Involvement of mitochondrial ribosomal proteins in ribosomal RNA-mediated protein folding

The peptidyl transferase center of the domain V of large ribosomal RNA in the prokaryotic and eukaryotic cytosolic ribosomes acts as general protein folding modulator. We showed earlier that one part of the domain V (RNA1 containing the peptidyl transferase loop) binds unfolded protein and directs it to a folding competent state (FCS) that is released by the other part (RNA2) to attain the folded native state by itself. Here we show that the peptidyl transferase loop of the mitochondrial ribosome releases unfolded proteins in FCS extremely slowly despite its lack of the rRNA segment analogous to RNA2. The release of FCS can be hastened by the equivalent activity of RNA2 or the large subunit proteins of the mitochondrial ribosome. The RNA2 or large subunit proteins probably introduce some allosteric change in the peptidyl transferase loop to enable it to release proteins in FCS.     

Evidence for a highly specific protein kinase phosphorylating two strongly acidic proteins of yeast 60 S ribosomal subunit

Two distinct, cyclic AMP-independent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) from yeast have been isolated and highly purified. The first of the enzymes, protein kinase 1 A, phosphorylates casein and phosvitin, and its cellular protein substrate is unknown. The second enzyme, protein kinase 1 B, phosphorylates two strongly acidic proteins, L44 and L45, of the 60 S ribosomal subunit.     

Nucleolin protein interacts with microprocessor complex to affect biogenesis of microRNAs 15a and 16

MicroRNAs (miRNA) are endogenous, short, non-coding RNA that undergo a multistep biogenesis before generating the functional, mature sequence. The core components of the microprocessor complex, consisting of Drosha and DGCR8, are both necessary and sufficient for this process, although accessory proteins have been found that modulate the biogenesis of a subset of miRNA. Curiously, many of the proteins involved in miRNA biogenesis are also needed for ribosomal RNA processing. Here we show that nucleolin, another protein critical for rRNA processing, is involved in the biogenesis of microRNA 15a/16 (miR-15a/16), specifically at the primary to precursor stage of processing. Through overexpression and knockdown studies, we show that miR-15a/16 levels are directly correlated to nucleolin expression. Furthermore, we found that cellular localization is critical for the proper functioning of nucleolin in this pathway and that nucleolin directly interacts with DGCR8 and Drosha in the nucleus. Nucleolin can bind to the primary miRNA both directly and specifically. Finally, we show that in the absence of nucleolin, cell extracts are unable to process miR-15a/16 in vitro and that this can be rescued by the addition of nucleolin. Our findings offer a new protein component in the microRNA biogenesis pathway and lend insight into miRNA dysregulation in certain cancers.     

The Arf family GTPase Arl4A complexes with ELMO proteins to promote actin cytoskeleton remodeling and reveals a versatile Ras-binding domain in the ELMO proteins family

The prototypical DOCK protein, DOCK180, is an evolutionarily conserved Rac regulator and is indispensable during processes such as cell migration and myoblast fusion. The biological activity of DOCK180 is tightly linked to its binding partner ELMO. We previously reported that autoinhibited ELMO proteins regulate signaling from this pathway. One mechanism to activate the ELMO-DOCK180 complex appears to be the recruitment of this complex to the membrane via the Ras-binding domain (RBD) of ELMO. In the present study, we aimed to identify novel ELMO-interacting proteins to further define the molecular events capable of controlling ELMO recruitment to the membrane. To do so, we performed two independent interaction screens: one specifically interrogated an active GTPase library while the other probed a brain cDNA library. Both methods converged on Arl4A, an Arf-related GTPase, as a specific ELMO interactor. Biochemically, Arl4A is constitutively GTP-loaded, and our binding assays confirm that both wild-type and constitutively active forms of the GTPase associate with ELMO. Mechanistically, we report that Arl4A binds the ELMO RBD and acts as a membrane localization signal for ELMO. In addition, we report that membrane targeting of ELMO via Arl4A promotes cytoskeletal reorganization including membrane ruffling and stress fiber disassembly via an ELMO-DOCK1800-Rac signaling pathway. We conclude that ELMO is capable of interacting with GTPases from Rho and Arf families, leading to the conclusion that ELMO contains a versatile RBD. Furthermore, via binding of an Arf family GTPase, the ELMO-DOCK180 is uniquely positioned at the membrane to activate Rac signaling and remodel the actin cytoskeleton.     

Balanced Production of Ribosome Components Is Required for Proper G1/S Transition in Saccharomyces cerevisiae

Cell cycle regulation is a very accurate process that ensures cell viability and the genomic integrity of daughter cells. A fundamental part of this regulation consists in the arrest of the cycle at particular points to ensure the completion of a previous event, to repair cellular damage, or to avoid progression in potentially risky situations. In this work, we demonstrate that a reduction in nucleotide levels or the depletion of RNA polymerase I or III subunits generates a cell cycle delay at the G₁/S transition in Saccharomyces cerevisiae. This delay is concomitant with an imbalance between ribosomal RNAs and proteins which, among others, provokes an accumulation of free ribosomal protein L5. Consistently with a direct impact of free L5 on the G₁/S transition, rrs1 mutants, which weaken the assembly of L5 and L11 on pre-60S ribosomal particles, enhance both the G₁/S delay and the accumulation of free ribosomal protein L5. We propose the existence of a surveillance mechanism that couples the balanced production of yeast ribosomal components and cell cycle progression through the accumulation of free ribosomal proteins. This regulatory pathway resembles the p53-dependent nucleolar-stress checkpoint response described in human cells, which indicates that this is a general control strategy extended throughout eukaryotes.     

Characterization of a central Ca2+/calmodulin-dependent protein kinase IIalpha/beta binding domain in densin that selectively modulates glutamate receptor subunit phosphorylation

The densin C-terminal domain can target Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) in cells. Although the C-terminal domain selectively binds CaMKIIα in vitro, full-length densin associates with CaMKIIα or CaMKIIβ in brain extracts and in transfected HEK293 cells. This interaction requires a second central CaMKII binding site, the densin-IN domain, and an "open" activated CaMKII conformation caused by Ca(2+)/calmodulin binding, autophosphorylation at Thr-286/287, or mutation of Thr-286/287 to Asp. Mutations in the densin-IN domain (L815E) or in the CaMKIIα/β catalytic domain (I205/206K) disrupt the interaction. The amino acid sequence of the densin-IN domain is similar to the CaMKII inhibitor protein, CaMKIIN, and a CaMKIIN peptide competitively blocks CaMKII binding to densin. CaMKII is inhibited by both CaMKIIN and the densin-IN domain, but the inhibition by densin is substrate-selective. Phosphorylation of a model peptide substrate, syntide-2, or of Ser-831 in AMPA receptor GluA1 subunits is fully inhibited by densin. However, CaMKII phosphorylation of Ser-1303 in NMDA receptor GluN2B subunits is not effectively inhibited by densin in vitro or in intact cells. Thus, densin can target multiple CaMKII isoforms to differentially modulate phosphorylation of physiologically relevant downstream targets.     

Quantitative analysis of ERK2 interactions with substrate proteins: roles for kinase docking domains and activity in determining binding affinity

Extracellular signal-regulated kinase-1 and -2 (ERK1/2) proteins regulate a variety of cellular functions, including cell proliferation and differentiation, by interacting with and phosphorylating substrate proteins. Two docking sites, common docking (CD/ED) domain and F-site recruitment site (FRS), on ERK proteins have been identified. Specific interactions with the CD/ED domain and the FRS occur with substrates containing a docking site for ERK and JNK, LXL (DEJL) motif (D-domain) and a docking site for ERK, FXF (DEF) motif (F-site), respectively. However, the relative contributions of the ERK docking sites in mediating substrate interactions that allow efficient phosphate transfer are largely unknown. In these studies, we provide a quantitative analysis of ERK2 interactions with substrates using surface plasmon resonance to measure real time protein-protein interactions. ERK2 interacted with ELK-1 (DEF and DEJL motifs), RSK-1 (DEJL motif), and c-Fos (DEF motif) with K(D) values of 0.25, 0.15, and 0.97 μM, respectively. CD/ED domain mutations inhibited interactions with ELK-1 and RSK-1 by 6-fold but had no effect on interactions with c-Fos. Select mutations in FRS residues differentially inhibited ELK-1 or c-Fos interactions with ERK2 but had little effect on RSK-1 interactions. Mutations in both the ED and FRS docking sites completely inhibited ELK-1 interactions but had no effect on interactions with stathmin, an ERK substrate whose docking site is unknown. The phosphorylation status of ERK2 did not affect interactions with RSK-1 or c-Fos but did inhibit interactions with ELK-1 and stathmin. These studies provide a quantitative evaluation of specific docking domains involved in mediating interactions between ERK2 and protein substrates and define the contributions of these interactions to phosphate transfer.     

Suppression of the ribosomal L2 gene reveals a novel mechanism for stress adaptation in soybean

Pseudomonas syringae pv. glycinea bacteria or zoospores of the fungus Phytophthora sojae were used to trigger a hypersensitive reaction (HR) in cell cultures of soybean (Glycine max [L.] Merr. cv. Williams 82). During a screen for genes that show an altered expression as a response to dying neighbour cells we have identified a gene fragment that is specifically but transiently down-regulated in an HR. The corresponding cDNA codes for the ribosomal protein L2 (rpL2) of 80S ribosomes, which is essential for the peptidyl-transferase activity. Two gene copies of rpL2 exist in soybean and both genes are transcribed. The temporary down-regulation of the rpL2 genes is followed by a transient block in the synthesis of new proteins as visualised by pulse-labelling experiments using ³⁵S-amino acids. The same basic phenomenon was also found after treatment of soybean cells with other stress-causing compounds such as elicitors or heavy metals. It is suggested that the transient block in protein synthesis allows a more rapid depletion of, for example, signal molecules with a short half-life time and thus leads to a faster adaptation of the cellular protein inventory to the new environmental conditions. Received: 10 May 2000 / Accepted: 21 July 2000     

Identification of the Residues in the Extracellular Domain of Thrombopoietin Receptor Involved in the Binding of Thrombopoietin and a Nuclear Distribution Protein (Human NUDC)

Thrombopoietin (TPO) and its receptor (Mpl) have long been associated with megakaryocyte proliferation, differentiation, and platelet formation. However, studies have also shown that the extracellular domain of Mpl (Mpl-EC) interacts with human (h) NUDC, a protein previously characterized as a human homolog of a fungal nuclear migration protein. This study was undertaken to further delineate the putative binding domain on the Mpl receptor. Using the yeast two-hybrid system assay and co-immunoprecipitation, we identified that within the Mpl-EC domain 1 (Mpl-EC-D1), amino acids 102–251 were strongly involved in ligand binding. We subsequently expressed five subdomains within this region with T7 phage display. Enzyme-linked immunosorbent binding assays identified a short stretch of peptide located between residues 206 and 251 as the minimum binding domain for both TPO and hNUDC. A series of sequential Ala replacement mutations in the region were subsequently used to identify the specific residues most involved in ligand binding. Our results point to two hydrophobic residues, Leu²²⁸ and Leu²³⁰, as having substantial effects on hNUDC binding. For TPO binding, mutations in residues Asp²³⁵ and Leu²³⁹ had the largest effect on binding efficacy. In addition, deletion of the conservative motif WGSWS reduced binding capacity for hNUDC but not for TPO. These separate binding sites on the Mpl receptor for TPO and hNUDC raise interesting implications for the cytokine-receptor interactions.     

MYCBP2 Is a Guanosine Exchange Factor for Ran Protein and Determines Its Localization in Neurons of Dorsal Root Ganglia

The small GTPase Ran coordinates retrograde axonal transport in neurons, spindle assembly during mitosis, and the nucleo-cytoplasmic transport of mRNA. Its localization is tightly regulated by the GTPase-activating protein RanGAP1 and the nuclear guanosine exchange factor (GEF) RCC1. We show that loss of the neuronal E3 ubiquitin ligase MYCBP2 caused the up-regulation of Ran and RanGAP1 in dorsal root ganglia (DRG) under basal conditions and during inflammatory hyperalgesia. SUMOylated RanGAP1 physically interacted with MYCBP2 and inhibited its E3 ubiquitin ligase activity. Stimulation of neurons induced a RanGAP1-dependent translocation of MYCBP2 to the nucleus. In the nucleus of DRG neurons MYCBP2 co-localized with Ran and facilitated through its RCC1-like domain the GDP/GTP exchange of Ran. In accordance with the necessity of a GEF to promote GTP-binding and nuclear export of Ran, the nuclear localization of Ran was strongly increased in MYCBP2-deficient DRGs. The finding that other GEFs for Ran besides RCC1 exist gives new insights in the complexity of the regulation of the Ran signaling pathway.     

Disease-causing mutation in PKR2 receptor reveals a critical role of positive charges in the second intracellular loop for G-protein coupling and receptor trafficking

Prokineticins are a pair of signal factors involved in many physiological processes by binding to two closely related G-protein-coupled receptors, PKR1 and PKR2. Recently, mutations in prokineticin 2 (PK2) and PKR2 are found to be associated with Kallmann syndrome and/or idiopathic hypogonadotropic hypogonadism, disorders characterized by delayed puberty and infertility. However, little is known how PKRs interact and activate G-proteins to elicit signal transduction. In the present study, we took advantage of one disease-associated mutation (R164Q) located in the second intracellular (IL2) loop of PKR2, to investigate the role of IL2 loop in the cell signaling, G-protein binding and receptor trafficking. R164Q mutant PKR2 showed normal cell surface expression and ligand binding capacity. However, the PKR2 signaling was abolished by R164Q mutation. We demonstrated that R164Q mutation disrupted the interaction of IL2 loop to the Gα(q), Gα(i), and Gα(16)-proteins. A positive-charged amino acid at this position is required for proper function, and the signaling efficacy and potency depend on the net amount of positive charges. We also demonstrated that the interactive partner of Arg-164 may localize in the C-terminal five residues of Gα(q)-protein. A series of mutation analysis indicated that the basic amino acids at the C terminus of IL2 loop may function cooperatively in GPCRs. Furthermore, R164Q mutation also results in minimal ligand-induced endocytosis of PKR2. As many GPCRs share structural homology in the C terminus of IL2 loop, our findings may have general application in understanding structure and function of GPCRs.     

The Saccharomyces cerevisiae genes ( CMP1 and CMP2 ) encoding calmodulin-binding proteins homologous to the catalytic subunit of mammalian protein phosphatase 2B

Summary Saccharomyces cerevisiae genomic clones that encode calmodulin-binding proteins were isolated by screening a λgt11 expression library using¹²⁵I-labeled calmodulin as probe. Among the cloned yeast genes, we found two closely related genes (CMP1 andCMP2) that encode proteins homologous to the catalytic subunit of phosphoprotein phosphatase. The presumed CMP1 protein (62999 Da) and CMP2 protein (68496 Da) contain a 23 amino acid sequence very similar to those identified as calmodulin-binding sites in many calmodulin-regulated proteins. The yeast genes encode proteins especially homologous to the catalytic subunit of mammalian phosphoprotein phosphatase type 213 (calcineurin). The products of theCMP1 andCMP2 genes were identified by immunoblot analysis of cell extracts as proteins of 62000 and 64000 Da, respectively. Gene disruption experiments demonstrated that elimination of either or both of these genes had no effect on cell viability, indicating that these genes are not essential for normal cell growth.     

Serotonin acts as a novel regulator of interleukin-6 secretion in osteocytes through the activation of the 5-HT2B receptor and the ERK1/2 signalling pathway

Interleukin-6 (IL-6) is a potent stimulator of osteoclastic bone resorption. Osteocyte secretion of IL-6 plays an important role in bone metabolism. Serotonin (5-HT) has recently been reported to regulate bone metabolism. The aim of this study was to evaluate the effect of serotonin on osteocyte expression of IL-6. The requirement for the 5-HT receptor(s) and the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) in serotonin-induced IL-6 synthesis were examined. In this study, real-time PCR and ELISA were used to analyse IL-6 gene and protein expression in serotonin-stimulated MLO-Y4 cells. ERK1/2 pathway activation was determined by Western blot. We found that serotonin significantly activated the ERK1/2 pathway and induced IL-6 mRNA expression and protein synthesis in cultured MLO-Y4 cells. However, these effects were abolished by pre-treatment of MLO-Y4 cells with a 5-HT_2B receptor antagonist, RS127445 or the ERK1/2 inhibitor, PD98059. Our results indicate that serotonin stimulates osteocyte secretion of IL-6 and that this effect is associated with activation of 5-HT_2B receptor and the ERK1/2 pathway. These findings provide support for a role of serotonin in bone metabolism by indicating serotonin regulates bone remodelling by mediating an inflammatory cytokine.     

Identification of the 5-Hydroxytryptamine2C Receptor as a 60-kDa N-Glycosylated Protein in Choroid Plexus and Hippocampus

Abstract: The rat 5-hydroxytryptamine_2C (5-HT_2C) receptor was identified as N -glycosylated polypeptide of 60-kDa apparent molecular mass using antibodies against its putative third and fourth (C-terminal) cytoplasmic domain. To show that the polypeptides detected on western blots and by immunoprecipitation represent the 5-HT_2C receptor, binding studies of the 5-HT_2C ligand [³H]-mesulergine to immunoprecipitates from extracts of pig choroid plexus were performed. We demonstrate the presence of a signal sequence that was cleaved off during membrane insertion resulting in a 38-kDa polypeptide. During further maturation, the receptor was N -glycosylated at two sites via a 48-kDa intermediate. This intermediate was far more abundant in choroid plexus than in hippocampus and may represent an intracellular receptor reserve. After transfection of 5-HT_2C cDNAs into cultured cells, polypeptides were observed that differed from the ones found in vivo due to abnormal N -glycosylation and possibly other alterations depending on the system used. Thus the 5-HT_2C receptor expressed in cell lines may also differ in function from the receptor in its native tissue.     

The Role of Sodium as a Surfactant and Suppressor of Non‐Radiative Recombination at Internal Surfaces in Cu2ZnSnS4

It is well‐known that sodium improves the performance of Cu₂ZnSnS₄ (CZTS) devices, yet the mechanism of the enhancement is still not fully understood. This work aims to present a unified account of the relationships between grain boundaries in CZTS, sodium content at these boundaries, non‐radiative recombination, and surfactant effects that produce large microstructural changes. Using temperature‐dependent photoluminescence measurements, it is demonstrated that samples containing dramatically different grain sizes display identical radiative and non‐radiative decay characteristics when sufficient sodium is present in the film. It is also shown that the sodium concentration needed to efficiently passivate non‐radiative defects is significantly less that the quantity needed to obtain micrometer‐sized CZTS grains. Finally, the high densities of donor‐acceptor pairs that are observed in CZTS films appear to reside within the grains themselves, rather than at grain boundaries.