Dissociation of NGF Induced Signal Transduction from Neurite Elongation by Expression of a Mutant Adaptor Protein v-Crk in PC12 Cells (original) (raw)
Related papers
The adapter protein SH2B1 (SH2-B, PSM) is recruited to multiple ligand-activated receptor tyrosine kinases, including the receptors for nerve growth factor (NGF), insulin, and IGF-I as well as the cytokine receptor-associated Janus kinase family kinases. In this study, we examine SH2B1's function in NGF signaling. We show that depleting endogenous SH2B1 using short hairpin RNA against SH2B1 inhibits NGF-dependent neurite outgrowth, but not NGF-mediated phosphorylation of Akt or ERKs 1/2. SH2B1 has been hypothesized to localize and function at the plasma membrane. We identify a nuclear localization signal within SH2B1 and show that it is required for nuclear translocation of SH2B1. Mutation of the nuclear localization signal has no effect on NGF-induced activation of TrkA and ERKs 1/2 but prevents SH2B1 from enhancing NGF-induced neurite outgrowth. Disruption of SH2B1 nuclear import also prevents SH2B1 from enhancing NGF-induced transcription of genes important for neuronal differentiation, including those encoding urokinase plasminogen activator receptor, and matrix metalloproteinases 3 and 10. Disruption of SH2B1 nuclear export by mutation of its nuclear export sequence similarly prevents SH2B1 enhancement of NGF-induced transcription of those genes. Nuclear translocation of the highly homologous family member SH2B2(APS) was not observed. Together, these data suggest that rather than simply acting as an adapter protein linking signaling proteins to the activated TrkA receptor at the plasma membrane, SH2B1 must shuttle between the plasma membrane and nucleus to function as a critical component of NGF-induced gene expression and neuronal differentiation.
TrkA Tyrosine Residues Involved in NGF-induced Neurite Outgrowth of PC12 Cells
European Journal of Neuroscience, 1995
The proto-oncogene product gp140prototrk (TrkA) is the receptor tyrosine kinase that mediates nerve growth factor-induced neuronal survival and differentiation. In receptor tyrosine kinases, specific intracellular tyrosine residues become phosphorylated after ligand binding and the phosphorylated tyrosines induce the cascade of signal transduction. Here we have identified intracellular tyrosine residues of TrkA involved in nerve growth factor-induced neurite outgrowth of PC12 cells, using site-directed mutagenesis and a PC12 cell line expressing very low levels of endogenous TrkA (PCl Pnr5 cells). We analysed eight conserved intracellular tyrosine residues of TrkA while the three putative autophosphorylation sites conferring tyrosine kinase activity were lefi intact. Five tyrosine residues, Y499, Y643, Y704, Y760 and Y794, in rat TrkA were involved in nerve growth factor-induced neurite outgrowth. None of these tyrosines mediated the full activity of wild-type TrkA, and a pair of these tyrosines, Y760 and Y794, promoted neurite outgrowth in an additive manner. These data indicate that no single tyrosine is sufficient to induce complete neurite outgrowth but the five tyrosine residues Y499, Y643, Y704, Y760 and Y794 cooperate to exhibit the full activity of wild-type TrkA.
Journal of Biological Chemistry, 1999
Csk homologous kinase (CHK), a member of the Csk regulatory tyrosine kinase family, is expressed primarily in brain and hematopoietic cells. The role of CHK in the nervous system is as yet unknown. Using PC12 cells as a model system of neuronal cells, we show that CHK participates in signaling mediated by TrkA receptors. CHK was found to be associated with tyrosine-phosphorylated TrkA receptors in PC12 cells upon stimulation with NGF. Binding assays and far Western blotting analysis, using glutathione S-transferase fusion proteins containing the Src homology 2 (SH2) and SH3 domains of CHK, demonstrate that the SH2 domain of CHK binds directly to the tyrosine-phosphorylated TrkA receptors. Site-directed mutagenesis of TrkA cDNA, as well as phosphopeptide inhibition of the in vitro interaction of the CHK-SH2 domain or native CHK with TrkA receptors, indicated that the residue Tyr-785 on TrkA is required for its binding to the CHK-SH2 domain upon NGF stimulation. In addition, overexpression of CHK resulted in enhanced activation of the mitogen-activated protein kinase pathway upon NGF stimulation, and microinjection of anti-CHK antibodies, but not anti-Csk antibodies, inhibited neurite outgrowth of PC12 cells in response to NGF. Thus, CHK is a novel signaling molecule that participates in TrkA signaling, associates directly with TrkA receptors upon NGF stimulation, and is involved in neurite outgrowth of PC12 cells in response to NGF.
The adapter protein SH2-B has been shown to bind to activated nerve growth factor (NGF) receptor TrkA and has been implicated in NGF-induced neuronal differentiation and the survival of sympathetic neurons. However, the mechanism by which SH2-B enhances and maintains neurite outgrowth is unclear. We examined the ability of truncation mutants to regulate neuronal differentiation and observed that certain truncation mutants localized in the nucleus rather than in the cytoplasm or at the plasma membrane as reported for wild-type SH2-B. Addition of the nuclear export inhibitor leptomycin B caused both overexpressed wild-type and endogenous SH2-B to accumulate in the nucleus of both PC12 cells and COS-7 cells as did deletion of a putative nuclear export sequence (amino acids 224 to 233) or mutation of two critical lysines in that sequence. Deleting or mutating the nuclear export signal caused SH2-B to lose its ability to enhance NGF-induced differentiation of PC12 cells. Neither the NGF-induced phosphorylation of ERKs 1 and 2 nor their subcellular distribution was altered in PC12 cells stably expressing the nuclear export-defective SH2-B(L231A, L233A). These data provide strong evidence that SH2-B shuttles constitutively between the nucleus and cytoplasm. However, SH2-B needs continuous access to the cytoplasm and/or plasma membrane to participate in NGFinduced neurite outgrowth. These data also suggest that the stimulatory effect of SH2-B on NGF-induced neurite outgrowth of PC12 cells is either downstream of ERKs or via some other pathway yet to be identified.
Journal of Cellular Biochemistry, 1998
Activation of receptor tyrosine kinases stimulates a diverse array of cellular responses such as proliferation and differentiation. The first events in the signal transduction pathways mediated by different receptor tyrosine kinases are similar and include activation of the mitogen-activated protein kinase (MAPK) pathway and the induction of immediate early genes. The precise signaling pathways leading to each of the cellular responses mediated by receptor tyrosine kinases are still unknown, although it has been proposed that sustained activation of the MAPK pathway by receptor tyrosine kinases such as the nerve growth factor (NGF) receptor TrkA is sufficient to induce differentiation in PC12 cells. In the present study we examined the effect of NGF on mutant PC12 cells that were derived spontaneously in our cultures. NGF induced normal activation of immediate early genes in these cells, whereas the activation of some delayed response genes, as well as neurite outgrowth, was impaired. Furthermore, activation of the NGF-induced extracellular signal-regulated kinase (ERK) in these cells was transient, not sustained. These results support the hypothesis that sustained activation of ERK plays an important role in activating the induction of delayed response genes. However, sustained ERK activation is not a mandatory condition for the promotion of all the features of differentiated PC12 cells, as NGF could induce transcription of the delayed response gene, transin, in PC12 mutant cells. Taken together, our results suggest that NGF induces differentiation of PC12 cells via several signaling pathways, an important one of which is the MAPK pathway.
Journal of Neurochemistry, 2002
To investigate potential differences between the family of Trk receptors that might have differential consequences on cell signaling, we generated a rat TrkA homologue of the 14-amino acid kinase insert isoform of TrkC termed TrkAKi. Signal transduction by the TrkAKi receptor has been investigated and compared with the homologous signaling defective TrkC (Ku 4) receptor. Herein, we demonstrate that TrkAKi receptors show a decrease in the absolute amount of kinase activity relative to wild-type TrkA, yet retain normal patterns of receptor tyrosine phosphorylation, as determined by phosphopeptide mapping studies, unlike TrkC(Kil 4). nnr5 cell clones expressing TrkAKi receptors show a decrease in nerve growth factor (NGF)-mediated SHC tyrosine phosphorylation and a loss of high-affinity TrkA-SHC interaction comparable to those expressing TrkC(Ki14). Moreover, nnr5 cells expressing TrkAKi receptors fail to demonstrate NGF-dependent tyrosine phosphorylation of the signaling molecules phospholipase Cy-l, MAP kinase/ERK-1, and SNT. TrkAKi receptors internalize NGF comparable to wild-type TrkA, but do not stimulate neurite outgrowth. It is interesting that, unlike TrkC(Ki14), TrkAKi receptors retain phosphatidylinositol 3-kinase activity and nnr5 cells stably expressing TrkAKi receptors retain NGF-dependent cell survival under serum-free conditions. Lastly, TrkAKi receptors fail to stimulate three immediate-early genes (NGF1A, NGF1B, and c-fos), suggesting that these gene products are not required for NGF-dependent cell survival responses.