PAQR10 and PAQR11 mediate Ras signaling in the Golgi apparatus

Ras plays a pivotal role in many cellular activities, and its subcellular compartmentalization provides spatial and temporal selectivity. Here we report a mode of spatial regulation of Ras signaling in the Golgi apparatus by two highly homologous proteins PAQR10 and PAQR11 of the progestin and AdipoQ receptors family. PAQR10 and PAQR11 are exclusively localized in the Golgi apparatus. Overexpression of PAQR10/PAQR11 stimulates basal and EGF-induced ERK phosphorylation and increases the expression of ERK target genes in a dose-dependent manner. Overexpression of PAQR10/PAQR11 markedly elevates Golgi localization of HRas, NRas and KRas4A, but not KRas4B. PAQR10 and PAQR11 can also interact with HRas, NRas and KRas4A, but not KRas4B. The increased Ras protein at the Golgi apparatus by overexpression of PAQR10/PAQR11 is in an active state. Consistently, knockdown of PAQR10 and PAQR11 reduces EGF-stimulated ERK phosphorylation and Ras activation at the Golgi apparatus. Intriguingly, PAQR10 and PAQR11 are able to interact with RasGRP1, a guanine nucleotide exchange protein of Ras, and increase Golgi localization of RasGRP1. The C1 domain of RasGRP1 is both necessary and sufficient for the interaction of RasGRP1 with PAQR10/PAQR11. The simulation of ERK phosphorylation by overexpressed PAQR10/PAQR11 is abrogated by downregulation of RasGRP1. Furthermore, differentiation of PC12 cells is significantly enhanced by overexpression of PAQR10/PAQR11. Collectively, this study uncovers a new paradigm of spatial regulation of Ras signaling in the Golgi apparatus by PAQR10 and PAQR11.     

Characterization of the topology and functional domains of RKTG

RKTG (Raf kinase trapping to Golgi) is exclusively localized at the Golgi apparatus and functions as a spatial regulator of Raf-1 kinase by sequestrating Raf-1 to the Golgi. Based on the structural similarity with adiponectin receptors, RKTG was predicted to be a seven-transmembrane protein with a cytosolic N-terminus, distinct from classical GPCRs (G-protein-coupled receptors). We analysed in detail the topology and functional domains of RKTG in this study. We determined that the N-terminus of RKTG is localized on the cytosolic side. Two short stretches of amino acid sequences at the membrane proximal to the N- and C-termini (amino acids 61-71 and 299-303 respectively) were indispensable for Golgi localization of RKTG, but were not required for the interaction with Raf-1. The three loops facing the cytosol between the transmembrane domains had different roles in Golgi localization and Raf-1 interaction. While the first cytosolic loop was only important for Golgi localization, the third cytosolic loop was necessary for both Golgi localization and Raf-1 sequestration. Taken together, these findings suggest that RKTG is a type III membrane protein with its N-terminus facing the cytosol and multiple sequences are responsible for its localization at the Golgi apparatus and Raf-1 interaction. As RKTG is the first discovered Golgi protein with seven transmembrane domains, the knowledge derived from this study would not only provide structural information about the protein, but also pave the way for future characterization of the unique functions of RKTG in the regulation of cell signalling.     

Characterization of Golgi scaffold proteins and their roles in compartmentalizing cell signaling

Subcellular compartmentalization has become an important theme in cell signaling. In particular, the Golgi apparatus (GA) plays a prominent role in compartmentalizing signaling cascades that originate at the plasma membrane or other organelles. To precisely regulate this process, cells have evolved a unique class of organizer proteins, termed “scaffold proteins”. Sef, PAQR3, PAQR10 and PAQR11 are scaffold proteins that have recently been identified on the GA and are referred to as Golgi scaffolds. The major cell growth signaling pathways, such as Ras/MAPK, PI3K/AKT, insulin and VEGF (vascular endothelial growth factor), are tightly regulated spatially and temporally by these Golgi scaffolds to ensure a physiologically appropriate outcome. Here, we discuss the subcellular localization and characterization of the topology and functional domains of these Golgi scaffolds and summarize their roles in the compartmentalization of cell signaling. We also highlight the physiological and pathological roles of these Golgi scaffolds in tumorigenesis and developmental disorders.     

Pancreatic expression and mitochondrial localization of the progestin-adipoQ receptor PAQR10

Steroid hormones induce changes in gene expression by binding to intracellular receptors that then translocate to the nucleus. Steroids have also been shown to rapidly modify cell function by binding to surface membrane receptors. We identified a candidate steroid membrane receptor, the progestin and adipoQ receptor (PAQR) 10, a member of the PAQR family, in a screen for genes differentially expressed in mouse pancreatic beta-cells. PAQR10 gene expression was tissue restricted compared with other PAQRs. In the mouse embryonic pancreas, PAQR10 expression mirrored development of the endocrine lineage, with PAQR10 protein expression confined to endocrine islet-duct structures in the late embryo and neonate. In the adult mouse pancreas, PAQR10 was expressed exclusively in islet cells except for its reappearance in ducts of maternal islets during pregnancy. PAQR10 has a predicted molecular mass of 29 kDa, comprises seven transmembrane domains, and, like other PAQRs, is predicted to have an intracellular N-terminus and an extracellular C-terminus. In silico analysis indicated that three members of the PAQR family, PAQRs 9, 10, and 11, have a candidate mitochondrial localization signal (MLS) at the N-terminus. We showed that PAQR10 has a functional N-terminal MLS and that the native protein localizes to mitochondria. PAQR10 is structurally related to some bacterial hemolysins, pore-forming virulence factors that target mitochondria and regulate apoptosis. We propose that PAQR10 may act at the level of the mitochondrion to regulate pancreatic endocrine cell development/survival.     

HID-1 is a peripheral membrane protein primarily associated with the medial- and trans- Golgi apparatus

Caenorhabditis elegans hid-1 gene was first identified in a screen for mutants with a high-temperature-induced dauer formation (Hid) phenotype. Despite the fact that the hid-1 gene encodes a novel protein (HID-1) which is highly conserved from Caenorhabditis elegans to mammals, the domain structure, subcellular localization, and exact function of HID-1 remain unknown. Previous studies and various bioinformatic softwares predicted that HID-1 contained many transmembrane domains but no known functional domain. In this study, we revealed that mammalian HID-1 localized to the medial- and trans-Golgi apparatus as well as the cytosol, and the localization was sensitive to brefeldin A treatment. Next, we demonstrated that HID-1 was a peripheral membrane protein and dynamically shuttled between the Golgi apparatus and the cytosol. Finally, we verified that a conserved N-terminal myristoylation site was required for HID-1 binding to the Golgi apparatus. We propose that HID-1 is probably involved in the intracellular trafficking within the Golgi region.     

Regulation of the Ras‐GTPase activating protein neurofibromin by C‐tail phosphorylation: implications for protein kinase C/Ras/extracellular signal‐regulated kinase 1/2 pathway signaling and neuronal differentiation

PKC, Ras, and ERK1/2 signaling is pivotal to differentiation along the neuronal cell lineage. One crucial protein that may play a central role in this signaling pathway is the Ras GTPase‐activating protein, neurofibromin, a PKC substrate that may exert a positive role in neuronal differentiation. In this report, we studied the dynamics of PKC/Ras/ERK pathway signaling, during differentiation of SH‐SY5Y neuroblastoma cells upon treatment with the PKC agonist, phorbol ester 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). Surprisingly, we observed that, among other PKC‐dependent signaling events, TPA induced a rapid and sustained decrease of neurofibromin immunoreactivity which was not due to proteolysis. Instead, we identified a specific phosphorylation event at the C‐tail of neurofibromin. This phosphorylation was acute and correlated perfectly with the signaling dynamics of the Ras/ERK pathway. Moreover, it persisted throughout prolonged treatment and TPA‐induced differentiation of SH‐SY5Y cells, concurrently with sustained activation of ERK1/2. Most importantly, C‐tail phosphorylation of neurofibromin correlated with a shift of neurofibromin localization from the nucleus to the cytosol. We propose that PKC‐dependent, sustained C‐tail phosphorylation is a requirement for prolonged recruitment of neurofibromin from the nucleus to the cytosol in order for a fine regulation of Ras/ERK pathway activity to be achieved during differentiation.     

In vivo kinetics of protein targeting to the endoplasmic reticulum determined by site-specific phosphorylation

We have developed a novel assay to detect the cytosolic localization of protein domains by inserting a short consensus sequence for phosphorylation by protein kinase A. In transfected COS-1 cells, this sequence was labeled efficiently with [(32)P]phosphate only when exposed to the cytosol and not when translocated into the lumen of the endoplasmic reticulum. The phosphorylation state of this sequence can therefore be used to determine the topology of membrane proteins. This assay is sufficiently sensitive to detect even the transient cytosolic exposure of the N-terminal domain of a membrane protein with a reverse signal-anchor sequence. The extent of phosphorylation per newly synthesized polypeptide was shown to reflect the time of exposure to the cytosol, which depends on translation, targeting and translocation of the N-terminus. By altering the length of the N-terminal domain or manipulating the translation rate, it was determined that protein targeting is rapid and requires only a few seconds. The rate of N-terminal translocation was estimated to be approximately 1.6 times the rate of translation.     

Tyrosine 763 of the murine granulocyte colony-stimulating factor receptor mediates Ras-dependent activation of the JNK/SAPK mitogen-activated protein kinase pathway.

The receptor for granulocyte colony-stimulating factor (G-CSF) can mediate differentiation and proliferation of hemopoietic cells. A proliferative signal is associated with activation of the ERK mitogen-activated protein kinase (MAPK) pathway. To determine whether other MAPK pathways are activated by G-CSF signalling, we have investigated activation of JNK/SAPK in cells proliferating in response to G-CSF. Here we show that G-CSF and interleukin-3 activate JNK/SAPK in two hemopoietic cell lines. The region of the G-CSF receptor required for G-CSF-induced JNK/SAPK activation is located within the C-terminal 68 amino acids of the cytoplasmic domain, which contains Tyr 763. Mutation of Tyr 763 to Phe completely blocks JNK/SAPK activation. However, the C-terminal 68 amino acids are not required for ERK2 activation. We show that activation of JNK/SAPK, like that of ERK2, is dependent on Ras but that higher levels of Ras-GTP are associated with activation of JNK/SAPK than with activation of ERK2. Two separate functional regions of the G-CSF receptor contribute to activation of Ras. The Y763F mutation reduces G-CSF-induced Ras activation from 30 to 35% Ras-GTP to 10 to 13% Ras-GTP. Low levels of Ras activation (10 to 13% Ras-GTP), which are sufficient for ERK2 activation, require only the 100 membrane-proximal amino acids. High levels of Ras-GTP provided by expression of oncogenic Ras are not sufficient to activate JNK/SAPK. An additional signal, also mediated by Tyr 763, is required for activation of JNK/SAPK.     

Exchange factors of the RasGRP family mediate Ras activation in the Golgi

H-Ras and N-Ras become activated both at the plasma membrane and in endomembrane structures such as the Golgi apparatus. This compartmentalized activation is relevant from a signaling standpoint, because effector molecules can become activated differently depending on the region of the cell where Ras proteins are activated. An unsolved question in this new regulatory mechanism is the understanding of how Ras proteins become activated in endomembranes. To approach this problem, we have studied the subcellular distribution and activities of a number of Ras guanosine nucleotide exchange factors. Our results indicate that Ras activation at the plasma membrane and endoplasmic reticulum is an unspecific process that can be achieved by most Ras activators. In contrast, GTP loading of Ras at the Golgi is only induced by members of the Ras guanosine nucleotide releasing protein family. In agreement with these observations, Ras guanosine nucleotide releasing proteins are the only Ras activators showing localization in the Golgi. These results indicate that the compartmentalized activation of effector pathways by Ras proteins depends not only on the specific localization of the GTPases but also in the availability of GDP/GTP exchange factors capable of activating Ras proteins in specific subcellular compartments.     

Intracellular localization modulates targeting of ExoS,a type III cytotoxin,to eukaryotic signalling proteins

ExoS is a bifunctional type III cytotoxin produced by Pseudomonas aeruginosa. Residues 96-232 comprise the Rho GTPase activating protein (Rho GAP) domain, whereas residues 233-453 comprise the 14-3-3-dependent ADP-ribosyltransferase domain. Earlier studies showed that the N-terminus targeted ExoS to intracellular membranes within eukaryotic cells. This N-terminal targeting region is now characterized for cellular and biological contributions to intoxications by ExoS. An ExoS(1-107)-green fluorescent protein (GFP) fusion protein co-localized with alpha-mannosidase, which indicated that the fusion protein localized near the Golgi. Residues 51-72 of ExoS (termed the membrane localization domain, MLD) were necessary and sufficient for membrane localization within eukaryotic cells. Deletion of the MLD did not inhibit type III secretion of ExoS from P. aeruginosa or type III delivery of ExoS into eukaryotic cells. Type III-delivered ExoS(DeltaMLD) localized within the cytosol of eukaryotic cells, whereas type III-delivered ExoS was membrane associated. Although type III-delivered ExoS(DeltaMLD) stimulated the reorganization of the actin cytoskeleton (a Rho GAP activity), it did not ADP-ribosylate Ras. Type III-delivered ExoS(DeltaMLD) and ExoS showed similar capacities for eliciting a cytotoxic response in CHO cells, which uncoupled the ADP-ribosylation of Ras from the cytotoxicity elicited by ExoS.     

Nucleotide sugar transporters of the Golgi apparatus

Glycosylation, sulfation and phosphorylation of proteins, proteoglycans and lipids occur in the lumen of the Golgi apparatus. The nucleotide substrates of these reactions must be first transported from the cytosol into the Golgi lumen by specific transporters. The topology and structure of these hydrophobic, multi-transmembrane-spanning proteins are beginning to be understood.     

Localization and trafficking of an isoform of the AtPRA1 family to the Golgi apparatus depend on both N- and C-terminal sequence motifs

Prenylated Rab acceptors (PRAs) bind to prenylated Rab proteins and possibly aid in targeting Rabs to their respective compartments. In Arabidopsis, 19 isoforms of PRA1 have been identified and, depending upon the isoforms, they localize to the endoplasmic reticulum (ER), Golgi apparatus and endosomes. Here, we investigated the localization and trafficking of AtPRA1.B6, an isoform of the Arabidopsis PRA1 family. In colocalization experiments with various organellar markers, AtPRA1.B6 tagged with hemagglutinin (HA) at the N-terminus localized to the Golgi apparatus in protoplasts and transgenic plants. The valine residue at the C-terminal end and an EEE motif in the C-terminal cytoplasmic domain were critical for anterograde trafficking from the ER to the Golgi apparatus. The N-terminal region contained a sequence motif for retention of AtPRA1.B6 at the Golgi apparatus. In addition, anterograde trafficking of AtPRA1.B6 from the ER to the Golgi apparatus was highly sensitive to the HA:AtPRA1.B6 level. The region that contains the sequence motif for Golgi retention also conferred the abundance-dependent trafficking inhibition. On the basis of these results, we propose that AtPRA1.B6 localizes to the Golgi apparatus and its ER-to-Golgi trafficking and localization to the Golgi apparatus are regulated by multiple sequence motifs in both the C- and N-terminal cytoplasmic domains.     

Intracellular localization and processing of Pseudomonas aeruginosa ExoS in eukaryotic cells

ExoS is a type III cytotoxin of Pseudomonas aeruginosa, which modulates two eukaryotic signalling pathways. The N-terminus (residues 1-234) is a GTPase activating protein (GAP) for RhoGTPases, while the C-terminus (residues 232-453) encodes an ADP-ribosyltransferase. Utilizing a series of N-terminal deletion peptides of ExoS and an epitope-tagged full-length ExoS, two independent domains have been identified within the N-terminus of ExoS that are involved in intracellular localization and expression of GAP activity. N-terminal peptides of ExoS localized to the perinuclear region of CHO cells, and a membrane localization domain was localized between residues 36 and 78 of ExoS. The capacity to elicit CHO cell rounding and express GAP activity resided within residues 90-234 of ExoS, which showed that membrane localization was not required to elicit actin reorganization. ExoS was present in CHO cells as a full-length form, which fractionated with membranes, and as an N-terminally processed fragment, which localized to the cytosol. Thus, ExoS localizes in eukaryotic cells to the perinuclear region and is processed to a soluble fragment, which possesses both the GAP and ADP-ribosyltransferase activities.     

The SH3 domain of Lck modulates T-cell receptor-dependent activation of extracellular signal-regulated kinase through activation of Raf-1

Engagement of the T-cell antigen receptor (TCR) results in the proximal activation of the Src family tyrosine kinase Lck. The activation of Lck leads to the downstream activation of the Ras/Raf/MEK/ERK signaling pathway (where ERK is extracellular signal-related kinase). Under conditions of weak, but not strong, stimulation through the TCR, a version of Lck that contains a single point mutation in the SH3 (Src homology 3) domain (W97ALck) fails to support the activation of ERK, despite initiating signaling through the TCR, as demonstrated by the robust activation of ZAP-70, PLC-γ, and Ras. We determined that the signaling lesion in W97ALck-expressing cells lies at the level of Raf-1 activation and is dependent on the presence of tyrosines 340/341 in the Raf-1 sequence. These data demonstrate a second function for Lck in TCR-mediated signaling to ERK. Additionally, we found that a significant fraction of Lck is localized to the Golgi apparatus and that, compared with wild-type Lck, W97ALck displays aberrant Golgi membrane localization. Our results support a model where under conditions of weak stimulation through the TCR, in addition to activated Ras, Golgi apparatus-localized Lck is needed for the full activation of Raf-1.     

Luteolin downregulates IL-1β-induced MMP-9 and -13 expressions in osteoblasts via inhibition of ERK signalling pathway

The inhibitory effect of four structurally related flavonoids, apigenin, baicalein, luteolin and quercetin on the matrix metalloproteinase (MMP)-9 and -13 expressions in osteoblasts was investigated. Treatment with IL-1β induced both MMP-9 and -13 mRNA expressions as measured by quantitative real-time PCR. Luteolin and apigenin decreased IL-1β-induced MMP-9 and -13 mRNA expressions, whereas baicalein and quercetin showed little effects. Related to signalling, treatment with IL-1β induced ERK phosphorylation as measured by Western blotting. Further studies showed that transfection with a constitutively active form of the Ras protein (Ras(V12)) induced stronger ERK phosphorylation and upregulated MMP-9 and -13 mRNA expressions. However, transfection with a dominant-negative form of the Ras protein (Ras(N17)) inhibited the ERK activation and MMP-9 and -13 mRNA expressions induced by IL-1β, which supported the involvement of ERK signalling in IL-1β-induced MMP-9 and -13 expressions. Treatment with luteolin effectively inhibited the IL-1β-induced ERK activation in dose-dependent manner. Taken together, luteolin might inhibit IL-1β-induced MMP-9 and -13 expressions, in part, via inhibition of ERK signalling.     

Identification and characterization of AtCASP,a plant transmembrane Golgi matrix protein

Golgins are a family of coiled-coil proteins that are associated with the Golgi apparatus. They are necessary for tethering events in membrane fusion and may act as structural support for Golgi cisternae. Here we report on the identification of an Arabidopsis golgin which is a homologue of CASP, a known transmembrane mammalian and yeast golgin. Similar to its homologues, the plant CASP contains a long N-terminal coiled-coil region protruding into the cytosol and a C-terminal transmembrane domain with amino acid residues which are highly conserved across species. Through fluorescent protein tagging experiments, we show that plant CASP localizes at the plant Golgi apparatus and that the C-terminus of this protein is sufficient for its localization, as has been shown for its mammalian counterpart. In addition, we demonstrate that the plant CASP is able to localize at the mammalian Golgi apparatus. However, mutagenesis of a conserved tyrosine in the transmembrane domain revealed that it is necessary for ER export and Golgi localization of the Arabidopsis CASP in mammalian cells, but is not required for its correct localization in plant cells. These data suggest that mammalian and plant cells have different mechanisms for concentrating CASP in the Golgi apparatus.†These authors have contributed equally to the work     

A membrane-proximal region of the interleukin-2 receptor gamma c chain sufficient for Jak kinase activation and induction of proliferation in T cells.

The interleukin-2 (IL-2) receptor (IL-2R) consists of three distinct subunits (alpha, beta, and gamma c) and regulates proliferation of T lymphocytes. Intracellular signalling results from ligand-mediated heterodimerization of the cytoplasmic domains of the beta and gamma c chains. To identify the residues of gamma c critical to this process, mutations were introduced into the cytoplasmic domain, and the effects on signalling were analyzed in the IL-2-dependent T-cell line CTLL2 and T-helper clone D10, using chimeric IL-2R chains that bind and are activated by granulocyte-macrophage colony-stimulating factor. Whereas previous studies of fibroblasts and transformed T cells have suggested that signalling by gamma c requires both membrane-proximal and C-terminal subdomains, our results for IL-2-dependent T cells demonstrate that the membrane-proximal 52 amino acids are sufficient to mediate a normal proliferative response, including induction of the proto-oncogenes c-myc and c-fos. Although gamma c is phosphorylated on tyrosine upon receptor activation and could potentially interact with downstream molecules containing SH2 domains, cytoplasmic tyrosine residues were dispensable for mitogenic signalling. However, deletion of a membrane-proximal region conserved among other cytokine receptors (cytoplasmic residues 5 to 37) or an adjacent region unique to gamma c (residues 40 to 52) abrogated functional interaction of the receptor chain with the tyrosine kinase Jak3. This correlated with a loss of all signalling events analyzed, including phosphorylation of the IL-2R beta-associated kinase Jak1, expression of c-myc and c-fos, and induction of the proliferative response. Thus, it appears in T cells that Jak3 is a critical mediator of mitogenic signaling by the gamma c chain.     

Decoupling Polarization of the Golgi Apparatus and GM1 in the Plasma Membrane

Cell polarization is a process of coordinated cellular rearrangements that prepare the cell for migration. GM1 is synthesized in the Golgi apparatus and localized in membrane microdomains that appear at the leading edge of polarized cells, but the mechanism by which GM1 accumulates asymmetrically is unknown. The Golgi apparatus itself becomes oriented toward the leading edge during cell polarization, which is thought to contribute to plasma membrane asymmetry. Using quantitative image analysis techniques, we measure the extent of polarization of the Golgi apparatus and GM1 in the plasma membrane simultaneously in individual cells subject to a wound assay. We find that GM1 polarization starts just 10 min after stimulation with growth factors, while Golgi apparatus polarization takes 30 min. Drugs that block Golgi polarization or function have no effect on GM1 polarization, and, conversely, inhibiting GM1 polarization does not affect Golgi apparatus polarization. Evaluation of Golgi apparatus and GM1 polarization in single cells reveals no correlation between the two events. Our results indicate that Golgi apparatus and GM1 polarization are controlled by distinct intracellular cascades involving the Ras/Raf/MEK/ERK and the PI3K/Akt/mTOR pathways, respectively. Analysis of cell migration and invasion suggest that MEK/ERK activation is crucial for two dimensional migration, while PI3K activation drives three dimensional invasion, and no cumulative effect is observed from blocking both simultaneously. The independent biochemical control of GM1 polarity by PI3K and Golgi apparatus polarity by MEK/ERK may act synergistically to regulate and reinforce directional selection in cell migration.