PC12 Cell Extracts Research Articles

Anti-Myelin Proteolipid Protein Peptide Monoclonal Antibodies Recognize Cell Surface Proteins on Developing Neurons and Inhibit Their Differentiation

Using a panel of monoclonal antibodies (mAbs) to myelin proteolipid protein (PLP) peptides, we found that in addition to CNS myelin, mAbs to external face but not cytoplasmic face epitopes immunostained neurons in immature human CNS tissues and in adult hippocampal dentate gyrus and olfactory bulbs, that is neural stem cell niches (NSCN). To explore the pathobiological significance of these observations, we assessed the mAb effects on neurodifferentiation in vitro. The mAbs to PLP 50–69 (IgG1κ and IgG2aκ), and 178–191 and 200–219 (both IgG1κ) immunostained live cell surfaces and inhibited neurite outgrowth of E18 rat hippocampal precursor cells and of PC12 cells, which do not express PLP. Proteins immunoprecipitated from PC12 cell extracts and captured by mAb-coated magnetic beads were identified by GeLC-MS/MS. Each neurite outgrowth-inhibiting mAb captured a distinct set of neurodifferentiation molecules including sequence-similar M6 proteins and other unrelated membrane and extracellular matrix proteins, for example integrins, Eph receptors, NCAM-1, and protocadherins. These molecules are expressed in adult human NSCN and are implicated in the pathogenesis of many chronic CNS disease processes. Thus, diverse anti-PLP epitope autoantibodies may inhibit neuronal precursor cell differentiation via multispecific recognition of cell surface molecules thereby potentially impeding endogenous neuroregeneration in NSCN and in vivo differentiation of exogenous neural stem cells.

Multispecific recognition of developmental neuronal antigens and neurite outgrowth inhibition by an anti-myelin proteolipid protein (PLP) peptide monoclonal antibody (mAb)

Abstract CNS injury results in the generation of antibodies to self-antigens, including epitopes of PLP, the most abundant CNS myelin protein. We previously found that despite similar recognition of CNS vertebrate myelin, anti-PLP50–69 mAbs F4.4C2 (IgG1κ) and F3.6E9 (IgG2aκ) differ in their recognition of neurons (Greenfield et al, 2006). To identify the neuronal mAb targets and determine the potential pathobiological significance of this finding, we used rat hippocampal precursor cells (RHPC) and PC12 cells, a nerve growth factor (NGF)-responsive neuronal cell line that does not express PLP. Both mAbs bound RHPC and PC12 cells and inhibited their in vitro neurite outgrowth; they differed in recognition of extracted PC12 cell proteins on Western blot and in extent of their recognition of human CNS neurons of different ages and in adult neural stem cell niches (NSCN). Thus, epitope sequence alone did not predict mAb neuronal reactivity. We immunoprecipitated proteins from NGF-treated and -untreated PC12 cell extracts using F4.4C2- and F3.9E9-coated magnetic beads; SDS-PAGE-separated bands of eluates from the beads were cut from gels and analyzed by GeLC-MS/MS. mAb F4.4C2 consistently captured the proteolipid gene family M6 neuronal protein. M6b and PLP50–69 have 45% sequence identity. M6b is required for neurite outgrowth and axon guidance and is expressed in developing human CNS neurons and NSCN. F4.4C2 also immunoprecipitated EphA family proteins and other cell surface growth and differentiation molecules expressed in human NSCN. Thus, certain anti-PLP autoantibodies generated after myelin injury may inhibit neuronal repair in the developing and adult human CNS by multispecific binding to neuronal growth and differentiation molecules.

Interaction of Mint2 with TrkA Is Involved in Regulation of Nerve Growth Factor-induced Neurite Outgrowth

TrkA receptor signaling is essential for nerve growth factor (NGF)-induced survival and differentiation of sensory neurons. To identify possible effectors or regulators of TrkA signaling, yeast two-hybrid screening was performed using the intracellular domain of TrkA as bait. We identified muc18-1-interacting protein 2 (Mint2) as a novel TrkA-binding protein and found that the phosphotyrosine binding domain of Mint2 interacted with TrkA in a phosphorylation- and ligand-independent fashion. Coimmunoprecipitation assays showed that endogenous TrkA interacted with Mint2 in rat tissue homogenates, and immunohistochemical evidence revealed that Mint2 and TrkA colocalized in rat dorsal root ganglion neurons. Furthermore, Mint2 overexpression inhibited NGF-induced neurite outgrowth in both PC12 and cultured dorsal root ganglion neurons, whereas inhibition of Mint2 expression by RNA interference facilitated NGF-induced neurite outgrowth. Moreover, Mint2 was found to promote the retention of TrkA in the Golgi apparatus and inhibit its surface sorting. Taken together, our data provide evidence that Mint2 is a novel TrkA-regulating protein that affects NGF-induced neurite outgrowth, possibly through a mechanism involving retention of TrkA in the Golgi apparatus.

Hermansky-Pudlak Syndrome Protein Complexes Associate with Phosphatidylinositol 4-Kinase Type II α in Neuronal and Non-neuronal Cells

The Hermansky-Pudlak syndrome is a disorder affecting endosome sorting. Disease is triggered by defects in any of 15 mouse gene products, which are part of five distinct cytosolic molecular complexes: AP-3, homotypic fusion and vacuole protein sorting, and BLOC-1, -2, and -3. To identify molecular associations of these complexes, we used in vivo cross-linking followed by purification of cross-linked AP-3 complexes and mass spectrometric identification of associated proteins. AP-3 was co-isolated with BLOC-1, BLOC-2, and homotypic fusion and vacuole protein sorting complex subunits; clathrin; and phosphatidylinositol-4-kinase type II alpha (PI4KIIalpha). We previously reported that this membrane-anchored enzyme is a regulator of AP-3 recruitment to membranes and a cargo of AP-3 ( Craige, B., Salazar, G., and Faundez, V. (2008) Mol. Biol. Cell 19, 1415-1426 ). Using cells deficient in different Hermansky-Pudlak syndrome complexes, we identified that BLOC-1, but not BLOC-2 or BLOC-3, deficiencies affect PI4KIIalpha inclusion into AP-3 complexes. BLOC-1, PI4KIIalpha, and AP-3 belong to a tripartite complex, and down-regulation of either PI4KIIalpha, BLOC-1, or AP-3 complexes led to similar LAMP1 phenotypes. Our analysis indicates that BLOC-1 complex modulates the association of PI4KIIalpha with AP-3. These results suggest that AP-3 and BLOC-1 act, either in concert or sequentially, to specify sorting of PI4KIIalpha along the endocytic route.

Myosin Va phosphorylated on Ser1650 is found in nuclear speckles and redistributes to nucleoli upon inhibition of transcription

Nuclear actin and nuclear myosins have been implicated in the regulation of gene expression in vertebrate cells. Myosin V is a class of actin-based motor proteins involved in cytoplasmic vesicle transport and anchorage, spindle-pole alignment and mRNA translocation. In this study, myosin-Va, phosphorylated on a conserved serine in the tail domain (phospho-ser(1650) MVa), was localized to subnuclear compartments. A monoclonal antibody, 9E6, raised against a peptide corresponding to phosphoserine(1650) and flanking regions of the murine myosin Va sequence, was immunoreactive to myosin Va heavy chain in cellular and nuclear extracts of HeLa cells, PC12 cells and B16-F10 melanocytes. Immunofluorescence microscopy with this antibody revealed discrete irregular spots within the nucleoplasm that colocalized with SC35, a splicing factor that earmarks nuclear speckles. Phospho-ser(1650) MVa was not detected in other nuclear compartments, such as condensed chromatin, Cajal bodies, gems and perinucleolar caps. Although nucleoli also were not labeled by 9E6 under normal conditions, inhibition of transcription in HeLa cells by actinomycin D caused the redistribution of phospho-ser(1650) MVa to nucleoli, as well as separating a fraction of phospho-ser(1650) MVa from SC35 into near-neighboring particles. These observations indicate a novel role for myosin Va in nuclear compartmentalization and offer a new lead towards the understanding of actomyosin-based gene regulation.

JNK phosphorylates synaptotagmin-4 and enhances Ca2+-evoked release

Ca2+ influx induced by membrane depolarization triggers the exocytosis of secretory vesicles in various cell types such as endocrine cells and neurons. Peptidyl growth factors enhance Ca2+-evoked release, an effect that may underlie important adaptive responses such as the long-term potentiation of synaptic transmission induced by growth factors. Here, we show that activation of the c-Jun N-terminal kinase (JNK) plays an essential role in nerve growth factor (NGF) enhancement of Ca2+-evoked release in PC12 neuroendocrine cells. Moreover, JNK associated with phosphorylated synaptotagmin-4 (Syt 4), a key mediator of NGF enhancement of Ca2+-evoked release in this system. NGF treatment led to phosphorylation of endogenous Syt 4 at Ser135 and translocation of Syt 4 from immature to mature secretory vesicles in a JNK-dependent manner. Furthermore, mutation of Ser135 abrogated enhancement of Ca2+-evoked release by Syt 4. These results provide a molecular basis for the effect of growth factors on Ca2+-mediated secretion.

FXYD6 Is a Novel Regulator of Na,K-ATPase Expressed in the Inner Ear

The exquisite sensitivity of the cochlea, which mediates the transduction of sound waves into nerve impulses, depends on the endolymph ionic composition and the endocochlear potential. A key protein in the maintenance of the electrochemical composition of the endolymph is the Na,K-ATPase. In this study, we have looked for the presence in the rat inner ear of members of the FXYD protein family, recently identified as tissue-specific modulators of Na,K-ATPase. Only FXYD6 is detected at the protein level. FXYD6 is expressed in various epithelial cells bordering the endolymph space and in the auditory neurons. FXYD6 co-localizes with Na,K-ATPase in the stria vascularis and can be co-immunoprecipitated with Na,K-ATPase. After expression in Xenopus oocytes, FXYD6 associates with Na,K-ATPase alpha1-beta1 and alpha1-beta2 isozymes, which are preferentially expressed in different regions of the inner ear and also with gastric and non-gastric H,K-ATPases. The apparent K(+) and Na(+) affinities of alpha1-beta1 and alpha1-beta2 isozymes are different. Association of FXYD6 with Na,K-ATPase alpha1-beta1 isozymes slightly decreases their apparent K(+) affinity and significantly decreases their apparent Na(+) affinity. On the other hand, association with alpha1-beta2 isozymes increases their apparent K(+) and Na(+) affinity. The effects of FXYD6 on the apparent Na(+) affinity of Na,K-ATPase and the voltage dependence of its K(+) effect are distinct from other FXYD proteins. In conclusion, this study defines the last FXYD protein of unknown function as a modulator of Na,K-ATPase. Among FXYD protein, FXYD6 is unique in its expression in the inner ear, suggesting a role in endolymph composition.

Ataxin-10 Interacts with O-Linked β-N-Acetylglucosamine Transferase in the Brain

Modification by O-GlcNAc involves a growing number of eucaryotic nuclear and cytosolic proteins. Glycosylation of intracellular proteins is a dynamic process that in several cases competes with and acts as a reciprocal modification system to phosphorylation. O-Linked beta-N-acetylglucosamine transferase (OGT) levels are highest in the brain, and neurodegenerative disorders such as Alzheimer disease have been shown to involve abnormally phosphorylated key proteins, probably as a result of hypoglycosylation. Here, we show that the neurodegenerative disease protein ataxin-10 (Atx-10) is associated with cytoplasmic OGT p110 in the brain. In PC12 cells and pancreas, this association is competed by the shorter OGT p78 splice form, which is down-regulated in brain. Overexpression of Atx-10 in PC12 cells resulted in the reconstitution of the Atx-10-OGT p110 complex and enhanced intracellular glycosylation activity. Moreover, in an in vitro enzyme assay using PC12 cell extracts, Atx-10 increased OGT activity 2-fold. These data indicate that Atx-10 might be essential for the maintenance of a critical intracellular glycosylation level and homeostasis in the brain.

Detection of 6-nitrotryptophan in proteins by Western blot analysis and its application for peroxynitrite-treated PC12 cells

We have previously reported on the formation of 6-nitrotryptophan by the reaction of reactive nitrogen species with a tryptophan residue in human Cu, Zn-superoxide dismutase (SOD) (F. Yamakura et al., J. Biochem. 138 (2005) 57–69). Here, we report on the preparation of anti-6-nitrotryptophan antiserum by using synthesized 6-nitrotryptophan-conjugated keyhole limpet hemocyanin as an antigen and the purification of the antibody by using a 6-nitrotryptophan-conjugated affinity column. The purified antibody was immunoreactive with 6-nitrotryptophan residue containing Cu, Zn-SOD but not immunoreactive with Cu, Zn-SOD, Mn-SOD, bovine serum albumin, and 3-nitrotyrosine residue containing Mn-SOD. Nitro group of 6-nitrotryptophan was reduced by sodium hydrosulfite to form 6-aminotryptophan as a major product. The reduced 6-nitrotryptophan residues lost its immunoreactivity with the antibody. We detected different immunoreactive bands between using antibody for 6-nitrotryptophan residues and that for 3-nitrotyrosine residues in crude extracts of neuron-like PC12 cells treated with peroxynitrite by a Western blot analysis. Western blot analysis for two-dimensional gel electrophoresis showed nine intensively stained immunoreactive spots for 6-nitrotryptophan residues in the peroxynitrite-treated PC12 cells, which were subjected to trypsin digestion and LC–ESI-MS/MS analysis. We identified M2 pyruvate kinase, elongation factor 2, mitochondrial aconitase, pyruvate carboxylase, and heat shock protein HSP90α as candidates for 6-nitrotryptophan residues containing proteins, with peptide coverage over 10%, in crude extracts of peroxynitrite-treated PC12 cells.

Microtubule-Associated Protein 1B-Light Chain 1 Enhances Activation of Rap1 by Exchange Protein Activated by Cyclic AMP but Not Intracellular Targeting

We have previously demonstrated that EPAC1 interacts with light chain (LC) 2 of microtubule-associated protein (MAP) 1A. In the present study, we investigated whether the structurally related LC1 of MAP1B also interacts with EPAC1. We demonstrate that LC1 copurifies with EPAC1 from extracts of PC-12 cells, using cyclic AMP-agarose. Using recombinant LC1 and LC2 in pull-down and solid phase binding assays, we demonstrate direct interaction with a glutathione S-transferase-fusion of the cyclic AMP-binding (CAMP) domain of EPAC1. We also tested whether LC1 directed intracellular targeting of EPAC1 through its interaction with the CAMP domain. EPAC1 was found be in the soluble and particulate, nuclear/perinuclear fractions of cells. We found that the catalytic (CAT) domain of EPAC1, and not the CAMP domain, was responsible for recruitment to the nuclear/perinuclear fraction of cells. The targeting sequence responsible was located between amino acids 764 and 838 of EPAC1. Overexpresssion of an isolated CAT domain in COS1 cells was found to displace endogenous EPAC1 from the nuclear/perinuclear fraction, thereby inhibiting EPAC-activated Rap1 in this compartment. In contrast, LC1 was not able to compete for the binding of EPAC1 to this fraction. LC1, however, was able to enhance interaction of EPAC1 with cyclic AMP and heightened the ability of EPAC to activate Rap1. Antibody disruption of EPAC1/LC1 interaction in PC-12 cells ablated the ability of cyclic AMP to activate Rap1. LC1 is therefore not involved in intracellular targeting of EPAC1, but it is rather a molecular chaperone of EPAC activity toward Rap1.

A molecular basis underlying differences in the toxicity of botulinum serotypes A and E.

Botulinum neurotoxins (BoNTs) block neurotransmitter release through their specific proteolysis of the proteins responsible for vesicle exocytosis. Paradoxically, two serotypes of BoNTs, A and E, cleave the same molecule, synaptosome-associated protein with relative molecular mass 25K (SNAP-25), and yet they cause synaptic blockade with very different properties. Here we compared the action of BoNTs A and E on the plasma membrane fusion machinery composed of syntaxin and SNAP-25. We now show that the BoNT/A-cleaved SNAP-25 maintains its association with two syntaxin isoforms in vitro, which is mirrored by retention of SNAP-25 on the plasma membrane in vivo. In contrast, BoNT/E severely compromises the ability of SNAP-25 to bind the plasma membrane syntaxin isoforms, leading to dissociation of SNAP-25. The distinct properties of botulinum intoxication, therefore, can result from the ability of shortened SNAP-25 to maintain its association with syntaxins-in the case of BoNT/A poisoning resulting in unproductive syntaxin/SNAP-25 complexes that impede vesicle exocytosis.

Simultaneous determination of catecholamines and polyamines in PC-12 cell extracts by micellar electrokinetic capillary chromatography with ultraviolet absorbance detection

A method for simultaneous determination of polyamines and catecholamines in cell extracts by micellar electrokinetic capillary chromatography with UV detection at 254 nm was established at the first time. The polyamines (putrescine, spermidine and spermine) and catecholamines (dopamine, serotonin, norepinephrine and epinephrine) were extracted from PC-12 cells and were derivatized with 6-aminoquinolyl- N-hydroxysuccinimidyl carbamate. Different derivatization conditions such as temperature, ratio of derivatization reagents and incubation time were investigated to find the best reaction condition which gave the highest detection sensitivity for polyamines and catecholamines. The influence of running buffer and additives on the separation such as pH, sodium dodecyl sulfate (SDS) concentrations and various additives was also investigated. Separation was achieved within 20 min with good repeatability in a 100 mM boric acid buffer containing 10 mM SDS and 10 mM 18-crown-6 at a pH of 9.5. The detection limit ranged from 1.0×10 −7 to 9.0×10 −7 M, which is sufficient for determination of polyamines and catecholamines in many cell extracts. This technique can be easily applied to polyamine-related anticancer drug studies or clinical follow-ups after each dosage of these anticancer drugs, since these drugs not only have great inhibition on polyamine levels in blood, but also have a large influence on catecholamine levels in blood.

Induction of the expression of heme oxygenase gene in PC12 cells by hypoxia.

To investigate the expression of the HO-1 gene in PC12 cells in hypoxic environment and gain further insight to the role of HO-1 in cerebral ischemia, PC12 cells were exposed to hypoxia environment (95% N2, 5% CO2) for 0.5 h, 1 h, 4 h, 8 h, 12 h, 24 h respectively. The level of HO-1 mRNA was examined by reverse transcriptase polymerase chain reaction (RT-PCR); the volume of COHb in the media were measured spectrophotometrically and the cGMP concentration of PC12 cell extracts was determined by radioimmunoassay. We found that after exposure to hypoxia for 1 h, 4 h, 8 h, 12 h, 24 h, HO-1 mRNA increased by 3%, 4%, 17%, 31% 36% as compared with that in control group respectively (P < 0.01 or P < 0.05); the COHb increased by 12%, 29%, 59%, 88%, 94% as compared with that in control group respectively (P < 0.01 or P < 0.05), and the cGMP concentration were 2.2, 3.4, 5.2, 8.1, 10.9-fold as that of the control group (P < 0.01). We are led to conclude that hypoxia induced the expression of HO-1 gene, the production of endogenous CO, and the concentration of cGMP was elevated as well.

Ritonavir inhibition of calcium-activated neutral proteases

Calpains (EC 3.4.22.17) are intracellular calcium-activated cysteine proteases that mediate tissue injury following post-ischemic and post-traumatic stress. Both human HIV protease and calpains share a similar secondary structure, where the active site is flanked by hydrophobic regions. The present study demonstrates that ritonavir, a hydrophobic HIV protease inhibitor, also inhibits calpain activity. In PC12 cell extracts assayed for calpain at maximal activity (2 mM calcium), ritonavir exhibited competitive inhibition with a K i of 11±7.0 μM. Experiments with purified enzymes showed inhibition for both m- and μ-calpain isoforms (m-calpain, K i =9.2±1.2 μM; μ-calpain, K i =5.9±1.4 μM). Ritonavir also inhibited calcium-stimulated calpain activity in PC12 cells in situ. These results suggest that ritonavir or analogues of the drug should be investigated as cytoprotective agents in conditions where cell death or injury is mediated via calpain activation.

Peptide substrates for ERK1/2: structure-function studies of serine 31 in tyrosine hydroxylase

The first physiological substrate identified for the extracellular signal-regulated protein kinases (ERKs) is serine 31 in tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. In the present studies, several synthetic peptides modeled after Ser31 in TH were evaluated as in vitro substrates for the ERKs. The phosphorylation of Ser31-containing peptides from type 1 human TH by activated, recombinant ERK2 was found to exhibit catalytic efficiencies ( V max/ K m) up to 4-fold higher than that of a synthetic myelin basic protein (MBP)-based peptide. Both types of peptides were also tested using extracts from PC12 cells (untreated or treated with nerve growth factor (NGF)). Although, the phosphorylation of the MBP peptide by extracts of PC12 cells was higher than that of the Ser31 peptide, the relative treatment-dependent increase was much greater for the Ser31 peptide and more closely mimicked the pattern of ERK phosphorylation, suggesting that the latter peptide may be a more specific substrate for the ERKs.

Phosphorylation-dependent Regulation of Phospholipase D2 by Protein Kinase Cδ in Rat Pheochromocytoma PC12 Cells

Many studies have shown that protein kinase C (PKC) is an important physiological regulator of phospholipase D (PLD). However, the role of PKC in agonist-induced PLD activation has been mainly investigated with a focus on the PLD1, which is one of the two PLD isoenzymes (PLD1 and PLD2) cloned to date. Since the expression of PLD2 significantly enhanced phorbol 12-myristate 13-acetate (PMA)- or bradykinin-induced PLD activity in rat pheochromocytoma PC12 cells, we investigated the regulatory mechanism of PLD2 in PC12 cells. Two different PKC inhibitors, GF109203X and Ro-31-8220, completely blocked PMA-induced PLD2 activation. In addition, specific inhibition of PKC delta by rottlerin prevented PLD2 activation in PMA-stimulated PC12 cells. Concomitant with PLD2 activation, PLD2 became phosphorylated upon PMA or bradykinin treatment of PC12 cells. Moreover, rottlerin blocked PMA- or bradykinin-induced PLD2 phosphorylation in PC12 cells. Expression of a kinase-deficient mutant of PKC delta using adenovirus-mediated gene transfer inhibited the phosphorylation and activation of PLD2 induced by PMA in PC12 cells, suggesting the phosphorylation-dependent regulation of PLD2 mediated by PKC delta kinase activity in PC12 cells. PKC delta co-immunoprecipitated with PLD2 from PC12 cell extracts, and associated with PLD2 in vitro in a PMA-dependent manner. Phospho-PLD2 immunoprecipitated from PMA-treated PC12 cells and PLD2 phosphorylated in vitro by PKC delta were resolved by two-dimensional phosphopeptide mapping and compared. At least seven phosphopeptides co-migrated, indicating the direct phosphorylation of PLD2 by PKC delta inside the cells. Immunocytochemical studies of PC12 cells revealed that after treatment with PMA, PKC delta was translocated from the cytosol to the plasma membrane where PLD2 is mainly localized. These results suggest that PKC delta-dependent direct phosphorylation plays an important role in the regulation of PLD2 activity in PC12 cells.

Nerve growth factor-mediated increases in protein methylation occur predominantly at type I arginine methylation sites and involve protein arginine methyltransferase 1.

Nerve growth factor (NGF)-specific signal transduction leads to changes in protein methylation during neuronal differentiation of PC12 cells (Cimato et al. [1997] J. Cell Biol. 138:1089-1103). In the present work, we demonstrate that, among NGF-regulated proteins, arginine methylation is more prevalent than carboxylmethylation. Type I protein arginine methyltransferase (PRMT) activity produces asymmetric dimethylation of the terminal guanidinonitrogen of arginines in substrate proteins, particularly glycine and arginine-rich (GAR) segments of proteins. Several GAR peptides were used to assay for methyltransferase activity and to compete with endogenous cellular proteins for the PRMT activity in PC12 cell extracts. Peptides derived from fibrillarin and nucleolin, as well as a synthetic GAR peptide containing a repetitive GRG motif, are each extremely effective at blocking in vitro methylation of the NGF-regulated PC12 cell methylated proteins. Myelin basic protein, a substrate for type II PRMT, selectively inhibits a 45 kDa protein but is a much less effective inhibitor of total methylation at an equimolar concentration. In addition, the fibrillarin- and nucleolin-derived peptides were used to detect elevated PRMT activity in homogenates of NGF-treated PC12 cells. Finally, immunoprecipitation of PRMT1 from PC12 cells provides the first demonstration of an NGF-activated methyltransferase and implicates PRMT1 in NGF signal transduction.

Microtubule-associated protein 1B phosphorylation by glycogen synthase kinase 3beta is induced during PC12 cell differentiation.

In recent studies we have demonstrated that glycogen synthase kinase 3beta (GSK3beta) and its substrate microtubule-associated protein 1B (MAP1B) regulate the microtubule cytoskeleton during axon outgrowth. To further examine the role GSK3beta plays in axon outgrowth we investigated the expression of GSK3beta and its activity towards MAP1B during nerve growth factor (NGF)-stimulated PC12 cell differentiation. Levels of GSK3beta expression increase relatively little during the course of differentiation. However, the expression of a novel GSK3beta isoform characterised by a reduced mobility on SDS gels is induced by NGF. Expression of this isoform and the GSK3beta-phosphorylated isoform of MAP1B (MAP1B-P) are induced in parallel in response to NGF. This increase lags behind initial neurite formation and the expression of MAP1B in these cells by about two days and coincides with a period when the majority of cells are extending existing neurites. MAP1B and GSK3beta are expressed throughout the PC12 cell but MAP1B-P expression is restricted to the growth cones and neurites. Consistent with these observations, we find that neurite extension is more sensitive to the GSK3 inhibitor Li+ than neurite formation and that this correlates with an inhibition of MAP1B phosphorylation. Additionally, GSK3beta from PC12 cells not exposed to NGF can not phosphorylate MAP1B in vitro. However, a soluble factor in differentiated PC12 cell extracts depleted of GSK3beta can activate MAP1B phosphorylation from undifferentiated cell extracts otherwise devoid of kinase activity. These experiments provide evidence for an NGF-mediated regulation of MAP1B phosphorylation in growing neurites by the induction of a novel isoform of GSK3beta.

Origin binding protein-containing protein-DNA complex formation at herpes simplex virus type 1 oriS: role in oriS-dependent DNA replication.

Initiation of herpes simplex virus type 1 (HSV-1) DNA replication during productive infection of fibroblasts and epithelial cells requires attachment of the origin binding protein (OBP), one of seven essential virus-encoded DNA replication proteins, to specific sequences within the two viral origins, oriL and oriS. Whether initiation of DNA replication during reactivation of HSV-1 from neuronal latency also requires OBP is not known. A truncated protein, consisting of the C-terminal 487 amino acids of OBP, termed OBPC, is the product of the HSV UL8.5 gene and binds to origin sequences, although OBPC's role in HSV DNA replication is not yet clear. To characterize protein-DNA complex formation at oriS in cells of neural and nonneural lineage, we used nuclear extracts of HSV-infected nerve growth factor-differentiated PC12 and Vero cells, respectively, as the source of protein in gel shift assays. In both cell types, three complexes (complexes A, B, and C) which contain either OBP or OBPC were shown to bind specifically to a probe which contains the highest-affinity OBP binding site in oriS, site 1. Complex A was shown to contain OBPC exclusively, whereas complexes B and C contained OBP and likely other cellular proteins. By fine-mapping the binding sites of these three complexes, we identified single nucleotides which, when mutated, eliminated formation of all three complexes, or complexes B and C, but not A. In transient DNA replication assays, both mutations significantly impaired oriS-dependent DNA replication, demonstrating that formation of OBP-containing complexes B and C is required for efficient initiation of oriS-dependent DNA replication, whereas formation of the OBPC-containing complex A is insufficient for efficient initiation.

Activation pathways of 5-fluorouracil in rat organs and in PC12 cells

Activation of the pyrimidine analogue 5-fluorouracil (5-FU) to the ribonucleotide level may occur through one of the following three pathways: 1) the 5-phosphoribosyl 1-pyrophosphate (PRPP)-mediated direct transfer of ribose 5-phosphate to 5-FU as catalysed by orotate phosphoribosyltransferase; 2) the ribose 1-phosphate (Rib1-P)-mediated addition of ribose by uridine phosphorylase, followed by the action of uridine kinase; and 3) the 2′-deoxyribose 1-phosphate (deoxyRib1-P)-mediated addition of deoxyribose, thought to be catalysed by thymidine phosphorylase, followed by the action of thymidine kinase. Many of the conclusions as to the precise pathways by which normal tissues and different cell lines activate uracil are indirectly derived from drug interactions affecting the availability of the substrates of the three pathways, or from measurement of activities of the enzymes metabolising 5-FU in normal tissues and tumours. In previous papers (Cappiello et al. Biochim Biophys Acta 1998;1425:273–81; Mascia et al. Biochim Biophys Acta 1999;1472:93–8), we assessed the molecular mechanisms by which the natural base uracil is salvaged in vitro to uracil ribonucleotides and deoxyribonucleotides in rat liver and brain. In this paper, we investigated the pathways of 5-FU activation to cytotoxic ribonucleotide and deoxyribonucleotide levels in normal rat tissues and PC12 cell extracts. The results clearly showed that normal rat tissues activated 5-FU mainly via the Rib1-P pathway, and to a lesser extent via the PRPP pathway. The deoxyRib1-P pathway was absent. PC12 cells activated 5-FU mainly via the PRPP pathway and to a lesser extent by the other two pathways.