on the apoptotic pathway. In endothelial cells, tumor necrosis factor alpha (TNF-alpha) induces dephosphorylation and subsequent ubiquitin-dependent degradation of the antiapoptotic protein Bcl-2. Here, we investigate the role of different putative phosphorylation sites to facilitate Bcl-2 degradation. Mutation of the consensus protein kinase B/Akt site or of potential protein kinase C or cyclic AMP-dependent protein kinase sites does not affect Bcl-2 stability. In contrast, inactivation of the three consensus mitogen-activated protein (MAP) kinase sites leads to a Bcl-2 protein that is ubiquitinated and subsequently degraded by the 26S proteasome. Inactivation of these sites within Bcl-2 revealed that dephosphorylation of Ser87 appears to play a major role. A Ser-to-Ala substitution at this position results in 50% degradation, whereas replacement of Thr74 with Ala leads to 25% degradation, as assessed by pulse-chase studies. We further demonstrated that incubation with TNF-alpha induces dephosphorylation of Ser87 of Bcl-2 in intact cells. Furthermore, MAP kinase triggers phosphorylation of Bcl-2, whereas a reduction in Bcl-2 phosphorylation was observed in the presence of MAP kinase-specific phosphatases or the MAP kinase-specific inhibitor PD98059. Moreover, we show that oxidative stress mediates TNF-alpha-stimulated proteolytic degradation of Bcl-2 by reducing MAP kinase activity. Taken together, these results demonstrate a direct protective role for Bcl-2 phosphorylation by MAP kinase against apoptotic challenges to endothelial cells and other cells.     

JNK1-mediated phosphorylation of Bcl-2 regulates starvation-induced autophagy

Starvation induces autophagy to preserve cellular homeostasis in virtually all eukaryotic organisms. However, the mechanisms by which starvation induces autophagy are not completely understood. In mammalian cells, the antiapoptotic protein, Bcl-2, binds to Beclin 1 during nonstarvation conditions and inhibits its autophagy function. Here we show that starvation induces phosphorylation of cellular Bcl-2 at residues T69, S70, and S87 of the nonstructured loop; Bcl-2 dissociation from Beclin 1; and autophagy activation. In contrast, viral Bcl-2, which lacks the phosphorylation site-containing nonstructured loop, fails to dissociate from Beclin 1 during starvation. Furthermore, the stress-activated signaling molecule, c-Jun N-terminal protein kinase 1 (JNK1), but not JNK2, mediates starvation-induced Bcl-2 phosphorylation, Bcl-2 dissociation from Beclin 1, and autophagy activation. Together, our findings demonstrate that JNK1-mediated multisite phosphorylation of Bcl-2 stimulates starvation-induced autophagy by disrupting the Bcl-2/Beclin 1 complex. These findings define a mechanism that cells use to regulate autophagic activity in response to nutrient status.     

Ion binding properties of the dehydrin ERD14 are dependent upon phosphorylation

The ERD14 protein (early response to dehydration) is a member of the dehydrin family of proteins which accumulate in response to dehydration-related environmental stresses. Here we show the Arabidopsis dehydrin, ERD14, possesses ion binding properties. ERD14 is an in vitro substrate of casein kinase II; the phosphorylation resulting both in a shift in apparent molecular mass on SDS-PAGE gels and increased calcium binding activity. The phosphorylated protein bound significantly more calcium than the nonphosphorylated protein, with a dissociation constant of 120 microm and 2.86 mol of calcium bound per mol of protein. ERD14 is phosphorylated by extracts of cold-treated tissues, suggesting that the phosphorylation status of this protein might be modulated by cold-regulated kinases or phosphatases. Calcium binding properties of ERD14 purified from Arabidopsis extracts were comparable with phosphorylated Escherichia coli-expressed ERD14. Approximately 2 mol of phosphate were incorporated per mol of ERD14, indicating a minimum of two phosphorylation sites. Western blot analyses confirmed that threonine and serine are possible phosphorylation sites on ERD14. Utilizing matrix assisted laser desorption ionization-time of flight/mass spectrometry we identified five phosphorylated peptides that were present in both in vivo and in vitro phosphorylated ERD14. Our results suggest that the polyserine (S) domain is most likely the site of phosphorylation in ERD14 responsible for the activation of calcium binding.     

Opposing effects of Bad phosphorylation at two distinct sites by Akt1 and JNK1/2 on ischemic brain injury

Increasing evidence suggests that the Bcl-2 family proteins play pivotal roles in regulation of the mitochondria cell-death pathway on transient cerebral ischemia. Bad, a BH3-only proapoptotic Bcl-2 family protein, has been shown to be phosphorylated extensively on serine by kinds of kinases. However, the exact mechanisms of the upstream kinases in regulation of Bad signaling pathway remain unknown. Here, we reported that Bad could be phosphorylated not only by Akt1 but also by JNK1/2 after transient global ischemia in rat hippocampal CA1 region. Our data demonstrated that Akt1 mediated the phosphorylation of Bad at serine 136, which increased the interaction of serine 136-phosphorylated Bad with 14-3-3 proteins and prevented the dimerization of Bad with Bcl-Xl, inhibited the release of cytochrome c to the cytosol and the death effector caspase-3 activation, leading to the survival of neuron. In contrast, JNK1/2 induced the phosphorylation of Bad at a novel site of serine 128 after brain ischemia/reperfusion, which inhibited the interaction of PI3K/Akt-induced serine 136-phosphorylated Bad with 14-3-3 proteins, thereby promoted the apoptotic effect of Bad. In addition, activated Akt1 inhibited the activation of Bad(S128) through downregulating JNK1/2 activation, thus inhibiting JNK-mediated Bad apoptosis pathway. Furthermore, the fate of cell to survive or to die was determined by a balance between prosurvival and proapoptotic signals. Taken together, our studies reveal that Bad phosphorylation at two distinct sites induced by Akt1 and JNK1/2 have opposing effects on ischemic brain injury, and present the possibility of Bad as a potential therapeutic target for stroke treatment.     

Dual Role of JNK1-mediated phosphorylation of Bcl-2 in autophagy and apoptosis regulation

Autophagy and apoptosis are fundamental cellular pathways that are both regulated by JNK-mediated Bcl-2 phosphorylation. Several years ago, JNK-mediated Bcl-2 phosphorylation was shown to interfere with its binding to pro-apoptotic BH3 domain-containing proteins such as Bax and recently, our laboratory demonstrated that JNK1-mediated Bcl-2 phosphorylation interferes with its binding to the pro-autophagy BH3 domain-containing protein Beclin 1. Here, we examined the kinetic relationship between Bcl-2 phosphorylation, Bcl-2-Beclin 1 interactions, Bcl-2-Bax interactions and caspase 3 activation during nutrient starvation. We found that after a short period of nutrient deprivation (4 hours), a small amount of Bcl-2 phosphorylation dissociates Bcl-2 from the Bcl-2-Beclin 1 complex but not from the Bcl-2-Bax complex. After 16 hours of nutrient deprivation, Bcl-2 phosphorylation reaches maximal levels, the Bcl-2-Bax complex is disrupted, and active caspase 3 is detected, indicating the initiation of apoptosis. Based on this result, we propose a speculative model for understanding the interrelationship between autophagy and apoptosis regulated by JNK1-mediated Bcl-2 phosphorylation. According to this model, rapid Bcl-2 phosphorylation may occur initially to promote cell survival by disrupting the Bcl-2-Beclin 1 complex and activating autophagy. At a certain point when autophagy is no longer able to keep the cell alive, Bcl-2 phosphorylation might then serve to inactivate its anti-apoptotic function.

Addendum to: Wei Y, Pattingre S, Sinha S, Bassik M, Levine B. JNK1-mediated phosphorylation of Bcl-2 regulates starvation-induced autophagy. Mol Cell 2008; 30:678-88.     

Dendrimer-based BH3 conjugate that targets human carcinoma cells

Our previous studies have demonstrated the efficacy of generation 5 poly(amidoamine) dendrimers (G5-PAMAM) as a platform for the targeted delivery of chemotherapeutics. However, anticancer therapy can be subverted by anti-apoptotic changes in cancer cells. Bcl-2 and several of its peptides are commonly overexpressed in a number of cancers. A means to reverse this anti-apoptotic mechanism is to expose the cells to BH3 peptide, which has a homology to the anti-apoptotic Bcl-2 protein. This cannot be done indiscriminately because it can induce apoptosis in normal cells. In order to specifically target BH3 peptides to cancer cells, we synthesized a trifunctional, G5-PAMAM-based nanodevice to which folic acid was conjugated as a targeting agent, along with fluorescein isothiocyanate as the reporter agent and BH3 peptides that were used to induce apoptosis. The results show, for the first time, the therapeutic potential of targeted BH3 peptides as a means of inducing apoptosis by interfering with anti-apoptotic proteins within specific cells.     

p21-Activated kinase 1 (Pak1) phosphorylates BAD directly at serine 111 in vitro and indirectly through Raf-1 at serine 112

Background

Cell survival depends on the balance between protective and apoptotic signals. When the balance of signals tips towards apoptosis, cells undergo programmed cell death. This balance has profound implications in diseases including cancer. Oncogenes and tumor suppressors are mutated to promote cell survival during tumor development, and many chemotherapeutic drugs kill tumor cells by stimulating apoptosis. BAD is a pro-apoptotic member of the Bcl-2 family of proteins, which can be phosphorylated on numerous sites to modulate binding to Bcl-2 and 14-3-3 proteins and inhibit its pro-apoptotic activities. One of the critical phosphorylation sites is the serine 112 (S112), which can be phosphorylated by several kinases including Pak1.

Methodology/Principal Findings

We mapped the Pak phosphorylation sites by making serine to alanine mutations in BAD and testing them as substrates in in vitro kinase assays. We found that the primary phosphorylation site is not S112 but serine 111 (S111), a site that is sometimes found phosphorylated in vivo. In transfection assays of HEK293T cells, we showed that Pak1 required Raf-1 to stimulate phosphorylation on S112. Mutating either S111 or S112 to alanine enhanced binding to Bcl-2, but the double mutant S111/112A bound better to Bcl-2. Moreover, BAD phosphorylation at S111 was observed in several other cell lines, and treating one of them with the Pak1 inhibitor 2,2′-Dihydroxy-1,1′-dinaphthyldisulfide (IPA-3) reduced phosphorylation primarily at S112 and to a smaller extent at S111, while Raf inhibitors only reduced phosphorylation at S112.

Conclusion/Significance

Together, these findings demonstrate that Pak1 phosphorylates BAD directly at S111, but phosphorylated S112 through Raf-1. These two sites of BAD serve as redundant regulatory sites for Bcl-2 binding.     

HIV-1 encoded virus protein U (Vpu) solution structure of the 41-62 hydrophilic region containing the phosphorylated sites Ser52 and Ser56

Degradation of the HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. To promote CD4 degradation Vpu has to be phosphorylated on a motif DSGXXS, which is conserved in several signalling proteins known to be degraded by the proteasome upon phosphorylation. Such phosphorylation is required for the interaction of Vpu with the ubiquitin ligase SCF-beta-TrCP that triggers CD4 degradation by the proteasome. In the present work, we used two peptides of 22 amino acids between residues 41 and 62 of Vpu. Vpu41-62 was predicted to form an alpha-helix-flexible-alpha-helix including the phosphorylation motif DS52GNES56 and Vpu_P41-62 was phosphorylated at the two sites Ser52 and Ser56. We analysed the conformational change induced by the phosphorylation of this peptide on the residues Ser52 and Ser56. Homo- and heteronuclear NMR techniques were used to assess the structural influence of phosphorylation. The spectra of the free peptides, Vpu_P41-62 and Vpu41-62, in both H2O (at pH 3.5 and 7.2) and a 1:1 mixture of H2O and trifluoroethanol were completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide segment (42-49) shows a less well-defined helix propensity. The Vpu_P41-62 domain of residues 50-62 forms a loop with the phosphate group pointing away, a short beta-strand and a flexible extended 'tail' of residues 60-62. Residues 50-60 exhibit alpha-proton NMR secondary chemical shift changes from random coil toward more beta-like structure with the combined (temperature, solvent and pH) NMR and molecular calculation experiments. Differences in this molecular region 50-62 suggest that conformational changes of Vpu_P play an important role in Vpu_P-induced degradation of CD4 molecules.