Eukaryotic Signal Transduction Research Articles

The Protein Kinase C Inhibitor Bisindolyl Maleimide 2 Binds with Reversed Orientations to Different Conformations of Protein Kinase A

As the key mediators of eukaryotic signal transduction, the protein kinases often cause disease, and in particular cancer, when disregulated. Appropriately selective protein kinase inhibitors are sought after as research tools and as therapeutic drugs; several have already proven valuable in clinical use. The AGC subfamily protein kinase C (PKC) was identified early as a cause of cancer, leading to the discovery of a variety of PKC inhibitors. Despite its importance and early discovery, no crystal structure for PKC has yet been reported. Therefore, we have co-crystallized PKC inhibitor bisindolyl maleimide 2 (BIM2) with PKA variants to study its binding interactions. BIM2 co-crystallized as an asymmetric pair of kinase-inhibitor complexes. In this asymmetric unit, the two kinase domains have different lobe configurations, and two different inhibitor conformers bind in different orientations. One kinase molecule (A) is partially open with respect to the catalytic conformation, the other (B) represents the most open conformation of PKA reported so far. In monomer A, the BIM2 inhibitor binds tightly via an induced fit in the ATP pocket. The indole moieties are rotated out of the plane with respect to the chemically related but planar inhibitor staurosporine. In molecule B a different conformer of BIM2 binds in a reversed orientation relative to the equivalent maleimide atoms in molecule A. Also, a critical active site salt bridge is disrupted, usually indicating the induction of an inactive conformation. Molecular modeling of the clinical phase III PKC inhibitor LY333531 into the electron density of BIM2 reveals the probable binding mechanism and explains selectivity properties of the inhibitor.

Making protein immunoprecipitates.

A wide variety of methods used in the study of signal transduction in eukaryotes rely on the ability to precipitate proteins from whole cell extracts. Immunoprecipitation and related methods of affinity purification are routinely used to assess binding partner interactions and enzyme activity in addition to the size of a protein, rates of protein synthesis and turnover, and protein abundance, thus making it a mainstay of a wide variety of protocols. This chapter will provide starting-point methods for immunoprecipitation of proteins under denaturing and nondenaturing conditions and the detection of protein-protein interactions by co-precipitation. The Notes section gives recommendations on how to troubleshoot potential problems that can arise while doing these methodologies.

Isozymes delta of phosphoinositide-specific phospholipase C and their role in signal transduction in the cell.

Phospholipase C (PLC, EC 3.1.4.11) is an enzyme crucial for the phosphoinositol pathway and whose activity is involved in eukaryotic signal transduction as it generates two second messengers: diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). There are four major types of phospholipase C named: beta, gamma, delta and the recently discovered epsilon, but this review will focus only on the recent advances for the delta isozymes of PLC. So far, four delta isozymes (named delta1-4) have been discovered and examined. They differ with regard to cellular distribution, activities, biochemical features and involvement in human ailments.

Isozymes delta of phosphoinositide-specific phospholipase C and their role in signal transduction in the cell.

Phospholipase C (PLC, EC 3.1.4.11) is an enzyme crucial for the phosphoinositol pathway and whose activity is involved in eukaryotic signal transduction as it generates two second messengers: diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). There are four major types of phospholipase C named: beta, gamma, delta and the recently discovered epsilon, but this review will focus only on the recent advances for the delta isozymes of PLC. So far, four delta isozymes (named delta1-4) have been discovered and examined. They differ with regard to cellular distribution, activities, biochemical features and involvement in human ailments.

Regulatory proteins with a sense of direction: cell cycle signalling network in Caulobacter.

Localization of kinases and other signalling molecules at discrete cellular locations is often an essential component of signal transduction in eukaryotes. Caulobacter crescentus is a small, single-celled bacterium that presumably lacks intracellular organelles. Yet in Caulobacter, the subcellular distribution of several two-component signal transduction proteins involved in the control of polar morphogenesis and cell cycle progression changes from a fairly dispersed distribution to a tight accumulation at one or both poles in a spatial and temporal pattern that is reproduced during each cell cycle. This cell cycle-dependent choreography suggests that similarly to what happens in eukaryotes, protein localization provides a means of modulating signal transduction in bacteria. Recent studies have provided important insights into the biological role and the mechanisms for the differential localization of these bacterial signalling proteins during the Caulobacter cell cycle.

Structure Determination and Dynamics of Peptides Overlapping the Catalytic Hairpin of the Ras-Specific GEF Cdc25Mm

Ras proteins are small G proteins playing a major role in eukaryotic signal transduction. Guanine nucleotide exchange factors (GEF) stimulate GDP/GTP exchange, resulting in the formation of the active Ras-GTP complex. In mammalian cells, two major Ras-specific GEF exist: Sos-like and Cdc25-like. To date, structural data are available only for Cdc25(Mm). We designed and synthesized Cdc25(Mm)-derived peptides spanning residues corresponding to the hSos1 HI helical hairpin that has been implicated in the GEF catalytic mechanism. NMR experiments on a chemically synthesized Cdc25(Mm)(1178-1222) peptide proved that helix I readily reaches a conformation very similar to the corresponding helix in hSos1, while residues corresponding to helix H in hSos1 show higher conformational flexibility. Molecular dynamics studies with the appropriate solvent model showed that different conformational spaces are available for the peptide. Since helix H is making several contacts with Ras and a Cdc25(Mm)(1178-1222) peptide is able to bind nucleotide-free Ras in a BIAcore assay, the peptide must be able to obtain the proper Ras-interacting conformation, at least transiently. These results indicate that rational design and improvement of the Ras-interacting peptides should take into account conformational and flexibility features to obtain molecules with the appropriate biochemical properties.

Stress-induced Protein Phosphatase 2C Is a Negative Regulator of a Mitogen-activated Protein Kinase

Protein phosphatases of type 2C (PP2Cs) play important roles in eukaryotic signal transduction. In contrast to other eukaryotes, plants such as Arabidopsis have an unusually large group of 69 different PP2C genes. At present, little is known about the functions and substrates of plant PP2Cs. We have previously shown that MP2C, a wound-induced alfalfa PP2C, is a negative regulator of mitogen-activated protein kinase (MAPK) pathways in yeast and plants. In this report, we provide evidence that alfalfa salt stress-inducible MAPK (SIMK) and stress-activated MAPK (SAMK) are activated by wounding and that MP2C is a MAPK phosphatase that directly inactivates SIMK but not the wound-activated MAPK, SAMK. SIMK is inactivated through threonine dephosphorylation of the pTEpY motif, which is essential for MAPK activity. Mutant analysis indicated that inactivation of SIMK depends on the catalytic activity of MP2C. A comparison of MP2C with two other PP2Cs, ABI2 and AtP2CHA, revealed that although all three phosphatases have similar activities toward casein as a substrate, only MP2C is able to dephosphorylate and inactivate SIMK. In agreement with the notion that MP2C interacts directly with SIMK, the MAPK was identified as an interacting partner of MP2C in a yeast two-hybrid screen. MP2C can be immunoprecipitated with SIMK in a complex in vivo and shows direct binding to SIMK in vitro in protein interaction assays. Wound-induced MP2C expression correlates with the time window when SIMK is inactivated, corroborating the notion that MP2C is involved in resetting the SIMK signaling pathway.

Calcium-induced conformational switching of Paramecium calmodulin provides evidence for domain coupling.

Calmodulin (CaM) is an intracellular calcium-binding protein essential for many pathways in eukaryotic signal transduction. Although a structure of Ca(2+)-saturated Paramecium CaM at 1.0 A resolution (1EXR.pdb) provides the highest level of detail about side-chain orientations in CaM, information about an end state alone cannot explain driving forces for the transitions that occur during Ca(2+)-induced conformational switching and why the two domains of CaM are saturated sequentially rather than simultaneously. Recent studies focus attention on the contributions of interdomain linker residues. Electron paramagnetic resonance showed that Ca(2+)-induced structural stabilization of residues 76-81 modulates domain coupling [Qin and Squier (2001) Biophys. J. 81, 2908-2918]. Studies of N-domain fragments of Paramecium CaM showed that residues 76-80 increased thermostability of the N-domain but lowered the Ca(2+) affinity of sites I and II [Sorensen et al. (2002) Biochemistry 41, 15-20]. To probe domain coupling during Ca(2+) binding, we have used (1)H-(15)N HSQC NMR to monitor more than 40 residues in Paramecium CaM. The titrations demonstrated that residues Glu78 to Glu84 (in the linker and cap of helix E) underwent sequential phases of conformational change. Initially, they changed in volume (slow exchange) as sites III and IV titrated, and subsequently, they changed in frequency (fast exchange) as sites I and II titrated. These studies provide evidence for Ca(2+)-dependent communication between the domains, demonstrating that spatially distant residues respond to Ca(2+) binding at sites I and II in the N-domain of CaM.

Molecular cloning and characterization of a rice phosphoinositide-specific phospholipase C gene, OsPI-PLC1, that is activated in systemic acquired resistance

Phospholipid signaling is an important component in eukaryotic signal transduction pathways; in plants, it plays a key role in growth and development as well as in responses to environmental stress. We cloned and characterized a gene from rice encoding phosphoinositide-specific phospholipase C ( OsPI-PLC1, Oryza sativa L. phosphoinositide-specific phospholipase C1). OsPI-PLC1 encodes a 599 amino acid protein containing the catalytic X and Y domains, as well as a C2-like domain, characteristics of this class of enzymes. Expression of OsPI-PLC1 was induced by various chemical and biological inducers of plant defence pathways, including benzothiadiazole (BTH), salicylic acid (SA), dichloroisonicotinic acid, probenazole (PB), jasmonic acid (JA) and its methyl ester, Pseudomonas syringae pv. syringae, and wounding. All of these treatments were shown to induce resistance in rice against blast disease caused by Magnaporthe grisea. OsPI-PLC1 was activated within 6 h after inoculation with the blast fungus in BTH-treated rice seedlings and in the incompatible interaction between a resistant genotype of rice and M. grisea , whereas the expression in the BTH-untreated seedlings and in the compatible interaction was only induced to a relatively low level at later time points (30 h) after inoculation, indicating that expression of OsPI-PLC1 is associated with the resistance response and/or the incompatible interaction. In addition, BTH treatment also induced systemic expression of OsPI-PLC1. These results suggest that OsPI-PLC1 may be involved in the signaling pathways leading to disease resistance in rice.

Molecular cloning and characterization of a rice phosphoinositide-specific phospholipase C gene, OsPI-PLC1, that is activated in systemic acquired resistance,

Phospholipid signaling is an important component in eukaryotic signal transduction pathways; in plants, it plays a key role in growth and development as well as in responses to environmental stress. We cloned and characterized a gene from rice encoding phosphoinositide-specific phospholipase C ( OsPI-PLC1 , Oryza sativa L. phosphoinositide-specific phospholipase C1). OsPI-PLC1 encodes a 599 amino acid protein containing the catalytic X and Y domains, as well as a C2-like domain, characteristics of this class of enzymes. Expression of OsPI-PLC1 was induced by various chemical and biological inducers of plant defence pathways, including benzothiadiazole (BTH), salicylic acid (SA), dichloroisonicotinic acid, probenazole (PB), jasmonic acid (JA) and its methyl ester, Pseudomonas syringae pv. syringae , and wounding. All of these treatments were shown to induce resistance in rice against blast disease caused by Magnaporthe grisea . OsPI-PLC1 was activated within 6 h after inoculation with the blast fungus in BTH-treated rice seedlings and in the incompatible interaction between a resistant genotype of rice and M. grisea , whereas the expression in the BTH-untreated seedlings and in the compatible interaction was only induced to a relatively low level at later time points (30 h) after inoculation, indicating that expression of OsPI-PLC1 is associated with the resistance response and/or the incompatible interaction. In addition, BTH treatment also induced systemic expression of OsPI-PLC1 . These results suggest that OsPI-PLC1 may be involved in the signaling pathways leading to disease resistance in rice.

An EVH1/WH1 domain as a key actor in TGFβ signalling

EVH1 (enabled VASP (vasodilator-stimulated protein) homology 1)/WH1 (WASP (Wiskott–Aldrich syndrome protein) homology 1) domains, present in Ena VASP and WASP, are protein interaction modules specialised in binding proline-rich ligands. An EVH1/WH1 domain is here identified in the recently cloned SMIF protein, a key protein in transforming growth factor-β (TGFβ) signalling which was not yet related to defined domains. The SMIF EVH1/WH1 domain interacts with the proline-rich Smad4 activation domain, leading to translocation of so-formed complex to the nucleus where SMIF possesses strong intrinsic TGFβ-inducible transcriptional activity. This finding highlights the pivotal role that the EVH1/WH1 family of domains play in multiple eukaryotic signal transduction pathways.

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Pharmacological analysis of nod factor-induced calcium spiking in Medicago truncatula. Evidence for the requirement of type IIA calcium pumps and phosphoinositide signaling.

Bacterial Nod factors trigger a number of cellular responses in root hairs of compatible legume hosts, which include periodic, transient increases in cytosolic calcium levels, termed calcium spiking. We screened 13 pharmaceutical modulators of eukaryotic signal transduction for effects on Nod factor-induced calcium spiking. The purpose of this screening was 2-fold: to implicate enzymes required for Nod factor-induced calcium spiking in Medicago sp., and to identify inhibitors of calcium spiking suitable for correlating calcium spiking to other Nod factor responses to begin to understand the function of calcium spiking in Nod factor signal transduction. 2-Aminoethoxydiphenylborate, caffeine, cyclopiazonic acid (CPA), 2,5-di-(t-butyl)-1,4-hydroquinone, and U-73122 inhibit Nod factor-induced calcium spiking. CPA and U-73122 are inhibitors of plant type IIA calcium pumps and phospholipase C, respectively, and implicate the requirement for these enzymes in Nod factor-induced calcium spiking. CPA and U-73122 inhibit Nod factor-induced calcium spiking robustly at concentrations with no apparent toxicity to root hairs, making CPA and U-73122 suitable for testing whether calcium spiking is causal to subsequent Nod factor responses.

Structural Basis for E2-Mediated SUMO Conjugation Revealed by a Complex between Ubiquitin-Conjugating Enzyme Ubc9 and RanGAP1

E2 enzymes catalyze attachment of ubiquitin and ubiquitin-like proteins to lysine residues directly or through E3-mediated reactions. The small ubiquitin-like modifier SUMO regulates nuclear transport, stress response, and signal transduction in eukaryotes and is essential for cell-cycle progression in yeast. In contrast to most ubiquitin conjugation, the SUMO E2 enzyme Ubc9 is sufficient for substrate recognition and lysine modification of known SUMO targets. Crystallographic analysis of a complex between mammalian Ubc9 and a C-terminal domain of RanGAP1 at 2.5 Å reveals structural determinants for recognition of consensus SUMO modification sequences found within SUMO-conjugated proteins. Structure-based mutagenesis and biochemical analysis of Ubc9 and RanGAP1 reveal distinct motifs required for substrate binding and SUMO modification of p53, IκBα, and RanGAP1.

Phospholipase D1 is required for efficient mating projection formation in Saccharomyces cerevisiae.

Phospholipase D1 (PLD1) is an important enzyme involved in lipid signal transduction in eukaryotes. A role for PLD1 in signaling in Saccharomyces cerevisiae was examined. Pheromone response in yeast is controlled by a well-characterized protein kinase cascade. Loss of PLD1 activity was found to impair pheromone-induced changes in cellular morphology that result in formation of mating projections. The rate at which projections appeared following pheromone treatment was delayed, suggesting that PLD1 facilitates the execution of a rate-limiting step in morphogenesis. Mutants were found to be less sensitive to pheromone, again arguing that PLD1 is acting at a rate-limiting step. The fact that morphogenesis is most dramatically affected indicates that PLD1 functions primarily in the morphogenic branch of the pheromone response pathway.

An Src homology 3-like domain is responsible for dimerization of the repressor protein KorB encoded by the promiscuous IncP plasmid RP4.

KorB is a regulatory protein encoded by the conjugative plasmid RP4 and a member of the ParB family of bacterial partitioning proteins. The protein regulates the expression of plasmid genes whose products are involved in replication, transfer, and stable inheritance of RP4 by binding to palindromic 13-bp DNA sequences (5'-TTTAGC(G/C)GCTAAA-3') present 12 times in the 60-kb plasmid. Here we report the crystal structure of KorB-C, the C-terminal domain of KorB comprising residues 297-358. The structure of KorB-C was solved in two crystal forms. Quite unexpectedly, we find that KorB-C shows a fold closely resembling the Src homology 3 (SH3) domain, a fold well known from proteins involved in eukaryotic signal transduction. From the arrangement of molecules in the asymmetric unit, it is concluded that two molecules form a functionally relevant dimer. The detailed analysis of the dimer interface and a chemical cross-linking study suggest that the C-terminal domain is responsible for stabilizing the dimeric form of KorB in solution to facilitate binding to the palindromic operator sequence. The KorB-C crystal structure extends the range of protein-protein interactions known to be promoted by SH3 and SH3-like domains.

The Arabidopsis AHK4 histidine kinase is a cytokinin-binding receptor that transduces cytokinin signals across the membrane.

Common histidine-to-aspartate (His-->Asp) phosphorelay is a paradigm of signal transduction in both prokaryotes and eukaryotes for the propagation of certain environmental stimuli, in which histidine (His)-kinases play central roles as sensors for environmental signals. For the higher plant, Arabidopsis thaliana, it was recently suggested that the His-kinase (AHK4 / CRE1 / WOL) is a sensor for cytokinins, which are a class of plant hormones important for the regulation of cell division and differentiation. Interestingly, AHK4 is capable of functioning as a cytokinin sensor in the eubacterium, Escherichia coli (Suzuki et al. 2001, Plant Cell Physiol. 42: 107). Here we further show that AHK4 is a primary receptor that directly binds a variety of natural and synthetic cytokinins (e.g. not only N(6)-substituted aminopurines such as isopentenyl-adenine, trans-zeatin, benzyl-adenine, but also diphenylurea derivatives such as thidiazuron), in a highly specific manner (K(d) = 4.55+/-0.48x10(-9) M). AHK4 has a presumed extracellular domain, within which a single amino acid substitution (Thr-301 to Ile) was shown to result in loss of its ability to bind cytokinins. This particular mutation corresponds to the previously reported wol allele (wooden leg) that causes a striking phenotype defective in vascular morphogenesis. Collectively, evidence is presented that AHK4 and its homologues (AHK3 and possibly AHK2) are receptor kinases that can transduce cytokinin signals across the plasma membrane of A. thaliana.

The Rop GTPase switch controls multiple developmental processes in Arabidopsis.

G proteins are universal molecular switches in eukaryotic signal transduction. The Arabidopsis genome sequence reveals no RAS small GTPase and only one or a few heterotrimeric G proteins, two predominant classes of signaling G proteins found in animals. In contrast, Arabidopsis possesses a unique family of 11 Rop GTPases that belong to the Rho family of small GTPases. Previous studies indicate that Rop controls actin-dependent pollen tube growth and H(2)O(2)-dependent defense responses. In this study, we tested the hypothesis that the Rop GTPase acts as a versatile molecular switch in signaling to multiple developmental processes in Arabidopsis. Immunolocalization using a general antibody against the Rop family proteins revealed a ubiquitous distribution of Rop proteins in all vegetative and reproductive tissues and cells in Arabidopsis. The cauliflower mosaic virus 35S promoter-directed expression of constitutively active GTP-bound rop2 (CA-rop2) and dominant negative GDP-bound rop2 (DN-rop2) mutant genes impacted many aspects of plant growth and development, including embryo development, seed dormancy, seedling development, lateral root initiation, morphogenesis of lateral organs in the shoot, shoot apical dominance and growth, phyllotaxis, and lateral organ orientation. The rop2 transgenic plants also displayed altered responses to the exogenous application of several hormones, such as abscisic acid-mediated seed dormancy, auxin-dependent lateral shoot initiation, and brassinolide-mediated hypocotyl elongation. CA-rop2 and DN-rop2 expression had opposite effects on most of the affected processes, supporting a direct signaling role for Rop in regulating these processes. Based on these observations and previous results, we propose that Rop2 and other members of the Rop family participate in multiple distinct signaling pathways that control plant growth, development, and responses to the environment.

Structure of the N-terminal domain of Yersinia pestis YopH at 2.0 A resolution.

Yersinia pestis, the causative agent of bubonic plague, injects effector proteins into the cytosol of mammalian cells that enable the bacterium to evade the immune response of the infected organism by interfering with eukaryotic signal transduction pathways. YopH is a modular effector composed of a C-terminal protein tyrosine phosphatase (PTPase) domain and a multifunctional N-terminal domain that not only orchestrates the secretion and translocation of YopH into eukaryotic cells but also binds tyrosine-phosphorylated target proteins to mediate substrate recognition. The crystal structure of the N-terminal domain of YopH (YopH(N); residues 1-130) has been determined at 2.0 A resolution. The amino-acid sequences that target YopH for secretion from the bacterium and translocation into eukaryotic cells form integral parts of this compactly folded domain. The structure of YopH(N) bears no resemblance to eukaryotic phosphotyrosine-binding domains, nor is it reminiscent of any known fold. Residues that have been implicated in phosphotyrosine-dependent protein binding are clustered together on one face of YopH(N), but the structure does not suggest a mechanism for protein-phosphotyrosine recognition.

Domain formation in a fluid mixed lipid bilayer modulated through binding of the C2 protein motif.

The role and mechanism of formation of lipid domains in a functional membrane have generally received limited attention. Our approach, based on the hypothesis that thermodynamic coupling between lipid-lipid and protein-lipid interactions can lead to domain formation, uses a combination of an experimental lipid bilayer model system and Monte Carlo computer simulations of a simple model of that system. The experimental system is a fluid bilayer composed of a binary mixture of phosphatidylcholine (PC) and phosphatidylserine (PS), containing 4% of a pyrene-labeled anionic phospholipid. Addition of the C2 protein motif (a structural domain found in proteins implicated in eukaryotic signal transduction and cellular trafficking processes) to the bilayer first increases and then decreases the excimer/monomer ratio of the pyrene fluorescence. We interpret this to mean that protein binding induces anionic lipid domain formation until the anionic lipid becomes saturated with protein. Monte Carlo simulations were performed on a lattice representing the lipid bilayer to which proteins were added. The important parameters are an unlike lipid-lipid interaction term and an experimentally derived preferential protein-lipid interaction term. The simulations support the experimental conclusion and indicate the existence of a maximum in PS domain size as a function of protein concentration. Thus, lipid-protein coupling is a possible mechanism for both lipid and protein clustering on a fluid bilayer. Such domains could be precursors of larger lipid-protein clusters ('rafts'), which could be important in various biological processes such as signal transduction at the level of the cell membrane.