Crystal Structure of the DH/PH Fragment of Dbs without Bound GTPase

Dbl proteins are guanine nucleotide exchange factors for Rho GTPases, containing adjacent Dbl homology (DH) and pleckstrin homology (PH) domains. This domain architecture is virtually invariant and typically required for full exchange potential. Several structures of DH/PH fragments bound to GTPases implicate the PH domain in nucleotide exchange. To more fully understand the functional linkage between DH and PH domains, we have determined the crystal structure of the DH/PH fragment of Dbs without bound GTPase. This structure is generally similar to previously determined structures of Dbs bound to GTPases albeit with greater apparent mobility between the DH and PH domains. These comparisons suggest that the DH and PH domains of Dbs are spatially primed for binding GTPases and small alterations in intradomain conformations that may be elicited by subtle biological responses, such as altered phosphoinositide levels, are sufficient to enhance exchange by facilitating interactions between the PH domain and GTPases

A negative-feedback loop regulating ERK1/2 activation and mediated by RasGPR2 phosphorylation

The dynamic regulation of ERK1 and −2 (ERK1/2) is required for precise signal transduction controlling cell proliferation, differentiation, and survival. However, the underlying mechanisms regulating the activation of ERK1/2 are not completely understood. In this study, we show that phosphorylation of RasGRP2, a guanine nucleotide exchange factor (GEF), inhibits its ability to activate the small GTPase Rap1 that ultimately leads to decreased activation of ERK1/2 in cells. ERK2 phosphorylates RasGRP2 at Ser394 located in the linker region implicated in its autoinhibition. These studies identify RasGRP2 as a novel substrate of ERK1/2 and define a negative-feedback loop that regulates the BRaf–MEK–ERK signaling cascade. This negative-feedback loop determines the amplitude and duration of active ERK1/2

Electrostatic and Functional Analysis of the Seven-Bladed WD β-Propellers

β-propeller domains composed of WD repeats are highly ubiquitous and typically used as multi-site docking platforms to coordinate and integrate the activities of groups of proteins. Here, we have used extensive homology modelling of the WD40-repeat family of seven-bladed β-propellers coupled with subsequent structural classification and clustering of these models to define subfamilies of β-propellers with common structural, and probable, functional characteristics. We show that it is possible to assign seven-bladed WD β-propeller proteins into functionally different groups based on the information gained from homology modelling. We examine general structural diversity within the WD40-repeat family of seven-bladed β-propellers and demonstrate that seven-bladed β-propellers composed of WD-repeats are structurally distinct from other seven-bladed β-propellers. We further provide some insights into the multifunctional diversity of the seven-bladed WD β-propeller surfaces. This report once again reinforces the importance of structural data and the usefulness of homology models in functional classification

Role of the C-Terminal SH3 Domain and N-Terminal Tyrosine Phosphorylation in Regulation of Tim and Related Dbl-Family Proteins †

Dbl-related oncoproteins are guanine nucleotide exchange factors (GEFs) specific for Rho-family GTPases and typically possess tandem Dbl (DH) and pleckstrin homology (PH) domains that act in concert to catalyze exchange. Although the exchange potential of many Dbl-family proteins is constitutively activated by truncation, the precise mechanisms of regulation for many Dbl-family proteins are unknown. Tim and Vav are distantly related Dbl-family proteins that are similarly regulated; their Dbl homology (DH) domains interact with N-terminal helices to exclude and prevent activation of Rho GTPases. Phosphorylation, substitution, or deletion of the blocking helices relieves this autoinhibition. Here we show that two other Dbl-family proteins, Ngef and Wgef, which like Tim contain a C-terminal SH3 domain, are also activated by tyrosine phosphorylation of a blocking helix. Consequently, basal autoinhibition of DH domains by direct steric exclusion using short N-terminal helices likely represents a conserved mechanism of regulation for the large family of Dbl-related proteins. N-Terminal truncation or phosphorylation of many other Dbl-family GEFs leads to their activation; similar autoinhibition mechanisms could explain some of these events. In addition, we show that the C-terminal SH3 domain binding to a polyproline region N-terminal to the DH domain of the Tim subgroup of Dbl-family proteins provides a unique mechanism of regulated autoinhibition of exchange activity that is functionally linked to the interactions between the autoinhibitory helix and the DH domain

PENERAPAN MODEL PEMBELAJARAN KOOPERATIF TIPE THINK PAIR SHARE UNTUK MENINGKATKAN HASIL BELAJAR SISWA PADA MATERI LUAS PERMUKAAN DAN VOLUME PRISMA DI KELAS VIII SMP NEGERI 18 PALU

Abstrak: Tujuan penelitian ini adalah untuk memperoleh deskripsi tentang penerapan model pembelajaran kooperatif tipe Think Pair Share (TPS) yang dapat meningkatkan hasil belajar siswa pada materi  luas permukaan dan volume prisma di kelas VIII SMP Negeri 18 Palu. Jenis penelitian ini adalah Penelitian Tindakan Kelas (PTK). Rancangan penelitian ini mengacu pada desain penelitian Kemmis dan Mc. Taggart yaitu 1) perencanaan, 2) pelaksanaan tindakan, 3) observasi dan 4) refleksi. Subjek penelitian ini adalah siswa kelas VIII SMP Negeri 18 Palu yang berjumlah 24 siswa. Penelitian ini dilakukan dalam dua siklus. Data pada penelitian ini dikumpulkan melalui lembar observasi, wawancara, catatan lapangan dan tes. Hasil penelitian menunjukkan bahwa penerapan model pembelajaran koopertif tipe think pair share dapat meningkatkan hasil belajar siswa pada materi luas permukaan dan volume prisma, dengan mengikuti fase-fase sebagai berikut: 1) mempersiapkan siswa dan meyampaikan tujuan pembelajaran, 2) think, 3) pair, 4) share, 5) membuat kesimpulan.Kata kunci: think pair share, hasil belajar, luas permukaan prisma, volume prisma

Activation of Human Phospholipase C-η2 by Gβγ †

Phospholipase C-η2 (PLC-η2) was recently identified as a novel broadly expressed phosphoinositide-hydrolyzing isozyme [Zhou, Y., et al. (2005) Biochem. J. 391, 667–676; Nakahara, M., et al. (2005) J. Biol. Chem. 280, 29128–29134]. In this study, we investigated the direct regulation of PLC-η2 by Gβγ subunits of heterotrimeric G proteins. Coexpression of PLC-η2 with Gβ1γ2, as well as with certain other Gβγ dimers, in COS-7 cells resulted in increases in inositol phosphate accumulation. Gβ1γ2-dependent increases in phosphoinositide hydrolysis also were observed with a truncation mutant of PLC-η2 that lacks the long alternatively spliced carboxy-terminal domain of the isozyme. To begin to define the enzymatic properties of PLC-η2 and its potential direct activation by Gβγ, a construct of PLC-η2 encompassing the canonical domains conserved in all PLCs (PH domain through C2 domain) was purified to homogeneity after expression from a baculovirus in insect cells. Enzyme activity of purified PLC-η2 was quantified after reconstitution with PtdIns(4,5)P2-containing phospholipid vesicles, and values for Km (14.4 µM) and Vmax [12.6 µmol min−1 (mg of protein)−1] were similar to activities previously observed with purified PLC-β or PLC-ε isozymes. Moreover, purified Gβ1γ2 stimulated the activity of purified PLC-η2 in a concentration-dependent manner similar to that observed with purified PLC-β2. Activation was dependent on the presence of free Gβ1γ2 since its sequestration in the presence of Gαi1 or GRK2-ct reversed Gβ1γ2-promoted activation. The PH domain of PLC-η2 is not required for Gβ1γ2-mediated regulation since a purified fragment encompassing the EF-hand through C2 domains but lacking the PH domain nonetheless was activated by Gβ1γ2. Taken together, these studies illustrate that PLC-η2 is a direct downstream effector of Gβγ and, therefore, of receptor-activated heterotrimeric G proteins

Crystal Structures of the Type III Effector Protein AvrPphF and Its Chaperone Reveal Residues Required for Plant Pathogenesis

The avrPphF locus from Pseudomonas syringae pv. phaseolicola, the causative agent of bean halo-blight disease, encodes proteins which either enhance virulence on susceptible hosts or elicit defense responses on hosts carrying the R1 resistance gene. Here we present the crystal structures of the two proteins from the avrPphF operon. The structure of AvrPphF ORF1 is strikingly reminiscent of type III chaperones from bacterial pathogens of animals, indicating structural conservation of these specialized chaperones, despite high sequence divergence. The AvrPphF ORF2 effector adopts a novel "mushroom"-like structure containing "head" and "stalk" subdomains. The head subdomain possesses limited structural homology to the catalytic domain of bacterial ADP-ribosyltransferases (ADP-RTs), though no ADP-RT activity was detected for AvrPphF ORF2 in standard assays. Nonetheless, this structural similarity identified two clusters of conserved surface-exposed residues important for both virulence mediated by AvrPphF ORF2 and recognition of this effector by bean plants expressing the R1 resistance gene

Molecular architecture of Gαo and the structural basis for RGS16-mediated deactivation

Heterotrimeric G proteins relay extracellular cues from heptahelical transmembrane receptors to downstream effector molecules. Composed of an α subunit with intrinsic GTPase activity and a βγ heterodimer, the trimeric complex dissociates upon receptor-mediated nucleotide exchange on the α subunit, enabling each component to engage downstream effector targets for either activation or inhibition as dictated in a particular pathway. To mitigate excessive effector engagement and concomitant signal transmission, the Gα subunit's intrinsic activation timer (the rate of GTP hydrolysis) is regulated spatially and temporally by a class of GTPase accelerating proteins (GAPs) known as the regulator of G protein signaling (RGS) family. The array of G protein-coupled receptors, Gα subunits, RGS proteins and downstream effectors in mammalian systems is vast. Understanding the molecular determinants of specificity is critical for a comprehensive mapping of the G protein system. Here, we present the 2.9 Å crystal structure of the enigmatic, neuronal G protein Gαo in the GTP hydrolytic transition state, complexed with RGS16. Comparison with the 1.89 Å structure of apo-RGS16, also presented here, reveals plasticity upon Gαo binding, the determinants for GAP activity, and the structurally unique features of Gαo that likely distinguish it physiologically from other members of the larger Gαi family, affording insight to receptor, GAP and effector specificity

Correction: Type III Effector Activation via Nucleotide Binding, Phosphorylation, and Host Target Interaction

The Pseudomonas syringae type III effector protein avirulence protein B (AvrB) is delivered into plant cells, where it targets the Arabidopsis RIN4 protein (resistance to Pseudomonas maculicula protein 1 [RPM1]–interacting protein). RIN4 is a regulator of basal host defense responses. Targeting of RIN4 by AvrB is recognized by the host RPM1 nucleotide-binding leucine-rich repeat disease resistance protein, leading to accelerated defense responses, cessation of pathogen growth, and hypersensitive host cell death at the infection site. We determined the structure of AvrB complexed with an AvrB-binding fragment of RIN4 at 2.3 Å resolution. We also determined the structure of AvrB in complex with adenosine diphosphate bound in a binding pocket adjacent to the RIN4 binding domain. AvrB residues important for RIN4 interaction are required for full RPM1 activation. AvrB residues that contact adenosine diphosphate are also required for initiation of RPM1 function. Nucleotide-binding residues of AvrB are also required for its phosphorylation by an unknown Arabidopsis protein(s). We conclude that AvrB is activated inside the host cell by nucleotide binding and subsequent phosphorylation and, independently, interacts with RIN4. Our data suggest that activated AvrB, bound to RIN4, is indirectly recognized by RPM1 to initiate plant immune system function

A Fluorogenic, Small Molecule Reporter for Mammalian Phospholipase C Isozymes

Phospholipase C isozymes (PLCs) catalyze the conversion of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. This family of enzymes are key signaling proteins that regulate the physiological responses of many extracellular stimuli such as hormones, neurotransmitters, and growth factors. Aberrant regulation of PLCs has been implicated in various diseases including cancer and Alzheimer’s disease. How, when, and where PLCs are activated under different cellular contexts are still largely unknown. We have developed a fluorogenic PLC reporter, WH-15, that can be cleaved in a cascade reaction to generate fluorescent 6-aminoquinoline. When applied in enzymatic assays with either pure PLCs or cell lysates, this reporter displays more than a 20-fold fluorescence enhancement in response to PLC activity. Under assay conditions, WH-15 has comparable Km and Vmax with the endogenous PIP2. This novel reporter will likely find broad applications that vary from imaging PLC activity in live cells to high throughput screening of PLC inhibitors