The FERONIA Receptor Kinase Maintains Cell-Wall Integrity during Salt Stress through Ca2+ Signaling

1. Download high-res image (193KB)
2. Download full-size image

     

Structure of a Signaling Cannabinoid Receptor 1-G Protein Complex

1. Download high-res image (195KB)
2. Download full-size image

     

Scaffold State Switching Amplifies,Accelerates, and Insulates Protein Kinase C Signaling

Scaffold proteins localize two or more signaling enzymes in close proximity to their downstream effectors. A-kinase-anchoring proteins (AKAPs) are a canonical family of scaffold proteins known to bind protein kinase A (PKA) and other enzymes. Several AKAPs have been shown to accelerate, amplify, and specify signal transduction to dynamically regulate numerous cellular processes. However, there is little theory available to mechanistically explain how signaling on protein scaffolds differs from solution biochemistry. In our present study, we propose a novel kinetic mechanism for enzymatic reactions on protein scaffolds to explain these phenomena, wherein the enzyme-substrate-scaffold complex undergoes stochastic state switching to reach an active state. This model predicted anchored enzymatic reactions to be accelerated, amplified, and insulated from inhibition compared with those occurring in solution. We exploited a direct interaction between protein kinase C (PKC) and AKAP7α as a model to validate these predictions experimentally. Using a genetically encoded PKC activity reporter, we found that both the strength and speed of substrate phosphorylation were enhanced by AKAP7α. PKC tethered to AKAP7α was less susceptible to inhibition from the ATP-competitive inhibitor Gö6976 and the substrate-competitive inhibitor PKC 20-28, but not the activation-competitive inhibitor calphostin C. Model predictions and experimental validation demonstrated that insulation is a general property of scaffold tethering. Sensitivity analysis indicated that these findings may be applicable to many other scaffolds as well. Collectively, our findings provide theoretical and experimental evidence that scaffold proteins can amplify, accelerate, and insulate signal transduction.     

Structure of the Sensor Domain of Mycobacterium tuberculosis PknH Receptor Kinase Reveals a Conserved Binding Cleft

Since their discovery over 20 years ago, eukaryotic-like transmembrane receptor Ser/Thr protein kinases (STPKs) have been shown to play critical roles in the virulence, growth, persistence, and reactivation of many bacteria. Information regarding the signals transmitted by these proteins, however, remains scarce. To enhance understanding of the basis for STPK receptor signaling, we determined the 1.7-Å-resolution crystal structure of the extracellular sensor domain of the Mycobacterium tuberculosis receptor STPK, PknH (Rv1266c). The PknH sensor domain adopts an unanticipated fold containing two intramolecular disulfide bonds and a large hydrophobic and polar cleft. The residues lining the cleft and those surrounding the disulfide bonds are conserved. These results suggest that PknH binds a small-molecule ligand that signals by changing the location or quaternary structure of the kinase domain.     

利用序列保守模体和局部构象信息预测转录因子结合位点

转录因子结合位点的计算预测是研究基因转录调控的重要环节,但常用的位置特异得分矩阵方法预测特异性偏低.通过深入分析结合位点的生物特征,提出了一种综合利用序列保守模体和局部构象信息的结合位点预测方法,以极大相关得分矩阵作为保守模体的描述模型,并根据二苷参数模型计算位点序列的局部构象,将两类信息得分组合为多维特征向量,在二次判别分析的框架下进行训练和滑动预测.预测过程中还引入了位置信息量以优化似然得分和过滤备选结果.针对大肠杆菌CRP和Fis结合位点数据的留一法测试结果表明,描述模型的改进和多种信息的融合能有效地改善预测方法的性能,大幅度提高特异性.     

Protease Cleavage Sites in HIV-1 gp120 Recognized by Antigen Processing Enzymes Are Conserved and Located at Receptor Binding Sites

The identification of vaccine immunogens able to elicit broadly neutralizing antibodies (bNAbs) is a major goal in HIV vaccine research. Although it has been possible to produce recombinant envelope glycoproteins able to adsorb bNAbs from HIV-positive sera, immunization with these proteins has failed to elicit antibody responses effective against clinical isolates of HIV-1. Thus, the epitopes recognized by bNAbs are present on recombinant proteins, but they are not immunogenic. These results led us to consider the possibility that changes in the pattern of antigen processing might alter the immune response to the envelope glycoprotein to better elicit protective immunity. In these studies, we have defined protease cleavage sites on HIV gp120 recognized by three major human proteases (cathepsins L, S, and D) important for antigen processing and presentation. Remarkably, six of the eight sites identified in gp120 were highly conserved and clustered in regions of the molecule associated with receptor binding and/or the binding of neutralizing antibodies. These results suggested that HIV may have evolved to take advantage of major histocompatibility complex (MHC) class II antigen processing enzymes in order to evade or direct the antiviral immune response.A major goal of HIV vaccine development is the development of immunogens that elicit protective antiviral antibody and cellular immune responses. However, after more than 25 years of research, vaccine immunogens able to elicit protective immunity in humans have yet to be described (11, 31). Although it has been possible to produce recombinant envelope proteins (gp120 and gp140) with many of the features of native virus proteins (e.g., complex glycosylation and the ability to bind CD4, chemokine receptors, and neutralizing antibodies), these antigens have not been able to elicit broadly neutralizing antibodies (bNAbs) or protective immune responses when used as immunogens (11, 32, 43, 50, 56, 74, 79). The fact that recombinant proteins can adsorb virus bNAbs from HIV-1-positive sera (59, 91) indicates that many recombinant envelope proteins are correctly folded but that the epitopes recognized by bNAbs are simply not immunogenic. Over the last decade, several different approaches have been employed to create immunogens able to elicit broadly neutralizing antibodies. These strategies have included efforts to duplicate and/or stabilize the oligomeric structure of HIV envelope proteins (5, 26, 87), the creation of minimal antigenic structures lacking epitopes that conceal important neutralizing sites (27, 46, 70, 89), and prime/boost strategies combining protein immunization with DNA immunization or infection with recombinant viruses in order to stimulate the endogenous synthesis and presentation of HIV immunogens (15, 29, 30, 83). However, none of these approaches has resulted in a clinically significant improvement in antiviral immunity or HIV vaccine efficacy. Efforts to elicit protective cellular immune responses (e.g., cytotoxic lymphocytes) by use of recombinant virus vaccines have likewise been disappointing (10, 61). In fact, such vaccines may have promoted HIV infection rather than inhibiting it (22, 23).In the present study, we describe the first steps in a new approach to reengineering the immunogenicity of HIV envelope proteins in order to improve the potency and specificity of humoral and cellular immune responses. The approach is based on defining the determinants of antigen processing and presentation of HIV envelope glycoproteins. Both humoral and cellular immune responses depend on proteolytic degradation of protein antigens prior to antigen presentation, mediated by professional antigen-presenting cells (APCs) such as macrophages, dendritic cells, and B cells (97). Normally, proteins of intracellular origin are processed by the proteasome, a 14- to 17-subunit protein complex located in the cytosol. Proteins of extracellular origin are processed in lysosomes or late endosomes of APCs. The resulting peptide epitopes are then loaded into major histocompatibility complex (MHC) class I or class II molecules and presented on the surfaces of APCs to CD8 or CD4 T cells. Within the endosomes and lysosomes of APCs, there are cathepsins, acid thiol reductase, and aspartyl endopeptidase. The enzymes perform two activities: degrading endocytosed protein antigens to liberate peptides for MHC class II binding (99) and removing the invariant chain chaperone (6, 94). Although all cathepsins can liberate epitopes from a diverse range of antigens (16), only cathepsins S and L have nonredundant roles in antigen processing in vivo (reviewed by Hsing and Rudensky [45]). Cathepsin L is expressed in thymic cortical epithelial cells but not in B cells or dendritic cells, while cathepsin S is found in all three types of APCs. Unlike cathepsins L and S, which are cysteine proteases and active at neutral pH, cathepsin D is an aspartic protease, is active at acidic pH, and participates in proteolysis and antigen presentation in connection with MHC class I and class II antigen presentation pathways established for CD4 and CD8 T cells. In considering the use of envelope proteins as potential vaccines, the route of immunization, formulation (e.g., adjuvants), protein folding, disulfide bonding, and glycosylation pattern all determine which peptides are available for MHC-restricted presentation.Previous studies provided evidence that gp120 was sensitive to digestion by cathepsins B, D, and L, but the specific cleavage sites were not defined (18). In the present study, we (i) describe the locations of eight protease cleavage sites on HIV-1 gp120 recognized by cathepsins L, S, and D, involved in antigen processing; (ii) determine the extent to which they are conserved; and (iii) evaluate the effect of cathepsin cleavage on the binding of gp120 to CD4-IgG and neutralizing antibodies. The results obtained provide new insights into the basis of envelope immunogenicity that may prove to be useful in the development of HIV vaccine antigens.     

The Pim-1 Protein Kinase Is an Important Regulator of MET Receptor Tyrosine Kinase Levels and Signaling

MET, the receptor for hepatocyte growth factor (HGF), plays an important role in signaling normal and tumor cell migration and invasion. Here, we describe a previously unrecognized mechanism that promotes MET expression in multiple tumor cell types. The levels of the Pim-1 protein kinase show a positive correlation with the levels of MET protein in human tumor cell lines and patient-derived tumor materials. Using small interfering RNA (siRNA), Pim knockout mice, small-molecule inhibitors, and overexpression of Pim-1, we confirmed this correlation and found that Pim-1 kinase activity regulates HGF-induced tumor cell migration, invasion, and cell scattering. The novel biochemical mechanism for these effects involves the ability of Pim-1 to control the translation of MET by regulating the phosphorylation of eukaryotic initiation factor 4B (eIF4B) on S406. This targeted phosphorylation is required for the binding of eIF4B to the eIF3 translation initiation complex. Importantly, Pim-1 action was validated by the evaluation of patient blood and bone marrow from a phase I clinical trial of a Pim kinase inhibitor, AZD1208. These results suggest that Pim inhibitors may have an important role in the treatment of patients where MET is driving tumor biology.     

Allosteric Coupling of Drug Binding and Intracellular Signaling in the A2A Adenosine Receptor

1. Download high-res image (173KB)
2. Download full-size image

     

Reassembly of JIP1 Scaffold Complex in JNK MAP Kinase Pathway Using Heterologous Protein Interactions

Formation of signaling protein complexes is crucial for proper signal transduction. Scaffold proteins in MAP kinase pathways are thought to facilitate complex assembly, thereby promoting efficient and specific signaling. To elucidate the assembly mechanism of scaffold complexes in mammals, we attempted to rationally rewire JIP1-dependent JNK MAP kinase pathway via alternative assembly of JIP1 complex. When JIP1-JNK docking interaction in the complex was replaced with heterologous protein interaction domains, such as PDZ domains and JNK-binding domains, a functional scaffold complex was reconstituted, and JNK signaling was rescued. Reassembly of JIP1 complex using heterologous protein interactions was sufficient for restoring of JNK MAP kinase pathway to induce signaling responses, including JNK activation and cell death. These results suggest a simple yet modular mechanism for JIP1 scaffold assembly in mammals.     

Interaction between Connexin50 and Mitogen-activated Protein Kinase Signaling in Lens Homeostasis

Both connexins and signal transduction pathways have been independently shown to play critical roles in lens homeostasis, but little is known about potential cooperation between these two intercellular communication systems. To investigate whether growth factor signaling and gap junctional communication interact during the development of lens homeostasis, we examined the effect of mitogen-activated protein kinase (MAPK) signaling on coupling mediated by specific lens connexins by using a combination of in vitro and in vivo assays. Activation of MAPK signaling pathways significantly increased coupling provided by Cx50, but not Cx46, in paired Xenopus laevis oocytes in vitro, as well as between freshly isolated lens cells in vivo. Constitutively active MAPK signaling caused macrophthalmia, cataract, glucose accumulation, vacuole formation in differentiating fibers, and lens rupture in vivo. The specific removal or replacement of Cx50, but not Cx46, ameliorated all five pathological conditions in transgenic mice. These results indicate that MAPK signaling specifically modulates coupling mediated by Cx50 and that gap junctional communication and signal transduction pathways may interact in osmotic regulation during postnatal fiber development.     

Disabled Is a Putative Adaptor Protein That Functions during Signaling by the Sevenless Receptor Tyrosine Kinase

DRK, the Drosophila homolog of the SH2-SH3 domain adaptor protein Grb2, is required during signaling by the sevenless receptor tyrosine kinase (SEV). One role of DRK is to provide a link between activated SEV and the Ras1 activator SOS. We have investigated the possibility that DRK performs other functions by identifying additional DRK-binding proteins. We show that the phosphotyrosine-binding (PTB) domain-containing protein Disabled (DAB) binds to the DRK SH3 domains. DAB is expressed in the ommatidial clusters, and loss of DAB function disrupts ommatidial development. Moreover, reduction of DAB function attenuates signaling by a constitutively activated SEV. Our biochemical analysis suggests that DAB binds SEV directly via its PTB domain, becomes tyrosine phosphorylated upon SEV activation, and then serves as an adaptor protein for SH2 domain-containing proteins. Taken together, these results indicate that DAB is a novel component of the SEV signaling pathway.     

Nucleus-Specific and Cell Cycle-Regulated Degradation of Mitogen-Activated Protein Kinase Scaffold Protein Ste5 Contributes to the Control of Signaling Competence

Saccharomyces cerevisiae cells are capable of responding to mating pheromone only prior to their exit from the G₁ phase of the cell cycle. Ste5 scaffold protein is essential for pheromone response because it couples pheromone receptor stimulation to activation of the appropriate mitogen-activated protein kinase (MAPK) cascade. In naïve cells, Ste5 resides primarily in the nucleus. Upon pheromone treatment, Ste5 is rapidly exported from the nucleus and accumulates at the tip of the mating projection via its association with multiple plasma membrane-localized molecules. We found that concomitant with its nuclear export, the rate of Ste5 turnover is markedly reduced. Preventing nuclear export destabilized Ste5, whereas preventing nuclear entry stabilized Ste5, indicating that Ste5 degradation occurs mainly in the nucleus. This degradation is dependent on ubiquitin and the proteasome. We show that Ste5 ubiquitinylation is mediated by the SCF^Cdc4 ubiquitin ligase and requires phosphorylation by the G₁ cyclin-dependent protein kinase (cdk1). The inability to efficiently degrade Ste5 resulted in pathway activation and cell cycle arrest in the absence of pheromone. These findings reveal that maintenance of this MAPK scaffold at an appropriately low level depends on its compartment-specific and cell cycle-dependent degradation. Overall, this mechanism provides a novel means for helping to prevent inadvertent stimulus-independent activation of a response and for restricting and maximizing the signaling competence of the cell to a specific cell cycle stage, which likely works hand in hand with the demonstrated role that G₁ Cdk1-dependent phosphorylation of Ste5 has in preventing its association with the plasma membrane.Scaffold proteins play a pivotal role in spatial and temporal regulation of multitiered mitogen-activated protein kinase (MAPK) cascades (8, 30, 107). Scaffold protein function can be controlled at several different levels, including phosphorylation, oligomerization, and subcellular localization, which can dramatically influence signaling (5, 21, 61).A well-characterized scaffold-dependent MAPK pathway drives the mating pheromone response in budding yeast Saccharomyces cerevisiae (15). The occupancy of a heterotrimeric G-protein-coupled receptor by pheromone results in release of its associated membrane-tethered Gβγ (Ste4-Ste18) complex. Ste5 scaffold protein (917 residues) is recruited to the plasma membrane via its association with this freed Gβγ (106) and by additional multivalent contacts with membrane phospholipids mediated by an N-terminal amphipathic α-helix (PM motif) (111) and an internal PH domain (34). Because Ste5 is also able to bind a MAPK kinase kinase (Ste11), a MAPK kinase (Ste7), and two MAPKs (Fus3 and Kss1) (102), membrane recruitment of Ste5 delivers these components to the plasma membrane. Membrane localization of Ste5 juxtaposes its passenger kinases to Ste20, a p21-activated protein kinase that also interacts with membrane phospholipids (94) and requires plasma membrane-tethered and GTP-loaded Cdc42 for its activation (56, 58, 60). GTP-bound Cdc42 is generated in this vicinity via other Gβγ-recruited effectors, especially Far1, which binds the Cdc42 guanine nucleotide exchange factor, Cdc24 (14, 98). Once activated, Ste20 directly phosphorylates and activates the Ste11 MAPK kinase kinase, triggering the MAPK cascade (24, 114).In naïve haploid cells, Ste5 undergoes continuous nucleocytoplasmic shuttling but is located predominantly in the nucleus (53, 66). In response to pheromone, this flux is dramatically shifted in favor of export, elevating the cytosolic pool of Ste5, thereby raising the number of molecules available for membrane recruitment (66, 79). Pheromone-induced nuclear export of Ste5 requires the exportin, Msn5/Ste21 (66).Little is known about why Ste5 is located in the nucleus in unstimulated cells. It has been suggested that passage of Ste5 through the nucleus modifies it in an as yet undefined manner to make it “competent” to subsequently promote signaling at the membrane (66, 103). However, other evidence indicates that nuclear shuttling of Ste5 is not necessary for its translocation to the plasma membrane or its function (34, 79, 111) and that reimport into the nucleus contributes to pathway downregulation following initial stimulation (53). It has remained obscure, mechanistically speaking, how nuclear localization of Ste5 contributes to the regulation of pathway activation and signal flux.Given that Ste5 is the least abundant component of this entire signaling system (≤500 molecules per haploid cell) (38), we suspected that dynamic regulation of the location and level of this scaffold protein provides a critically important control point for influencing the timing, potency, duration, and specificity of signaling in this pathway. Indeed, as described here, we found that the subcellular localization of Ste5 and cell cycle progression have dramatic effects in controlling the stability of Ste5. Our findings provide new insights about the physiological importance of Ste5 nuclear localization and G₁ cyclin-dependent protein kinase 1 (CDK1) action in establishment and maintenance of the conditions that preserve signaling fidelity in this system.     

Differential Regulation of Cysteinyl Leukotriene Receptor Signaling by Protein Kinase C in Human Mast Cells

Cysteinyl leukotrienes (cys-LTs) are a group of lipid mediators that are potent bronchoconstrictors, powerful inducers of vascular leakage and potentiators of airway hyperresponsiveness. Cys-LTs play an essential role in asthma and are synthesized as well as activated in mast cells (MCs). Cys-LTs relay their effects mainly through two known GPCRs, CysLT₁R and CysLT₂R. Although protein kinase C (PKC) isoforms are implicated in the regulation of CysLT₁R function, neither the role of PKCs in cys-LT-dependent MC inflammatory signaling nor the involvement of specific isoforms in MC function are known. Here, we show that PKC inhibition augmented LTD₄ and LTE₄-induced calcium influx through CysLT₁R in MCs. In contrast, inhibition of PKCs suppressed c-fos expression as well MIP1β generation by cys-LTs. Interestingly, cys-LTs activated both PKCα and PKCε isoforms in MC. However, knockdown of PKCα augmented cys-LT mediated calcium flux, while knockdown of PKCε attenuated cys-LT induced c-fos expression and MIP1β generation. Taken together, these results demonstrate for the first time that cys-LT signaling downstream of CysLT₁R in MCs is differentially regulated by two distinct PKCs which modulate inflammatory signals that have significant pathobiologic implications in allergic reactions and asthma pathology.     

JSAP1, a Novel Jun N-Terminal Protein Kinase (JNK)-Binding Protein That Functions as a Scaffold Factor in the JNK Signaling Pathway

The major components of the mitogen-activated protein kinase (MAPK) cascades are MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Recent rapid progress in identifying members of MAPK cascades suggests that a number of such signaling pathways exist in cells. To date, however, how the specificity and efficiency of the MAPK cascades is maintained is poorly understood. Here, we have identified a novel mouse protein, termed Jun N-terminal protein kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), by a yeast two-hybrid screen, using JNK3 MAPK as the bait. Of the mammalian MAPKs tested (JNK1, JNK2, JNK3, ERK2, and p38alpha), JSAP1 preferentially coprecipitated with the JNKs in cotransfected COS-7 cells. JNK3 showed a higher binding affinity for JSAP1, compared with JNK1 and JNK2. In similar cotransfection studies, JSAP1 also interacted with SEK1 MAPKK and MEKK1 MAPKKK, which are involved in the JNK cascades. The regions of JSAP1 that bound JNK, SEK1, and MEKK1 were distinct from one another. JNK and MEKK1 also bound JSAP1 in vitro, suggesting that these interactions are direct. In contrast, only the activated form of SEK1 associated with JSAP1 in cotransfected COS-7 cells. The unstimulated SEK1 bound to MEKK1; thus, SEK1 might indirectly associate with JSAP1 through MEKK1. Although JSAP1 coprecipitated with MEK1 MAPKK and Raf-1 MAPKKK, and not MKK6 or MKK7 MAPKK, in cotransfected COS-7 cells, MEK1 and Raf-1 do not interfere with the binding of SEK1 and MEKK1 to JSAP1, respectively. Overexpression of full-length JSAP1 in COS-7 cells led to a considerable enhancement of JNK3 activation, and modest enhancement of JNK1 and JNK2 activation, by the MEKK1-SEK1 pathway. Deletion of the JNK- or MEKK1-binding regions resulted in a significant reduction in the enhancement of the JNK3 activation in COS-7 cells. These results suggest that JSAP1 functions as a scaffold protein in the JNK3 cascade. We also discuss a scaffolding role for JSAP1 in the JNK1 and JNK2 cascades.     

Evidence for a Reduction of Coupling between GABA<Subscript>A</Subscript> Receptor Agonist and Ionophore Binding Sites by Inorganic Phosphate

[³⁵S]TBPS binding to the GABA_A receptor ionophore binding site is anion dependent. Using autoradiography on rat brain sections, we show that permeabilities of anions through the receptor channel correlate with their efficiencies to promote basal [³⁵S]TBPS binding. Phosphate made an exception as it induced more binding than expected from its permeability. Well-permeable anions (chloride, nitrate, formate) allowed [³⁵S]TBPS binding to be effectively displaced by 1 mM GABA, whereas low-permeable anions (acetate, phosphate, propionate) markedly prevented this GABA effect, especially in the thalamus, the transition from the high to the low GABA effect being between formate and acetate. In the presence of phosphate, GABA enhanced [³H]flunitrazepam binding to benzodiazepine site of recombinant α1β2γ2 receptors with the same efficacy but lower potency as compared to the presence of chloride, whereas [³⁵S]TBPS binding was abnormally modulated by GABA. These results suggest that inorganic phosphate affects coupling between agonist and ionophore sites in GABA_A receptors. Special issue dedicated to Simo S. Oja