Export of proteins from the endoplasmic reticulum (ER) in COPII-coated vesicles occurs at defined sites, which contain the scaffolding protein Sec16. We identify TFG-1, a new conserved regulator of protein secretion that interacts directly with SEC-16 and controls the export of cargoes from the ER in C. elegans. Hydrodynamic studies indicate that TFG-1 forms hexamers, which facilitate the co-assembly of Sec16 with COPII subunits. Consistent with these findings, TFG-1 depletion leads to a dramatic decline in both SEC-16 and COPII levels at ER exit sites. The amino-terminus of human TFG was identified previously as a fusion partner of two protein kinases, creating a pair of oncogenes. We propose that fusion of these kinases to TFG relocalizes their activities to ER exit sites, where they prematurely phosphorylate substrates during ER export. Our findings provide a mechanism by which translocations involving TFG can result in cellular transformation and oncogenesis.
Uncoordinated movement in Rab2 mutants is caused by impaired retention of cargo on dense core vesicles, not by defective synaptic vesicle release. (Also see the companion article by Edwards et al. in this issue.)
Cytokinesis requires the spatio-temporal coordination of cell-cycle control and cytoskeletal reorganization. Members of the Rho-family of GTPases are crucial regulators of this process and assembly of the contractile ring depends on local activation of Rho signalling. Here, we show that the armadillo protein p0071, unlike its relative p120(ctn), is localized at the midbody during cytokinesis and is essential for cell division. Both knockdown and overexpression of p0071 interfered with normal cell growth and survival due to cytokinesis defects with formation of multinucleated cells and induction of apoptosis. This failure of cytokinesis seemingly correlated with the deregulation of Rho activity in response to altered p0071 expression. The function of p0071 in regulating Rho activity occurred through an association of p0071 with RhoA, as well as the physical and functional interaction of p0071 with Ect2, the one Rho guanine-nucleotide exchange factor (GEF) essential for cytokinesis. These findings support an essential role for p0071 in spatially regulating restricted Rho signalling during cytokinesis.
Human CC chemokine receptor 5 (CCR5), a member of the superfamily of G protein-coupled receptors, regulates the activation and directed migration of leukocytes and serves as the main coreceptor for the entry of R5 tropic strains of human immunodeficiency viruses. We have previously shown that RANTES/CCL5 binding to CCR5 induces GPCR kinase (GRK)-and protein kinase C (PKC)-mediated phosphorylation of four distinct C-terminal serine residues. To study these phosphorylation events in vivo, we have generated monoclonal antibodies, which specifically react only with either phosphorylated or nonphosphorylated CCR5. These phosphosite-specific antibodies reveal that following ligand stimulation of the receptor serine 337 is exclusively phosphorylated by a PKC-mediated mechanism, while GRKs phosphorylate serine 349. GRK-mediated receptor phosphorylation proceeds in a regular time-dependent manner (t1 ⁄2 ϳ2 min) with an apparent EC 50 of 5 nM. In contrast, PKC phosphorylates serine 337 at 50-fold lower concentrations and in a very rapid, albeit transient manner. Protein phosphatases that are active at neutral pH and are inhibited by okadaic acid rapidly dephosphorylate phosphoserine 337, but less efficiently phosphoserine 349, in intact cells and in an in vitro assay. Immunofluorescence microscopy demonstrates that phosphorylated receptors accumulate in a perinuclear compartment, which resembles recycling endosomes. This study is the first to analyze in detail the spatial and temporal dynamics of GRK-versus PKCmediated phosphorylation of a G protein-coupled receptor and its subsequent dephosphorylation on the level of individual phosphorylation sites. G protein-coupled receptors (GPCR)1 comprise the largest known family of signal-transducing proteins and respond to a large variety of external stimuli (1, 2). The receptors relay the information encoded by the ligand through the activation of heterotrimeric guanine nucleotide-binding proteins and intracellular effector molecules. Many GPCR undergo a process of rapid desensitization, which involves ligand-induced phosphorylation of serine and threonine residues located in the third intracellular loop or C-terminal domain by two different families of protein kinases. (i) GPCR kinases (GRKs) specifically phosphorylate only the agonist-occupied GPCR and thus mediate agonist-specific or homologous receptor phosphorylation (3, 4). (ii) In contrast, the second messenger-activated kinases, such as cyclic AMP-dependent protein kinase and protein kinase C (PKC), potentially phosphorylate both the ligand-bound GPCR and multiple other receptors in a heterologous manner. Receptor phosphorylation enhances the affinity of the agonistoccupied receptor for interaction with arrestin which interdicts signal transduction between the receptor and G proteins by steric mechanisms. The nonvisual arrestins, -arrestin-1 and -arrestin-2, also promote clathrin-mediated endocytosis of phosphorylated receptors and have been implicated in crosstalk with other signaling pathways. Once internalized, GPCR...
Early endothelial outgrowth cells (eEOCs) reproducibly have been shown to act protectively in acute ischemic kidney injury (AKI) and chronic kidney injury. Bone morphogenetic protein-5 (BMP-5) acted antifibrotically in human hypertensive nephropathy. The aim of the current study was to analyze effects of BMP-5 treatment in an eEOC-based therapy of murine AKI and 5/6-nephrectomy. Male C57/Bl6N mice were either subjected to unilateral renal artery clamping postuninephrectomy or to 5/6-nephrectomy. Untreated or BMP-5-pretreated murine eEOCs were injected into recipient animals at the time of reperfusion (AKI) or at 2 and 5 days after 5/6-nephrectomy. Analysis of renal function and morphology was performed at 48 h and at 6 wk (AKI) or at 8 wk (5/6 model). Cellular consequences of eEOC treatment were evaluated using different in vitro assays. AKI was mitigated significantly by injecting BMP-5-pretreated eEOCs. Renal function was improved at 48 h [corrected] after cell therapy. In 5/6-nephrectomy, the cells failed to act renoprotectively, [corrected] but proteinuria was reduced after administering untreated eEOCs." Next, the original version read as "BMP-5 acts as a potent eEOC agonist in murine AKI in the short [corrected] term. Cell effects in 5/6-nephrectomy are heterogenous, but untreated cells act antifibrotically [corrected] without any impact on EnMT.
Ang-2 acts as modulator of eEOCs in AKI. The migration analysis indicates that the Ang-2 significantly alters indirect (paracrine) activity of eEOCs, thus promoting renoprotection in a dose-dependent manner.
Diabetic nephropathy is the most frequent single cause of end-stage renal disease in our society. Microvascular damage is a key event in diabetes-associated organ malfunction. Early endothelial outgrowth cells (eEOCs) act protective in murine acute kidney injury. The aim of the present study was to analyze consequences of eEOC treatment of murine diabetic nephropathy with special attention on endothelial-to-mesenchymal transdifferentiation, autophagy, senescence, and apoptosis. Male C57/Bl6N mice (8-12 wk old) were treated with streptozotocin for 5 consecutive days. Animals were injected with untreated or bone morphogenetic protein (BMP)-5-pretreated syngeneic murine eEOCs on days 2 and 5 after the last streptozotocin administration. Four, eight, and twelve weeks later, animals were analyzed for renal function, proteinuria, interstitial fibrosis, endothelial-to-mesenchymal transition, endothelial autophagy, and senescence. In addition, cultured mature murine endothelial cells were investigated for autophagy, senescence, and apoptosis in the presence of glycated collagen. Diabetes-associated renal dysfunction (4 and 8 wk) and proteinuria (8 wk) were partly preserved by systemic cell treatment. At 8 wk, antiproteinuric effects were even more pronounced after the injection of BMP-5-pretreated cells. The latter also decreased mesenchymal transdifferentiation of the endothelium. At 8 wk, intrarenal endothelial autophagy (BMP-5-treated cells) and senescence (native and BMP-5-treated cells) were reduced. Autophagy and senescence in/of cultured mature endothelial cells were dramatically reduced by eEOC supernatant (native and BMP-5). Endothelial apoptosis decreased after incubation with eEOC medium (native and BMP-5). eEOCs act protective in diabetic nephropathy, and such effects are significantly stimulated by BMP-5. The cells modulate endothelial senescence, autophagy, and apoptosis in a protective manner. Thus, the renal endothelium could serve as a therapeutic target in diabetes-associated kidney dysfunction.
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