| 1. |
- Edsjö, Anders, et al.
(författare)
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Differences in early and late responses between neurotrophin-stimulated trkA- and trkC-transfected SH-SY5Y neuroblastoma cells
- 2001
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Ingår i: Cell Growth & Differentiation. - 1044-9523. ; 12:1, s. 39-50
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Tidskriftsartikel (refereegranskat)abstract
- Despite their sympathetic neuroblast origin, highly malignant neuroblastoma tumors and derived cell lines have no or low expression of the neurotrophin receptor genes, trkA and trkC. Expression of exogenous trkA in neuroblastoma cells restores their ability to differentiate in response to nerve growth factor (NGF). Here we show that stable expression of trkC in SH-SY5Y neuroblastoma cells resulted in morphological and biochemical differentiation upon treatment with neurotrophin-3 (NT-3). To some extent, trkA- and trkC-transfected SH-SY5Y (SH-SY5Y/trkA and SH-SY5Y/trkC) cells resembled one another in terms of early signaling events and neuronal marker gene expression, but important differences were observed. Although induced Erk 1/2 and Akt/PKB phosphorylation was stronger in NT-3-stimulated SH-Y5Y/trkC cells, activation of the immediate-early genes tested was more prominent in NGF-treated SH-SY5Y/ trkA cells. In particular, c-fos was not induced in the SH-SY5Y/trkC cells. There were also phenotypic differences. The concentrations of norepinephrine, the major sympathetic neurotransmitter, and growth cone-located synaptophysin, a neurosecretory granule protein, were increased in NGF-treated SH-SY5Y/trkA but not in NT-3-treated SH-SY5Y/trkC cells. Our data suggest that NT-3/p145trkC and NGF/p140trkA signaling differ in some aspects in neuroblasoma cells, and that this may explain the phenotypic differences seen in the long-term neurotrophin-treated cells.
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| 2. |
- Edsjö, Anders, et al.
(författare)
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Expression of trkB in Human Neuroblastoma in Relation to MYCN Expression and Retinoic Acid Treatment.
- 2003
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Ingår i: Laboratory Investigation. - 1530-0307. ; 83:6, s. 813-823
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Tidskriftsartikel (refereegranskat)abstract
- Expression of full-length trkB can be found in some highly malignant neuroblastoma tumors with an amplified MYCN gene. This contrasts sympathetic neuroblasts, from which neuroblastomas are thought to arise, which neither express trkB nor are dependent on the p145trkB ligands, brain-derived neurotrophic factor (BDNF) or neurotrophin-4/5, for their normal development. In this study we show that trkB was expressed in two out of five neuroblastoma tumors with amplified MYCN, while no trkB expression was observed when the MYCN gene was overexpressed in a non–MYCN-amplified neuroblastoma cell line. This shows that MYCN overexpression per se is not sufficient to induce trkB expression. trkB expression and BDNF responsiveness in neuroblastoma cells can be induced by all-trans-retinoic acid (RA). When SH-SY5Y cells were stimulated with a combination of RA and BDNF, norepinephrine and tyrosine hydroxylase levels were unaltered, showing that the cells did not change toward a more catecholaminergic sympathetic phenotype. However, expression of growth-associated protein 43, indicative of a neuronal phenotype, was elevated. Vesicular acetylcholine transporter, choline acetyl transferase, and neuropeptide tyrosine mRNA levels also increased in RA-BDNF–treated cells, which could suggest that these cells develop into a sympathetic cholinergic phenotype. In addition, treatment with RA-induced expression of the platelet-derived growth factor receptor-alpha. As previously shown for BDNF, platelet-derived growth factor stimulated growth of the RA-treated cells, findings that could have clinical relevance. If these receptors mediate a mitogenic signal in vivo also, this might limit the effect of RA treatment on neuroblastoma patients.
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| 3. |
- Larsen, EC, et al.
(författare)
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A role for PKC-epsilon in Fc gamma R-mediated phagocytosis by RAW 264.7 cells
- 2002
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Ingår i: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 159:6, s. 939-944
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Tidskriftsartikel (refereegranskat)abstract
- Protein kinase C (PKC) plays a prominent role in immune signaling, and the paradigms for isoform selective signaling are beginning to be elucidated. Real-time microscopy was combined with molecular and biochemical approaches to demonstrate a role for PKC-epsilon in Fcgamma receptor (FcgammaR)-dependent phagocytosis. RAW 264.7 macrophages were transfected with GFP-conjugated PKC isoforms, and GFP movement was followed during phagocytosis of fluorescent IgG-opsonized beads. PKC-epsilon, but not PKC-delta, concentrated around the beads. PKC-epsilon accumulation was transient; apparent as a "flash" on target ingestion. Similarly, endogenous PKC-epsilon was specifically recruited to the nascent phagosomes in a time-dependent manner. Overexpression of PKC-epsilon, but not PKC-alpha, PKC-delta, or PKC-gamma enhanced bead uptake 1.8-fold. Additionally, the rate of phagocytosis in GFP PKC-epsilon expressors was twice that of cells expressing GFP PKC-delta. Expression of the regulatory domain (ERD) and the first variable region (epsilonV1) of PKC-epsilon inhibited uptake, whereas the corresponding PKC-delta region had no effect. Actin polymerization was enhanced on expression of GFP PKC-epsilon and ERD, but decreased in cells expressing epsilonV1, suggesting that the epsilonRD and epsilonV1 inhibition of phagocytosis is not due to effects on actin polymerization. These results demonstrate a role for PKC-epsilon in FcgammaR-mediated phagocytosis that is independent of its effects on actin assembly.
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| 4. |
- Lennartsson, Johan, 1972-
(författare)
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Stem Cell Factor Induced Signal Transduction
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Stem Cell Factor (SCF) can function as a survival factor, a mitogen or a chemoattractant depending on cell type. Binding of SCF to c-Kit induces dimerization and subsequent autophosphorylation of the receptor. This thesis describes the intracellular signal transduction elicited by c-Kit. In the search for signal transduction molecules binding to activated c-Kit, we identified the adaptor proteins Grb2 and Grb7 as interacting partners. Grb2 could associate to Tyr-703 in the kinase insert as well as to Tyr-936 in the C-terminal tail of c-Kit. However, Grb7 could only bind to Tyr-936. Tyr-568 in c-Kit is essential for SCF induced association and activation of Src family kinases (SFK). A mutated receptor that could not activate SFK (Y568F or Y568/570F) showed reduced Shc phosphorylation, Ras GTP loading, Erk activation and induction of c-fos. However, activation of SFK is not essential for the mitogenic response as measured by DNA synthesis.Tyr-900 in c-Kit was identified as a SFK dependent phosphorylation site. The adaptor protein Crk and the p85 subunit of PI3’-kinase could associate with phosphorylated Tyr-900. In addition, we could demonstrate a constitutive complex between Crk and p85, suggesting indirect binding of Crk to Tyr-900 via p85. Mutation of Tyr-900 (Y900F) led to a reduced phosphorylation of Crk-II, loss of the second wave of Erk phosphorylation and a reduced mitogenic response. In addition the mutated receptor showed an increased ligand-induced degradation as compared to the wild-type receptor.There exist two splice forms of c-Kit that differ in the presence or absence of four amino acids in the extracellular juxtamembrane region. These splice forms bind SCF with similar affinity but display striking differences in signaling characteristics, e.g. in phosphorylation kinetics, ligand-induced c-Kit degradation and activation of Erks. However, other pathways are activated similarly by both splice forms, such as the Ser/Thr kinase Akt which lies downstream of PI3’-kinase. In this study we show that differential phosphorylation of the various tyrosine residues occurs. Interestingly, Tyr-568 is more efficiently phosphorylated in the shorter form leading to stronger binding of SFK, whereas the PI3’-kinase binding site showed a similar degree of phosphorylation consistent with the data on Akt activation.
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| 5. |
- Ling, Mia, et al.
(författare)
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Induction of neurites by the regulatory domains of PKCdelta and epsilon is counteracted by PKC catalytic activity and by the RhoA pathway.
- 2004
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 1090-2422 .- 0014-4827. ; 292:1, s. 135-150
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Tidskriftsartikel (refereegranskat)abstract
- We have shown that protein kinase C (PKC) var epsilon, independently of its kinase activity, via its regulatory domain (RD), induces neurites in neuroblastoma cells. This study was designed to evaluate whether the same effect is obtained in nonmalignant neural cells and to dissect mechanisms mediating the effect. Overexpression of PKCvar epsilon resulted in neurite induction in two immortalised neural cell lines (HiB5 and RN33B). Phorbol ester potentiated neurite outgrowth from PKCvar epsilon-overexpressing cells and led to neurite induction in cells overexpressing PKCδ. The effects were potentiated by blocking the PKC catalytic activity with GF109203X. Furthermore, kinase-inactive PKCδ induced more neurites than the wild-type isoform. The isolated regulatory domains of novel PKC isoforms also induced neurites. Experiments with PKCδ-overexpressing HiB5 cells demonstrated that phorbol ester, even in the presence of a PKC inhibitor, led to a decrease in stress fibres, indicating an inactivation of RhoA. Active RhoA blocked PKC-induced neurite outgrowth, and inhibition of the RhoA effector ROCK led to neurite outgrowth. This demonstrates that neurite induction by the regulatory domain of PKCδ can be counteracted by PKCδ kinase activity, that PKC-induced neurite outgrowth is accompanied by stress fibre dismantling indicating an inactivation of RhoA, and that the RhoA pathway suppresses PKC-mediated neurite outgrowth.
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| 6. |
- Massoumi, Ramin, et al.
(författare)
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Leukotriene D-4 induces stress-fibre formation in intestinal epithelial cells via activation of RhoA and PKC delta
- 2002
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Ingår i: Journal of Cell Science. - 0021-9533. ; 115:17, s. 3509-3515
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Tidskriftsartikel (refereegranskat)abstract
- The intestinal epithelial barrier, which is regulated by the actin cytoskeleton, exhibits permeability changes during inflammation. Here we show that activation of the CysLT(1) receptor by the inflammatory mediator leukotriene D-4 (LTD4) causes a rapid increase in stress-fibre formation in intestinal epithelial cells. This effect was mimicked by cytotoxic necrotising factor-1 (CNF-1)-induced activation of RhoA, overexpression of constitutively active RhoA (L63-RhoA) and phorbol-ester-induced activation of protein kinase C (PKC). In accordance, inhibition of RhoA, by C3 exoenzyme or by dominant-negative RhoA (N19-RhoA), as well as GF109203X-induced inhibition of PKC, suppressed the LTD4-induced stress-fibre formation. Introduction of the dominant-negative regulatory domain of PKCdelta, but not the corresponding structures from PKCalpha, betaII or epsilon, blocked the LTD4-induced stress-fibre formation. Evaluating the relationship between PKCdelta and RhoA in LTD4-induced stress-fibre formation, we found that C3 exoenzyme inhibited the rapid LTD4-elicited translocation of PKCdelta to the plasma membrane. Furthermore, CNF-1-induced stress-fibre formation was blocked by GF109203X and by overexpression of the regulatory domain of PKC-delta, whereas PKC-induced stress-fibre production was not affected by N19-RhoA. We conclude that PKC-delta is located downstream of RhoA and that active RhoA and PKCdelta are both necessary for LTD4-induced stress-fibre formation.
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| 7. |
- Paruchuri, Sailaja, et al.
(författare)
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Leukotriene D-4 activates MAPK through a Ras-independent but PKC epsilon-dependent pathway in intestinal epithelial cells
- 2002
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Ingår i: Journal of Cell Science. - 0021-9533. ; 115:9, s. 1883-1893
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Tidskriftsartikel (refereegranskat)abstract
- We have recently shown that leukotriene D-4 (LTD4) increases cell survival in intestinal epithelial cells. Here we report and explore the complementary finding that LTD4 also enhances proliferation in these cells. This proliferative response was approximately half of that induced by epidermal growth factor (EGF) and its required activation of protein kinase C (PKC), Ras and the mitogen-activated protein kinase (MAPK) Erk-1/2. EGF also activated Erk-1/2 in these cells; however the EGF-receptor inhibitor PD153035 did not affect the LTD4-induced activation of Erk-1/2. In addition, LTD4 did not induce phosphorylation of the EGF receptor, nor did pertussis toxin (PTX) block EGF-induced activation of Erk-1/2, thus refuting a possible crosstalk between the receptors. Furthermore, LTD4-induced, but not EGF-Induced, activation of Erk-1/2 was sensitive to PTX, PKC inhibitors and downregulation of PKCepsilon. A definite role for PKCepsilon in LTD4-induced stimulation of Erk-1/2 was documented by the inability of LTD4 to activate Erk-1/2 in cells transfected with either the regulatory domain of PKCepsilon (an isoform specific dominant-negative inhibitor) or a kinase-dead PKCepsilon Although Ras and Raf-1 were both transiently activated by LTD4, only Raf-1 activation was abolished by abrogation of the PKC signal. Furthermore, the LTD4-induced activation of Erk-1/2 was unaffected by transfection with dominant-negative N17 Ras but blocked by transfection with kinase-dead Raf-1. Consequently, LTD4 regulates the proliferative response by a distinct Ras-independent, PKCepsilon-dependent activation of Erk-1/2 and a parallel Ras-dependent signaling pathway.
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| 8. |
- Schultz, Anna, et al.
(författare)
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Identification of an amino acid residue in the PKC C1b domain crucial for its localisation to the Golgi network.
- 2004
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 279:30, s. 31750-31760
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Tidskriftsartikel (refereegranskat)abstract
- Protein kinase C (PKC) isoforms have been reported to be targeted to the Golgi complex via their C1 domains. We have shown recently that the regulatory domain of PKCtheta induces apoptosis in neuroblastoma cells and that this effect is correlated to Golgi localization via the C1b domain. This study was designed to identify specific residues in the C1 domains that mediate Golgi localization. We demonstrate that the isolated C1b domains from PKCalpha, -delta, -epsilon, -eta, and -theta are targeted to the Golgi complex, whereas the corresponding C1a domains localize throughout the cell. Sequence alignment showed that amino acid residues corresponding to Glu-246 and Met-267 in PKCtheta are conserved among C1b but absent from C1a domains. Mutation of Met-267, but not of Glu-246, to glycine abolished the Golgi localization of the isolated C1b domain and the regulatory domain of PKCtheta. The mutated PKCtheta regulatory domain constructs lacking Golgi localization were unable to induce apoptosis, suggesting a direct correlation between Golgi localization and apoptotic activity of PKCtheta regulatory domain. Mutation of analogous residues in the C1b domain of PKCepsilon abrogated its Golgi localization, demonstrating that this effect is not restricted to one PKC isoform. The abolished Golgi localization did not affect neurite induction by PKCepsilon. However, the PKCepsilon mutant did not relocate to the Golgi network in response to ceramide and ceramide did not suppress the neurite-inducing capacity of the protein. Thus, the specific mutations in the C1b domain influence both the localization and function of full-length PKCepsilon.
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| 9. |
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| 10. |
- Stensman, Helena, et al.
(författare)
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Autophosphorylation suppresses, whereas kinase inhibition augments, the translocation of PKCa in response to diacylglycerol.
- 2004
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 279:39, s. 40576-40583
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Tidskriftsartikel (refereegranskat)abstract
- We have seen that protein kinase Calpha (PKCalpha) is transiently translocated to the plasma membrane by carbachol stimulation of neuroblastoma cells. This is induced by the Ca2+ increase, and PKCalpha does not respond to diacylglycerol (DAG). The unresponsiveness is dependent on structures in the catalytic domain of PKCalpha. This study was designed to investigate if and how the kinase activity and autophosphorylation are involved in regulating the translocation. PKCalpha enhanced green fluorescent protein translocation was studied in living neuroblastoma cells by confocal microscopy. Carbachol stimulation induced a transient translocation of PKCalpha to the plasma membrane and a sustained translocation of kinase-dead PKCalpha. In cells treated with the PKC inhibitor GF109203X, wild-type PKCalpha also showed a sustained translocation. The same effects were seen with PKCbetaI, PKCbetaII, and PKCdelta. Only kinase-dead and not wild-type PKCalpha translocated in response to 1,2-dioctanoylglycerol. To examine whether autophosphorylation regulates relocation to the cytosol, the autophosphorylation sites in PKCalpha were mutated to glutamate, to mimic phosphorylation, or alanine, to mimic the nonphosphorylated protein. After stimulation with carbachol, glutamate mutants behaved like wild-type PKCalpha, whereas alanine mutants behaved like kinase-dead PKCalpha. When the alanine mutants were treated with 1,2-dioctanoylglycerol, all cells showed a sustained translocation of the protein. However, neither carbachol nor GF109203X had any major effects on the level of autophosphorylation, and GF109203X potentiated the translocation of the glutamate mutants. We, therefore, hypothesize that 1) autophosphorylation of PKCalpha limits its sensitivity to DAG and 2) that kinase inhibitors augment the DAG sensitivity of PKCalpha, perhaps by destabilizing the closed conformation.
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