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Biomedical subjects

D R Kaplan

Publications and source records attributed to D R Kaplan.

At least 37 records · Page 2Linked to original sources
PubMed1998

Mobility of TrkA is regulated by phosphorylation and interactions with the low-affinity NGF receptor.

The nerve growth factor (NGF) receptor is a complex of two proteins, gp75 and the tyrosine kinase TrkA. Using fluorescence recovery after photobleaching, we have studied the diffusion properties of the TrkA receptor. For PC12 cells that express both gp75 and TrkA, TrkA was relatively immobile with only 28 +/- 1% of receptor molecules free to diffuse with D = (3.64 +/- 0.23) x 10(-9) cm2/s. Addition of NGF decreased the mobile fraction to 21 +/- 1% with D = (4.11 +/- 0.18) x 10(-9) cm2/s. Using the Sf9 baculovirus expression system, we were able to study TrkA in the absence and presence of gp75. On Sf9 cells, TrkA showed a mobile fraction of 46 +/- 2% with D = (2.64 +/- 0.21) x 10(-9) cm2/s in the absence of gp75 and 43 +/- 2% with D = (2.31 +/- 0.25) x 10(-9) cm2/s in its presence. Thus, gp75 did not alter TrkA mobility. Addition of NGF to the medium approximately halved the mobile fraction for TrkA in both the absence and presence of gp75. However, using a kinase-deficient mutant of TrkA, we found that ligand-induced immobilization requires an active kinase in the absence of gp75 but not in its presence. In addition, using point mutations at specific TrkA autophosphorylation sites, we determined that mobility is controlled by multiple phosphorylation sites, but the SHC binding site at Y490 may be particularly important for ligand-induced immobilization of TrkA. Therefore, two mechanisms lead to NGF-induced immobilization of TrkA--the first resulting from autophosphorylation of TrkA and the second occurring through TrkA's association with gp75.

Animals↗
PubMed1998

Neurotrophins and neuronal versus glial differentiation in medulloblastomas and other pediatric brain tumors.

Medulloblastomas are highly malignant and poorly understood childhood neoplasms. To determine if neurotrophins might influence the phenotypic properties of medulloblastoma in a paracrine or autocrine manner, 51 pediatric brain tumors including 20 biopsy specimens of these primitive neuroectodermal tumors (PNETs) and 31 other pediatric brain tumors were studied. Immunohistochemistry was used with antibodies to nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and NT-3, their cognate high affinity receptors as well as to neuronal and glial markers. TrkA, TrkB, and TrkC were observed in 5 (25%), 8 (40%), and 17 (85%), respectively, of these medulloblastomas while NGF, BDNF, and NT-3 were observed in 6 (30%), 8 (40%), and 3 (15%), respectively, and antibodies to neurofilament (NF) and glial fibrillary acidic proteins (GFAP) stained 16 (80%) and 11 (55%), respectively. TrkA and NGF were not observed in the same biopsy samples, while TrkB and BDNF were co-distributed in 6 of the cases, all of which expressed NF proteins. TrkC and NT-3 were co-distributed in 3 of the medulloblastomas, and these areas overlapped with NF protein-positive tumor cells in all 3 cases. In contrast to medulloblastomas, TrkA and NGF co-distributed in other pediatric brain tumors, and both Trk receptors and their neurotrophins co-distributed with GFAP-positive tumor cells in 13 (42%) of the non-PNET pediatric brain tumors. The absence of medulloblastomas that contain NGF and TrkA is consistent with in vitro data demonstrating that NGF-mediated TrkA signaling induces apoptosis. Finally, this study also suggests that BDNF and NT-3 may act in a paracrine or autocrine manner through TrkB and TrkC receptors, respectively, to induce neuronal differentiation in medulloblastomas.

Adolescent↗
PubMed1998

Distribution of the neurotrophins brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 in the postnatal rat brain: an immunocytochemical study.

The neurotrophin family of trophic factors influences survival and function of neurons in both the peripheral and central nervous systems. Critical information regarding physiological function of these factors may be gained by examining their localization in the brain. Here we report the immunocytochemical characterization of antisera directed against brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin 4/5. These antisera provide important tools to localize the bioactive neurotrophin proteins. Correspondence between protein distribution and previously determined messenger RNA expression was observed in some brain regions, such as hippocampus and cortex. However, neurotrophin proteins were also detected in neurons which have no apparent corresponding messenger RNA, indicating that the proteins may be transported from the sites of synthesis in certain populations. Immunocytochemical double-labelling analysis also indicated that a sub-population of neurotrophin-positive cells were labelled with an astrocyte marker (glial fibrillary acidic protein) as well, demonstrating that trophic molecules are localized to glial cells as well as neurons in vivo. Thus, the use of antisera specific for individual neurotrophic factors has indicated potential cellular sites of action.

Animals↗
PubMed1997

Interleukin 3-dependent survival by the Akt protein kinase.

Interleukin 3 (IL-3)-dependent survival of hematopoietic cells is known to rely on the activity of multiple signaling pathways, including a pathway leading to activation of phosphoinositide 3-kinase (PI 3-kinase), and protein kinase Akt is a direct target of PI 3-kinase. We find that Akt kinase activity is rapidly induced by the cytokine IL-3, suggesting a role for Akt in PI 3-kinase-dependent signaling in hematopoetic cells. Dominant-negative mutants of Akt specifically block Akt activation by IL-3 and interfere with IL-3-dependent proliferation. Overexpression of Akt or oncogenic v-akt protects 32D cells from apoptosis induced by IL-3 withdrawal. Apoptosis after IL-3 withdrawal is accelerated by expression of dominant-negative mutants of Akt, indicating that a functional Akt signaling pathway is necessary for cell survival mediated by the cytokine IL-3. Thus Akt appears to be an important mediator of anti-apoptotic signaling in this system.

Animals↗
PubMed1997

Autophosphorylation of activation loop tyrosines regulates signaling by the TRK nerve growth factor receptor.

Many receptor tyrosine kinases possess an "activation loop" containing three similarly placed tyrosine autophosphorylation sites. To examine their roles in the TRK NGF receptor, these residues (Tyr-670, Tyr-674, and Tyr-675) were mutated singly and in all combinations to phenylalanine and stably expressed in Trk-deficient PC12nnr5 cells. All mutant receptors showed significantly diminished nerve growth factor (NGF)-stimulated autophosphorylation, indicating impaired catalytic activity. NGF-induced neurite outgrowth exhibited dose-responsive behavior when transfectants were compared by relative receptor expression and exhibited a functional hierarchy: wild type > Y670F >/= Y674F >> Y675F >/= YY670/674FF = YY670/675FF >> YY674/675FF > YYY670/674/675FFF. NGF-induced tyrosine phosphorylation of Shc, ERKs, and SNT and immediate early gene inductions generally paralleled neurogenic potential. However, activation of phosphatidylinositol 3'-kinase and tyrosine phosphorylation of phospholipase Cgamma-1 was essentially abolished. The latter effect appears due to selective inability of the mutated TRKs to autophosphorylate the tyrosine residue (Tyr-785) required for binding phospholipase Cgamma-1 and indicates that the "activation loop" tyrosines participate in NGF-dependent changes in receptor conformation. Our findings stress the importance that expression levels play in assessing the consequences of receptor mutations and that all three activation loop tyrosines have roles regulating both overall and specific NGF-mediated signaling through TRK.

Amino Acid Sequence↗
PubMed1997

Regulation of neuronal survival by the serine-threonine protein kinase Akt.

A signaling pathway was delineated by which insulin-like growth factor 1 (IGF-1) promotes the survival of cerebellar neurons. IGF-1 activation of phosphoinositide 3-kinase (PI3-K) triggered the activation of two protein kinases, the serine-threonine kinase Akt and the p70 ribosomal protein S6 kinase (p70(S6K)). Experiments with pharmacological inhibitors, as well as expression of wild-type and dominant-inhibitory forms of Akt, demonstrated that Akt but not p70(S6K) mediates PI3-K-dependent survival. These findings suggest that in the developing nervous system, Akt is a critical mediator of growth factor-induced neuronal survival.

Androstadienes↗
PubMed1997

Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate.

The regulation of the serine-threonine kinase Akt by lipid products of phosphoinositide 3-kinase (PI 3-kinase) was investigated. Akt activity was found to correlate with the amount of phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P2) in vivo, and synthetic PtdIns-3,4-P2 activated Akt both in vitro and in vivo. Binding of PtdIns-3,4-P2 occurred within the Akt pleckstrin homology (PH) domain and facilitated dimerization of Akt. Akt mutated in the PH domain was not activated by PI 3-kinase in vivo or by PtdIns-3, 4-P2 in vitro, and it was impaired in binding to PtdIns-3,4-P2. Examination of the binding to other phosphoinositides revealed that they bound to the Akt PH domain with much lower affinity than did PtdIns-3,4-P2 and failed to increase Akt activity. Thus, Akt is apparently regulated by the direct interaction of PtdIns-3,4-P2 with the Akt PH domain.

3T3 Cells↗
PubMed1997

Nerve growth factor induces apoptosis in human medulloblastoma cell lines that express TrkA receptors.

Neurotrophins act through their cognate receptors to promote the differentiation and/or survival of neuronal progenitor cells, immature neurons, and other cells. Here, we examined the effects of nerve growth factor (NGF) and its cognate receptor (Trk or TrkA) on the survival of a common childhood brain tumor, i.e., medulloblastoma, a tumor that resembles CNS neuroepithelial progenitor cells. To do this, we engineered two human medulloblastoma cell lines (i.e., D283MED and DAOY cells) to express human TrkA using a retroviral expression vector. Surprisingly, NGF-treated medulloblastoma cells expressing the TrkA receptor (D283trk and DAOYtrk cells) grown in the presence or absence of serum underwent massive apoptosis, but similar treatment did not induce apoptosis in wild-type uninfected cells, cells expressing an empty vector, or cells expressing the TrkC receptor. Furthermore, D283MED cells engineered to express the human p75 NGF receptor (D283p75) also did not undergo apoptosis. Significantly, NGF-induced apoptosis in D283trk and DAOYtrk cells can be inhibited by anti-NGF antibodies and by K-252a, an inhibitor of TrkA tyrosine phosphorylation and mimicked by high concentrations of NT3. Because NGF treatment primarily eliminated D283trk cells from the S phase of the cell cycle, this form of NGF-mediated apoptosis is cell cycle-dependent. These findings suggest that a NGF/TrkA signal transduction pathway could activate apoptotic cell death programs in CNS neuroepithelial progenitor cells and in childhood brain tumors.

3T3 Cells↗
PubMed1997

Truncated trkB receptors on nonneuronal cells inhibit BDNF-induced neurite outgrowth in vitro.

The function of truncated trkB receptors during nervous system plasticity and regeneration is currently unknown. The extensive nonneuronal localization of truncated trkB-T1 receptors, coupled with their up-regulation by CNS glial cells in response to injury, has led to the speculation that these receptors may sequester BDNF and NT-4/5 to reduce their local availability and, thus, limit axonal sprouting. Conversely, trkB-T1 receptors could bind and present neurotrophins to injured axons and facilitate their regeneration in a manor analogous to that proposed for p75(NTR) receptors on Schwann cells. To address this issue, we used an in vitro coculture paradigm in which wild-type 3T3 NIH fibroblasts or two different 3T3 cell clones stably expressing trkB-T1 receptors served as monolayer substrates upon which to evaluate the effect of trkB-T1 receptors on nonneuronal cells to influence neurotrophin (NGF, BDNF, NT-3, and NT-4/5)-induced neurite outgrowth from retinoic acid (RA)-treated SY5Y neuroblastoma cells. In these experiments, BDNF and NT-4/5 produce a strong phosphorylation of trk receptors on the RA-SY5Y cells and induce differentiation of the SY5Y cells (as measured by the development of neurofilament-positive neuritic processes). This ability of the trkB ligands to stimulate neurite outgrowth is dose dependent since increasing concentrations of BDNF (5, 25, and 100 ng/ml) result in an increased percentage of SY5Y cells developing neurites and in progressively longer neurites from SY5Y cells on the control 3T3 monolayers. In these experiments, BDNF and NT-4/5 induce the strongest neurite outgrowth, followed by NT-3 and then NGF. When trkB-T1 receptors are present on the 3T3 cell substratum both BDNF- and NT-4/5-induced neurite extension from the SY5Y cells are strongly inhibited. In contrast, NGF-induced neurite growth is unaffected and NT-3-associated growth is somewhat reduced. These results suggest that the inhibitory effect of the trkB-T1 receptors on the nonneuronal cell substrates is selective for neurite outgrowth that is mediated via the trkB-kinase receptors on the neuroblastoma cells. This ability of trkB-T1 receptors on the nonneuronal substratum to inhibit BDNF-induced neurite outgrowth can be overcome by the addition of high concentrations of BDNF (1 microg/ml). Binding assays using 125I-BDNF suggest that this inhibitory effect could be mediated via binding and internalization of BDNF by the trkB-T1 receptors on the 3T3 cells. These results provide strong support for the hypothesis that the up-regulation of trkB-T1 receptors on astrocytes following CNS lesions enhances the sequestration of the trkB ligands, BDNF and NT- 4/5, at the site of reactive gliosis and, thus, contributes to the inhibition of CNS axonal regeneration from neurons expressing trkB-kinase receptors by removing their ligands from the extracellular environment.

3T3 Cells↗
PubMed1997

Ligand-induced down-regulation of Trk messenger RNA, protein and tyrosine phosphorylation in rat cortical neurons.

Chronic exposure of brain neurons to nerve growth factor in vitro and in vivo results in increased levels of the nerve growth factor receptor TrkA. In contrast, in the present study, we have found that chronic exposure of rat embryonic cortical neurons to brain-derived neurotrophic factor (BDNF) leads to a pronounced reduction of the levels of protein and messenger RNA for the full-length but not the truncated BDNF receptor TrkB. Similar effects were observed with the other TrkB ligands neurotrophin-3 and neurotrophin-4/5. After pretreatment with BDNF, neurotrophin-3 or neurotrophin-4/5, subsequent tyrosine phosphorylation responses of the remaining Trks to the same factors were greatly reduced. Three days exposure of rat embryonic cortical neurons to BDNF induced an absolute refractory period of several hours, with no subsequent response to the same factor. Similar but less pronounced refractory effects were observed with neurotrophin-3 and neurotrophin-4/5. Our results suggest a negative regulatory effect of BDNF and other TrkB ligands on TrkB receptors. Down-regulation of the TrkB response by its ligands might play a role in the control of BDNF action during early development, when BDNF levels significantly increase. Our findings are also of potential clinical relevance, since the possibility of ligand-induced down-regulation of the receptor response needs to be addressed when considering BDNF or other neurotrophins for the therapy of neurodegeneration.

Animals↗
PubMed1997

Signal transduction by the neurotrophin receptors.

The neurotrophins signal cell survival, differentiation, growth cessation, and apoptosis through two cell surface receptors, the Trks and p75NTR (p75 neurotrophin receptor). Recent advances indicate that the particular events that are mediated by neurotrophins are dependent upon the cell type and the expression pattern of each neurotrophin receptor. For example, TrkA activation induces cell death of neural tumor cells, and survival and differentiation of neurons. Likewise, p75NTR, when activated in the absence of a strong Trk signal, induces apoptosis of neurons, while in the presence of Trk it enhances responses to neurotrophin. These differing responses point to a complex interplay between neurotrophin-stimulated survival, differentiation, and apoptosis pathways.

Apoptosis↗
PubMed1996

Developmental and mature expression of full-length and truncated TrkB receptors in the rat forebrain.

The neurotrophins brain-derived neurotrophic factor (BDNF) and NT-4/5 exert their trophic effects on the nervous system via signaling through trkB receptors. These receptors occur as splice variants of the trkB gene that encodes a full-length receptor containing the signal transducing tyrosine kinase domain as well as truncated forms lacking this domain. Because the importance of the trkB isoforms for development and maturation of the nervous system is unknown, we have examined the expression of trkB receptor isoforms during development of the rat forebrain using 1) a sensitive ribonuclease protection assay to distinguish full-length and truncated trkB transcripts, 2) western blot analysis to characterize developmental changes in trkB proteins, and 3) immunohistochemistry to determine the cellular localization of trkB receptors. In the rat forebrain, adult mRNA levels for full-length trkB are reached by birth, whereas truncated trkB message does not peak until postnatal days 10-15. Western blot analysis indicates that full-length trkB protein is the major form during early development, whereas truncated trkB protein predominates in all forebrain regions of late postnatal and adult rats. These data also suggest that the glycosylation state of these receptors changes during postnatal maturation. TrkB immunoreactivity is present predominately within neurons, where it is localized to axons, cell soma, and dendrites. Strong dendritic immunostaining is particularly evident in certain neuronal populations, such as pyramidal neurons in the hippocampus and in layer V of the neocortex. The dendritic localization of trkB receptors supports the hypothesis that dendrites, as well as axons, are important sites for neurotrophin actions in the central nervous system.

Animals↗
PubMed1996

Differential utilization of Trk autophosphorylation sites.

Tyrosine autophosphorylation controls the catalytic and signaling activities of the neurotrophin receptors, the Trks. To analyze the regulation of distinct tyrosine sites, we generated a panel of antibodies that report the phosphorylation state of individual tyrosines within the Trk cytoplasmic domain. Using pheochromocytoma-derived cell lines, we show that individual tyrosines within the nerve growth factor receptor TrkA are phosphorylated in a non-coordinate fashion following receptor activation. The non-coordinate response of these tyrosines reflects their separate functions in regulating the catalytic and signaling activities of Trk receptors. The differential utilization of distinct sites on Trk receptor tyrosine kinases suggests that the receptor can specify both the timing and the nature of neurotrophin-stimulated signal transduction pathways. Moreover, we show that these Trk autophosphorylation sites, which have hitherto been mapped and characterized only in non-neuronal cell lines, are activated in normal neurons in response to ligand stimulation.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Enzyme-linked immunosorbent assay for trkA tyrosine kinase activity.

A 96-well microtiter enzyme-linked immunosorbent assay was developed to assay the activity of the cytoplasmic domain of trkA tyrosine kinase. The assay involves immobilization of phospholipase C- gamma/glutathione S-transferase fusion protein on a microtiter plate, addition of the kinase reaction mixture, and detection by an antibody to phosphotyrosine followed by an alkaline phosphatase-conjugated second antibody. The substrate used in this system, phospholipase C-gamma, is one of several biologically important substrates for the phosphorylation reaction of receptor-linked tyrosine kinases. The assay was then used to characterize kinase inhibitory activities of various small molecules including analogs of K-252a.

Adenosine Triphosphate↗
PubMed1996

TrkC isoforms with inserts in the kinase domain show impaired signaling responses.

The genetic locus for the TrkC/neurotrophin 3 (NT-3) receptor tyrosine kinase encodes multiple isoforms including receptors with inserts in the catalytic domain. This study examines the signaling capabilities of TrkC and related kinase insert isoforms TrkC14 and TrkC25. We show that in PC12 cells expressing both TrkC and TrkA/nerve growth factor (NGF) receptors, different morphological changes occur upon addition of NGF or NT-3. NT-3-treated cells exhibit longer neurites and larger cell bodies as compared to NGF-treated cells. Both TrkC and TrkA mediate qualitatively similar increases in the tyrosine phosphorylation of phospholipase C (PLC)-gamma1, Shc, SNT, and MAPK and the transcription of the c-fos, c-jun, NGFI-A, and NGFI-B immediate early genes. However, the TrkC kinase insert forms fail to stimulate these events. Furthermore, TrkC14 and TrkC25 have only a low intrinsic tyrosine kinase activity, and insertion of the TrkC14 kinase insert into TrkA at an equivalent position results in a dramatic reduction of the kinase activity and signaling capabilities of TrkA. The TrkC14 and -25 isoforms may fail to transmit signals due to their low intrinsic kinase activity and failure to activate and/or tyrosine phosphorylate targets shown to be involved in neurotrophin signal transduction pathways.

Animals↗