scholarly journals Resolution of regulated secretion into sequential MgATP-dependent and calcium-dependent stages mediated by distinct cytosolic proteins.

1992 ◽  
Vol 119 (1) ◽  
pp. 139-151 ◽  
Author(s):  
J C Hay ◽  
T F Martin
Keyword(s):  
Secretory Pathway ◽  
Kinase Inhibitors ◽  
Specific Activity ◽  
Gel Filtration ◽  
Protein Kinase Inhibitors ◽  
Active Components ◽  
Cytosolic Proteins ◽  
Regulated Secretion ◽  
Calcium Dependent ◽  
Regulated Secretory Pathway

The biochemical events and components responsible for ATP-dependent Ca(2+)-activated secretion remain to be identified. To simplify the molecular dissection of regulated secretion, we have resolved norepinephrine (NE) secretion from semi-intact PC12 cells into two kinetically distinct stages, each of which was studied separately to discern its molecular requirements. The first stage consisted of MgATP-dependent priming of the secretory apparatus in the absence of Ca2+. MgATP-dependent priming was readily reversible and inhibited by a broad range of protein kinase inhibitors. The second stage consisted of Ca(2+)-triggered exocytosis which, in contrast to priming, occurred in the absence of MgATP. Both priming and triggering were found to be dependent upon or stimulated by cytosolic proteins. The priming and triggering activities of cytosol were functionally distinct as indicated by differing thermolability. Furthermore, active components in cytosol resolved by gel filtration were found to support either priming or triggering, but not both. For both priming and triggering reactions, several peaks of activity were detected; one of each type of factor was partially purified from rat brain cytosol, and found to be enriched for stage-specific activity. Two partially purified factors exhibiting stage-specific activity, a approximately 20-kD priming factor and approximately 300-kD triggering factor, were able to support regulated secretion as effectively as crude cytosol when used sequentially in the partial reactions. Further characterization of stage-specific cytosolic factors should clarify the nature of MgATP- and Ca(2+)-dependent events in the regulated secretory pathway.

Multiple Forms of Phosphoglyceromutase from Chicken Breast Muscle

1972 ◽  
Vol 50 (10) ◽  
pp. 1132-1142 ◽  
Author(s):  
Eric James ◽  
R. O. Hurst ◽  
T. G. Flynn
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Diffusion Constant ◽  
Sedimentation Coefficient ◽  
Breast Muscle ◽  
Heat Inactivation ◽  
Chicken Breast ◽  
Multiple Forms ◽  
Active Components ◽  
Net Charges

Phosphoglyceromutase (2,3-diphospho-D-glycerate: 2-phospho-D-glycerate phosphotransferase, EC 2.7.5.3) has been purified from both frozen and fresh chicken breast muscle. During purification it was found that substrate, 3-phospho-D-glycerate stabilized the enzyme against heat inactivation to almost the same extent as did the cofactor 2,3-diphospho-D-glycerate.Phosphoglyceromutase prepared from frozen chicken breast muscle separated into three peaks of activity (I, II, and III) following chromatography on DEAE-Sephadex in 0.05 μ phosphate buffer, pH 8.0, using a 0.0–0.4 M NaCl gradient. Each peak of activity was shown by polyacrylamide disc gel electrophoresis at pH 9.3 to contain two enzymically active components (isoenzymes Ia Ib, IIa IIb, and IIIa IIIb). Isoenzymes in the same peak had the same specific activity. Phosphoglyceromutase prepared from fresh chicken breast muscle yielded only one peak of activity following chromatography on DEAE-Sephadex. This peak contained two enzymically active components corresponding to isoenzymes Ia and Ib. Additional peaks of activity were not produced when phosphoglyceromutase from fresh muscle was subjected to freezing and thawing.Isoenzyme Ia and mixtures of Ia and Ib, IIa and IIb, and IIIa and IIIb were homogeneous in the ultra-centrifuge sedimenting as single peaks. The sedimentation coefficient obtained for isoenzyme Ia and for Ia and Ib combined was 4.15 S, the diffusion constant 6.62 × 10−7 cm2/s, and the molecular weight calculated from both gel filtration and sedimentation data was of the order of 59 000. These results were confirmed by charge isomer studies which also showed that the isoenzymes of phosphoglyceromutase from frozen chicken breast muscle were proteins of the same size but different net charges.

scholarly journals Passive Sorting in Maturing Granules of AtT-20 Cells: The Entry and Exit of Salivary Amylase and Proline-rich Protein

1997 ◽  
Vol 138 (1) ◽  
pp. 45-54 ◽  
Author(s):  
Anna M. Castle ◽  
Amy Y. Huang ◽  
J. David Castle
Keyword(s):  
Secretory Pathway ◽  
Critical Role ◽  
Endogenous Hormones ◽  
Salivary Amylase ◽  
Regulated Secretion ◽  
J 13 ◽  
Regulated Secretory Pathway ◽  
Granule Maturation ◽  
Proline Rich Protein

Previous studies have suggested that salivary amylase and proline-rich protein are sorted differently when expressed in AtT-20 cells (Castle, A.M., L.E. Stahl, and J.D. Castle. 1992. J. Biol. Chem. 267:13093– 13100; Colomer, V., K. Lal, T.C. Hoops, and M.J. Rindler. 1994.EMBO (Eur. Mol. Biol. Organ.) J. 13:3711– 3719). We now show that both exocrine proteins behave similarly and enter the regulated secretory pathway as judged by immunolocalization and secretagogue- dependent stimulation of secretion. Analysis of stimulated secretion of newly synthesized proline-rich protein, amylase, and endogenous hormones indicates that the exogenous proteins enter the granule pool with about the same efficiency as the endogenous hormones. However, in contrast to the endogenous hormones, proline-rich protein and amylase are progressively removed from the granule pool during the process of granule maturation such that only small portions remain in mature granules where they colocalize with the stored hormones. The exogenous proteins that are not stored are recovered from the incubation medium and are presumed to have undergone constitutive-like secretion. These results point to a level of sorting for regulated secretion after entry of proteins into forming granules and indicate that retention is essential for efficient storage. Consequently, the critical role of putative sorting receptors for regulated secretion may be in retention rather than in granule entry.

PKA inhibitor, H-89, affects the intracellular transit of regulated secretory proteins in rat lacrimal glands

1998 ◽  
Vol 274 (1) ◽  
pp. C262-C271 ◽  
Author(s):  
P. Robin ◽  
B. Rossignol ◽  
M. N. Raymond
Keyword(s):  
Protein Kinase ◽  
Secretory Pathway ◽  
Kinase Inhibitors ◽  
Secretory Granules ◽  
Protein Kinase Inhibitors ◽  
Secretory Proteins ◽  
Lacrimal Glands ◽  
Calphostin C ◽  
A 23187 ◽  
Golgi Network

We tested the effect of H-89, a protein kinase A (PKA) inhibitor, on the intracellular transit of the regulated secretory proteins in rat lacrimal glands. We show that H-89, by itself, induces the secretion of newly synthesized proteins trafficking in its presence but not of proteins already stored in the mature secretory granules. This secretion does not depend on the presence of extracellular Ca2+. The proteins released are identical to those secreted after cholinergic stimulation or under the action of the ionophore A-23187, but the secretion level is ∼40% lower. The effect of H-89 seems to be due to PKA inhibition because other protein kinase inhibitors (calphostin C, chelerythrine, H-85) do not induce secretion. We further show that H-89 does not modify the rate of glycoprotein galactosylation but induces the secretion of newly galactosylated glycoproteins. Finally, we used a “20°C block” procedure to show that H-89 affects a trans-Golgi network (TGN) or post-TGN step of the secretory pathway. Our results demonstrate that, in lacrimal cells, H-89 affects the intracellular trafficking of secretory proteins, suggesting a role for PKA in this process.

scholarly journals Partial purification and characterization of a wortmannin-sensitive and insulin-stimulated protein kinase that activates heart 6-phosphofructo-2-kinase

2000 ◽  
Vol 347 (1) ◽  
pp. 305-312 ◽  
Author(s):  
Johan DEPREZ ◽  
Luc BERTRAND ◽  
Dario R. ALESSI ◽  
Ulrike KRAUSE ◽  
Louis HUE ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Insulin Signalling ◽  
Gel Filtration ◽  
Protein Kinase Inhibitors ◽  
Partial Purification ◽  
Dependent Manner ◽  
Rat Hearts ◽  
Insulin Signalling Pathway ◽  
Perfused Rat Hearts

A wortmannin-sensitive and insulin-stimulated protein kinase (WISK), which phosphorylates and activates cardiac 6-phosphofructo-2-kinase (PFK-2), was partially purified from perfused rat hearts. Immunoblotting showed that WISK was devoid of protein kinase B (PKB), serum- and glucocorticoid-regulated protein kinase and protein kinase Cζ (PKCζ). Comparison of the inhibition of WISK, PKCα and PKCζ by different protein kinase inhibitors suggested that WISK was not a member of the PKC family. In addition, WISK contained no detectable phosphoinositide-dependent protein kinase-1 (PDK1) activity. WISK phosphorylated recombinant heart PFK-2 in a time-dependent manner to the extent of 0.4 mol of phosphate incorporated/mol of enzyme subunit, and increased the Vmax of PFK-2 twofold, without affecting the Km for fructose 6-phosphate. WISK phosphorylated Ser-466 to a greater extent than Ser-483 in recombinant heart PFK-2, and both sites were demonstrated to be phosphorylated to the same extent by PKB. Gel filtration and in-gel kinase analysis indicated that WISK was a monomer with a Mr of 56500. Treatment of WISK with protein phosphatase 2A (PP2A) catalytic subunits reversed the effect of insulin, suggesting the involvement of an upstream activating kinase. Indeed, PDK1 was able to partially reactivate the PP2A-treated WISK and this reactivation was not enhanced by PtdIns(3,4,5)P3-containing vesicles. Moreover, a single 57000-Mr band was labelled on incubation of the dephosphorylated WISK preparation with PDK1 and [γ-32P]ATP. These findings provide evidence for the existence of a new protein kinase in the insulin signalling pathway, probably downstream of PDK1.

scholarly journals Minireview: How Peptide Hormone Vesicles Are Transported to the Secretion Site for Exocytosis

2008 ◽  
Vol 22 (12) ◽  
pp. 2583-2595 ◽  
Author(s):  
Joshua J. Park ◽  
Y. Peng Loh
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Hormone Secretion ◽  
Release Site ◽  
Peptide Hormone ◽  
Adaptor Proteins ◽  
Transport Systems ◽  
Cytosolic Proteins ◽  
Readily Releasable Pool ◽  
Regulated Secretory Pathway

Abstract Post-Golgi transport of peptide hormone-containing vesicles from the site of genesis at the trans-Golgi network to the release site at the plasma membrane is essential for activity-dependent hormone secretion to mediate various endocrinological functions. It is known that these vesicles are transported on microtubules to the proximity of the release site, and they are then loaded onto an actin/myosin system for distal transport through the actin cortex to just below the plasma membrane. The vesicles are then tethered to the plasma membrane, and a subpopulation of them are docked and primed to become the readily releasable pool. Cytoplasmic tails of vesicular transmembrane proteins, as well as many cytosolic proteins including adaptor proteins, motor proteins, and guanosine triphosphatases, are involved in vesicle budding, the anchoring of the vesicles, and the facilitation of movement along the transport systems. In addition, a set of cytosolic proteins is also necessary for tethering/docking of the vesicles to the plasma membrane. Many of these proteins have been identified from different types of (neuro)endocrine cells. Here, we summarize the proteins known to be involved in the mechanisms of sorting various cargo proteins into regulated secretory pathway hormone-containing vesicles, movement of these vesicles along microtubules and actin filaments, and their eventual tethering/docking to the plasma membrane for hormone secretion.

scholarly journals Differential expression of the regulated catecholamine secretory pathway in different hereditary forms of pheochromocytoma

2008 ◽  
Vol 295 (5) ◽  
pp. E1223-E1233 ◽  
Author(s):  
Graeme Eisenhofer ◽  
Thanh-Truc Huynh ◽  
Abdel Elkahloun ◽  
John C. Morris ◽  
Gennady Bratslavsky ◽  
...  
Keyword(s):  
Differential Expression ◽  
Secretory Pathway ◽  
Tumor Tissue ◽  
Signs And Symptoms ◽  
Catecholamine Release ◽  
Catecholamine Secretion ◽  
Von Hippel Lindau ◽  
Calcium Dependent ◽  
Men 2 ◽  
Regulated Secretory Pathway

Pheochromocytomas in patients with von Hippel-Lindau (VHL) syndrome and multiple endocrine neoplasia type 2 (MEN 2) differ in the types and amounts of catecholamines produced and the resulting signs and symptoms. We hypothesized the presence of different processes of catecholamine release reflecting differential expression of components of the regulated secretory pathway among the two types of hereditary tumors. Differences in catecholamine secretion from tumors in patients with VHL syndrome ( n = 47) and MEN 2 ( n = 32) were examined using measurements of catecholamines in tumor tissue, urine, and plasma, the last of which was under baseline conditions in all subjects and in a subgroup of patients who received intravenous glucagon to provoke catecholamine release. Microarray and proteomics analyses, quantitative PCR, and Western blotting were used to assess expression of tumor tissue secretory pathway components. The rate constant for baseline catecholamine secretion was 20-fold higher in VHL than in MEN 2 tumors (0.359 ± 0.094 vs. 0.018 ± 0.009 day−1), but catecholamine release was responsive only to glucagon in MEN 2 tumors. Compared with tumors from MEN 2 patients, those from VHL patients were characterized by reduced expression of numerous components of the regulated secretory pathway (e.g., SNAP25, syntaxin, rabphilin 3A, annexin A7, calcium-dependent secretion activator). The mutation-dependent differences in expression of secretory pathway components indicate a more mature regulated secretory pathway in MEN 2 than VHL tumors. These data provide a unique mechanistic link to explain how variations in the molecular machinery governing exocytosis may contribute to clinical differences in the secretion of neurotransmitters or hormones and the subsequent presentation of a disease.

Perturbation of regulated secretion in the pancreatic acinar cell line, AR42J

1992 ◽  
Vol 262 (2) ◽  
pp. G257-G266
Author(s):  
E. Sachs ◽  
J. D. Jamieson
Keyword(s):  
Secretory Pathway ◽  
Secretory Granules ◽  
Pancreatic Acinar Cell ◽  
Secretory Proteins ◽  
Regulated Secretion ◽  
Golgi Transport ◽  
Test Conditions ◽  
Intracellular Compartments ◽  
Regulated Secretory Pathway ◽  
Acidic Compartments

The regulated secretory pathway comprises accelerated discharge of proteins in response to hormonal stimuli, their presence in secretory granules (SG), and a long intracellular residence time. Dexamethasone induction of AR42J results in an increase in granule content and responsiveness to cholecystokinin (CCK). We studied the effects of conditions implicated in sorting of secretory proteins into the regulated pathway using [35S]methionine pulse-chase protocols that examine transport of secretory proteins from the rough endoplasmic reticulum (RER)----SG and specifically from the Golgi complex (GC)----SG. The latter uses a chase at 20 degrees C to allow accumulation of labeled proteins in the trans-Golgi, followed by a shift to 37 degrees C that initiates their transport to SG under test conditions. Quantitation of CCK-8-stimulated discharge of prestored amylase and of newly synthesized labeled proteins that have entered SG during the chase enables us to examine the effect of perturbants over selected parts of the pathway. The effects of acidic intracellular compartments, the cytoskeleton, protein synthesis, ATP, and temperature on pre- and post-Golgi entry of proteins into the regulated pathway were studied. NH4Cl, monensin, Na azide, incubation at 20 degrees C, and pertussis toxin retarded RER----SG transport without affecting amylase discharge. Only incubation with 20 mM NH4Cl or 1 microM monensin inhibited transfer of newly synthesized proteins from the late GC----SG. RER----Golgi or intra-Golgi transport thus appears to require ATP and possibly guanosine 5'-triphosphate (GTP)-binding proteins. Acidic compartments appear to be essential for sorting of secretory proteins from the GC----SG.

scholarly journals Neurotrophins induce release of neurotrophins by the regulated secretory pathway

1998 ◽  
Vol 95 (16) ◽  
pp. 9614-9619 ◽  
Author(s):  
Alex Krüttgen ◽  
J. Carsten Möller ◽  
John V. Heymach ◽  
Eric M. Shooter
Keyword(s):  
Neurotrophic Factor ◽  
Secretory Pathway ◽  
Brain Derived Neurotrophic Factor ◽  
Tyrosine Kinase Receptor ◽  
Simultaneous Application ◽  
Regulated Secretion ◽  
Neurotrophin 3 ◽  
Regulated Secretory Pathway ◽  
Activity Dependent ◽  
Dependent Processes

Recent studies have established that neurotrophin synthesis and secretion are regulated by activity and that these factors are involved in activity-dependent processes in the nervous system. Neurotrophins also are known to induce increases in intracellular calcium, a trigger for regulated secretion. This finding raises the possibility that neurotrophins themselves may stimulate regulated secretion of neurotrophins. To address this question, we studied the release of neurotrophins from transfected PC12 cells, a widely used model for neuronal secretion and neurotrophin signal transduction. We found that neurotrophins induced the regulated secretion of brain-derived neurotrophic factor, neurotrophin-3 (NT-3), and neurotrophin-4/5. The effect of brain-derived neurotrophic factor on release of NT-3 could be abolished by REX, a p75 blocking antibody, but not by K252a, an inhibitor of neurotrophin tyrosine kinase receptor (Trk) signaling. The nerve growth factor effect on release of NT-3 could be blocked only by simultaneous application of REX and K252a, suggesting that they are mediated by TrkA as well as p75. Our data show that neurotrophins are able to induce the regulated secretion of neurotrophins and suggest a signal-transducing role for both TrkA and p75 in this process. The neurotrophin-induced release of neurotrophins may be relevant for activity-dependent processes such as synaptic plasticity and memory formation.

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