The nucleotide exchange factor Ric-8A is a chaperone for the conformationally dynamic nucleotide-free state of Gαi1

Heterotrimeric G protein α subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of Gα, catalyzed by guanine nucleotide exchange factors (GEFs). In addition to transmembrane G protein-coupled receptors (GPCRs), which act on G protein heterotrimers, members of the family cytosolic proteins typified by mammalian Ric-8A are GEFs for Gi/q/12/13-class Gα subunits. Ric-8A binds to Gα?GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free Gαi1 is stable, but dissociates upon binding of GTP to Gαi1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release from Gαi1, experiments were conducted to characterize the physical state of nucleotide-free Gαi1 (hereafter referred to as Gαi1[ ]) in solution, both as a monomeric species, and in the complex with Ric-8A. We found that Ric-8A-bound, nucleotide-free Gαi1 is more accessible to trypsinolysis than Gαi1?GDP, but less so than Gαi1[ ] alone. The TROSY-HSQC spectrum of [(15)N]Gαi1[ ] bound to Ric-8A shows considerable loss of peak intensity relative to that of [(15)N]Gαi1?GDP. Hydrogen-deuterium exchange in Gαi1[ ] bound to Ric-8A is 1.5-fold more extensive than in Gαi1?GDP. Differential scanning calorimetry shows that both Ric-8A and Gαi1?GDP undergo cooperative, irreversible unfolding transitions at 47° and 52°, respectively, while nucleotide-free Gαi1 shows a broad, weak transition near 35°. The unfolding transition for Ric-8A:Gαi1[ ] is complex, with a broad transition that peaks at 50°, suggesting that both Ric-8A and Gαi1[ ] are stabilized within the complex, relative to their respective free states. The C-terminus of Gαi1 is shown to be a critical binding element for Ric-8A, as is also the case for GPCRs, suggesting that the two types of GEF might promote nucleotide exchange by similar mechanisms, by acting as chaperones for the unstable and dynamic nucleotide-free state of Gα.     

Ric-8A,a GEF,and a Chaperone for G Protein α-Subunits: Evidence for the Two-Faced Interface

Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a prominent non-receptor GEF and a chaperone of G protein α-subunits (Gα). Recent studies shed light on the structure of Ric-8A, providing insights into the mechanisms underlying its interaction with Gα. Ric-8A is composed of a core armadillo-like domain and a flexible C-terminal tail. Interaction of a conserved concave surface of its core domain with the Gα C-terminus appears to mediate formation of the initial Ric-8A/GαGDP intermediate, followed by the formation of a stable nucleotide-free complex. The latter event involves a large-scale dislocation of the Gα α5-helix that produces an extensive primary interface and disrupts the nucleotide-binding site of Gα. The distal portion of the C-terminal tail of Ric-8A forms a smaller secondary interface, which ostensibly binds the switch II region of Gα, facilitating binding of GTP. The two-site Gα interface of Ric-8A is distinct from that of GPCRs, and might have evolved to support the chaperone function of Ric-8A.     

Nucleobindin 1 Is a Calcium-regulated Guanine Nucleotide Dissociation Inhibitor of Gαi1

Nucleobindin 1 (NUCB1) is a widely expressed multidomain calcium-binding protein whose precise physiological and biochemical functions are not well understood. We engineered and heterologously expressed a soluble form of NUCB1 (sNUCB1) and characterized its biophysical and biochemical properties. We show that sNUCB1 exists as a dimer in solution and that each monomer binds two divalent calcium cations. Calcium binding causes conformational changes in sNUCB1 as judged by circular dichroism and fluorescence spectroscopy experiments. Earlier reports suggested that NUCB1 might interact with heterotrimeric G protein α subunits. We show that dimeric calcium-free sNUCB1 binds to expressed Gα_i1 and that calcium binding inhibits the interaction. The binding of sNUCB1 to Gα_i1 inhibits its basal rate of GDP release and slows its rate and extent of GTPγS uptake. Additionally, our tissue culture experiments show that sNUCB1 prevents receptor-mediated Gα_i-dependent inhibition of adenylyl cyclase. Thus, we conclude that sNUCB1 is a calcium-dependent guanine nucleotide dissociation inhibitor (GDI) for Gα_i1. To our knowledge, sNUCB1 is the first example of a calcium-dependent GDI for heterotrimeric G proteins. We also show that the mechanism of GDI activity of sNUCB1 is unique and does not arise from the consensus GoLoco motif found in RGS proteins. We propose that cytoplasmic NUCB1 might function to regulate heterotrimeric G protein trafficking and G protein-coupled receptor-mediated signal transduction pathways.     

Localization of the Translational Guanine Nucleotide Exchange Factor eIF2B: A Common Theme for GEFs?

The eukaryotic initiation factor 2B (eIF2B) serves an essential recycling function in protein synthesis. As the guanine nucleotide exchange factor for eIF2, it recycles eIF2 from a GDP to a GTP bound form that is competent for translation initiation. Stressdependent controls target this eIF2B-recycling step allowing a re-programming of the global gene expression profile. In addition, a human disease, leukoencephalopathy with vanishing white matter (VWM), is caused by mutations in the eIF2B subunit genes. Recently, we have found that the eIF2B guanine nucleotide exchange factor resides in a specific cytoplasmic focus in the yeast, Saccharmoyces cerevisiae. eIF2B is a resident feature of this focus, whereas eIF2 shuttles to and fro. Moreover, the in vivo rate of eIF2 shuttling correlates with changes in guanine nucleotide exchange activity implicating this large cytoplasmic focus as a site of guanine nucleotide exchange. In this perspective, we discuss these findings in the wider context of the assortment of guanine nucleotide exchange factors.     

The Guanine Nucleotide Exchange Factor RIC8 Regulates Conidial Germination through Gα Proteins in Neurospora crassa

Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia) germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth.     

Amino Acid Region 1000–1008 of Factor V Is a Dynamic Regulator for the Emergence of Procoagulant Activity

Single chain factor V (fV) circulates as an M_r 330,000 quiescent pro-cofactor. Removal of the B domain and generation of factor Va (fVa) are vital for procoagulant activity. We investigated the role of the basic amino acid region 1000–1008 within the B domain of fV by constructing a recombinant mutant fV molecule with all activation cleavage sites (Arg⁷⁰⁹/Arg¹⁰¹⁸/Arg¹⁵⁴⁵) mutated to glutamine (fV^Q3), a mutant fV molecule with region 1000–1008 deleted (fV^ΔB9), and a mutant fV molecule containing the same deletion with activation cleavage sites changed to glutamine (fV^ΔB9/Q3). The recombinant molecules along with wild type fV (fV^WT) were transiently expressed in COS-7L cells, purified, and assessed for their ability to bind factor Xa (fXa) prior to and following incubation with thrombin. The data showed that fV^Q3 was severely impaired in its interaction with fXa before and after incubation with thrombin. In contrast, K_D(app) values for fV^ΔB9 (0.9 nm), fVa^ΔB9 (0.4 nm), and fV^ΔB9/Q3 (0.7 nm) were similar to the affinity of fVa^WT for fXa (0.3 nm). Two-stage clotting assays revealed that although fV^Q3 was deficient in its clotting activity, fV^ΔB9/Q3 had clotting activity comparable with fVa^WT. The k_cat value of prothrombinase assembled with fV^ΔB9/Q3 was minimally affected, whereas the K_m value of the reaction was increased 57-fold compared with the K_m value obtained with prothrombinase assembled with fVa^WT. These findings strongly suggest that amino acid region 1000–1008 of fV is a regulatory sequence protecting the organisms from spontaneous binding to fXa and unnecessary prothrombinase complex formation, which in turn results in catastrophic physiological consequences.     

Structure,Function, and Dynamics of the Gα Binding Domain of Ric-8A

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Importin-�� Is a GDP-to-GTP Exchange Factor of Ran: IMPLICATIONS FOR THE MECHANISM OF NUCLEAR IMPORT*

Ran-GTP interacts strongly with importin-β, and this interaction promotes the release of the importin-α-nuclear localization signal cargo from importin-β. Ran-GDP also interacts with importin-β, but this interaction is 4 orders of magnitude weaker than the Ran-GTP·importin-β interaction. Here we use the yeast complement of nuclear import proteins to show that the interaction between Ran-GDP and importin-β promotes the dissociation of GDP from Ran. The release of GDP from the Ran-GDP-importin-β complex stabilizes the complex, which cannot be dissociated by importin-α. Although Ran has a higher affinity for GDP compared with GTP, Ran in complex with importin-β has a higher affinity for GTP. This feature is responsible for the generation of Ran-GTP from Ran-GDP by importin-β. Ran-binding protein-1 (RanBP1) activates this reaction by forming a trimeric complex with Ran-GDP and importin-β. Importin-α inhibits the GDP exchange reaction by sequestering importin-β, whereas RanBP1 restores the GDP nucleotide exchange by importin-β by forming a tetrameric complex with importin-β, Ran, and importin-α. The exchange is also inhibited by nuclear-transport factor-2 (NTF2). We suggest a mechanism for nuclear import, additional to the established RCC1 (Ran-guanine exchange factor)-dependent pathway that incorporates these results.Ran (Gsp1p in yeast) is a Ras-like GTPase that regulates diverse cellular processes, including nuclear transport, mitotic spindle assembly, and post-mitotic nuclear assembly (1, 2). Like other GTPases, Ran can bind GTP and GDP. Ran-GTP is generated in the nucleus by the guanine exchange factor RCC1 (regulator of chromosome condensation 1), which is associated with the chromatin (3). Ran-GDP is produced in the cytoplasm by the activation of the intrinsic GTPase activity of Ran by RanGAP1 (GTPase-activating protein) (4) and RanBP1 (Ran-binding protein-1, Yrb1p in yeast). The compartmentalization of RanGAP1 (cytoplasm) and RCC1 (nucleus) gives rise to the asymmetric distribution of Ran-GDP (cytoplasm) and Ran-GTP (nucleus) across the nuclear envelope. This asymmetric distribution of Ran-GDP and Ran-GTP plays a central role in nucleocytoplasmic transport by mediating assembly and disassembly of import and export complexes through interaction with the nuclear import machinery (for reviews, see Refs. 5–9).The passage of molecules into the nucleus occurs through the nuclear pore complexes (NPCs)⁶ (10). Nucleocytoplasmic transport is driven by a series of protein-protein interactions and involves several soluble carriers named β-karyopherins. Import carriers are called importins and export carriers are called exportins. The classical nuclear import pathway involves importin-β (Kap95p in yeast) and the adaptor protein importin-α (Kap60p in yeast). In the cytoplasm importin-β binds to importin-α. Their interaction triggers a conformational change of importin-α that increases its affinity for cargo proteins containing a nuclear localization signal (NLS) (11, 12). The translocation of the resulting complex (importin-β·importin-α·NLS) involves interactions with the NPC proteins (nucleoporins), particularly the FXFG-repeat domains (11). The protein cargo is released in the nucleus by the action of Ran-GTP, which induces the dissociation of importin-α from importin-β by forming a stable complex with importin-β. The importins are then recycled to the cytoplasm. Importin-β transfers to the cytoplasm associated with Ran-GTP, and importin-α is exported by CAS (exportin2; Cse1p in yeast) in the form of an importin-α·CAS·Ran-GTP complex (13). Importin-β and importin-α are released from their complexes in the cytoplasm by the combined action of RanBP1 and RanGAP1. Importin-β and importin-α are then able to function in a new cycle of transport, whereas Ran-GDP is transported into the nucleus by NTF2 (nuclear-transport factor-2, Ntf2p in yeast) (14). In the nucleus Ran-GDP is transformed to Ran-GTP by the action of RCC1 (3).The complexity of the nuclear import mechanism is highlighted by the fact that it involves the active participation of soluble factors other than Ran-GTP, importin-β, and importin-α. Indeed, Ran-GDP, RanBP1, and NTF2 have been shown to be involved in the docking and translocation events of nuclear import. Chi et al. (15) have demonstrated that Ran-GDP forms a stable complex with RanBP1 and importin-β; they suggested a role for Ran-GDP in the association of the importin-β·importin-α·NLS complex with the nuclear pore and speculated that the importin-β·importin-α·NLS·Ran-GDP·RanBP1 pentameric complex was the actual translocation complex that moved through the pore. This model has also been adopted by others (16–18) who have proposed that a stable Ran-GDP-containing complex was created on nucleoporin Nup358 (also called RanBP2) and that upon displacement of the importin-β·importin-α·Ran-GDP complex from the RBH (domain homologous to RanBP1) domains of Nup358 by RanBP1, binding of NTF2 triggered translocation to the nucleus. The role of NTF2 as the factor responsible for the translocation of the transport complex through the nuclear envelope has also been proposed by Paschal et al. (19). The role of Ran-GDP and RanBP1 in nuclear import has been demonstrated by a single mutation of a cysteine residue of importin-β; the mutation was required for binding Ran-GDP·RanBP1, but not Ran-GTP·RanBP1, and inhibited the nuclear import in permeabilized cells (20). The active role of RanBP1 in nuclear import has been further demonstrated by Künzler et al. (21), who showed that mutations in the Yrb1 gene encoding the yeast ortholog of RanBP1 impair nucleocytoplasmic transport.Despite considerable evidence for the involvement of Ran-GDP, RanBP1, and NTF2 in nuclear protein import, the precise mechanism by which these molecules regulate this process has been unknown. Here we characterize the interaction between Kap95p and Gsp1p-GDP. We show that this interaction results in GDP-to-GTP exchange on Gsp1p. Furthermore, we demonstrate that Gsp1p, Kap60p, Kap95p, Yrb1p, and Ntf2p interact to regulate the GDP-to-GTP exchange on Gsp1p. We suggest a mechanism of nuclear import additional to the RCC1-dependent pathway that incorporates our observations.     

G protein-coupled receptors and resistance to inhibitors of cholinesterase-8A (Ric-8A) both regulate the regulator of g protein signaling 14 RGS14·Gαi1 complex in live cells

Regulator of G protein Signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates both conventional and unconventional G protein signaling pathways. Like other RGS (regulator of G protein signaling) proteins, RGS14 acts as a GTPase accelerating protein to terminate conventional Gα(i/o) signaling. However, unlike other RGS proteins, RGS14 also contains a G protein regulatory/GoLoco motif that specifically binds Gα(i1/3)-GDP in cells and in vitro. The non-receptor guanine nucleotide exchange factor Ric-8A can bind and act on the RGS14·Gα(i1)-GDP complex to play a role in unconventional G protein signaling independent of G protein-coupled receptors (GPCRs). Here we demonstrate that RGS14 forms a Gα(i/o)-dependent complex with a G(i)-linked GPCR and that this complex is regulated by receptor agonist and Ric-8A (resistance to inhibitors of cholinesterase-8A). Using live cell bioluminescence resonance energy transfer, we show that RGS14 functionally associates with the α(2A)-adrenergic receptor (α(2A)-AR) in a Gα(i/o)-dependent manner. This interaction is markedly disrupted after receptor stimulation by the specific agonist UK14304, suggesting complex dissociation or rearrangement. Agonist-mediated dissociation of the RGS14·α(2A)-AR complex occurs in the presence of Gα(i/o) but not Gα(s) or Gα(q). Unexpectedly, RGS14 does not dissociate from Gα(i1) in the presence of stimulated α(2A)-AR, suggesting preservation of RGS14·Gα(i1) complexes after receptor activation. However, Ric-8A facilitates dissociation of both the RGS14·Gα(i1) complex and the Gα(i1)-dependent RGS14·α(2A)-AR complex after receptor activation. Together, these findings indicate that RGS14 can form complexes with GPCRs in cells that are dependent on Gα(i/o) and that these RGS14·Gα(i1)·GPCR complexes may be substrates for other signaling partners such as Ric-8A.     

A Bacteriophage-Encoded J-Domain Protein Interacts with the DnaK/Hsp70 Chaperone and Stabilizes the Heat-Shock Factor ��32 of Escherichia coli

The universally conserved J-domain proteins (JDPs) are obligate cochaperone partners of the Hsp70 (DnaK) chaperone. They stimulate Hsp70''s ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene 057w of the T4-related enterobacteriophage RB43 encodes a bona fide JDP protein, named Rki, which specifically interacts with the Escherichia coli host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ³², which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ³² facilitates RB43 bacteriophage proliferation is discussed.     

Coordination of Eukaryotic Translation Elongation Factor 1A (eEF1A) Function in Actin Organization and Translation Elongation by the Guanine Nucleotide Exchange Factor eEF1B��

Eukaryotic translation elongation factor 1A (eEF1A) both shuttles aminoacyl-tRNA (aa-tRNA) to the ribosome and binds and bundles actin. A single domain of eEF1A is proposed to bind actin, aa-tRNA and the guanine nucleotide exchange factor eEF1Bα. We show that eEF1Bα has the ability to disrupt eEF1A-induced actin organization. Mutational analysis of eEF1Bα F163, which binds in this domain, demonstrates effects on growth, eEF1A binding, nucleotide exchange activity, and cell morphology. These phenotypes can be partially restored by an intragenic W130A mutation. Furthermore, the combination of F163A with the lethal K205A mutation restores viability by drastically reducing eEF1Bα affinity for eEF1A. This also results in a consistent increase in actin bundling and partially corrected morphology. The consequences of the overlapping functions in this eEF1A domain and its unique differences from the bacterial homologs provide a novel function for eEF1Bα to balance the dual roles in actin bundling and protein synthesis.The final step of gene expression takes place at the ribosome as mRNA is translated into protein. In the yeast Saccharomyces cerevisiae, elongation of the polypeptide chain requires the orchestrated action of three soluble factors. The eukaryotic elongation factor 1 (eEF1)² complex delivers aminoacyl-tRNA (aa-tRNA) to the empty A-site of the elongating ribosome (1). The eEF1A subunit is a classic G-protein that acts as a “molecular switch” for the active and inactive states based on whether GTP or GDP is bound, respectively (2). Once an anticodon-codon match occurs, the ribosome acts as a GTPase-activating factor to stimulate GTP hydrolysis resulting in the release of inactive GDP-bound eEF1A from the ribosome. Because the intrinsic rate of GDP release from eEF1A is extremely slow (3, 4), a guanine nucleotide exchange factor (GEF) complex, eEF1B, is required (5, 6). The yeast S. cerevisiae eEF1B complex contains two subunits, the essential catalytic subunit eEF1Bα (5) and the non-essential subunit eEF1Bγ (7).The co-crystal structures of eEF1A:eEF1Bα C terminus:GDP: Mg²⁺ and eEF1A:eEF1Bα C terminus:GDPNP (8, 9) demonstrated a surprising structural divergence from the bacterial EF-Tu-EF-Ts (10) and mammalian mitochondrial EF-Tu_mt-EF-Ts_mt (11). While the G-proteins have a similar topology and consist of three well-defined domains, a striking difference was observed in binding sites for their GEFs. The C terminus of eEF1Bα interacts with domain I and a distinct pocket of domain II eEF1A, creating two binding interfaces. In contrast, the bacterial counterpart EF-Ts and mammalian mitochondrial EF-Ts_mt, make extensive contacts with domain I and III of EF-Tu and EF-Tu_mt, respectively. The altered binding interface of eEF1Bα to domain II of eEF1A is particularly unexpected given the functions associated with domain II of eEF1A and EF-Tu. The crystal structure of the EF-Tu:GDPNP:Phe-tRNA^Phe complex reveals aa-tRNA binding to EF-Tu requires only minor parts of both domain II and tRNA to sustain stable contacts (12). That eEF1A employs the same aa-tRNA binding site is supported by genetic and biochemical data (13-15). Interestingly, eEF1Bα contacts many domain II eEF1A residues in the region hypothesized to be involved in the binding of the aa-tRNA CCA end (8). Because, the shared binding site of eEF1Bα and aa-tRNA on domain II of eEF1A is significantly different between the eukaryotic and bacterial/mitochondrial systems, eEF1Bα may play a unique function aside from guanine nucleotide release in eukaryotes.In eukaroytes, eEF1A is also an actin-binding and -bundling protein. This noncanonical function of eEF1A was initially observed in Dictyostelium amoebae (16). It is estimated that greater than 60% of Dictyostelium eEF1A is associated with the actin cytoskeleton (17). The eEF1A-actin interaction is conserved among species from yeast to mammals, suggesting the importance of eEF1A for cytoskeleton integrity. Using a unique genetic approach, multiple eEF1A mutations were identified that altered cell growth and morphology, and are deficient in bundling actin in vitro (18, 19). Intriguingly, most mutations localized to domain II, the shared aa-tRNA and eEF1Bα binding site. Previous studies have demonstrated that actin bundling by eEF1A is significantly reduced in the presence of aa-tRNA while eEF1A bound to actin filaments is not in complex with aa-tRNA (20). Therefore, actin and aa-tRNA binding to eEF1A is mutually exclusive. In addition, overexpression of yeast eEF1A or actin-bundling deficient mutants do not affect translation elongation (18, 19, 21), suggesting eEF1A-dependent cytoskeletal organization is independent of its translation elongation function (18, 20). Thus, while aa-tRNA binding to domain II is conserved between EF-Tu and eEF1A, this actin bundling function associated with eEF1A domain II places greater importance on its relationship with the “novel” binding interface between eEF1A domain II and eEF1Bα.Based on this support for an overlapping actin bundling and eEF1Bα binding site in eEF1A domain II, we hypothesize that eEF1Bα modulates the equilibrium between actin and translation functions of eEF1A and is perhaps the result of evolutionary selective pressure to balance the eukaryotic-specific role of eEF1A in actin organization. Here, we present kinetic and biochemical evidence using a F163A mutant of eEF1Bα for the importance of the interactions between domain II of eEF1A and eEF1Bα to prevent eEF1A-dependent actin bundling as well as promoting guanine nucleotide exchange. Furthermore, altered affinities of eEF1Bα mutants for eEF1A support that this complex formation is a determining factor for eEF1A-induced actin organization. Interestingly, the F163A that reduces eEF1A affinity is an intragenic suppressor of the lethal K205A eEF1Bα mutant that displays increased affinity for eEF1A. This, along with a consistent change in the actin bundling correlated with the affinity of eEF1Bα for eEF1A, indicates that eEF1Bα is a balancer, directing eEF1A to translation elongation and away from actin, and alterations in this balance result in detrimental effects on cell growth and eEF1A function.     

The Protein Kinase C�� Catalytic Fragment Is Critical for Maintenance of the G2/M DNA Damage Checkpoint

Protein kinase Cδ (PKCδ) is an essential component of the intrinsic apoptotic program. Following DNA damage, such as exposure to UV radiation, PKCδ is cleaved in a caspase-dependent manner, generating a constitutively active catalytic fragment (PKCδ-cat), which is necessary and sufficient for keratinocyte apoptosis. We found that in addition to inducing apoptosis, expression of PKCδ-cat caused a pronounced G₂/M cell cycle arrest in both primary human keratinocytes and immortalized HaCaT cells. Consistent with a G₂/M arrest, PKCδ-cat induced phosphorylation of Cdk1 (Tyr¹⁵), a critical event in the G₂/M checkpoint. Treatment with the ATM/ATR inhibitor caffeine was unable to prevent PKCδ-cat-induced G₂/M arrest, suggesting that PKCδ-cat is functioning downstream of ATM/ATR in the G₂/M checkpoint. To better understand the role of PKCδ and PKCδ-cat in the cell cycle response to DNA damage, we exposed wild-type and PKCδ null mouse embryonic fibroblasts (MEFs) to UV radiation. Wild-type MEFs underwent a pronounced G₂/M arrest, Cdk1 phosphorylation, and induction of apoptosis following UV exposure, whereas PKCδ null MEFs were resistant to these effects. Expression of PKCδ-green fluorescent protein, but not caspase-resistant or kinase-inactive PKCδ, was able to restore G₂/M checkpoint integrity in PKCδ null MEFs. The function of PKCδ in the DNA damage-induced G₂/M cell cycle checkpoint may be a critical component of its tumor suppressor function.     

βArrestin1 Regulates the Guanine Nucleotide Exchange Factor RasGRF2 Expression and the Small GTPase Rac-mediated Formation of Membrane Protrusion and Cell Motility

βArrestin proteins shuttle between the cytosol and nucleus and have been shown to regulate G protein-coupled receptor signaling, actin remodeling, and gene expression. Here, we tested the hypothesis that βarrestin1 regulates actin remodeling and cell migration through the small GTPase Rac. Depletion of βarrestin1 promotes Rac activation, leading to the formation of multipolar protrusions and increased cell circularity, and overexpression of a dominant negative form of Rac reverses these morphological changes. Small interfering RNA library screen identifies RasGRF2 as a target of βarrestin1. RasGRF2 gene and protein expression levels are elevated following depletion of βarrestin1, and the consequent activation of Rac results in dephosphorylation of cofilin that can promote actin polymerization and formation of multipolar protrusions, thereby retarding cell migration and invasion. Together, these results suggest that βarrestin1 regulates rasgrf2 gene expression and Rac activation to affect membrane protrusion and cell migration and invasion.     

Evidence for a Second, High Affinity G�¦� Binding Site on G��i1(GDP) Subunits

It is well known that Gα_i1(GDP) binds strongly to Gβγ subunits to form the Gα_i1(GDP)-Gβγ heterotrimer, and that activation to Gα_i1(GTP) results in conformational changes that reduces its affinity for Gβγ subunits. Previous studies of G protein subunit interactions have used stoichiometric amounts of the proteins. Here, we have found that Gα_i1(GDP) can bind a second Gβγ subunit with an affinity only 10-fold weaker than the primary site and close to the affinity between activated Gα_i1 and Gβγ subunits. Also, we find that phospholipase Cβ2, an effector of Gβγ, does not compete with the second binding site implying that effectors can be bound to the Gα_i1(GDP)-(Gβγ)₂ complex. Biophysical measurements and molecular docking studies suggest that this second site is distant from the primary one. A synthetic peptide having a sequence identical to the putative second binding site on Gα_i1 competes with binding of the second Gβγ subunit. Injection of this peptide into cultured cells expressing eYFP-Gα_i1(GDP) and eCFP-Gβγ reduces the overall association of the subunits suggesting this site is operative in cells. We propose that this second binding site serves to promote and stabilize G protein subunit interactions in the presence of competing cellular proteins.The plasma membranes of cells are organized as a series of protein-rich and lipid-rich domains (1–3). Many of the protein-rich domains, in particular those organized by caveolin proteins, are thought to be complexes of functionally related proteins that transduce extracellular signals (2). There is increasing evidence that heterotrimeric G proteins exist in pre-formed membrane complexes with their receptors and their intracellular effectors (4–8).The G protein signaling system is initiated when an extracellular agonist binds to its specific G protein-coupled receptor (for review see Refs. 9–12). The ligand-bound receptor will then catalyze the exchange of GTP for GDP on the Gα subunit in the G protein heterotrimer. In the basal state, Gα(GDP) binds strongly to Gβγ, but in the GTP-bound state this affinity is reduced, allowing Gα(GTP) and Gβγ subunits to individually bind to a host of specific intracellular enzymes and change their catalytic activity.Although the interactions between G protein subunits have been studied extensively in vitro, their behavior in cells may differ. For example, in pure or semi-pure systems, activation of Gα(GDP) sufficiently weakens its affinity for Gβγ resulting in dissociation (13). However, in cells separation of the heterotrimer is observed under some circumstances, but not others (7, 14–17). The reason for these differences in behavior is not clear. There are four families of Gα subunits that each contain several members, and, additionally, there are many subtypes of Gβγ subunits (18). It is possible that differences in dissociation behavior reflect differences in affinity between G protein subunit subtypes (19), the presence of various protein partners, and/or differences in post-synthetic modifications of the subunits (20).The mechanism that allows activated G proteins to remain bound is not apparent from the crystal structure (21, 22). If G protein subunits do not dissociate in cells, then their interaction must change in such a manner as to expose the effector interaction site(s). We have found that phospholipase Cβ1 (PLCβ1),⁴ an important effector of Gα_q (23), is bound to Gα_q prior to activation and throughout the activation cycle (6) implying that Gα_q(GDP) interacts with PLCβ1 in a non-functional manner.We have evidence that signaling complexes are stabilized by a series of secondary interactions. Using purified proteins and model membranes, we have found that membranes of the Gα_q-Gβγ/PLCβ1/RGS4 signaling system have secondary, weaker binding sites to members of this signaling system in addition to their high affinity site(s) to their functional partner(s). We speculate that secondary contacts allow for self-scaffolding of signaling proteins. To understand the nature of these secondary contacts, we have studied the ability of the Gα_i1(GDP)-Gβγ heterotrimer to remain complexed through the activation cycle (24). Here, we present evidence that Gα_i1(GDP) has two distinct Gβγ binding sites that only differ in affinity by an order of magnitude and may allow for continued association between the subunits upon activation. We also find that this site plays an important role in stabilizing G protein associations in cells and provides a mechanism of self-scaffolding.     

The Risk Factors for Failure of an Upper Extremity Replantation: Is the Use of Cigarettes/Tobacco a Significant Factor?

Background

The purpose of this study was to explore the potential risk factors associated with the failure of an upper extremity replantation with a focus on cigarette or tobacco use.

Patients and Methods

A cohort of 102 patients with 149 replants (6 extremities, 143 digits) and a mean age of 41 years (range 5 to 72 years) was enrolled in this study. The data collected included age, gender, tobacco/cigarettes use, trauma mechanism, underlying disease (e.g., hypertension (HTN), diabetes mellitus (DM), etc.), and vein graft use. An analysis with a multivariable regression was conducted to identify the risk factors of replant failure and their respective odds ratios (ORs).

Results

Multilevel generalized linear mixed models (GLMMs) with a binomial distribution and logit link showed that smoking did not increase the risk of replant failure (p = 0.234). In addition, the survival of replants was not affected by DM or HTN (p = 0.285 and 0.938, respectively). However, the replantation results were significantly affected by the age of the patients and the mechanism of injury. Patients older than 50 years and those with avulsion or crush injuries tended to have a higher risk of replant failure (OR = 2.29, 6.45, and 5.42, respectively; p = 0.047, 0.028, and 0.032, respectively).

Conclusions

This study showed that the use of cigarettes/tobacco did not affect the replantation outcome. The main risks for replant failure included being older than 50 years and the trauma mechanism (avulsion or crush injuries).