A conserved function in phosphatidylinositol metabolism for mammalian Vps13 family proteins.

The Vps13 protein family is highly conserved in eukaryotic cells. In humans, mutations in the gene encoding the family member VPS13A lead to the neurodegenerative disorder chorea-acanthocytosis. In the yeast Saccharomyces cerevisiae, there is just a single version of VPS13, thereby simplifying the task of unraveling its molecular function(s).

Recruitment of actin modifiers to TrkA endosomes governs retrograde NGF signaling and survival.

The neurotrophins NGF and NT3 collaborate to support development of sympathetic neurons. Although both promote axonal extension via the TrkA receptor, only NGF activates retrograde transport of TrkA endosomes to support neuronal survival. Here, we report that actin depolymerization is essential for initiation of NGF/TrkA endosome trafficking and that a Rac1-cofilin signaling module associated with TrkA early endosomes supports their maturation to retrograde transport-competent endosomes.

Trk retrograde signaling requires persistent, Pincher-directed endosomes.

Target-derived neurotrophins use retrogradely transported Trk-signaling endosomes to promote survival and neuronal phenotype at the soma. Despite their critical role in neurotrophin signaling, the nature and molecular composition of these endosomes remain largely unknown, the result of an inability to specifically identify the retrograde signaling entity. Using EGF-bound nanoparticles and chimeric, EGF-binding TrkB receptors, we elucidate Trk-endosomal events involving their formation, processing, retrograde transport, and somal signaling in sympathetic neurons.

Trk activation in the secretory pathway promotes Golgi fragmentation.

Activation of nascent receptor tyrosine kinases within the secretory pathway has been reported, yet the consequences of intracellular activation are largely unexplored. We report that overexpression of the Trk neurotrophin receptors causes accumulation of autoactivated receptors in the ER-Golgi intermediate compartment. Autoactivated receptors exhibit inhibited Golgi-mediated processing and they inhibit Golgi-mediated processing of other co-expressed transmembrane proteins, apparently by inducing fragmentation of the Golgi apparatus.

Pincher-generated Nogo-A endosomes mediate growth cone collapse and retrograde signaling.

Nogo-A is one of the most potent myelin-associated inhibitors for axonal growth, regeneration, and plasticity in the adult central nervous system. The Nogo-A-specific fragment NogoDelta20 induces growth cone collapse, and inhibits neurite outgrowth and cell spreading by activating RhoA. Here, we show that NogoDelta20 is internalized into neuronal cells by a Pincher- and rac-dependent, but clathrin- and dynamin-independent, mechanism.

Trk-signaling endosomes are generated by Rac-dependent macroendocytosis.

Why neurotrophins and their Trk receptors promote neuronal differentiation and survival whereas receptor tyrosine kinases for other growth factors, such as EGF, do not, has been a long-standing question in neurobiology. We provide evidence that one difference lies in the selective ability of Trk to generate long-lived signaling endosomes. We show that Trk endocytosis is distinguished from the classical clathrin-based endocytosis of EGF receptor (EGFR).

Regulation of ras signaling dynamics by Sos-mediated positive feedback.

The RTK-Ras-ERK cascade is a central signaling module implicated in the control of diverse biological processes including cell proliferation, differentiation, and survival. The coupling of RTK to Ras is mediated by the Ras-specific nucleotide-exchange factor Son of Sevenless (Sos), which activates Ras by inducing the exchange of GDP for GTP . Considerable evidence indicates that the duration and amplitude of Ras signals are important determinants in controlling the biological outcome .

Pincher-mediated macroendocytosis underlies retrograde signaling by neurotrophin receptors.

Retrograde signaling by neurotrophins is crucial for regulating neuronal phenotype and survival. The mechanism responsible for retrograde signaling has been elusive, because the molecular entities that propagate Trk receptor tyrosine kinase signals from the nerve terminal to the soma have not been defined. Here, we show that the membrane trafficking protein Pincher defines the primary pathway responsible for neurotrophin retrograde signaling in neurons.

Pincher, a pinocytic chaperone for nerve growth factor/TrkA signaling endosomes.

A central tenet of nerve growth factor (NGF) action that is poorly understood is its ability to mediate cytoplasmic signaling, through its receptor TrkA, that is initiated at the nerve terminal and conveyed to the soma. We identified an NGF-induced protein that we termed Pincher (pinocytic chaperone) that mediates endocytosis and trafficking of NGF and its receptor TrkA. In PC12 cells, overexpression of Pincher dramatically stimulated NGF-induced endocytosis of TrkA, unexpectedly at sites of clathrin-independent macropinocytosis within cell surface ruffles.

Sustained signaling by phospholipase C-gamma mediates nerve growth factor-triggered gene expression.

In contrast to conventional signaling by growth factors that requires their continual presence, a 1-min pulse of nerve growth factor (NGF) is sufficient to induce electrical excitability in PC12 cells due to induction of the peripheral nerve type 1 (PN1) sodium channel gene. We have investigated the mechanism for this triggered signaling pathway by NGF in PC12 cells. Mutation of TrkA at key autophosphorylation sites indicates an essential role for the phospholipase C-gamma (PLC-gamma) binding site, but not the Shc binding site, for NGF-triggered induction of PN1.

Fibroblast growth factor receptor 3 induces gene expression primarily through Ras-independent signal transduction pathways.

Fibroblast growth factor receptors (FGFR) are widely expressed in many tissues and cell types, and the temporal expression of these receptors and their ligands play important roles in the control of development. There are four FGFR family members, FGFR-1-4, and understanding the ability of these receptors to transduce signals is central to understanding how they function in controlling differentiation and development. We have utilized signal transduction by FGF-1 in PC12 cells to compare the ability of FGFR-1 and FGFR-3 to elicit the neuronal phenotype.

Identification of the cytoplasmic regions of fibroblast growth factor (FGF) receptor 1 which play important roles in induction of neurite outgrowth in PC12 cells by FGF-1.

Fibroblast growth factor 1 (FGF-1) induces neurite outgrowth in PC12 cells. Recently, we have shown that the FGF receptor 1 (FGFR-1) is much more potent than FGFR-3 in induction of neurite outgrowth. To identify the cytoplasmic regions of FGFR-1 that are responsible for the induction of neurite outgrowth in PC12 cells, we took advantage of this difference and prepared receptor chimeras containing different regions of the FGFR-1 introduced into the FGFR-3 protein.

Identification of PN1, a predominant voltage-dependent sodium channel expressed principally in peripheral neurons.

Membrane excitability in different tissues is due, in large part, to the selective expression of distinct genes encoding the voltage-dependent sodium channel. Although the predominant sodium channels in brain, skeletal muscle, and cardiac muscle have been identified, the major sodium channel types responsible for excitability within the peripheral nervous system have remained elusive. We now describe the deduced primary structure of a sodium channel, peripheral nerve type 1 (PN1), which is expressed at high levels throughout the peripheral nervous system and is targeted to nerve terminals of cultured dorsal root ganglion neurons.

Activation of codependent transcription factors is required for transcriptional induction of the vgf gene by nerve growth factor and Ras.

Nerve growth factor (NGF) treatment of PC12 cells leads to the elaboration of a neuronal phenotype, including the induction of neuronally expressed genes such as vgf. To study vgf transcription, we have created chimeric vgf/beta-globin genes in which vgf promoter sequences drive the expression of the beta-globin reporter gene or of a chimeric beta-globin gene fused to 3' untranslated vgf gene sequences. We have found that the level of inducibility of the latter construct by NGF resembles that of the endogenous vgf gene.

The fibroblast growth factor receptor-1 is necessary for the induction of neurite outgrowth in PC12 cells by aFGF.

The PC12 subclone, fnr-PC12 cells, is defective in neurite outgrowth in response to acidic fibroblast growth factor (aFGF); however, its response to nerve growth factor (NGF) is normal. Examination of the expression of FGF receptors (FGFRs) revealed that although PC12 cells express FGFR-1, -3, and -4, fnr-PC12 cells have a reduced level of expression of FGFR-1 but not FGFR-3 and -4. Transfection of FGFR-1 into fnr-PC12 cells efficiently restored aFGF-induced neurite outgrowth, whereas transfection of FGFR-3 was much less efficient.

Calcium influx induces neurite growth through a Src-Ras signaling cassette.

We find that calcium influx through voltage-dependent calcium channels causes extensive neurite outgrowth in PC12 cells. The calcium signal transduction pathway promoting neurite outgrowth causes the rapid activation of protein tyrosine kinases, which include Src. Protein tyrosine phosphorylation results in the formation of an Shc/Grb2 complex, leading to Ras activation, MAP kinase activation, and the subsequent induction of the immediate early gene NGFI-A.

A single pulse of nerve growth factor triggers long-term neuronal excitability through sodium channel gene induction.

The continuous presence of nerve growth factor (NGF) is thought to be required for the elaboration of neuronal-like traits in PC12 cells. Surprisingly, we find that a 1 min exposure to NGF is sufficient to engage a longer-term genetic program leading to the acquisition of membrane excitability. Whereas continuous exposure to NGF causes the induction of a family of sodium channels, the effect of a brief exposure is to induce selectively expression of the peripheral nerve-type sodium channel gene PN1, through a distinct signaling pathway requiring immediate-early genes.

Nerve growth factor regulates the abundance and distribution of K+ channels in PC12 cells.

We examined the effect of nerve growth factor (NGF) treatment on expression of a neuronal delayed rectifler K+ channel subtype, Kv2.1 (drk1), in PC12 cells. Anti-Kv2.

Neuronal growth factor regulation of two different sodium channel types through distinct signal transduction pathways.

Neuronal growth factors regulate the expression of voltage-activated sodium current in differentiating sympathetic neurons and PC12 cells. We show that, in PC12 cells, the NGF- and FGF-induced sodium current results from increased expression of two distinct sodium channel types. Sodium current results from the rapid induction of a novel sodium channel transcript, also found in peripheral neurons, and from the long term induction of brain type II/IIA mRNA.

A branched signaling pathway for nerve growth factor is revealed by Src-, Ras-, and Raf-mediated gene inductions.

A myriad of gene induction events underlie nerve growth factor (NGF)-induced differentiation of PC12 cells. To dissect the signal transduction pathways which lead to NGF actions, we have assessed the relative roles of NGF receptor, Src, Ras, and Raf activities in mediating specific gene inductions. We have used the PC12 cell line as well as sublines which inducibly express activated forms of either Src, Ras, or Raf or a dominant inhibitory form of Ras (p21N17 Ras) to study the expression of multiple NGF-inducible mRNAs.

The cytoplasmic raf oncogene induces a neuronal phenotype in PC12 cells: a potential role for cellular raf kinases in neuronal growth factor signal transduction.

The neuron-like differentiation of PC12 cells is induced by nerve growth factor (NGF) through stimulation of a membrane-bound protooncoprotein signaling pathway containing the NGF receptor Trk, the tyrosine kinase Src, and the GTP-binding protein Ras. The Raf-1 and B-raf protooncogenes encode cytoplasmic serine/threonine kinases that are stimulated by NGF in a Ras-dependent manner. To investigate the possible roles of cytoplasmic Raf kinases in eliciting neuronal differentiation, we have expressed the activated Raf-1 oncogene in PC12 cells.

Signal transduction pathways in neuronal differentiation.

New insights into the signal transduction pathways for neuronal growth factors and cell adhesion molecules are affording us a better understanding of the intracellular mechanisms for neuronal differentiation, and of the ways in which the various signals are integrated during this process.

Rapid fibroblast growth factor-induced increases in protein phosphorylation and ornithine decarboxylase activity: regulation by heparin and comparison to nerve growth factor-induced increases.

Fibroblast growth factors (FGFs), like nerve growth factor (NGF), induce morphological differentiation of PC12 cells. This activity of FGF is regulated by glycosaminoglycans. To further understand the mechanisms of FGF and glycosaminoglycan actions in PC12 cells, we studied the regulation of protein phosphorylation and ornithine decarboxylase (ODC) activity by FGF in the presence and absence of heparin.

Ras is essential for nerve growth factor- and phorbol ester-induced tyrosine phosphorylation of MAP kinases.

Treatment of PC12 cells with nerve growth factor (NGF) induces a rapid increase in tyrosine phosphorylation of multiple cellular proteins. Expression of a dominant inhibitory Ras mutant specifically blocked NGF- and TPA-induced tyrosine phosphorylation of two proteins of approximately 42 and 44 kd. Conversely, expression of an oncogenic variant of Ras induced tyrosine phosphorylation of the same 42 and 44 kd proteins.