Nerve growth factor regulates both the phosphorylation and steady-state levels of microtubule-associated protein 1.2 (MAP1.2)

This study characterizes effects of nerve growth factor (NGF) on the steady-state level and phosphorylation of a high molecular mass microtubule-associated protein in PC12 rat pheochromocytoma cells. Past work showed that NGF significantly raises the relative levels of this phosphoprotein, designated MAP1.2, with a time course similar to that of neurite outgrowth. To study this in greater detail, MAP1.2 in PC12 cell lysates was resolved by SDS-PAGE in gels containing 3.25% acrylamide/4 M urea and identified by comigration with material immunoprecipitated from the lysates by MAP1 antibodies. Quantification by metabolic radiolabeling with [35S]methionine or by silver staining revealed a 3.0-3.5-fold increase in MAP1.2 levels relative to total cell protein after NGF treatment for 2 wk or longer. A partial increase was detectable after 3 d, but not after 2 h of NGF exposure. As measured by incorporation of [32P]phosphate, NGF had a dual effect on MAP1.2. Within 15 min to 2 h, NGF enhanced the incorporation of phosphate into MAP1.2 by two- to threefold relative to total cell phosphoproteins. This value slowly increased thereafter so that by 2 wk or more of NGF exposure, the average enhancement of phosphate incorporation per MAP1.2 molecule was over fourfold. The rapid action of NGF on MAP1.2 could not be mimicked by either epidermal growth factor, a permeant cAMP derivative, phorbol ester, or elevated K+, each of which alters phosphorylation of other PC12 cell proteins. SDS-PAGE revealed multiple forms of MAP1.2 which, based on the effects of alkaline phosphatase on their electrophoretic mobilities, differ, at least in part, in extent of phosphorylation. Before NGF treatment, most PC12 cell MAP1.2 is in more rapidly migrating, relatively poorly phosphorylated forms. After long-term NGF exposure, most is in more slowly migrating, more highly phosphorylated forms. The effects of NGF on the rapid phosphorylation of MAP1.2 and on the long-term large increase in highly phosphorylated MAP1.2 forms could play major functional roles in NGF-mediated neuronal differentiation. Such roles may include effects on microtubule assembly, stability, and cross-linking and, possibly for the rapid effects, nuclear signaling.     

Nerve growth factor and fibroblast growth factor selectively activate a protein kinase that phosphorylates high molecular weight microtubule-associated proteins. Detection, partial purification, and characterization in PC12 cells

A cell-free assay has been developed to detect and characterize a nerve growth factor (NGF)-stimulated protein kinase activity in PC12 cells that phosphorylates high molecular weight microtubule-associated proteins (HMW-MAPs). The activity was partially purified and separated from other endogenous nonregulated HMW-MAP kinase activities by chromatography on heparin-Sepharose and Mono-Q resin. Characterization of the NGF-activated kinase (designated HMK) revealed the following features. 1) Both MAP1 and MAP2 are phosphorylated with approximately equal efficiencies. 2) Activation reaches a plateau within 3 min of NGF treatment and persists for approximately 60 min; subsequently, a substantial decline occurs by 5 h. 3) Maximal activation reaches 15-20-fold; activation is nearly as high with fibroblast growth factor, an agent that mimics NGF in promoting PC12 cell neuronal differentiation. 4) Epidermal growth factor and depolarizing levels of K+ stimulate HMK activity by only 2-4-fold; additional agents without PC12 cell differentiation activity (insulin, phorbol ester, and a permeant cAMP analogue) do not stimulate HMK activity. 5) The divalent cation requirement shows a preference for Mn2+ over Mg2+. 6) There is inhibition by 10 mM 2-aminopurine but not by 6-thioguanine, heparin, or NaF. 7) HMW-MAPs and myelin basic protein are effective substrates while histones IIIs and H1, dephospho-beta-casein, and S6 protein are not phosphorylated by HMK. These and other features appear to distinguish HMK from a variety of other well-characterized protein kinases as well as from other previously described NGF-activated kinases. The properties of HMK indicate that it could play a role in the signaling pathway for growth-factor-promoted neuronal differentiation.     

The phosphorylation of stathmin by MAP kinase

Stathmin, a ubiquitous cytosolic phosphoprotei which may play a role in integrating the effects of diverse signals regulating proliferation, differentiation and other cell functions, was found to be phosphorylated rapidly and stoichiometrically by mitogen-activated protein (MAP) kinasein vitro. Ser-25 was identified as the major site and Ser-38 as a minor site of phosphorylation, while the p42 and p44 isoforms of MAP kinase were the only significant stathmin kinases detected in PC12 cells after stimulation by nerve growth factor (NGF). The results suggest that MAP kinases are the enzymes responsible for increasing the level of phosphorylation of Ser-25, which has been observed previously in PC12 cells following stimulation by NGF.Submitted February 1993.     

ATP enhances neuronal differentiation of PC12 cells by activating PKCα interactions with cytoskeletal proteins

PKCα is a key mediator of the neuronal differentiation controlled by NGF and ATP. However, its downstream signaling pathways remain to be elucidated. To identify the signaling partners of PKCα, we analyzed proteins coimmunoprecipitated with this enzyme in PC12 cells differentiated with NGF and ATP and compared them with those obtained with NGF alone or growing media. Mass spectrometry analysis (LC-MS/MS) identified plectin, peripherin, filamin A, fascin, and β-actin as potential interacting proteins. The colocalization of PKCα and its interacting proteins increased when PC12 cells were differentiated with NGF and ATP. Peripherin and plectin organization and the cortical remodeling of β-actin were dramatically affected when PKCα was down-regulated, suggesting that all three proteins might be functional targets of ATP-dependent PKCα signaling. Taken together, these data demonstrate that PKCα is essential for controlling the neuronal development induced by NGF and ATP and interacts with the cytoskeletal components at two levels: assembly of the intermediate filament peripherin and organization of cortical actin.     

The beta-PDGF receptor induces neuronal differentiation of PC12 cells.

Expression of the mouse beta-PDGF receptor by gene transfer confers PDGF-dependent and reversible neuronal differentiation of PC12 pheochromocytoma cells similar to that observed in response to NGF and basic FGF. A common property of the PDGF, NGF, and basic FGF-induced differentiation response is the requirement for constant exposure of cells to the growth factor. To test the hypothesis that a persistent level of growth factor receptor signaling is required for the maintenance of the neuronal phenotype, we examined the regulation of the serine/threonine-specific MAP kinases after either short- (10 min) or long-term (24 h) stimulation with growth factors. Mono Q FPLC resolved two peaks of growth factor-stimulated MAP kinase activity that coeluted with tyrosine phosphorylated 41- and 43-kDa polypeptides. MAP kinase activity was markedly stimulated (approximately 30-fold) within 5 min of exposure to several growth factors (PDGF, NGF, basic FGF, EGF, and IGF-I), but was persistently maintained at 10-fold above basal activity after 24 h only by the growth factors that also induce PC12 cell differentiation (PDGF, NGF, and basic FGF). Thus the beta-PDGF receptor is in a subset of tyrosine kinase-encoded growth factor receptors that are capable of maintaining continuous signals required for differentiation of PC12 cells. These signals include the constitutive activation of cytoplasmic serine/threonine protein kinases.     

Peripherin Is Tyrosine-Phosphorylated at Its Carboxyl-Terminal Tyrosine

Abstract: Peripherin is a type III intermediate filament present in peripheral and certain CNS neurons. We report here that peripherin contains a phosphotyrosine residue and, as such, is the only identified intermediate filament protein known to be modified in this manner. Antiserum specific for phosphotyrosine recognizes peripherin present in PC12 cells (with or without nerve growth factor treatment) and in rat sciatic nerve as well as that expressed in Sf-9 cells and SW-13 cl. 2 vim⁻ cells. The identity of peripherin as a tyrosine-phosphorylated protein in PC12 cells was confirmed by immunoprecipitation, two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and phosphoamino acid analysis. Unlike serine/threonine phosphorylation, tyrosine phosphorylation of peripherin is not regulated by depolarization or nerve growth factor treatment. To identify the site of tyrosine phosphorylation, rat peripherin was mutated at several tyrosine residues and expressed in SW-13 cl. 2 vim⁻ cells. Tyrosine phosphorylation was selectively lost only for peripherin mutants in which the carboxy-terminal tyrosine (Y474) was mutated. Indirect immunofluorescence staining indicated that both wild-type peripherin and peripherin Y474F form a filamentous network in SW-13 cl. 2 vim⁻ cells. This indicates that tyrosine phosphorylation of the peripherin C-terminal residue is not required for assembly and leaves open the possibility that this modification serves other functions.     

Interleukin-6 Induces Expression of Peripherin and Cooperates with Trk Receptor Signaling to Promote Neuronal Differentiation in PC12 Cells

Abstract: In contrast to the intensively studied nerve growth factor (NGF)-related family of cytokines, relatively little is known about the mechanisms of neurotrophic activity elicited by the cytokine interleukin-6 (IL-6). We have examined the mechanisms of IL-6-induced neuronal differentiation of the pheochromocytoma cell line PC12. IL-6 independently induced the expression of peripherin , identifying this gene as the first neuronal-specific target of IL-6. However, IL-6 alone failed to elicit neurite outgrowth in PC12 cells and instead required low levels of Trk/NGF receptor tyrosine kinase activity to induce neuronal differentiation. The cooperating Trk signal could be provided by either overexpression of Trk or exposure to low concentrations of NGF. IL-6 also functioned cooperatively with basic fibroblast growth factor to promote PC12 differentiation. IL-6 and Trk/NGF synergized in enhancing tyrosine phosphorylation of the Erk-1 mitogen-activated protein kinase and in activating expression of certain NGF target genes. NGF also induced expression of the gp80 subunit of the IL-6 receptor, providing another potential mechanism of cooperativity between NGF and IL-6 signaling. We propose that IL-6 functions as an enhancer of NGF signaling rather than as an autonomous neuronal differentiation signal. Moreover, our results demonstrate that a Trk receptor-specific cellular response can be achieved in the absence of NGF through amplification of its basal signaling activity by the IL-6 receptor system.     

Thrombopoietin inhibits nerve growth factor-induced neuronal differentiation and ERK signalling

Thrombopoietin (TPO), a hematopoietic growth factor regulating platelet production, and its receptor (TPOR) were recently shown to be expressed in the brain where they exert proapoptotic activity. Here we used PC12 cells, an established model of neuronal differentiation, to investigate the effects of TPO on neuronal survival and differentiation. These cells expressed TPOR mRNA. TPO increased cell death in neuronally differentiated PC12 cells but had no effect in undifferentiated cells. Surprisingly, TPO inhibited nerve growth factor (NGF)-induced differentiation of PC12 cells in a dose- and time-dependent manner. This inhibition was dependent on the activity of Janus kinase-2 (JAK2). Using phospho-kinase arrays and Western blot we found downregulation of the NGF-stimulated phosphorylation of the extracellular signal-regulated kinase p42ERK by TPO with no effect on phosphorylation of Akt or stress kinases. NGF-induced phosphorylation of ERK-activating kinases, MEK1/2 and C-RAF was also reduced by TPO while NGF-induced RAS activation was not attenuated by TPO treatment. In contrast to its inhibitory effects on NGF signalling, TPO had no effect on epidermal growth factor (EGF)-stimulated ERK phosphorylation or proliferation of PC12 cells. Our data indicate that TPO via activation of its receptor-bound JAK2 delays the NGF-dependent acquisition of neuronal phenotype and decreases neuronal survival by suppressing NGF-induced ERK activity.     

Identification of peripherin as a Akt substrate in neurons

Activation of Akt-mediated signaling pathways is crucial for survival and regeneration of injured neurons. In this study, we attempted to identify novel Akt substrates by using an antibody that recognized a consensus motif phosphorylated by Akt. PC12 cells that overexpressed constitutively active Akt were used. Using two-dimensional PAGE, we identified protein spots that exhibited increased immunostaining of the antibody. Mass spectrometry revealed several major spots as the neuronal intermediate filament protein, peripherin. Using several peripherin fragments, the phosphorylation site was determined as Ser(66) in its head domain in vitro. Furthermore, a co-immunoprecipitation experiment revealed that Akt interacted with the head domain of peripherin in HEK 293T cells. An antibody against phosphorylated peripherin was raised, and induction of phosphorylated peripherin was observed not only in Akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. These results suggest that peripherin is a novel substrate for Akt in vivo and that its phosphorylation may play a role in motor nerve regeneration.     

SNT, a differentiation-specific target of neurotrophic factor-induced tyrosine kinase activity in neurons and PC12 cells.

To elucidate the signal transduction mechanisms used by ligands that induce differentiation and the cessation of cell division, we utilized p13suc1-agarose, a reagent that binds p34cdc2/cdk2. By using this reagent, we identified a 78- to 90-kDa species in PC12 pheochromocytoma cells that is rapidly phosphorylated on tyrosine following treatment with the differentiation factors nerve growth factor (NGF) and fibroblast growth factor but not by the mitogens epidermal growth factor or insulin. This species, called SNT (suc-associated neurotrophic factor-induced tyrosine-phosphorylated target), was also phosphorylated on tyrosine in primary rat cortical neurons treated with the neurotrophic factors neurotrophin-3, brain-derived neurotrophic factor, and fibroblast growth factor but not in those treated with epidermal growth factor. In neuronal and fibroblast cells, where NGF can also act as a mitogen, SNT was tyrosine phosphorylated to a much greater extent during NGF-induced differentiation than during NGF-induced proliferation. SNT was phosphorylated in vitro on serine, threonine, and tyrosine in p13suc1-agarose precipitates from NGF-treated PC12 cells, indicating that this protein may be a substrate of kinase activities associated with p13suc1-p34cdc2/cdk2 complexes. In addition, SNT was associated predominantly with nuclear fractions following subcellular fractionation of NGF-treated PC12 cells. Finally, in PC12 cells, NGF-stimulated tyrosine phosphorylation of SNT was dependent on the levels of Trk tyrosine kinase activity and was constitutively induced by expression of pp60v-src. However, Ras was not required for constitutive SNT tyrosine phosphorylation, suggesting that this protein functions distally to Trk and pp60v-src but in a pathway parallel to that of Ras. SNT is the first identified specific target of differentiation factor-induced tyrosine kinase activity in neuronal cells.     

Cross talk among tyrosine kinase receptors in PC12 cells: desensitization of mitogenic epidermal growth factor receptors by the neurotrophic factors, nerve growth factor and basic fibroblast growth factor.

We have studied the effects of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) on epidermal growth factor (EGF) binding to PC12 cells. We show that NGF and bFGF rapidly induce a reduction in 125I-EGF binding to PC12 cells in a dose-dependent manner. This decrease amounts to 50% for NGF and 35% for bFGF. Both factors appear to act through a protein kinase C(PKC)-independent pathway, because their effect persists in PKC-downregulated PC12 cells. Scatchard analysis indicates that NGF and bFGF decrease the number of high affinity EGF binding sites. In addition to their effect on EGF binding, NGF and bFGF activate in intact PC12 cells one or several serine/threonine kinases leading to EGF receptor threonine phosphorylation. Using an in vitro phosphorylation system, we show that NGF- or bFGF-activated extracellular regulated kinase 1 (ERK1) is able to phosphorylate a kinase-deficient EGF receptor. Phosphoamino acid analysis indicates that this phosphorylation occurs mainly on threonine residues. Furthermore, two comparable phosphopeptides are observed in the EGF receptor, phosphorylated either in vivo after NGF treatment or in a cell-free system by NGF-activated ERK1. Finally, a good correlation was found between the time courses of ERK1 activation and 125I-EGF binding inhibition after NGF or bFGF treatment. In conclusion, in PC12 cells the NGF- and bFGF-stimulated ERK1 appears to be involved in the induction of the threonine phosphorylation of the EGF receptor and the decrease in the number of high affinity EGF binding sites.     

Relationship Between the Nerve Growth Factor-Regulated Clone 73 Gene Product and the 58-Kilodalton Neuronal Intermediate Filament Protein (Peripherin)

Exposure of PC12 cells to nerve growth factor (NGF) has been shown to induce an mRNA that encodes a novel neuronal intermediate filament protein. The findings presented here concern the identity of this filament protein. The major protein in NGF-treated PC12 cell cytoskeletons derived by extraction with 1% Triton X-100 is of apparent Mr = 58,000, focuses by isoelectric focusing as several closely spaced spots of pl 5.6-5.8, and is elevated relative to non-NGF-treated cells. Partial microsequencing of this material reveals 2 internal sequences that are identical to a 14-residue sequence encoded by the NGF-regulated clone 73 mRNA, but not to sequences of other known proteins. An antiserum raised against a 19-residue synthetic peptide corresponding to the deduced C-terminus of the protein encoded by the NGF-regulated clone 73 mRNA specifically recognizes the 58,000-Mr protein. Properties of the 58-kilodalton protein strongly suggest that it corresponds to an intermediate filament protein (peripherin) previously identified in PC12 cells and in peripheral and certain CNS neurons. Identification of the intermediate filament protein encoded by an NGF-induced message should facilitate studies of its regulation and function.     

Phosphorylation of peripherin, an intermediate filament protein, in mouse neuroblastoma NIE 115 cell line and in sympathetic neurons

Peripherin, an intermediate filament protein, described recently, is expressed in well defined neuronal populations. We studied the phosphorylation, in vivo, of this protein in mouse neuroblastoma NIE 115 cell line and in sympathetic neurons labelled with [32P]-orthophosphate. The autoradiograms of proteins separated on two-dimensional polyacrylamide gels were compared with the Coomassie-blue stainings. The results show that peripherin occurs as a mixture of phosphorylated and non-phosphorylated isoforms, and that these forms coexist in both differentiated and non-differentiated cells. We demonstrate by cleavage at the unique tryptophan residue, a characteristic shared by most other intermediate filament proteins (IFP), that the phosphorylation sites are located on the amino-terminal half of peripherin as it is for vimentin and desmin. These results are discussed in relation to the organization of the filamentous network constituted by peripherin.     

Nerve growth factor-induced phosphorylation of SNAP-25 in PC12 cells: a possible involvement in the regulation of SNAP-25 localization

Synaptosomal-associated protein of 25 kDa (SNAP-25), a t-SNARE protein essential for neurotransmitter release, is phosphorylated at Ser187 following activation of cellular protein kinase C by treatment with phorbol 12-myristate 13-acetate. However, it remains unclear whether neuronal activity or an endogenous ligand induces the phosphorylation of SNAP-25. Here we studied the phosphorylation of SNAP-25 in PC12 cells using a specific antibody for SNAP-25 phosphorylated at Ser187. A small fraction of SNAP-25 was phosphorylated when cells were grown in the absence of nerve growth factor (NGF). A brief treatment with NGF that was enough to activate the mitogen-activated protein kinase signal transduction pathway did not increase the phosphorylation of SNAP-25; however, phosphorylation was up-regulated after a prolonged incubation with NGF. Up-regulation was transitory, and maximum phosphorylation (a fourfold increase over basal phosphorylation) was achieved between 36 and 48 h after the addition of NGF. Immunofluorescent microscopy showed that SNAP-25 was localized primarily in the plasma membrane, although a significant population was also present in the cytoplasm. Quantitative microfluorometry revealed that prolonged treatment with NGF resulted in a preferential localization of SNAP-25 in the plasma membrane. A mutational study using a fusion protein with green fluorescent protein as a tag indicated that the point mutation of Ser187 to Ala abolished the NGF-dependent relocalization. A population of SNAP-25 in the plasma membrane was not increased by a point mutation at Ser187 to Glu; however, it was increased by prolonged treatment with NGF, indicating that the SNAP-25 phosphorylation is essential, but not sufficient, for the NGF-induced relocation to the plasma membrane. Our results suggest a close temporal relationship between the up-regulation of SNAP-25 phosphorylation and its relocation, and NGF-induced differentiation of PC12 cells.     

Characterization of a Nerve Growth Factor-Stimulated Protein Kinase in PC12 Cells Which Phosphorylates Microtubule-Associated Protein 2 and pp250

Treatment of PC12 cells with nerve growth factor (NGF) resulted in the rapid, but transient, activation of a protein kinase which specifically phosphorylated an endogenous 250-kDa cytoskeletal protein (pp250). We report that the microtubule-associated protein, MAP2, is an alternative substrate for the NGF-activated kinase. NGF treatment maximally activated the kinase within 5 min; however, the activity declined with longer exposure to NGF. The enzyme was localized predominantly in microsomal and soluble fractions and phosphorylated MAP2 on serine and threonine residues. The soluble enzyme was fractionated by DEAE chromatography and gel filtration and had an apparent Mr of 45,000. The enzyme was purified to near homogeneity by chromatofocussing and had a pI of 4.9. Kinetic analysis revealed that NGF treatment caused a sevenfold increase in Vmax for MAP2. The Km with respect to the MAP2 substrate was approximately 50 nM and was not altered by NGF treatment. A novel feature of the NGF-stimulated enzyme was its sharp dependence on Mn2+ concentration. The active enzyme is likely to be phosphorylated, because inclusion of phosphatase inhibitors was required for recovery of optimal activity and the activity was lost on treatment of the enzyme with alkaline phosphatase. Histones, tubulin, casein, bovine serum albumin, and the ribosomal subunit protein S-6 were not phosphorylated by this enzyme. The NGF-stimulated kinase was distinct from A kinase, C kinase, or other NGF-stimulated kinases. The rapid and transient activation of the protein kinase upon NGF treatment suggests that the enzyme may play a role in signal transduction in PC12 cells.     

Phosphorylation of translation initiation factor eIF-4E is induced in a ras-dependent manner during nerve growth factor-mediated PC12 cell differentiation.

Translation initiation factor eIF-4E, which binds to the 5' cap structure of eukaryotic mRNAs, is believed to play an important role in the control of cell growth. Consistent with this, overexpression of eIF-4E in fibroblasts results in their malignant transformation. The activity of eIF-4E is thought to be regulated by phosphorylation on a single serine residue (Ser-53). Treatment of rat pheochromocytoma (PC12) cells with nerve growth factor (NGF) strongly curtails their growth and causes their differentiation into cells that resemble sympathetic neurons. The present study shows that eIF-4E is rapidly phosphorylated in PC12 cells upon NGF treatment, resulting in a significant increase in the steady-state levels of the phosphorylated protein. In contrast, epidermal growth factor, a factor which elicits a weak mitogenic response in PC12 cells, did not significantly enhance eIF-4E phosphorylation. We also show that although the mitogen and tumor promoter, phorbol 12-myristate-13-acetate, is able to induce phosphorylation of eIF-4E in PC12 cells, the NGF-mediated increase is primarily a protein kinase C-independent response. The NGF-induced enhancement of eIF-4E phosphorylation is abrogated in PC12 cells expressing a dominant inhibitory ras mutant (Ser-17 replaced by Asn), indicating that eIF-4E phosphorylation is dependent on a ras signalling pathway. As phosphorylation of eIF-4E effects translation initiation, these results suggest that NGF-mediated and ras-dependent eIF-4E phosphorylation may play a role in switching the pattern of gene expression during the differentiation of PC12 cells.