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Dephosphorylation
Selected AbstractsInactivation of phosphorylase is a major component of the mechanism by which insulin stimulates hepatic glycogen synthesisFEBS JOURNAL, Issue 13 2003Susan Aiston Multiple signalling pathways are involved in the mechanism by which insulin stimulates hepatic glycogen synthesis. In this study we used selective inhibitors of glycogen synthase kinase-3 (GSK-3) and an allosteric inhibitor of phosphorylase (CP-91149) that causes dephosphorylation of phosphorylase a, to determine the relative contributions of inactivation of GSK-3 and dephosphorylation of phosphorylase a as alternative pathways in the stimulation of glycogen synthesis by insulin in hepatocytes. GSK-3 inhibitors (SB-216763 and Li+) caused a greater activation of glycogen synthase than insulin (90% vs. 40%) but a smaller stimulation of glycogen synthesis (30% vs. 150%). The contribution of GSK-3 inactivation to insulin stimulation of glycogen synthesis was estimated to be less than 20%. Dephosphorylation of phosphorylase a with CP-91149 caused activation of glycogen synthase and translocation of the protein from a soluble to a particulate fraction and mimicked the stimulation of glycogen synthesis by insulin. The stimulation of glycogen synthesis by phosphorylase inactivation cannot be explained by either inhibition of glycogen degradation or activation of glycogen synthase alone and suggests an additional role for translocation of synthase. Titrations with the phosphorylase inactivator showed that stimulation of glycogen synthesis by insulin can be largely accounted for by inactivation of phosphorylase over a wide range of activities of phosphorylase a. We conclude that a signalling pathway involving dephosphorylation of phosphorylase a leading to both activation and translocation of glycogen synthase is a critical component of the mechanism by which insulin stimulates hepatic glycogen synthesis. Selective inactivation of phosphorylase can mimic insulin stimulation of hepatic glycogen synthesis. [source] Phosphorylation status of pyruvate dehydrogenase distinguishes metabolic phenotypes of cultured rat brain astrocytes and neuronsGLIA, Issue 10 2010Nader D. Halim Abstract Glucose metabolism in nervous tissue has been proposed to occur in a compartmentalized manner with astrocytes contributing largely to glycolysis and neurons being the primary site of glucose oxidation. However, mammalian astrocytes and neurons both contain mitochondria, and it remains unclear why in culture neurons oxidize glucose, lactate, and pyruvate to a much larger extent than astrocytes. The objective of this study was to determine whether pyruvate metabolism is differentially regulated in cultured neurons versus astrocytes. Expression of all components of the pyruvate dehydrogenase complex (PDC), the rate-limiting step for pyruvate entry into the Krebs cycle, was determined in cultured astrocytes and neurons. In addition, regulation of PDC enzymatic activity in the two cell types via protein phosphorylation was examined. We show that all components of the PDC are expressed in both cell types in culture, but that PDC activity is kept strongly inhibited in astrocytes through phosphorylation of the pyruvate dehydrogenase alpha subunit (PDH,). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorlyated PDH,. Dephosphorylation of astrocytic PDH, restores PDC activity and lowers lactate production. Our findings suggest that the glucose metabolism of astrocytes and neurons may be far more flexible than previously believed. © 2010 Wiley-Liss, Inc. [source] Topoisomerase inhibitor induced dephosphorylation of H1 and H3 histones as a consequence of cell cycle arrestJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2005Nicole Happel Abstract Posttranslational modifications of histones have an integral function in the structural and functional organization of chromatin. Several changes in the modification state of histones could be observed after induction of apoptosis with topoisomerase inhibitors and other inducers. Most of these studies include the analysis of the state of phosphorylation of histones, and the results are to some extent controversial, depending on cell lines and agents used. In the present study we compared the kinetics of the dephosphorylation of H1 and H3 histones with apoptosis markers after treatment of leukemic cell lines with topoisomerase inhibitors. In parallel, we determined cell cycle parameters in detail. Dephosphorylation of both histone classes started within 1 h of induction, and no direct correlation with timing and intensity of the investigated apoptotic features could be observed. In contrast, we show that the effect of topoisomerase inhibitors on the state of H1 and H3 phosphorylation is not directly related to apoptosis, but reflects the changes in the cell cycle distribution of cells treated with these inducers. © 2005 Wiley-Liss, Inc. [source] Dephosphorylation of pCREB by protein serine/threonine phosphatases is involved in inactivation of Aanat gene transcription in rat pineal glandJOURNAL OF NEUROCHEMISTRY, Issue 1 2003Marco Koch Abstract The rat pineal gland is a suitable model to investigate neurotransmitter-controlled gene expression, because it is well established that the stimulation of melatonin biosynthesis by norepinephrine (NE) depends on the activation of the gene that encodes arylalkylamine N -acetyltransferase (AANAT), the melatonin rhythm enzyme. The mechanisms responsible for downregulation of Aanat transcription are less clear. In this in vitro study we investigated the role of pCREB dephosphorylation for termination of Aanat gene transcription. Immunosignals for pCREB, strongly induced after NE stimulation, rapidly decreased after withdrawal of NE. The immunoreactivity of the inhibitory transcription factor ICER increased twofold after NE treatment for 6 h, but did not change within 30 min after removal of the stimulus. Application of protein serine/threonine phosphatase (PSP) inhibitors prevented pCREB dephosphorylation and blocked the decreases in Aanat mRNA levels, AANAT protein amount and melatonin biosynthesis all of which occurred rapidly after NE withdrawal. PSPs in the rat pineal gland were characterized by immunocytochemistry and immunoblotting. NE-stimulation for 8 h induced accumulation of PSP1-catalytic subunit (CSU) in pinealocyte nuclei, but did not affect the distribution of PSP2A-CSU. The results identify dephosphorylation of pCREB by PSPs as an essential mechanism for downregulation of Aanat transcription in the rat pineal gland. [source] Cholesterol-dependent modulation of dendrite outgrowth and microtubule stability in cultured neuronsJOURNAL OF NEUROCHEMISTRY, Issue 1 2002Qi-Wen Fan Abstract Microtubule-associated protein 2 (MAP2) is a neuron-specific cytoskeletal protein enriched in dendrites and cell bodies. MAP2 regulates microtubule stability in a phosphorylation-dependent manner, which has been implicated in dendrite outgrowth and branching. We have previously reported that cholesterol deficiency causes tau phosphorylation and microtubule depolymerization in axons (Fan et al. 2001). To investigate whether cholesterol also modulates microtubule stability in dendrites by modulating MAP2 phosphorylation, we examined the effect of compactin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, and TU-2078 (TU), a squalene epoxidase inhibitor, on these parameters using cultured neurons. We have found that cholesterol deficiency induced by compactin and TU, inhibited dendrite outgrowth, but not of axons, and attenuated axonal branching. Dephosphorylation of MAP2 and microtubule depolymerization accompanied these alterations. The amount of protein phosphatase 2 A (PP2A) and its activity in association with microtubules were decreased, while those unbound to microtubules were increased. The synthesized ceramide levels and the total ceramide content were increased in these cholesterol-deficient neurons. These alterations caused by compactin were prevented by concurrent treatment of cultured neurons with ,-migrating very-low-density lipoproteins (,-VLDL) or cholesterol. Taken together, we propose that cholesterol-deficiency causes a selective inhibition of dendrite outgrowth due to the decreased stability of microtubules as a result of inhibition of MAP2 phosphorylation. [source] Nitric Oxide-Sensitive Guanylyl Cyclase Activity Inhibition Through Cyclic GMP-Dependent DephosphorylationJOURNAL OF NEUROCHEMISTRY, Issue 5 2000Rut Ferrero Abstract: The soluble form of guanylyl cyclase (sGC) plays a pivotal role in the transduction of inter- and intracellular signals conveyed by nitric oxide. Here, a feedback inhibitory mechanism triggered by cyclic guanosine-3,,5,-monophosphate (cGMP)-dependent protein kinase (PKG) activation is described. Preincubation of chromaffin cells with C-type natriuretic peptide, which increased cGMP levels and activated PKG, or with cGMP-permeant analogue (which also activates PKG), in the presence of a broad-spectrum phosphodiesterase inhibitor, resulted in a decrease in subsequent sodium nitroprusside (SNP)-dependent cGMP elevations. This inhibitory effect was mimicked by activating a protein phosphatase and counteracted by the selective PKG inhibitor KT-5823 and by different protein phosphatase inhibitors. Immunoprecipitation of sGC from cells submitted to different treatments followed by immunodetection with antiphosphoserine antibodies (clone 4A9) showed changes in phosphorylation levels of the , subunit of sGC, and these changes correlated well with differences in SNP-elicited cGMP accumulations. Pretreatment of cells with several PKG inhibitors or protein phosphatase inhibitors produced an enhancement of SNP-stimulated cGMP rises without changing the SNP concentration required to produce half-maximal or maximal responses. Taken together, these results indicate that the catalytic activity of sGC is closely coupled to the phosphorylation state of its , subunit and that the tonic activity of PKG or its stimulation regulates sGC activity through dephosphorylation of the , subunit. [source] Effects of Fungal Phytase on Utilization of Dietary Protein and Minerals, and Dephosphorylation of Phytic Acid in the Alimentary Tract of Channel Catfish Ictalurus punctatus Fed an All-Plant-Protein DietJOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 1 2002Weibing Yan A feeding trial was conducted to quantify the effects of phytase at levels of 0, 500, 1,000, 2,000, 4,000, and 8,000 units (U) per kg diet on utilization of dietary protein and minerals by fingerling (12 g) channel catfish Ictalurus punctatus fed an all-plant-protein diet composed of soybean meal, corn, and wheat middlings. The effects of phytase on dephosphorylation of phytic acid (phytate) in the alimentary tract of catfish also were determined. After 14 wk, mean weight gains (30.2,43.9 g/fish), feed conversion ratios (2.27,2.40 g feed consumed/g weight gain), protein efficiency ratios (1.47,1.61 g weight gaid/g protein consumed), and dietary protein retentions (23.8,26.7%) did not differ significantly (P > 0.05) among treatment groups. A digestibility trial conducted after the feeding trial showed no difference (P > 0.05) in mean digestibility of diet dry matter (49.0,58.3%) or crude protein (85.4-88.5%) among treatment groups. Concentrations of ash (46.7,48.6%), calcium (Ca, 17.9,18.5%), phosphorus (P, 9.1,9.5%), and manganese (Mn, 65.5,74.1 mg/kg) were significantly higher (P , 0.05) in bone of fish fed , 500 U/kg than in bone of fish fed 0 U/kg (ash, 43.5%; Ca, 16.4%; P, 8.4%; and Mn, 49.0 ma/kg), but concentrations of these minerals did not differ (P > 0.05) in bone of fish fed , 500 Uk/g. The magnesium (Mg) content of bone did not differ (P > 0.05) between fish fed 0 U/kg (0.29%) or 500 U/kg (0.34%), but was significantly lower in fish fed 0 U/kg than in fish fed , 1,000 U/kg (0.35,37%). Bone Mg levels did not differ (P > 0.05) among fish fed , 500 U/kg. The amount of zinc (Zn) in bone of fish fed 8,000 U/kg (153.3 mg/kg) was significantly higher than that in fish fed 0 U/kg (115.7 mg/kg) or 500 U/kg (130.3 mg/ kg), but did not differ from Zn levels in bone of fish fed 1,000,4,000 U/kg (134.5,135.8 mg/ kg). Dephosphorylation of phytate occurred primarily in the stomach within 2,8 h after diet ingestion, depending on the level of phytase supplementation. Initial levels of total phytate in the diet decreased 32,94% in stomach contents of fish fed l,000,8,000 U/kg within 2 h after feeding. Eight hours after feeding, stomach contents of fish fed , 1,000 U/kg contained less than 6% of initial total dietary phytate. Stomach contents of fish fed 500 U/kg retained 92% of initial total dietary phytate 2 h after feeding and 15% of total dietary phytate 8 h after feeding. Results of this study indicate that phytase supplementation at levels up to 8,000 U/kg diet did not increase weight gain or improve dietary protein utilization of channel catfish fed an all-plant-protein diet. Addition of phytase at a level of 1,000 U/kg diet was sufficient to significantly increase the Ca, P, Mg, and Mn content of bone, relative to fish fed an unsupplemented diet, and significantly decrease the quantity of total phytate in feces. A phytase level of 8,000 U/kg diet significantly increased the bioavailability of naturally occurring Zn in feed ingredients and increased the rate of phytate dephosphorylation in the stomach, compared with a diet containing no added phytase. Increased utilization of naturally occurring minerals in feed ingredients reduces the need for mineral supplements in diets and results in decreased elimination of minerals in feces. Thus, use of phytase in catfish feeds can be expected to provide both economic and environmental benefits. [source] Dephosphorylation of p-ERK1/2 in relation to tumor remission after HER-2 and Raf1 blocking therapy in a conditional mouse tumor model,,MOLECULAR CARCINOGENESIS, Issue 5 2006Carolin K. Hausherr Abstract Several studies have shown that HER-2/neu (erbB-2) blocking therapy strategies can cause tumor remission. However, the responsible molecular mechanisms are not yet known. Both ERK1/2 and Akt/PKB are critical for HER-2-mediated signal transduction. Therefore, we used a mouse tumor model that allows downregulation of HER-2 in tumor tissue by administration of anhydrotetracycline (ATc). Switching-off HER-2 caused a rapid tumor remission by more than 95% within 7 d of ATc administration compared to the volume before switching-off HER-2. Interestingly, HER-2 downregulation caused a dephosphorylation of p-ERK1/2 by more than 80% already before tumor remission occurred. Levels of total ERK protein were not influenced. In contrast, dephosphorylation of p-Akt occurred later, when the tumor was already in remission. These data suggest that in our HER-2 tumor model dephosphorylation of p-ERK1/2 may be more critical for tumor remission than dephosphorylation of p-Akt. To test this hypothesis we used a second mouse tumor model that allows ATc controlled expression of BXB-Raf1 because the latter constitutively signals to ERK1/2, but cannot activate Akt/PKB. As expected, downregulation of BXB-Raf1 in tumor tissue caused a strong dephosphorylation of p-ERK1/2, but did not decrease levels of p-Akt. Interestingly, tumor remission after switching-off BXB-Raf1 was similarly efficient as the effect of HER-2 downregulation, despite the lack of p-Akt dephosphorylation. In conclusion, two lines of evidence strongly suggest that dephosphorylation of p-ERK1/2 and not that of p-Akt is critical for the rapid tumor remission after downregulation of HER-2 or BXB-Raf1 in our tumor model: (i) dephosphorylation of p-ERK1/2 but not that of p-Akt precedes tumor remission after switching-off HER-2 and (ii) downregulation of BXB-Raf1 leads to a similarly efficient tumor remission as downregulation of HER-2, although no p-Akt dephosphorylation was observed after switching-off BXB-Raf1. © 2006 Wiley-Liss, Inc. [source] AMP-activated protein kinase , a sensor of glycogen as well as AMP and ATP?ACTA PHYSIOLOGICA, Issue 1 2009A. McBride Abstract The classical role of the AMP-activated protein kinase (AMPK) is to act as a sensor of the immediate availability of cellular energy, by monitoring the concentrations of AMP and ATP. However, the , subunits of AMPK contain a glycogen-binding domain, and in this review we develop the hypothesis that this is a regulatory domain that allows AMPK to act as a sensor of the status of cellular reserves of energy in the form of glycogen. We argue that the pool of AMPK that is bound to the glycogen particle is in an active state when glycogen particles are fully synthesized, causing phosphorylation of glycogen synthase at site 2 and providing a feedback inhibition of further extension of the outer chains of glycogen. However, when glycogen becomes depleted, the glycogen-bound pool of AMPK becomes inhibited due to binding to ,1,6-linked branch points exposed by the action of phosphorylase and/or debranching enzyme. This allows dephosphorylation of site 2 on glycogen synthase by the glycogen-bound form of protein phosphatase-1, promoting rapid resynthesis of glycogen and replenishment of glycogen stores. This is an extension of the classical role of AMPK as a ,guardian of cellular energy', in which it ensures that cellular energy reserves are adequate for medium-term requirements. The literature concerning AMPK, glycogen structure and glycogen-binding proteins that led us to this concept is reviewed. [source] Intracellular sodium modulates the state of protein kinase C phosphorylation of rat proximal tubule Na+,K+ -ATPaseACTA PHYSIOLOGICA, Issue 2 2002F. R. IBARRA ABSTRACT The natriuretic hormone dopamine and the antinatriuretic hormone noradrenaline, acting on , -adrenergic receptors, have been shown to bidirectionally modulate the activity of renal tubular Na+,K+ -adenosine triphosphate (ATPase). Here we have examined whether intracellular sodium concentration influences the effects of these bidirectional forces on the state of phosphorylation of Na+,K+ -ATPase. Proximal tubules dissected from rat kidney were incubated with dopamine or the , -adrenergic agonist, oxymetazoline, and transiently permeabilized in a medium where sodium concentration ranged between 5 and 70 mM. The variations of sodium concentration in the medium had a proportional effect on intracellular sodium. Dopamine and protein kinase C (PKC) phosphorylate the catalytic subunit of rat Na+,K+ -ATPase on the Ser23 residue. The level of PKC induced Na+,K+ -ATPase phosphorylation was determined using an antibody that only recognizes Na+,K+ -ATPase, which is not phosphorylated on its PKC site. Under basal conditions Na+,K+ -ATPase was predominantly in its phosphorylated state. When intracellular sodium was increased, Na+,K+ -ATPase was predominantly in its dephosphorylated state. Phosphorylation of Na+,K+ -ATPase by dopamine was most pronounced when intracellular sodium was high, and dephosphorylation by oxymetazoline was most pronounced when intracellular sodium was low. The oxymetazoline effect was mimicked by the calcium ionophore A23187. An inhibitor of the calcium-dependent protein phosphatase, calcineurin, increased the state of Na+,K+ -ATPase phosphorylation. The results imply that phosphorylation of renal Na+,K+ -ATPase activity is modulated by the level of intracellular sodium and that this effect involves PKC and calcium signalling pathways. The findings may have implication for the regulation of salt excretion and sodium homeostasis. [source] Regulation of sperm flagellar motility activation and chemotaxis caused by egg-derived substance(s) in sea cucumberCYTOSKELETON, Issue 4 2009Masaya Morita Abstract The sea cucumber Holothuria atra is a broadcast spawner. Among broadcast spawners, fertilization occurs by means of an egg-derived substance(s) that induces sperm flagellar motility activation and chemotaxis. Holothuria atra sperm were quiescent in seawater, but exhibited flagellar motility activation near eggs with chorion (intact eggs). In addition, they moved in a helical motion toward intact eggs as well as a capillary filled with the water layer of the egg extracts, suggesting that an egg-derived compound(s) causes motility activation and chemotaxis. Furthermore, demembranated sperm flagella were reactivated in high pH (>7.8) solution without cAMP, and a phosphorylation assay using (,-32P)ATP showed that axonemal protein phosphorylation and dephosphorylation also occurred in a pH-dependent manner. These results suggest that the activation of sperm motility in holothurians is controlled by pH-sensitive changes in axonemal protein phosphorylation. Ca2+ concentration affected the swimming trajectory of demembranated sperm, indicating that Ca2+ -binding proteins present at the flagella may be associated with regulation of flagellar waveform. Moreover, the phosphorylation states of several axonemal proteins were Ca2+ -sensitive, indicating that Ca2+ impacts both kinase and phosphatase activities. In addition, in vivo sperm protein phosphorylation occurred after treatment with a water-soluble egg extract. Our results suggest that one or more egg-derived compounds activate motility and subsequent chemotactic behavior via Ca2+ -sensitive flagellar protein phosphorylation. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Protein tyrosine phosphatases in Chaetopterus egg activationDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5-6 2003Shantá D. Hinton Changes in protein tyrosine phosphorylation are an essential aspect of egg activation after fertilization. Such changes result from the net contributions of both tyrosine kinases and phosphatases (PTP). This study was conducted to determine what role(s) PTP may have in egg activation. We identified four novel PTP in Chaetopterus pergamentaceus oocytes, cpPTPNT6, cpPTPNT7, cpPTPR2B, and cpPTPR2A, that have significant homology to, respectively, human PTP,, -,, -D2 and -BAS. The first two are cytosolic and the latter two are transmembrane. Several PTP inhibitors were tested to see if they would affect Chaetopterus pergamentaceus fertilization. Eggs treated with ,-bromo-4-hydroxyacetophenone (PTP inhibitor 1) exhibited microvillar elongation, which is a sign of cortical changes resulting from activation. Those treated with Na3VO4 underwent full parthenogenetic activation, including polar body formation and pseudocleavage and did so independently of extracellular Ca2+, which is required for the Ca2+ oscillations that initiate development after fertilization. Fluorescence microscopy identified phosphotyrosine-containing proteins in the cortex and around the nucleus of vanadate-activated eggs, whereas in fertilized eggs they were concentrated only in the cortex. Immunoblots of vanadate-activated and fertilized eggs showed tyrosine hyperphosphorylation of approximately140 kDa protein. These results suggest that PTP most likely maintain the egg in an inactive state by dephosphorylation of proteins independent of the Ca2+ oscillations in the activation process. [source] A critical step for postsynaptic F-actin organization: Regulation of Baz/Par-3 localization by aPKC and PTENDEVELOPMENTAL NEUROBIOLOGY, Issue 9 2009Preethi Ramachandran Abstract Actin remodeling has emerged as a critical process during synapse development and plasticity. Thus, understanding the regulatory mechanisms controlling actin organization at synapses is exceedingly important. Here, we used the highly plastic Drosophila neuromuscular junction (NMJ) to understand mechanisms of actin remodeling at postsynaptic sites. Previous studies have suggested that the actin-binding proteins Spectrin and Coracle play a critical role in NMJ development and the anchoring of glutamate receptors most likely through actin regulation. Here, we show that an additional determinant of actin organization at the postsynaptic region is the PDZ protein Baz/Par-3. Decreasing Baz levels in postsynaptic muscles has dramatic consequences for the size of F-actin and spectrin domains at the postsynaptic region. In turn, proper localization of Baz at this site depends on both phosphorylation and dephosphorylation events. Baz phosphorylation by its binding partner, atypical protein kinase C (aPKC), is required for normal Baz targeting to the postsynaptic region. However, the retention of Baz at this site depends on its dephosphorylation mediated by the lipid and protein phosphatase PTEN. Misregulation of the phosphorylation state of Baz by genetic alterations in PTEN or aPKC activity has detrimental consequences for postsynaptic F-actin and spectrin localization, synaptic growth, and receptor localization. Our results provide a novel mechanism of postsynaptic actin regulation through Baz, governed by the antagonistic actions of aPKC and PTEN. Given the conservation of these proteins from worms to mammals, these results are likely to provide new insight into actin organization pathways. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009 [source] LAR protein tyrosine phosphatase receptor associates with TrkB and modulates neurotrophic signaling pathwaysDEVELOPMENTAL NEUROBIOLOGY, Issue 13 2006Tao Yang Abstract The identities of receptor protein tyrosine phosphatases (PTPs) that associate with Trk protein tyrosine kinase (PTK) receptors and modulate neurotrophic signaling are unknown. The leukocyte common antigen-related (LAR) receptor PTP is present in neurons expressing TrkB, and like TrkB is associated with caveolae and regulates survival and neurite outgrowth. We tested the hypothesis that LAR associates with TrkB and regulates neurotrophic signaling in embryonic hippocampal neurons. Coimmunoprecipitation and coimmunostaining demonstrated LAR interaction with TrkB that is increased by BDNF exposure. BDNF neurotrophic activity was reduced in LAR,/, and LAR siRNA-treated LAR+/+ neurons and was augmented in LAR-transfected neurons. In LAR,/, neurons, BDNF-induced activation of TrkB, Shc, AKT, ERK, and CREB was significantly decreased; while in LAR-transfected neurons, BDNF-induced CREB activation was augmented. Similarly, LAR+/+ neurons treated with LAR siRNA demonstrated decreased activation of Trk and AKT. LAR is known to activate the Src PTK by dephosphorylation of its negative regulatory domain and Src transactivates Trk. In LAR,/, neurons, or neurons treated with LAR siRNA, phosphorylation of the Src regulatory domain was increased (indicating Src inactivation), consistent with a role for Src in mediating LAR's ability to up-regulate neurotrophic signaling. Interactions between LAR, TrkB, and Src were further confirmed by the findings that Src coimmunoprecipitated with LAR, that the Src inhibitor PP2 blocked the ability of LAR to augment TrkB signaling, and that siRNA-induced depletion of Src decreased LAR interaction with TrkB. These studies demonstrate that receptor PTPs can associate with Trk complexes and promote neurotrophic signaling and point to receptor PTP-based strategies as a novel approach for modulating neurotrophin function. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] Focused proteomics: Monoclonal antibody-based isolation of the oxidative phosphorylation machinery and detection of phosphoproteins using a fluorescent phosphoprotein gel stainELECTROPHORESIS, Issue 15 2004James Murray Abstract We have raised monoclonal antibodies capable of immunocapturing all five complexes involved in oxidative phosphorylation for evaluating their post-translational modifications. Complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (F1F0 ATP synthase) from bovine heart mitochondria were obtained in good yield from small amounts of tissue in more than 90% purity in one step. The composition and purity of the complexes was evaluated by Western blotting using monoclonal antibodies against individual subunits of the five complexes. In this first study, the phosphorylation state of the proteins without inducing phosphorylation or dephosphorylation was identified by using the novel Pro-Q Diamond phosphoprotein gel stain. The major phosphorylated components were the same as described before in sucrose gradient enriched complexes. In addition a few additional potential phosphoproteins were observed. Since the described monoclonal antibodies show cross reactivity to human proteins, this procedure will be a fast and efficient way of studying post-translational modifications in control and patient samples using only small amounts of tissue. [source] Acetate inhibits NFAT activation in T cells via importin ,1 interferenceEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 8 2007Kazuhiro Ishiguro Dr. Abstract Acetate is a principal short chain fatty acid produced by bacterial fermentation in the colon and a major end product of alcohol metabolism. In the present study, we assessed the effects of acetate on T cell activation and found that acetate inhibited NFAT activation but not NF-,B activation. Moreover, acetate impaired the nuclear translocation of NFAT but not that of NF-,B. Unlike cyclosporin A (CsA), acetate did not affect the dephosphorylation of NFAT and calcineurin activity. Acetate impaired the binding of NFAT to importin ,1, which is involved in NFAT nuclear translocation. NFAT is a critical transcription factor in cytokine and early response gene expression in activated T cells. Agents targeting NFAT such as CsA are used to suppress harmful immune responses in inflammatory diseases. Therefore, we also evaluated the efficacy of acetate in murine models of inflammatory diseases, and found that acetate administration (as well as administration of dexamethasone) attenuated trinitrobenzenesulfonic acid-induced colitis and dinitrofluorobenzene-induced dermatitis. These findings indicate for the first time that acetate inhibits NFAT activation by interfering with the interaction between NFAT and importin ,1 in T cells and that acetate can potentially act as an anti-inflammatory agent. [source] IL-7 inhibits dexamethasone-induced apoptosis via Akt/PKB in mature, peripheral T cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2003Hadassah Sade Abstract We have investigated the mechanism of IL-7-mediated inhibition of dexamethasone-induced apoptosis in T cells. Broad-spectrum caspase inhibitors block dexamethasone-triggered nuclear fragmentation, but not the loss of mitochondrial transmembrane potential or membrane integrity in CD3+ mature T cells isolated from adult mouse spleens. IL-7 blocked dexamethasone-induced apoptosis and the processing of caspase-3 and caspase-7. IL-7 also blocked dexamethasone-triggered dephosphorylation of the serine-threonine kinase Akt/PKB and its target, the Ser136 residue in Bad. The loss of anti-apoptotic proteins Bcl-xL and inhibitor of apoptosis protein-2 (IAP-2) was also blocked by IL-7. The protective effect was attenuated by pharmacological inhibitors of phosphatidylinositol-3 kinase (PI3K) with one exception: inhibition of PI3K did not abrogate Bcl-xL expression in the presence of IL-7. The anti-apoptotic role of Akt suggested by these experiments was tested by overexpression of constitutively active Akt, which blocked dexamethasone-induced apoptosis and elevated IAP-2 but not Bcl-xL levels in a mature T cell line. Thus, IL-7 regulates IAP-2 expression and inhibits dexamethasone-induced apoptosis by activating Akt via PI3K-dependent signaling, but regulates Bcl-xL expression via a PI3K-independent pathway in mature T cells. [source] Altered subcellular location of phosphorylated Smads in Alzheimer's diseaseEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2006Uwe Ueberham Abstract A number of growth factors and cytokines, such as transforming growth factor beta 1 (TGF-,1), is elevated in Alzheimer's disease (AD), giving rise to activated intracellular mitogenic signaling cascades. Activated mitogenic signaling involving the mitogen-activated protein kinases (MAPKs) and other protein kinases might alter the phosphorylation states of structural proteins such as tau, resulting in hyperphosphorylated deposits. Many intracellular signaling proteins are potential targets of misregulated phosphorylation and dephosphorylation. Recently, a crosstalk between MAPKs and Smad proteins, both involved in mediating TGF-,1 signaling, has been reported. Although TGF-,1 has previously been shown to be involved in the pathogenesis of AD, the role of Smad proteins has not been investigated. In this study we thus analysed the subcellular distribution of phosphorylated Smad2 and Smad3 in the hippocampus of both normal and AD brains. Here we report on strong nuclear detection of phosphorylated Smad2 and Smad3 in neurons of control brains. In AD brains these phosphorylated proteins were additionally found in cytoplasmic granules in hippocampal neurons, within amyloid plaques and attached to neurofibrillary tangles. Our data suggest a critical role of Smad proteins in the pathogenesis of AD. [source] Homologous desensitization of guanylyl cyclase A, the receptor for atrial natriuretic peptide, is associated with a complex phosphorylation patternFEBS JOURNAL, Issue 11 2010Juliane Schröter Atrial natriuretic peptide (ANP), via its guanylyl cyclase A (GC-A) receptor and intracellular guanosine 3,,5,-cyclic monophosphate production, is critically involved in the regulation of blood pressure. In patients with chronic heart failure, the plasma levels of ANP are increased, but the cardiovascular actions are severely blunted, indicating a receptor or postreceptor defect. Studies on metabolically labelled GC-A-overexpressing cells have indicated that GC-A is extensively phosphorylated, and that ANP-induced homologous desensitization of GC-A correlates with receptor dephosphorylation, a mechanism which might contribute to a loss of function in vivo. In this study, tandem MS analysis of the GC-A receptor, expressed in the human embryonic kidney cell line HEK293, revealed unambiguously that the intracellular domain of the receptor is phosphorylated at multiple residues: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 and Thr513. MS quantification based on multiple reaction monitoring demonstrated that ANP-provoked desensitization was accompanied by a complex pattern of receptor phosphorylation and dephosphorylation. The population of completely phosphorylated GC-A was diminished. However, intriguingly, the phosphorylation of GC-A at Ser487 was selectively enhanced after exposure to ANP. The functional relevance of this observation was analysed by site-directed mutagenesis. The substitution of Ser487 by glutamate (which mimics phosphorylation) blunted the activation of the GC-A receptor by ANP, but prevented further desensitization. Our data corroborate previous studies suggesting that the responsiveness of GC-A to ANP is regulated by phosphorylation. However, in addition to the dephosphorylation of the previously postulated sites (Ser497, Thr500, Ser502, Ser506, Ser510), homologous desensitization seems to involve the phosphorylation of GC-A at Ser487, a newly identified site of phosphorylation. The identification and further characterization of the specific mechanisms involved in the downregulation of GC-A responsiveness to ANP may have important pathophysiological implications. Structured digital abstract ,,MINT-7713870, MINT-7713887: PMCA (uniprotkb:P20020) and GC-A (uniprotkb:P18910) colocalize (MI:0403) by fluorescence microscopy (MI:0416) [source] Betulinic acid-mediated inhibitory effect on hepatitis B virus by suppression of manganese superoxide dismutase expressionFEBS JOURNAL, Issue 9 2009Dachun Yao The betulinic acid (BetA) purified from Pulsatilla chinensis (PC) has been found to have selective inhibitory effects on hepatitis B virus (HBV). In hepatocytes from HBV-transgenic mice, we showed that BetA substantially inhibited HBV replication by downregulation of manganese superoxide dismutase (SOD2) expression, with subsequent reactive oxygen species generation and mitochondrial dysfunction. Also, the HBV X protein (HBx) is suppressed and translocated into the mitochondria followed by cytochrome c release. Further investigation revealed that SOD2 expression was suppressed by BetA-induced cAMP-response element-binding protein dephosphorylation at Ser133, which subsequently prevented SOD2 transcription through the cAMP-response element-binding protein-binding motif on the SOD2 promoter. SOD2 overexpression abolished the inhibitory effect of BetA on HBV replication, whereas SOD2 knockdown mimicked this effect, indicating that BetA-mediated HBV clearance was due to modulation of the mitochondrial redox balance. This observation was further confirmed in HBV-transgenic mice, where both BetA and PC crude extracts suppressed SOD2 expression, with enhanced reactive oxygen species generation in liver tissues followed by substantial HBV clearance. We conclude that BetA from PC could be a good candidate for anti-HBV drug development. [source] 7-Ketocholesterol-induced apoptosisFEBS JOURNAL, Issue 12 2005Involvement of several pro-apoptotic but also anti-apoptotic calcium-dependent transduction pathways Oxysterols, and particularly 7-ketocholesterol, appear to be strongly involved in the physiopathology of atherosclerosis. These molecules are suspected to be cytotoxic to the cells of the vascular wall and monocytes/macrophages, particularly by inducing apoptosis. Previous studies have demonstrated that 7-ketocholesterol-induced apoptosis is triggered by a sustained increase of cytosolic-free Ca2+, which elicits the mitochondrial pathway of apoptosis by activation of the calcium-dependent phosphatase calcineurin, leading to dephosphorylation of the ,BH3 only' protein BAD. However, thorough study of the results suggests that other pathways are implicated in 7-ketocholesterol-induced cytotoxicity. In this study, we demonstrate the involvement of two other calcium-dependent pathways during 7-ketocholesterol-induced apoptosis. The activation of the MEK,ERK pathway by the calcium-dependent tyrosine kinase PYK 2, a survival pathway which delays apoptosis as shown by the use of the MEK inhibitor U0126, and a pathway involving another pro-apoptotic BH3 only protein, Bim. Indeed, 7-ketocholesterol treatment of human monocytic THP-1 cells induces the release of Bim-LC8 from the microtubule-associated dynein motor complex, and its association with Bcl-2. Therefore, it appears that 7-ketocholesterol-induced apoptosis is a complex phenomenon resulting from calcium-dependent activation of several pro-apoptotic pathways and also one survival pathway. [source] Inactivation of phosphorylase is a major component of the mechanism by which insulin stimulates hepatic glycogen synthesisFEBS JOURNAL, Issue 13 2003Susan Aiston Multiple signalling pathways are involved in the mechanism by which insulin stimulates hepatic glycogen synthesis. In this study we used selective inhibitors of glycogen synthase kinase-3 (GSK-3) and an allosteric inhibitor of phosphorylase (CP-91149) that causes dephosphorylation of phosphorylase a, to determine the relative contributions of inactivation of GSK-3 and dephosphorylation of phosphorylase a as alternative pathways in the stimulation of glycogen synthesis by insulin in hepatocytes. GSK-3 inhibitors (SB-216763 and Li+) caused a greater activation of glycogen synthase than insulin (90% vs. 40%) but a smaller stimulation of glycogen synthesis (30% vs. 150%). The contribution of GSK-3 inactivation to insulin stimulation of glycogen synthesis was estimated to be less than 20%. Dephosphorylation of phosphorylase a with CP-91149 caused activation of glycogen synthase and translocation of the protein from a soluble to a particulate fraction and mimicked the stimulation of glycogen synthesis by insulin. The stimulation of glycogen synthesis by phosphorylase inactivation cannot be explained by either inhibition of glycogen degradation or activation of glycogen synthase alone and suggests an additional role for translocation of synthase. Titrations with the phosphorylase inactivator showed that stimulation of glycogen synthesis by insulin can be largely accounted for by inactivation of phosphorylase over a wide range of activities of phosphorylase a. We conclude that a signalling pathway involving dephosphorylation of phosphorylase a leading to both activation and translocation of glycogen synthase is a critical component of the mechanism by which insulin stimulates hepatic glycogen synthesis. Selective inactivation of phosphorylase can mimic insulin stimulation of hepatic glycogen synthesis. [source] Echistatin inhibits pp125FAK autophosphorylation, paxillin phosphorylation and pp125FAK,paxillin interaction in fibronectin-adherent melanoma cellsFEBS JOURNAL, Issue 16 2000Rossella Della Morte Echistatin, a snake-venom RGD-containing protein, was previously shown to disrupt cell-matrix adhesion by a mechanism that involves the reduction of pp125FAK tyrosine phosphorylation levels. The aim of this study was to establish the sequence of events downstream pp125FAK dephosphorylation that could be responsible for echistatin-induced disassembly of actin cytoskeleton and focal adhesions in fibronectin-adherent B16-BL6 melanoma cells. The results obtained show that echistatin induces a decrease of both autophosphorylation and kinase activity of pp125FAK. One hour of cell exposure to echistatin caused a 39% decrease of pp125FAK Tyr397 phosphorylation and a 31% reduction of pp125FAK autophosphorylation activity as measured by immune-complex kinase assay. Furthermore, 1 h of cell treatment by echistatin produced a 63% decrease of paxillin phosphorylation, as well as a reduction in the amount of paxillin bound to pp125FAK. Immunofluorescence analysis of echistatin treated cells showed the concomitant disappearance of both paxillin and pp125FAK from focal adhesions. The reduction of paxillin phosphorylation may represent a critical step in the pathway by which disintegrins exert their biological activity, including the inhibition of experimental metastasis in vivo. [source] Transmitting the signal of excess nitrogen in Saccharomyces cerevisiae from the Tor proteins to the GATA factors: connecting the dotsFEMS MICROBIOLOGY REVIEWS, Issue 3 2002Terrance G. Cooper Abstract Major advances have recently occurred in our understanding of GATA factor-mediated, nitrogen catabolite repression (NCR)-sensitive gene expression in Saccharomyces cerevisiae. Under nitrogen-rich conditions, the GATA family transcriptional activators, Gln3 and Gat1, form complexes with Ure2, and are localized to the cytoplasm, which decreases NCR-sensitive expression. Under nitrogen-limiting conditions, Gln3 and Gat1 are dephosphorylated, move from the cytoplasm to the nucleus, in wild-type but not rna1 and srp1 mutants, and increase expression of NCR-sensitive genes. ,Induction' of NCR-sensitive gene expression and dephosphorylation of Gln3 (and Ure2 in some laboratories) when cells are treated with rapamycin implicates the Tor1/2 signal transduction pathway in this regulation. Mks1 is posited to be a negative regulator of Ure2, positive regulator of retrograde gene expression and to be itself negatively regulated by Tap42. In addition to Tap42, phosphatases Sit4 and Pph3 are also argued by some to participate in the regulatory pathway. Although a treasure trove of information has recently become available, much remains unknown (and sometimes controversial) with respect to the precise biochemical functions and regulatory pathway connections of Tap42, Sit4, Pph3, Mks1 and Ure2, and how precisely Gln3 and Gat1 are prevented from entering the nucleus. The purpose of this review is to provide background information needed by students and investigators outside of the field to follow and evaluate the rapidly evolving literature in this exciting field. [source] Combination of Clk family kinase and SRp75 modulates alternative splicing of Adenovirus E1AGENES TO CELLS, Issue 3 2008Jun-ichiro Yomoda SR proteins are non-snRNP splicing factors harbouring a domain rich in Arg-Ser repeats, which are extensively phosphorylated by several kinases. We performed a comparative study of different SR kinases, including SRPK, Clk, PRP4 and DYRK, and found that only Clks efficiently altered 5, splice site selection of Adenovirus E1A. The phosphorylation state of SR proteins was examined using a phospho-SR specific antibody mAb1H4 and a 75 kDa protein was most evidently hyperphosphorylated by Clks. Administration of TG003, a specific inhibitor for the Clk family members, specifically and rapidly induced dephosphorylation of 75 kDa SR protein. Imaging with mRFP-SRp75 in living cells revealed that its nuclear distribution was rapidly altered upon inhibition of the Clk activity by TG003. Co-transfection experiments demonstrated that HA-tagged SRp75 was hyperphosphorylated by Clk family members, but not by other SR kinases. These results indicate that Clks specifically hyperphosphorylate SRp75. Furthermore, SRp75 over-expression promoted the selection of 12S 5, splice site in E1A pre-mRNA, which is stimulated by co-expression of Clks. These results suggest that the specific combination of SR protein and SR kinase plays a distinct role in alternative splicing through dynamic balance of phosphorylation. [source] Vinexin , regulates the phosphorylation of epidermal growth factor receptor on the cell surfaceGENES TO CELLS, Issue 9 2006Masaru Mitsushima Epidermal growth factor (EGF) regulates various cellular events, including proliferation, differentiation, migration and oncogenesis. In this study, we found that exogenous expression of vinexin , enhanced the phosphorylation of 180-kDa proteins in an EGF-dependent manner in Cos-7 cells. Western blot analysis using phospho-specific antibodies against EGFR identified EGFR as a phosphorylated 180-kDa protein. Vinexin , did not stimulate the phosphorylation of EGFR but suppressed the dephosphorylation, resulting in a sustained phosphorylation. Mutational analyses revealed that both the first and third SH3 domains were required for a sustained phosphorylation of EGFR. Small interfering RNA-mediated knockdown of vinexin , reduced the phosphorylation of EGFR on the cell surface in HeLa cells. The sustained phosphorylation of EGFR induced by vinexin , was completely abolished by adding the EGFR-specific inhibitor AG1478 even after EGF stimulation, suggesting that the kinase activity of EGFR is required for the sustained phosphorylation induced by vinexin ,. We also found that E3 ubiquitin ligase c-Cbl is a binding partner of vinexin , through the third SH3 domain. Expression of wild-type vinexin , but not a mutant containing a mutation in the third SH3 domain decreased the cytosolic pool of c-Cbl and increased the amount of membrane-associated c-Cbl. Furthermore, over-expression of c-Cbl suppressed the sustained phosphorylation of EGFR induced by vinexin ,. These results suggest that vinexin , plays a role in maintaining the phosphorylation of EGFR on the plasma membrane through the regulation of c-Cbl. [source] Pml and TAp73 interacting at nuclear body mediate imatinib-induced p53-independent apoptosis of chronic myeloid leukemia cellsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2009Jin-Hwang Liu Abstract Bcr-abl signals for leukemogenesis of chronic myeloid leukemia (CML) and activates ras. Since the function of promyelocytic leukemia protein (pml) is provoked by ras to promote apoptosis and senescence in untransformed cells, the function is probably masked in CML. Imatinib specifically inhibits bcr-abl and induces apoptosis of CML cells. As reported previously, p53wild CML was more resistant to imatinib than that lacking p53. Here, we searched for an imatinib-induced p53 independent proapoptotic mechanism. We found imatinib up-regulated phosphorylation of p38 mitogen-activated protein kinase (MAPK), checkpoint kinase 2 (chk2) and transactivation-competent (TA) p73; expression of pml and bax; formation of PML-nuclear body (NB); and co-localization of TAp73/PML-NB in p53-nonfunctioning K562 and p53mutant Meg-01 CML cells, but not in BCR-ABL - HL60 cells. In K562 cells, with short interfering RNAs (siRNAs), knockdown of pml led to dephosphorylation of TAp73. Knockdown of either pml or TAp73 abolished the imatinib-induced apoptosis. Inhibition of p38 MAPK with SB203580 led to dephosphorylation of TAp73, abolishment of TAp73/PML-NB co-localization, and the subsequent apoptosis. Conversely, interferon ,-2a (IFN,), which increased phosphrylated TAp73 and TAp73/PML-NB co-localization, increased additively apoptosis with imatinib. The imatinib-induced TAp73/PML-NB co-localization was accompanied by co-immpunoprecipitation of TAp73 with pml. The imatinib-induced co-localization was also found in primary CML cells from 3 of 6 patients, including 2 with p53mutant and one with p53wild. A novel p53-independent proapoptotic mechanism using p38 MAPK /pml/TAp73 axis with a step processing at PML-NB and probably with chk2 and bax being involved is hereby evident in some imatinib-treated CML cells. © 2009 UICC [source] PPAR, and PP2A are involved in the proapoptotic effect of conjugated linoleic acid on human hepatoma cell line SK-HEP-1INTERNATIONAL JOURNAL OF CANCER, Issue 11 2007Giuliana Muzio Abstract Conjugated linoleic acid (CLA), found in dairy products, in beef and lamb has been demonstrated to possess anticancer properties protecting several tissues from developing cancer. Moreover, it has been shown to modulate apoptosis in several cancer cell lines. The aim of this study was to investigate which signaling transduction pathways were modulated in CLA-induced apoptosis in human hepatoma SK-HEP-1 cells. The cells exposed to CLA were evaluated for PPAR,, PP2A, pro-apoptotic proteins Bak, Bad and caspases, and anti-apoptotic proteins Bcl-2 and Bcl-XL. Cells were also treated with okadaic acid, a PP2A inhibitor, or with Wy-14643, a specific PPAR, agonist. The CLA-induced apoptosis was concomitant to the increase of percentage of cells in the S phase, PPAR,, PP2A and pro-apoptotic proteins; simultaneously, antiapoptotic proteins decreased. Inhibition of PP2A prevented apoptosis, and PPAR, agonist showed similar effect as CLA. The increased PP2A could be responsible for the dephosphorylation of Bcl-2 and Bad, permitting apoptotic activity of Bax and Bad. The increase of caspase 8 and 9 suggested that both the intrinsic and extrinsic apoptotic pathways were induced. PP2A was probably increased by PPAR,, since putative PPRE sequences were found in genes encoding its subunits. In conclusion, CLA induces apoptosis in human hepatoma SK-HEP-1 cells, by increasing PPAR,, PP2A and pro-apoptotic proteins. © 2007 Wiley-Liss, Inc. [source] Cytosolic NADP phosphatases I and II from Arthrobacter sp. strain KM: Implication in regulation of NAD+/NADP+ balanceJOURNAL OF BASIC MICROBIOLOGY, Issue 3 2004Shigeyuki Kawai NADP phosphatase (NADPase) is an enzyme that converts NADP+ into NAD+ through dephosphorylation of NADP+, and is considered to be one of the possible candidates for regulation of the NAD+/NADP+ balance in vivo. In order to obtain an intrinsic NADPase, the NADP+ -degrading activity in a membrane-free cell extract of a Gram-positive bacterium, Arthrobacter sp. strain KM, was first assessed and demonstrated to be mainly achieved through the NADPase reaction, indicating NADPase is essential for degradation of NADP+ and therefore for regulation of the NAD+/NADP+ balance in cytosol. Then, the isolation of cytosolic NADPase was attempted using NADP+ as a substrate. Two NADPase isozymes, designated as NADPases I and II, were purified from the cell extract of the bacterium, and were indicated to be the sole cytosolic NADPases regulating the balance of NAD+/NADP+. NADPases I and II are homodimers of 32 and 30 kDa subunits, respectively, and most active at pH 7,8. The N-terminal amino acid sequences of the two enzymes are similar to each other. Among the biological substrates tested, both enzymes showed the highest activity toward NADP+ and NADPH. AMP, ADP, and pyridoxal 5,-phosphate were also dephosphorylated, but to lower extents. Comparison of the features of NADPases I and II with those of other acid phosphatases possessing NADPase activity suggested that NADPases I and II are novel enzymes participating in regulation of the NAD+/NADP+ balance in the cytosol. (© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Sustained BMP Signaling in Osteoblasts Stimulates Bone Formation by Promoting Angiogenesis and Osteoblast Differentiation,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2009Fengjie Zhang Abstract Angiogenesis and bone formation are tightly coupled during the formation of the skeleton. Bone morphogenetic protein (BMP) signaling is required for both bone development and angiogenesis. We recently identified endosome-associated FYVE-domain protein (endofin) as a Smad anchor for BMP receptor activation. Endofin contains a protein-phosphatase pp1c binding domain, which negatively modulates BMP signals through dephosphorylation of the BMP type I receptor. A single point mutation of endofin (F872A) disrupts interaction between the catalytic subunit pp1c and sensitizes BMP signaling in vitro. To study the functional impact of this mutation in vivo, we targeted expression of an endofin (F872A) transgene to osteoblasts. Mice expressing this mutant transgene had increased levels of phosphorylated Smad1 in osteoblasts and showed increased bone formation. Trabecular bone volume was significantly increased in the transgenic mice compared with the wildtype littermates with corresponding increases in trabecular bone thickness and number. Interestingly, the transgenic mice also had a pronounced increase in the density of the bone vasculature measured using contrast-enhanced ,CT imaging of Microfil-perfused bones. The vessel surface and volume were both increased in association with elevated levels of vascular endothelial growth factor (VEGF) in osteoblasts. Endothelial sprouting from the endofin (F872A) mutant embryonic metatarsals cultured ex vivo was increased compared with controls and was abolished by an addition of a VEGF neutralizing antibody. In conclusion, osteoblast targeted expression of a mutant endofin protein lacking the pp1c binding activity results in sustained signaling of the BMP type I receptor, which increases bone formation and skeletal angiogenesis. [source] |