共查询到20条相似文献,搜索用时 15 毫秒
1.
Eric V. Wong Andrew W. Schaefer †Gary Landreth Vance Lemmon 《Journal of neurochemistry》1996,66(2):779-786
Abstract: L1 is an axonal cell adhesion molecule found primarily on projection axons of both the CNS and PNS. It is a phosphorylated membrane-spanning glycoprotein that can be immunoprecipitated from rat brain membranes in association with protein kinase activities. Western blot analysis demonstrates that casein kinase II (CKII), a ubiquitous serine/threonine kinase enriched in brain, is present in these immunoprecipitates. CKII preparations partially purified from PC12 cells are able to phosphorylate recombinant L1 cytoplasmic domain (L1CD), which consists of residues 1,144–1,257. Using these as well as more highly purified kinase preparations, phosphorylation assays of small peptides derived from the L1CD were performed. CKII was able to phosphorylate a peptide encompassing amino acids (aa) 1,173–1,185, as well as a related peptide representing an alternatively spliced nonneuronal L1 isoform that lacks aa 1,177–1,180. Both peptides were phosphorylated with similar kinetic profiles. Serine to alanine substitutions in these peptides indicate that the CKII phosphorylation site is at Ser1,181 . This is consistent with experiments in which L1CD was phosphorylated by these kinase preparations, digested, and the radiolabeled fragments sequenced. Furthermore, when L1 immunoprecipitates were used to phosphorylate L1CD, one of the residues phosphorylated is the same residue phosphorylated by CKII. Finally, in vivo radiolabeling indicates that Ser1,181 is phosphorylated in newborn rat brain. These data show that CKII is associated with and able to phosphorylate L1. This phosphorylation may be important in regulating certain aspects of L1 function, such as adhesivity or signal transduction. 相似文献
2.
Jacques J. H. Hens Marina De Wit Frans Boomsma †‡ Marc Mercken A. Beate Oestreicher Willem Hendrik Gispen Pierre N. E. De Graan 《Journal of neurochemistry》1995,64(3):1127-1136
Abstract: B-50 (GAP-43) is a presynaptic protein kinase C (PKC) substrate implicated in the molecular mechanism of noradrenaline release. To evaluate the importance of the PKC phosphorylation site and calmodulin-binding domain of B-50 in the regulation of neurotransmitter release, we introduced two monoclonal antibodies to B-50 into streptolysin O-permeated synaptosomes isolated from rat cerebral cortex. NM2 antibodies directed to the N-terminal residues 39–43 of rat B-50 dose-dependently inhibited Ca2+ -induced radiolabeled and endogenous noradrenaline release from permeated synaptosomes. NM6 C-terminal-directed (residues 132–213) anti-B-50 antibodies were without effect in the same dose range. NM2 inhibited PKC-mediated B-50 phosphorylation at Ser41 in synaptosomal plasma membranes and permeated synaptosomes, inhibited 32 P-B-50 dephosphorylation by endogenous synaptosomal phosphatases, and inhibited the binding of calmodulin to synaptosomal B-50 in the absence of Ca2+ . Similar concentrations of NM6 did not affect B-50 phosphorylation or dephosphorylation or B-50/calmodulin binding. We conclude that the N-terminal residues 39–43 of the rat B-50 protein play an important role in the process of Ca2+ -induced noradrenaline release, presumably by serving as a local calmodulin store that is regulated in a Ca2+ - and phosphorylation-dependent fashion. 相似文献
3.
Abstract: The growth cone is responsible for axonal elongation and pathfinding by responding to various modulators for neurite growth, including neurotransmitters, although the sensor mechanisms are not fully understood. Among neurotransmitters, GABA is most likely to demonstrate activity in vivo because GABA and the GABAA receptor appear even in early stages of CNS development. We investigated the GABAA receptor-mediated signaling pathway in the growth cone using isolated growth cones (IGCs). Both the GABAA binding site and the benzodiazepine modulatory site were enriched in the growth cone membrane. In the intact IGC, GABA induced picrotoxin-sensitive Cl− flux (not influx but efflux) and increased the intracellular Ca2+ concentration in a picrotoxin- and verapamil-sensitive manner. Protein kinase C (PKC)-dependent phosphorylation of two proteins identified as GAP-43 and MARCKS protein was enhanced in the intact IGC stimulated by GABA, resulting in the release of MARCKS protein and GAP-43 from the membrane. Collectively, our results suggest the following scheme: activation of the functional GABAA receptor localized in the growth cone membrane → Cl− efflux induction through the GABAA -associated Cl− channel → Ca2+ influx through an L-type voltage-sensitive Ca2+ channel → Ca2+ -dependent phosphorylation of GAP-43 and MARCKS protein by PKC. 相似文献
4.
Tina K. Machu Jordan A. Firestone Michael D. Browning† 《Journal of neurochemistry》1993,61(1):375-377
Abstract: The γ2 subunit of the GABAA receptor (GABAA -R) is alternatively spliced. The long variant (γ2L ) contains eight additional amino acids that possess a consensus sequence site for protein phosphorylation. Previous studies have demonstrated that a peptide or fusion protein containing these eight amino acids is a substrate for protein kinase C (PKC), but not cyclic AMP-dependent protein kinase A (PKA)-stimulated phosphorylation. We have examined the ability of PKA, PKC, and Ca2+ /calmodulin-dependent protein kinase (CAM kinase II) to phosphorylate a synthetic peptide corresponding to residues 336–351 of the intracellular loop of the γ2L subunit and inclusive of the alternatively spliced phosphorylation consensus sequence site. PKC and CAM kinase II produced significant phosphorylation of this peptide, but PKA was ineffective. The K m values for PKC-and CAM kinase II-stimulated phosphorylation of this peptide were 102 and 35 μM , respectively. Maximal velocities of 678 and 278 nmol of phosphate/min/mg were achieved by PKC and CAM kinase II, respectively. The phosphorylation site in the eight-amino-acid insert of the γ2L subunit has been shown to be necessary for ethanol potentiation of the GABAA -R. Thus, our results suggest that PKC, CAM kinase II, or both may play a role in the effects of ethanol on GABAergic function. 相似文献
5.
Carlos-Alberto Gonçalves Amanda Hall Alistair T. R. Sim Stephen J. Bunn Philip D. Marley Tat B. Cheah Peter R. Dunkley 《Journal of neurochemistry》1997,69(6):2387-2396
Abstract: The protein kinases and protein phosphatases that act on tyrosine hydroxylase in vivo have not been established. Bovine adrenal chromaffin cells were permeabilized with digitonin and incubated with [γ-32 P]ATP, in the presence or absence of 10 µ M Ca2+ , 1 µ M cyclic AMP, 1 µ M phorbol dibutyrate, or various kinase or phosphatase inhibitors. Ca2+ increased the phosphorylation of Ser19 and Ser40 . Cyclic AMP, and phorbol dibutyrate in the presence of Ca2+ , increased the phosphorylation of only Ser40 . Ser31 and Ser8 were not phosphorylated. The Ca2+ -stimulated phosphorylation of Ser19 was incompletely reduced by inhibitors of calcium/calmodulin-stimulated protein kinase II (46% with KN93 and 68% with CaM-PKII 273–302), suggesting that another protein kinase(s) was contributing to the phosphorylation of this site. The Ca2+ -stimulated phosphorylation of Ser40 was reduced by specific inhibitors of protein kinase A (56% with H89 and 38% with PKAi 5–22 amide) and protein kinase C (70% with Ro 31-8220 and 54% with PKCi 19–31), suggesting that protein kinases A and C contributed to most of the phosphorylation of this site. Results with okadaic acid and microcystin suggested that Ser19 and Ser40 were dephosphorylated by PP2A. 相似文献
6.
Abstract: We have shown previously that a neurofilament (NF)-associated kinase (NFAK) extracted from chicken NF preparations phosphorylates selectively the middle molecular mass NF subunit (NF-M). Here we show that the major kinase activity in NFAK is indistinguishable from enzymes of the casein kinase I (CKI) family based on the following criteria: (1) inhibition of NFAK phosphorylation by the selective CKI inhibitor CKI-7, (2) the similarity in substrate specificity of NFAK and authentic CKI, (3) the correspondence of two-dimensional phosphopeptide maps of NF-M phosphorylated in vitro by NFAK with those generated by CKI under similar conditions, and (4) immunological cross-reactivity of NFAK with an antibody raised against CKI. We have also identified Ser502 , Ser528 , and Ser536 as phosphorylation sites by NFAK/CKI in vitro, each of which is also phosphorylated in vivo. All three serines are found in peptides with CKI phosphorylation consensus sequences, and Ser528 and Ser536 and flanking amino acids are highly conserved in higher vertebrate NF-M sequences. Neither Ser502 nor Ser536 has been identified previously as NF-M phosphorylation sites. 相似文献
7.
Souad Hilmi Michel Fournier Henri Valeins Jean Claude Gandar Jacques Bonnet 《Journal of neurochemistry》1995,64(2):902-907
Abstract: Myelin membrane prepared from mouse sciatic nerve possesses both kinase and substrates to incorporate [32 P]PO4 3− from [γ-32 P]ATP into protein constituents. Among these, P0 glycoprotein is the major phosphorylated species. To identify the phosphorylated sites, P0 protein was in vitro phosphorylated, purified, and cleaved by CNBr. Two 32 P-phosphopeptides were isolated by HPLC. The exact localization of the sequences around the phosphorylated sites was determined. The comparison with rat P0 sequence revealed, besides a Lys172 to Arg substitution, that in the first peptide, two serine residues (Ser176 and Ser181 ) were phosphorylated, Ser176 appearing to be modified subsequently to Ser181 . In the second peptide, Ser197 , Ser199 , and Ser204 were phosphorylated. All these serines are clustered in the C-terminal region of P0 protein. This in vitro study served as the basis for the identification of the in vivo phosphorylation sites of the C terminal region of P0 . We found that, in vivo, Ser181 and Ser176 are not phosphorylated, whereas Ser197 , Ser199 , Ser204 , Ser208 , and Ser214 are modified to various extents. Our results strongly suggest that the phosphorylation of these serine residues alters the secondary structure of this domain. Such a structural perturbation could play an important role in myelin compaction at the dense line level. 相似文献
8.
Stephen J. Bunn Alistair T. R. Sim Lynn M. Herd Leone M. Austin Peter R. Dunkley 《Journal of neurochemistry》1995,64(3):1370-1378
Abstract: Tyrosine hydroxylase (TOH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated by phosphorylation. Activation of histaminergic H1 receptors on cultured bovine adrenal chromaffin cells stimulated a rapid increase in TOH phosphorylation (within 5 s) that was sustained for at least 5 min. The initial increase in TOH phosphorylation (up to 1 min) was essentially unchanged by the removal of extracellular Ca2+ . In contrast, the H1 -mediated response was abolished by preloading the cells with BAPTA acetoxymethyl ester (50 µ M ) and significantly reduced by prior exposure to caffeine (10 m M for 10 min) to deplete intracellular Ca2+ . Trypticphosphopeptide analysis by HPLC revealed that the H1 response in the presence or absence of extracellular Ca2+ resulted in a major increase in the phosphorylation of Ser19 with smaller increases in that of Ser40 and Ser31 . In contrast, although a brief stimulation with nicotine (30 µ M for 60 s) also resulted in a major increase in Ser19 phosphorylation, this response was abolished in the absence of extracellular Ca2+ . These data indicate that the mobilization of intracellular Ca2+ plays a crucial role in supporting H1 -mediated TOH phosphorylation and may thus have a potentially important role in regulating catecholamine synthesis. 相似文献
9.
Steven R. Ennis Nobuyuki Kawai Xiao-dan Ren Galaleldin E. Abdelkarim Richard F. Keep 《Journal of neurochemistry》1998,71(6):2565-2573
Abstract: The aim of this study was to determine the effect of angiotensin II (AII) on tyrosine hydroxylase (TOH) activity and phosphorylation in bovine adrenal chromaffin cells (BACCs). We report here that stimulation of BACCs with AII (100 n M ) produced a significant increase in both TOH activity and phosphorylation over a period of 10 min. The increase in TOH activity was receptor-mediated. Tryptic phosphopeptide analysis by HPLC revealed that AII stimulated an increase in phosphorylation of three sites on TOH, Ser19 , Ser31 , and Ser40 , with the largest increase being observed for Ser31 phosphorylation. Pretreatment of the cells with the protein kinase C inhibitor Ro 31-8220 (10 µ M , 15 min) did not affect TOH activity or phosphorylation produced by AII. The inhibitor also did not affect the TOH activity or Ser40 phosphorylation produced by forskolin (10 µ M , 10 min). In contrast, Ro 31-8220 fully inhibited the TOH activation as well as Ser31 and Ser40 phosphorylation of TOH produced by phorbol 12, 13-dibutyrate (500 n M , 10 min). Removal of extracellular Ca2+ from the incubation medium inhibited the AII-induced TOH activity by 50% and significantly blocked Ser19 and Ser31 phosphorylation but did not affect Ser40 phosphorylation in response to AII. These results indicate that AII activates a complex and perhaps novel signaling pathway leading to the phosphorylation and activation of TOH. The TOH activation by AII appears to be partially independent of Ser40 phosphorylation, suggesting a potentially important role for Ser31 phosphorylation. 相似文献
10.
Cross-Reaction of Anti-Rat B-50: Characterization and Isolation of a "B-50 Phosphoprotein" from Bovine Brain 总被引:7,自引:6,他引:1
Anna Beate Oestreicher Marcel van Duin Henk Zwiers Willem Hendrik Gispen 《Journal of neurochemistry》1984,43(4):935-943
Abstract: Antibodies to the phosphoprotein B-50 of rat brain were used to trace cross-reacting brain proteins of vertebrates. With the SDS-gel-immunoperoxidase method, a cross-reacting protein (CP) of apparent Mr 53,000 was demonstrated in the homogenate and the synaptic plasma membrane fraction of bovine brain. Sequence 1–24 of adrenocorticotropin (ACTH1-24 ) (10−5 M and 10−4 M ) inhibited endogenous phosphorylation of CP in synaptic plasma membranes. The protein was partially characterized and purified to homogeneity from bovine brain by procedures previously described for rat B-50. CP was enriched in ammonium sulfate precipitated protein (ASP) fractions and phosphorylated by an endogenous protein kinase. Two-dimensional gel analysis of bovine and rat ASP showed that the cross-reacting protein had an isoelectric point less acidic than B-50. Limited proteolysis by Staphylococcus aureus protease yielded a "peptide map" analogous to B-50. Two major fragments of Mr 30,000 and 17,000 were produced. In addition, CP exhibited other similarities to rat B-50: phosphorylation by rat brain protein kinase C, microheterogeneity observed after isoelectric focusing, and possibly degradation by endogenous proteolysis. Cross-reaction of proteins in brain homogenates of other mammalian species and of chicken was demonstrated: the Mr of the proteins ranged from 47,000 to 53,000. We conclude that (1) the cross-reacting bovine protein is a "B-50 protein," and (2) the M r of the "B-50 protein" varies from species to species. 相似文献
11.
Benoit I. Giasson James A. Cromlish Eric S. Athlan Walter E. Mushynski 《Journal of neurochemistry》1996,66(3):1207-1213
Abstract: The activation of cyclic AMP-dependent protein kinase (PKA) in rat dorsal root ganglion (DRG) cultures increased phosphorylation of the low-molecular-mass neurofilament subunit (NFL) at a site previously identified as Ser55 but had no effect on neurofilament integrity. When PKA was activated in DRG cultures treated with 20–250 n M okadaic acid, neurofilament fragmentation was enhanced, and there was a corresponding increase in phosphorylation of NFL at a novel site. This site was also phosphorylated by PKA in vitro and was determined to be Ser2 by mass spectrometric analysis of the purified chymotryptic phosphopeptide. The PKA sites in NFL were dephosphorylated by the purified catalytic subunit of protein phosphatase-2A but not that of protein phosphatase-1, and phosphoserine-2 was a better substrate than phosphoserine-55. The phosphorylation and dephosphorylation of Ser2 and Ser55 in NFL may therefore be involved in the modulation of neurofilament dynamics through the antagonistic effects of PKA and protein phosphatase-2A. 相似文献
12.
Abstract: In this report, the phosphorylation sites of neurofilament protein of medium molecular mass (NF-M) by protein kinase FA /glycogen synthase kinase 3α (kinase FA /GSK-3α) were determined by two-dimensional electrophoresis/TLC, phosphoamino acid analysis, HPLC, Edman degradation, and peptide sequencing. Kinase FA /GSK-3α phosphorylates NF-M predominantly on serine, residue. Three major tryptic phosphopeptide peaks were resolved by C18 reverse-phase HPLC. Edman degradation and peptide sequence analysis revealed that AKS(p)PVSK is the phosphorylation site sequence for the first major peak. When mapping with the amino acid sequence of neurofilament, we finally demonstrate Ser603 -Pro, one of the in vivo sites in NF-M, as the major site phosphorylated by kinase FA /GSK-3α. By using the same approach, we also identified the in vivo sites of Ser502 -Pro, Ser506 -Pro, and Ser666 -Pro as the other three major sites in NF-M phosphorylated by kinase FA /GSK-3α. Taken together, the results provide initial evidence that kinase FA /GSK-3α may represent a physiologically relevant protein kinase involved in the in vivo phosphorylation of NF-M. Because Ser502 , Ser506 , Ser603 , and Ser666 are all flanked by a carboxyl-terminal proline residue, the results provide further evidence that FA /GSK-3α may represent a proline-directed protein kinase involved in the structure-function regulation of the neuronal cytoskeletal system. 相似文献
13.
F. M. J. Heemskerk L. H. Schrama W. E. J. M. Ghijsen P. N. E. De Graan F. H. Lopes da Silva W. H. Gispen 《Journal of neurochemistry》1991,56(6):1827-1835
Abstract: Recently we have shown that 4-aminopyridine (4-AP), a drug known to enhance transmitter release, stimulates the phosphorylation of the protein kinase C substrate B-50 (GAP-43) in rat brain synaptosomes and that this effect is dependent on the presence of extracellular Ca2+ . Hence, we were interested in the relationship between changes induced by 4-AP in the intracellular free Ca2+ concentration ([Ca2+ ]i ) and B-50 phosphorylation in synaptosomes. 4-AP (100 μ M ) elevates the [Ca2+ ]i (as determined with fura-2) to approximately the same extent as depolarization with 30 m M K+ (from an initial resting level of 240 n M to ∼480 n M after treatment). However, the underlying mechanisms appear to be different: In the presence of 4-AP, depolarization with K+ still evoked an increase in [Ca2+ ]i , which was additive to the elevation caused by 4-AP. Several Ca2+ channel antagonists (CdCl2 , LaCl3 , and diphenylhydantoin) inhibited the increase in B-50 phosphorylation by 4-AP. It is interesting that the increase in [Ca2+ ]i and the increase in B-50 phosphorylation by 4-AP were attenuated by tetrodotoxin, a finding pointing to a possible involvement of Na+ channels in this action. These results suggest that 4-AP (indirectly) stimulates both Ca2+ influx and B-50 phosphorylation through voltage-dependent channels by a mechanism dependent on Na+ channel activity. 相似文献
14.
Five strains of filamentous fungi belonging to the genera Mortierella and Cunninghamella were examined for the content of dihomo-γ-linolenic, arachidonic, eicosapentaenoic acids and prostaglandins (type E2 and F 2α ). Prostaglandins were detected using an ELISA method in mycelia of all tested strains (range 50–4800 ng g−1 of PGE2 and 6–30 ng g−1 of PG F 2α ). Several micro-organisms also produced prostaglandins in the culture medium (2·2–137·6 μg l−1 for PGE2 and 0·4–7·8 μg l−1 for PG F 2α ). 相似文献
15.
†Miroslav Kubes Jocelyne Cordier Jacques Glowinski Jean-Antoine Girault Hervé Chneiweiss 《Journal of neurochemistry》1998,71(3):1307-1314
Abstract: PEA-15 (phosphoprotein enriched in astrocytes, Mr = 15,000) is an acidic serine-phosphorylated protein highly expressed in the CNS, where it can play a protective role against cytokine-induced apoptosis. PEA-15 is a major substrate for protein kinase C. Endothelins, which are known to exert pleiotropic effects on astrocytes, were used to analyze further the processes involved in PEA-15 phosphorylation. Endothelin-1 or endothelin-3 (0.1 µ M ) induced a robust phosphorylation of PEA-15 that was abolished by the removal of extracellular calcium, but only diminished by inhibitors of protein kinase C. Microsequencing of phosphopeptides generated by digestion of PEA-15 following endothelin-1 treatment identified two phosphorylated residues: Ser104 , previously recognized as the protein kinase C site, and a novel phosphoserine, Ser116 , located in a consensus motif for either protein kinase casein kinase II or calcium/calmodulin-dependent protein kinase II (CaMKII). Partly purified PEA-15 was a substrate in vitro for CaMKII, but not for casein kinase II. Two-dimensional phosphopeptide mapping demonstrated that the site phosphorylated in vitro by CaMKII was also phosphorylated in intact astrocytes in response to endothelin. CaMKII phosphorylated selectively Ser116 and had no effect on Ser104 , but in vitro phosphorylation by CaMKII appeared to facilitate further phosphorylation by protein kinase C. Treatment of intact astrocytes with okadaic acid enhanced the phosphorylation of the CaMKII site. These results demonstrate that PEA-15 is phosphorylated in astrocytes by CaMKII (or a related kinase) and by protein kinase C in response to endothelin. 相似文献
16.
Abstract: Recent evidence suggests that β-amyloid peptide (β-AP) may induce tau protein phosphorylation, resulting in loss of microtubule binding capacity and formation of paired helical filaments. The mechanism by which β-AP increases tau phosphorylation, however, is unclear. Using a hybrid septal cell line, SN56, we demonstrate that aggregated β-AP1–40 treatment caused cell injury. Accompanying the cell injury, the levels of phosphorylated tau as well as total tau were enhanced as detected immunochemically by AT8, PHF-1, Tau-1, and Tau-5 antibodies. Alkaline phosphatase treatment abolished AT8 and PHF-1 immunoreactivity, confirming that the tau phosphorylation sites were at least at Ser199/202 and Ser396 . In association with the increase in tau phosphorylation, the immunoreactivity of cell-associated and secreted β-amyloid precursor protein (β-APP) was markedly elevated. Application of antisense oligonucleotide to β-APP reduced expression of β-APP and immunoreactivity of phosphorylated tau. Control peptide β-AP1–28 did not produce significant effects on tau phosphorylation, although it slightly increased cell-associated β-APP. These results suggest that βAP1–40 -induced tau phosphorylation may be associated with increased β-APP expression in degenerated neurons. 相似文献
17.
J. W. Haycock J. Y. Lew A. Garcia-Espana K. Y. Lee †K. Harada E. Meller M. Goldstein 《Journal of neurochemistry》1998,71(4):1670-1675
Abstract: The effects of depolarization by elevated potassium concentrations were studied in PC12 cells and in stably transfected AtT-20 cells expressing wild-type or [Leu19 ]-recombinant tyrosine hydroxylase (rTH). Changes in the phosphorylation states of Ser19 and Ser40 in tyrosine hydroxylase (TH) were determined immunochemically using antibodies specific for the phosphorylated state of each site and compared with changes in TH activity in PC12 cell lysates and with changes in l -DOPA biosynthesis rates in intact AtT-20 cells. Treatment of either PC12 cells or AtT-20 cells expressing wild-type rTH with elevated potassium produced a transient increase in the phosphorylation state of Ser19 (up to 0.7 mol of phosphate/mol of subunit) in concert with a more gradual and sustained increase in Ser40 phosphorylation. Elevated potassium treatment also increased TH activity in PC12 cell lysates, but these increases paralleled the temporal course of Ser40 , as opposed to Ser19 , phosphorylation. Similarly, increases in DOPA accumulation produced by elevated potassium in AtT-20 cells expressing wild-type rTH paralleled the increases in the phosphorylation state of Ser40 but not Ser19 . Moreover, elevated potassium produced comparable increases in DOPA accumulation in AtT-20 cells expressing rTH in which Ser19 phosphorylation had been eliminated (by substitution of Leu for Ser19 ). Thus, depolarization-induced increases in the stoichiometry of Ser19 phosphorylation do not appear to influence directly the activity of TH in situ. 相似文献
18.
Abstract: Phosphorylation of the nervous system-specific protein GAP-43 in growth cones in vivo increases as the growth cones near their targets, at a time when the gangliosides GM1 and GD1a are being accumulated in the growth cone membrane, thus raising the possibility that the gangliosides could modulate GAP-43 behavior. We used a subcellular fraction of intact isolated growth cones to show that both GM1 and GD1a affected the calcium- dependent posttranslational regulation of GAP-43 in several similar ways. Both gangliosides induced rapid incorporation of phosphate into GAP-43; however, the induction was undetectable with our antibody 2G12 that is specific for kinase C-phosphorylated GAP-43. Furthermore, neither ganglioside stimulated kinase C activity in isolated growth cones, suggesting that the rapid Phosphorylation may not be on Ser41 , the kinase C site. However, both gangliosides did induce a slower accumulation of GAP-43 phosphorylated on Ser41 , apparently by inhibiting a phosphatase. Finally, calcium-dependent proteolysis of GAP-43 was also stimulated by both GM1 and GD1a. In contrast, GD1a, but not GM1, caused the redistribution of GAP-43 into the isolated growth cone cytoskeleton. The results demonstrate that both gangliosides can modulate the calcium-dependent regulation of GAP-43. 相似文献
19.
Shiaw-Der Yang Jen-Shin Song Jau-Song Yu Shine-Gwo Shiah 《Journal of neurochemistry》1993,61(5):1742-1747
Abstract— Previously, we identified protein kinase FA /gly-cogen synthase kinase-3 (GSK-3) as a microtubule-associated protein kinase that can incorporate 4 mol of phosphates into 1 mol of protein and cause its electrophoretic mobility shift in sodium dodecyl sulfate gels, a unique property characteristic of paired helical filament-associated pathological (PHF-) in Alzheimer's disease brains. In this report, we identified TPPKS(p)PSAAK and SPVVSGDTS(p)PR as two phosphorylation site sequences phosphorylated by kinase FA /GSK-3 in using peptide sequence analysis and sequential manual Edman degradation for radiosequencing. When mapping with human brain sequence, we further identified Ser235 -Pro and Ser404 -Pro as the two major phosphorylation sites according to the numbering of the longest isoform. Ser235 and Ser404 have been reported as two of the major abnormal phosphorylation sites in PHF-. Taken together, the results provide initial evidence that protein kinase FA /GSK-3 may represent one of the Ser-Pro motif-directed kinases involved in the abnormal phosphorylation of pathological PHF- in Alzheimer's disease brain. 相似文献
20.
Abstract: De novo l -DOPA biosynthesis was studied in stably transfected AtT-20 cells expressing wild-type- or [Leu40 ]-recombinant tyrosine hydroxylase (rTH). Basal rates of DOPA accumulation were much higher by cells expressing rTH in which Leu was substituted for Ser40 (S40L-rTH) than by those expressing wild-type rTH (WT-rTH). Treatment of WT-rTH cells with forskolin produced an increase in DOPA accumulation and a concomitant increase in WT-rTH phospho-Ser40 content, whereas DOPA production by cells expressing S40L-rTH was entirely unaffected by forskolin. After forskolin treatment of 32 Pi -prelabeled cells, WT-rTH was phosphorylated at Ser8 , Ser19 , Ser31 , and Ser40 , whereas 32 P incorporation into S40L-rTH was restricted to Ser8 , Ser19 , and Ser31 . Relatively prolonged treatment of AtT-20 cells expressing WT-rTH with either a depolarizing agent (elevated potassium) or a phosphatase inhibitor (okadaic acid) increased DOPA production and increased the phosphorylation state of Ser40 ; but, unlike forskolin, these treatments also increased DOPA production by cells expressing S40L-rTH. Thus, the present studies demonstrate that Ser40 phosphorylation mediates forskolin-induced increases in DOPA biosynthesis directly but that mechanisms other than Ser40 phosphorylation can mediate the increases in DOPA biosynthesis produced either by depolarization or by protein phosphatase inhibition. 相似文献