Neurosciences, Biomedical Research Foundation of the Academy of Athens, Athens, Greece

PKC-dependent signaling pathways are integral to regulationof transcriptional programs towards neuronal differentiationand acquisition of neurotransmitter phenotypes. PKCs (EC2.7.11.13) are nodal components for the transductionmachinery of signals from G protein-coupled and tyrosinekinase receptors to the Ras/Raf/MEK/ERK pathway, havinglong been thought as the link between receptor-dependentgeneration of diacylglycerol (DAG) and Ras activation. Thephorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA)acts as a diacylglycerol analog (Griner and Kazanietz 2007)directly activating classic and novel isoforms of PKC.Indeed, an established model for the role of PKC in neuronaldifferentiation are the neuroblastoma SH-SY5Y cells, which,following activation of PKC by TPA, can be induced todifferentiate into a sympathetic phenotype characterized byincreased neuritic outgrowth, activation of neuron-specificgenes such as GAP-43, neuropeptide Y, tyrosine hydroxylase(Pahlman et al. 1981; Troller et al. 2001; Wernesson et al.1998; Olsson and Nanberg 2001), increases in noradrenaline

(Heikkila et al. 1993), and development of membraneexcitability (Jalonen and Akerman 1988). Similarly, we havepreviously shown that in the human neuroblastoma IMR32cells, PKCa as well as Ras are required for differentiationand expression of tyrosine hydroxylase (Mangoura et al.2006b).

Received October 20 2008; revised manuscript received December 3,2008; accepted February 3 2009.Address correspondence and reprint requests to Dr Dimitra Mangoura,

Neurosciences, Biomedical Research Foundation of the Academy ofAthens, Soranou Efesiou 4, 11527 Athens, Greece.E-mail: mangoura@bioacademy.grAbbreviations: CSRD, cysteine/serine-rich domain; CTD, C-terminal

domain; DAG, diacylglycerol; EGF, epidermal growth factor; EGFR,EGF receptor: PKA, protein kinase A; ERK, extracellular signal-regu-lated kinase; GAP, GTPase-activating protein; GEF, guanine-nucleotideexchange factor; GRP, guanine-nucleotide releasing protein; NF1, neu-rofibromatosis type 1; PKC, protein kinase C; SAP, shrimp alkalinephosphatase; SDS-PAGE, sodium dodecyl sulfate polyacrylamide elec-trophoresis; TPA, 12-O-tetradecanoyl-phorbol-13-acetate.

Abstract

PKC, Ras, and ERK1/2 signaling is pivotal to differentiation

along the neuronal cell lineage. One crucial protein that may

play a central role in this signaling pathway is the Ras

GTPase-activating protein, neurofibromin, a PKC substrate

that may exert a positive role in neuronal differentiation. In this

report, we studied the dynamics of PKC/Ras/ERK pathway

signaling, during differentiation of SH-SY5Y neuroblastoma

cells upon treatment with the PKC agonist, phorbol ester 12-

O-tetradecanoyl-phorbol-13-acetate (TPA). Surprisingly, we

observed that, among other PKC-dependent signaling events,

TPA induced a rapid and sustained decrease of neurofibromin

immunoreactivity which was not due to proteolysis. Instead,

we identified a specific phosphorylation event at the C-tail of

neurofibromin. This phosphorylation was acute and correlated

perfectly with the signaling dynamics of the Ras/ERK path-

way. Moreover, it persisted throughout prolonged treatment

and TPA-induced differentiation of SH-SY5Y cells, concur-

rently with sustained activation of ERK1/2. Most importantly,

C-tail phosphorylation of neurofibromin correlated with a shift

of neurofibromin localization from the nucleus to the cytosol.

We propose that PKC-dependent, sustained C-tail phos-

phorylation is a requirement for prolonged recruitment of

neurofibromin from the nucleus to the cytosol in order for a

fine regulation of Ras/ERK pathway activity to be achieved

during differentiation.

Keywords: adaptin, differentiation, drebrin, epitope masking,

metabolic labeling, neurofibromatosis, PKCa.

J. Neurochem. (2009) 109, 573–583.

JOURNAL OF NEUROCHEMISTRY | 2009 | 109 | 573–583 doi: 10.1111/j.1471-4159.2009.05975.x

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 109, 573–583 573

Mechanistically, PKC activation is necessary for theformation of Ras and activated Raf-1 complexes (Maraiset al. 1998) and thus activation of the three kinase module,consisting of Raf (MAPKKK), MAPK kinase (MAPKK orMEK), and extracellular signal-regulated kinase (ERK)(MAPK) (Raman et al. 2007). In these initial signalingevents, the direct targets of PKC/TPA signaling may includeRas itself, at least in the case of the K-Ras isoform (Bivonaet al. 2006), Ras activators that belong to the guanine-nucleotide releasing protein (GRP) and SOS-families ofguanine-nucleotide exchange factors (GEFs) (Kawakamiet al. 2003), and the Ras down-regulators, or GAPs, thatactivate the intrinsic GTPase activity of Ras and lead to GTPhydrolysis and inactivation of Ras.

A prominent RasGAP in neural cells is neurofibromin,the product of the tumor suppressor Nf1 gene (Rubin andGutmann 2005). Mutations or deletions of this gene causeNeurofibromatosis 1 (NF1), a dominantly inherited diseasewhich is characterized by symptoms that primarily derivefrom differentiation or proliferation defects in neural cells,notably learning disabilities often with quantifiable differ-ences in brain morphology (Kayl et al. 2000) and gliomas(Rubin and Gutmann 2005). Accordingly, an inducive roleof neurofibromin in differentiation has been suggested fromstudies in neuroblastoma cell lines (Mangoura et al.2006b), coupled to prior cell growth arrest in C6 gliomacells (Mangoura et al. 2006a) and PC12 pheochromocy-toma cells (Yunoue et al. 2003; Patrakitkomjorn et al.2008). Considering that activated Ras and Raf are alsopotent inducers of neuronal differentiation in severalcellular contexts, these studies have established thatneurofibromin is an important regulator of Ras signalingduring neural differentiation. Ras may control the durationof the downstream activation of ERK, an important effectorof the biological output of this signaling pathway. Theduration of ERK activation may determine the cellularresponse in neurons or neuron progenitors such as PC12cells: a transient (early and short) activation as byepidermal growth factor (EGF) generally induces cellproliferation whereas a sustained activation induces neuriteoutgrowth and differentiation (Marshall 1995; Stork 2002).The mechanisms, however, that mediate Ras regulation byneurofibromin are still not understood. Only recently, PKC-dependent phosphorylation of neurofibromin has beenhighlighted as a mechanism by which neurofibrominactivity is recruited in signalosomes to regulate Ras-GTPlevels upon growth factor signaling (Mangoura et al.2006a).

More specifically, while both acute PKCa- and PKCe-dependent phosphorylation of the proximal to N-terminuscysteine–serine rich domain of neurofibromin by TPA orEGF facilitates association of neurofibromin with the actincytoskeleton, phosphorylation by PKCa leads to enhance-ment of its GAP activity and to cell growth arrest in C62B

glioma cells (Mangoura et al. 2006a). The C-terminaldomain of neurofibromin (CTD), which has an importantrole in the nuclear localization of neurofibromin via itsbipartite nuclear localization signal (Li et al. 2001; Van-denbroucke et al. 2004) and in mediating interactions withproteins implicated in neuronal differentiation like CRMP-2(Patrakitkomjorn et al. 2008), is also a target for PKC atleast in vitro (Izawa et al. 1996). Yet, no specific neuro-fibromin phosphorylation site(s) that would correlate witha specific biological outcome in vivo has been identifiedthus far.

In this study, we have uncovered a novel aspect of TPA/PKC signaling which is associated with prolonged activationof the Ras/ERK pathway and commitment of SH-SY5Yneuroblastoma cells to differentiation. We present evidencefor a specific and sustained PKC-dependent phosphorylationof neurofibromin on serine residues at its C-tail, whichcorrelates with changes in the subcellular shuttling of theprotein from the nucleus to cytosolic structures. We proposethat this event is required for the RasGAP-dependent finetuning of the levels of activation of the Ras/ERK pathwayduring prolonged differentiation-associated signaling.

Materials and methods

Materials, Ras activation assays, metabolic labeling, immunocyto-

chemistry, and statistics are presented as supporting information.

Cell culture and treatmentsSH-SY5Y cells were grown in DMEM-F12 medium (1 : 1, v/v)

supplemented with 10% fetal bovine serum and 1% penicillin/

streptomycin, at 37�C and 7% CO2; for starvation, cells at �80%confluency were grown in 1% fetal bovine serum for at least 16 h.

Agonists and inhibitors or vehicle (DMSO) were added at

concentrations and times indicated in each experiment. To induce

differentiation cells plated at 35 000 cells/35 mm plate were

incubated 24 h later with vehicle or TPA (20 nmol/L), and medium

was changed every 2 days.

Cell lysis and western blot analysisCells were lysed in radioimmunoprecipitation (RIPA) buffer

supplemented with protease inhibitors and phosphatase inhibitors

(Mangoura et al. 2006a). For immunoprecipitations, unless stated

otherwise, cells were lysed in 50 mmol/L Tris buffer, pH7.2,

150 mmol/L NaCl, 5 mmol/L EDTA, 0.5% NP-40. Cell lysates

were centrifuged at 13 000 rpm (20 min, 4�C) and supernatants

were assayed for protein content. For western blot analysis, 10–

30 lg of protein, immunoprecipitates, or subcellular fractions (20–

40 lg) were separated by sodium dodecyl sulfate polyacrylamide

electrophoresis, electrotransferred to nitrocellulose membranes

(Schleicher and Schuell, Dassel, Germany), Western blotted with

the indicated antibodies and the appropriate species-specific

secondary antibodies and immunoreactivity was visualized as

described previously (Mangoura et al. 2006a). In pilot experiments

with total cell lysates, it was established that increases in

luminescence (ECL signals) were linear from 10–60 lg of

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 109, 573–583� 2009 The Authors

574 | G. Leondaritis et al.

protein/lane for 30 s of exposure for all antibodies used, except

for p-ERK1/2 mAb which was linear for 5–20 lg and 10 s of

exposure.

Cell fractionation experimentsFor membrane translocation of PKCa, cells were harvested by

scraping in ice-cold phosphate-buffered saline, washed, resuspen-

dend in homogenization buffer [20 mmol/L HEPES, pH7.5;

150 mmol/L NaCl; 2 mmol/L EDTA; 2 mmol/L EGTA] supple-

mented with protease and phosphatase inhibitors and Dounce-

homogenized by 40 strokes. The homogenate was cleared by

centrifugation at 1000 g ·10 min and cytosolic and membrane

fractions were obtained by centrifugation at 50 000 g ·1 h

at 4�C. For nuclear fractionation experiments, cells were

fractionated into cytosolic, total nuclei and subnuclei fractions

as described previously (Takemoto et al. 2004). The resulting

nuclei pellet after Triton X-100 extraction was vigorously

homogenized in RIPA with a 28-gauge needle and incubation

for 1 h at 4�C.

Immunoprecipitation and alkaline phosphatase treatmentsFor immunoprecipitation of neurofibromin, cell lysates (200–

1000 lg of protein) were first pre-cleared with normal rabbit

serum captured on protein-G-sepharose beads (1 h, 4�C) and then

incubated overnight with the GRP-N neurofibromin antibody or a

mixture of antibodies GRP-D and GRP-N on protein-G-agarose

beads; after extensive washing proteins were boiled in Laemmli

buffer. For in vitro phosphatase treatments, immunoprecipitates

were sequentially washed in NP-40 buffer without phosphatase

inhibitors and Tris 10 mmol/L pH7.2 at 4�C and divided into

three equal aliquots. For control reactions, samples were

quenched directly by boiling in Laemmli buffer; for mock

reactions, samples were incubated for 1 h (37�C) in alkaline

phosphatase buffer [50 mmol/L Tris, pH 8.8; 5 mmol/L MgCl2]

with or without 4U of heat-inactivated shrimp alkaline phospha-

tase (SAP), while for phosphatase reactions, samples were

incubated with 4U of SAP. Reactions were terminated by boiling

in Laemmli buffer and proteins were processed for western

blotting.

Western blot-phosphatase assay and quantification ofneurofibromin C-tail phosphorylationProteins from control and treated cell lysates or subcellular

fractions were analyzed with sodium dodecyl sulfate polyacryl-

amide electrophoresis on the same 6% or 8% gel as described

above. After transfer, nitrocellulose membranes were sequentially

washed in Tris-buffered saline plus 0.1% Tween20 and alkaline

phosphatase buffer. Then the membrane was cut into strips which

were incubated in alkaline phosphatase buffer in the absence or

presence of 10U/mL SAP for 4 h at 37�C. After treatment, the

strips were washed and processed for western blotting analysis with

the GRP-D antibody. For quantification of TPA-induced neurofi-

bromin C-tail phosphorylation in supernatant and nuclei fractions,

GRP-D immunoreactivity signals of TPA-treated SH-SY5Y sub-

cellular fractions blotted in duplicates on buffer- and SAP-treated

membranes were analyzed by densitometry with the NIH Image J

software and normalized to the respective values in control

(vehicle-treated) cells.

Results

Characterization of acute and long-term PKC-Ras-Raf-ERKsignaling and regulation of the RasGAP neurofibrominduring differentiation in neuroblastoma SH-SY5Y cellsLong-term treatment with the phorbol ester TPA (48 h), asexpected, induced differentiation of SH-SY5Y cells, whichwas readily observed as acquisition of a differentiatedmorphology, a three-fold increase in bIII-tubulin abundance,as well as increases in a-adaptin expression (Fig. S1)indicating a maturation of the secretory machinery(McLaughlin et al. 2006). TPA-dependent differentiationalso enhanced immunodetection of the developmentally-regulated, actin cytoskeleton-binding neuronal protein dre-brin. Drebrin was found to be enriched in the submembranouscortical cytoskeleton (Fig. 1a, arrowheads), and drasticallyshifted away from the actin-rich distal ends of the outgrownneurites (Fig. 1a, arrows in left versus arrows in right frame),as typically seen in mature neurons (Asada et al. 1994).

Then we determined the long-term activation and phos-phorylation status of the PKC-Ras-Raf-ERK signaling relay

Fig. 1 Prolonged treatment with the phorbol ester TPA results in dif-

ferentiation of SH-SY5Y neuroblastoma cells, down-regulation of

PKCa and neurofibromin immunoreactivity and sustained ERK1/2

activation. (a) Distribution of the actin cytoskeleton-associated protein

drebrin in control and differentiated SH-SY5Y cells. Arrows and

arrowheads indicate F-actin-rich growing tips of neurites and cortical

co-localization of drebrin and actin respectively. Insets show the

merged images of drebrin and Hoescht 33258 nuclear staining;

bar = 10 lm. (b) and (c) Abundance of the indicated proteins revealed

by western blotting.

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 109, 573–583

Neurofibromin C-tail phosphorylation | 575

molecules, all involved in the acute response to TPA and theonset of differentiation of SH-SY5Y neuroblastoma cells.Western blot analysis of the abundance of these moleculesrevealed that in differentiated SH-SY5Y cells TPA hadcaused down-regulation of PKCa, but not of c-Raf-1, ERK1/2 (Fig. 1b), or Ras (not shown). Moreover, we observed asustained phosphorylation of c-Raf-1, indicated by itsmobility shift, as well as of both MARCKS, a PKC-specificsubstrate, and ERK1/2 (Fig. 1b). Prolonged phosphorylationof c-Raf and ERK1/2 may require reoccurring activation ofRas and the obligatory involvement of RasGAPs, thus weassessed the levels of the two prominent RasGAPs, neuro-fibromin and p120GAP (Fig. 1c). Expression of p120GAPwas unaffected by TPA treatment, yet the levels of neuro-fibromin appeared significantly reduced when assessed byimmunoblotting with the GRP-D antibody that recognizesthe C-terminus of the protein. These results, at odds with theestablished up-regulation of neurofibromin in differentiatingneurons (Li et al. 2001) indicated that the RasGAP neuro-fibromin, as detected by its western blotting, was apparentlypreferentially down-regulated by prolonged TPA treatmentand differentiation in SH-SY5Y.

In order to further investigate this signaling pathwayinvolving PKC and neurofibromin in the regulation of Ras/ERK signaling, we assessed the acute events following

exposure to TPA. PKCa, the prominent classic PKC isoformin SH-SY5Y, was robustly activated, as evident by the acuteand maximal translocation of PKCa from the cytosolic to themembrane fractions (Fig. 2a) and the increased phosphory-lation of its major substrate, MARCKS (Fig. 2b). PKC maydirectly activate Ras, a prerequisite for Raf-MEK-ERKactivation, in a membrane receptor-independent manner(Marais et al. 1998) and we thus examined whether TPA-dependent activation of PKCa induced Ras activation in SH-SY5Y in comparison to activation by the GPCR muscarinicreceptor or the tyrosine kinase EGFR. Activation of Ras wasachieved by EGF or TPA, while carbachol induced only amodest activation Fig. 2c); subsequently, EGF- and carba-chol-induced ERK1/2 activation was transient and declinedafter 15 min, whereas TPA elicited a sustained activationwhich persisted for over 60 min (Fig. 2d). Pharmacologicalinhibition of classic and novel PKCs, MEK1/2, or EGFRkinases further established the specificity of signaling foreach agonist as PKC-dependent (Fig. S2). The down-regu-lation of neurofibromin, during TPA-induced differentiation,led us to correlate ERK activation and neurofibrominabundance in response to the same agonists. The sustainedERK activation by TPA was reflected in the time-dependentreduction of neurofibromin immunoreactivity (Fig. 2e), andwas dependent on specific activation of classic PKCs, as the

Fig. 2 Differential activation of PKCa and the Ras/ERK pathway by

TPA, EGF and carbachol in SH-SY5Y, and differential down-regula-

tion of neurofibromin immunoreactivity. Serum-starved SH-SY5Y cells

were incubated with 100 nmol/L TPA for the indicated times and PKCa

activation was assessed by its translocation to the membrane fraction

(a) or by the phosphorylation of MARCKS (b). Activation of H-Ras (c)

was assessed by GST-c-Raf-1-RBD affinity precipitation after treat-

ment with EGF (100 ng/mL), carbachol (1 mmol/L) or TPA (100 nmol/

L) for 5 min. SH-SY5Y cells were treated with the indicated agonists

for 5-60 min and activation of ERK1/2 (d) or abundance of neurofi-

bromin (e) was assessed by immunoblotting. (f) Cells were pre-incu-

bated with the PKC inhibitors Ro-31-8220 (2 lmol/L) and Go6976

(2 lmol/L) for 40 min, treated with TPA 100 nmol/L for 5 min and ly-

sates were immunoblotted for the indicated proteins.

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 109, 573–583� 2009 The Authors

576 | G. Leondaritis et al.

classic PKC inhibitor Go6976 reversed the reduction equallyto Ro-31-2188, an inhibitor of both classic and novel PKCs(Fig. 2f). Moreover, activation of PKA had no effects onneurofibromin immunoreactivity (Fig. S3).

Phorbol ester induces PKC-dependent phosphorylationof neurofibromin at its C-tailPKC-dependent phosphorylation often results in proteasomaldegradation of its substrates, while recent studies havesuggested that neurofibromin protein levels may also beregulated by growth factor-induced proteasomal proteolysis(Cichowski et al. 2003). We thus first examined the possi-bility that the TPA-induced reduction of neurofibrominimmunoreactivity was due to proteasomal proteolysis. Wefound that pre-incubation of SH-SY5Y cells with twoproteasome inhibitors, lactacystin or ALLN, did not attenuateor prevent acute or long-term TPA-induced loss of neurofi-bromin immunoreactivity (Fig. S4a and b). Similar resultswere obtained in GFPu-SH-SY5Y cells, a cell line whichstably expresses a proteasome peptide substrate fused to GFP(GFPu) (Bence et al. 2001) and allows for an internal controlof inhibition of proteasome activity (Fig. S4c and d).Consistent with the absence of proteasomal degradation ofneurofibromin, stoichiometric quantification of neurofibro-min protein levels using [35S]Met/Cys-metabolic labeling incontrol and TPA-treated cells showed no evidence ofneurofibromin degradation, as the [35S]neurofibromin levelsremained constant (Fig. 3a). These results established thatthe decrease of immunoreactivity observed with the GRP-D

antibody did not reflect actual decrease of neurofibrominprotein levels.

We thus reasoned that the observed differences inneurofibromin immunoreactivity in TPA-treated cells couldbe the result of epitope masking, because of phosphorylationon specific residues and we directly investigated thispossibility. First, use of another neurofibromin antibodyGRP-N that targets the N-terminus of the protein (aminoacids 509–528) did not report the reduction seen with theGRP-D antibody (amino acids 2798–2818), conclusivelysuggesting a specific epitope masking effect on the

Fig. 3 Phorbol ester induces PKC-dependent phosphorylation of

neurofibromin at its C-tail. (a) Cells were metabolically labeled by

[35S]Met/Cys for 20 h and then treated with 100 nmol/L TPA for the

indicated times. Neurofibromin was immunoprecipitated with a mixture

of antibodies and subjected to SDS-PAGE and gel autoradiography.

Arrow indicates the position of neurofibromin and left bar the Mr of a

pre-stained marker. (b) Total cell lysate from control or TPA-treated

SH-SY5Y cells was immunoblotted with GRP-D or GRP-N antibodies

(p120GAP was included as a loading control). (c) Effects of in vitro

alkaline phosphatase treatment on TPA-induced down-regulation of

neurofibromin GRP-D antibody immunoreactivity. Neurofibromin was

immunoprecipitated by a mixture of GRP-D and GRP-N antibodies

from control and TPA-treated SH-SY5Y cells (as in b) and immuno-

precipitates were washed and either boiled in Laemmli buffer (lanes 1

and 4) or incubated for 1 h at 37�C in the presence of buffer (lanes 2

and 5) or 4 U of shrimp alkaline phosphatase (lanes 3 and 6). Neu-

rofibromin GRP-D immunoreactivity was then assessed by immuno-

blotting (IgG signal is included as a loading control for phosphatase

assays). (d) Western blot-phosphatase assay of neurofibromin phos-

phorylation. Lysates from control or TPA-treated SH-SY5Y cells were

separated by SDS-PAGE and electrotransferred to nitrocellulose

membranes. Replicate strips were treated with buffer or alkaline

phosphatase and then immunoblotted with GRP-D antibody and

control antibodies (p120GAP, p-PKC and p-MARCKS).

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 109, 573–583

Neurofibromin C-tail phosphorylation | 577

C-terminus of the protein (Fig. 3b). Then, immunoprecipi-tates from vehicle- or TPA-treated SH-SY5Y cells, using amixture of GRP-N+GRP-D antibodies, were either eluted inLaemmli buffer (Fig. 3c, lanes 1, 4) or incubated for 1 h at37�C in the absence (lanes 2, 5), or presence (lanes 3, 6) ofalkaline phosphatase before they were processed for westernblot analysis with GRP-D; we found that alkaline phospha-tase treatment restored neurofibromin immunoreactivity toGRP-D antibodies in TPA-treated cells to levels comparableto controls (lanes 1 and 4 versus 3 and 6). Consistently,experiments using immobilized, denatured neurofibromin toavoid any interference of co-immunoprecipitated proteins onthe in vitro phosphatase reactions yielded similar results.Specifically, after proteins from control and TPA-treated SH-SY5Y total cell lysates were analyzed by SDS-PAGE andthen transferred onto nitrocellulose membranes, they weresubjected to a western blot-phosphatase assay, as described inMaterials and Methods. Under these conditions completerecovery of GRP-D immunoreactivity of neurofibromin inTPA-treated cell lysates was also achieved (Fig. 3d).

As an important internal control, we documented thatphosphatase treatment of other blotted proteins in the samesamples diminished or significantly reduced the immunore-activity signal from phosphorylation-sensitive antibodies,namely of MARCKS and PKC (p-MARCKS and p-PKC,Fig. 3d) without affecting the signal from phosphorylation-insensitive antibodies (p120GAP, Fig. 3d).

C-tail phosphorylation of neurofibromin correlates withenhanced signaling throughout the PKC/Ras/ERK1/2pathwayWe next assessed whether this reverse specificity of the GRP-D antibody for phosphorylated C-tail could be used to studythe signaling dynamics and regulation of neurofibrominphosphorylation in vivo. In this series of experiments, a15 min treatment with TPA was used to activate PKC andthen, specific inhibitors were added sequentially in order torapidly shut down PKC or protein phosphatase activity.Robust activation of PKC by a 15 min treatment with TPAresulted in marked phosphorylation of the RasGEF SOS-1and the Ras effector c-Raf-1, observed in both cases as amobility shift (Fig. 4a, lane 2), and of MARCKS (Fig. 4a,lane 2); these phosphorylation events continued for up to60 min treatment with TPA (lane 3). We found that inhibitionof PKC by Go6976 or Ro-31-8220 after this pulse of PKCactivation resulted in reversal of all PKC-dependent phos-phorylation events within 45 min: p-MARCKS levels werereduced to control values and Raf-1 mobility shift wasdiminished, as did the SOS-1 mobility shift (lane 3 vs. lanes4 and 5). On the contrary, incubation with the proteinphosphatases 1/2A inhibitor calyculin A resulted in either nochanges (e.g., Raf-1 and p-MARCKS) or slight potentiation(SOS-1) of the TPA effect (lane 6). Under these conditions,neurofibromin C-tail phosphorylation (seen as loss of GRP-D

immunoreactivity) was also reversed by PKC inhibition(Fig. 4a). These dynamic changes on phosphorylation of Rasregulators SOS-1, neurofibromin or the Ras effector c-Raf-1were readily translated into concomitant changes in theactivation levels of Ras and ERK1/2. Specifically, bothTPA-induced formation of H-Ras-GTP and ERK1/2 phos-phorylation were decreased with rapid kinetics with PKCinhibition after an initial activation as described above

Fig. 4 PKC-dependent, C-tail phosphorylation of neurofibromin cor-

relates with the signaling dynamics of the PKC/Ras/SOS/Raf/ERK

pathway. (a) SH-SY5Y cells were pretreated with 100 nmol/L TPA for

15 min (lane 2) and incubated for 45 min further with the following

treatments: addition of DMSO (lane 3), inhibition of PKC activity by Ro-

31-8220 (Ro, 4 lmol/L; lane 4) or by Go6976 (Go, 4 lmol/L; lane 5), or

inhibition of phosphatase activity by calyculin A (CalA, 2 nmol/L; lane

6). Total lysate proteins from control (lane 1) and treated cells were

immunoblotted as indicated. The line and double arrows in SOS-1 and

c-Raf-1 strips highlight the phosphorylation-induced mobility shift of

both proteins. (b) PKC activity was inhibited by Ro-31-8220 (4 lmol/L)

for 10 min after an initial activation by 100 nmol/L TPA for 15 min.

Phosphorylation of neurofibromin and activation of ERK1/2 were as-

sessed by immunoblotting; activation of H-Ras was assessed as de-

scribed in Fig. 2.

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 109, 573–583� 2009 The Authors

578 | G. Leondaritis et al.

(Fig. 4b). These results suggested that C-tail phosphorylationof neurofibromin is a specific event which correlates highlywith enhanced signaling throughout the PKC/SOS/neuro-fibromin/Ras/Raf/MEK/ERK module.

Differential C-tail phosphorylation of nuclear and non-nuclear pools of neurofibrominTaking together the established role of the neurofibromin-CTD for nuclear localization (Vandenbroucke et al. 2004)and the fact that CTD is phosphorylated by PKC, at leastin vitro (Izawa et al. 1996), we reasoned that PKC-dependentC-tail phosphorylation of neurofibromin might serve as aproximal regulatory input for the nucleocytoplasmic shuttlingof the protein. We first established by immunocytochemistry(Fig. 5a) a close association of neurofibromin with cytoplas-

mic structures (Fig. 5a, arrowheads) and the nucleus(Fig. 5a, arrows) and then the presence of a nuclear poolof neurofibromin was reaffirmed in subcellular fractionationexperiments (Fig. 5b). SH-SY5Y cells were fractionated intocytosol and non-nuclear membranes (‘‘supernatant’’) andnuclei, and then nuclei were further subfractionated intonuclear envelope (NE lane) and Triton X-100-insolublenuclei fractions (nuclear matrix and DNA binding proteins,‘‘pellet’’). In all nuclei containing fractions calnexin and b-COP, an ER and a Golgi marker, respectively, were barelydetectable, while in nuclei or nuclear pellet, but not nuclearenvelope fractions, the nuclear matrix protein marker, laminA/C was highly enriched (Fig. 5b).

Under regular western blotting conditions, neurofibrominwas detected in comparable amounts in both supernatant andnuclei fractions excluding the nuclear envelope (Fig. 5c,SAP minus lanes). In order to test possible C-tail phosphor-ylation of distinct subcellular pools of neurofibromin weperformed a western blot-phosphatase assay in thesefractions. C-tail phosphorylation was significantly higher inthe cytosolic pool of neurofibromin compared with thenuclear pool under basal conditions (Fig. 5c, SAP plus lanes)as revealed by densitometry analysis (phosphatase-to-buffer-treated GRP-D immunoreactivity: 2.24 ± 0.23 forsupernatant neurofibromin and 1.32 ± 0.26 for nuclearneurofibromin, n = 3 ± SD, p < 0.05). Thus, a predominatepercentage (estimated as high as 55%) of neurofibrominmolecules was C-tail phosphorylated under basal conditions.

Sustained PKC-induced C-tail phosphorylation ofneurofibromin correlates with cytoplasmic recruitmentof the proteinThe subcellular fractionation studies pointed out that phos-phorylation at the C-tail of neurofibromin may control itssubcellular shuttling. In TPA-treated SH-SY5Y cells thereduction of GRP-D immunoreactivity was observed in bothcytosolic and nuclear pools of neurofibromin (Fig. 6a, upperpanel, lanes 1, 2 versus lanes 3, 4). When, however, westernblot-phosphatase assays were performed to reveal C-tailphosphorylation, we observed that phosphorylation occurredonly in the cytosolic pool as suggested by the recovery ofimmunoreactivity of GRP-D (Fig. 6a, upper panel, lanes 5, 7compared with lanes 1, 3; quantification in lower panel). TheTPA-dependent reduction of nuclear GRP-D immunoreac-tivity remained unaffected by phosphatase treatment sug-gesting that there had been a depletion of the nuclear pool ofneurofibromin upon TPA treatment (Fig. 6a, upper panel,lanes 6 and 8 versus lanes 2 and 4; quantification in lowerpanel). Further evidence for the shift of neurofibromin fromnucleus to cytosol upon TPA-induced PKC activation wasobtained at the single cell level with immunolocalization ofneurofibromin in control and TPA-treated cells. In themajority of vehicle-treated cells, strong neurofibromin stain-ing in all nuclei and variable amounts in the cytosol was

Fig. 5 C-tail phosphorylation of nuclear and non-nuclear pools of

neurofibromin. (a) Nuclear localization of neurofibromin in SH-SY5Y

cells. Cells were fixed and stained for neurofibromin and nuclear DNA

as described in Materials and methods. (b) SH-SY5Y cells were

fractionated to nuclei supernatant, total nuclei (nuclei), nuclear enve-

lope (NE) and detergent-insoluble nuclei (pellet) fractions. Proteins

from all fractions were immunoblotted to validate the fractionation

protocol using the indicated markers. (c) Western blot-phosphatase

assay of neurofibromin in nuclei supernatant, nuclei, nuclear envelope

and pellet fractions, as described in Fig. 3d. In b and c, left lines

indicate the position of prestained molecular weight markers.

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 109, 573–583

Neurofibromin C-tail phosphorylation | 579

apparent (Fig. 6b, arrows in panel (i); a substantial translo-cation of neurofibromin from the nucleus to the rest of thecell body was readily observed upon treatment with TPA for1 h, with cells exhibiting intense cytoplasmic localizationand faint nuclear localization of neurofibromin (Fig. 6b,arrows in panels ii–iii). Importantly, the same changes inlocalization were also observed even after 4 days of treat-ment (Fig. S5). Collectively, these results suggested that theC-tail phosphorylation of neurofibromin by PKC, a sustainedphenomenon during TPA-dependent neuronal differentiationof SH-SY5Y cells, was coupled to the recruitment of theprotein in the cytosol and away from the nucleus.

Discussion

It has been long proposed that TPA-induced activation ofclassic PKCs in SH-SY5Y evokes an ERK-dependentneuronal differentiation, which we have now shown toinclude up-regulation of bIII-tubulin, drebrin and a-adaptin(Fig. 1 & Fig. S1). Our study provides specific evidence thatPKC directly activates Ras upstream of ERK and concur-rently regulates by phosphorylation both Ras-GTPase mod-ulators as well as Ras effectors that link Ras activation toERK activation, namely, the RasGEF SOS-1, the ERK kinasekinase (MAPKKK) c-Raf-1 and importantly, the tumorsuppressor RasGAP neurofibromin. Specifically, upon TPA-induced PKC activation, electrophoretic mobility of themajority of c-Raf-1 and SOS-1 molecules was readilyobserved. The mobility shift of c-Raf-1 (Figs. 1b and 4a) isattributed to direct phosphorylation by PKC and consequent

regulation of its kinase activity towards MEK1/2 (Soderholmet al. 2001). TPA-induced mobility shift of SOS-1 (Fig. 4a)is also due to phosphorylation and activation of its GEFactivity by an activated PKC (Rubio et al. 2006). Impor-tantly, when compared with physiological agonists, we alsofound that TPA treatment activated Ras with a potencysimilar to that of EGF and much greater than the M3muscarinic agonist carbachol (Fig. 2c). Interestingly, thesame three agonists differed significantly concerning theextent and duration of ERK1/2 activation (Fig. 2d), withTPA producing a delayed but sustained phosphorylation ofERK1/2 compared to EGF and carbachol. This differencewas also observed at the level of Ras activation (data notshown) and presumably reflects the stoichiometric activationof the majority of PKC molecules by TPA compared to EGFor carbachol. Overall, these results support a model whereTPA/PKC-induced Ras activation feeds into the c-Raf-1/MEK1/ERK1/2 pathway and moreover suggest that the TPA/PKC-induced differentiation is Ras-dependent, advancing theprevious implication that c-Raf-1 was the possible primarytarget of TPA/PKC signaling in SH-SY5Y (Soderholm et al.2001).

The specificity of PKC signaling events was verified usingpharmacological inhibition with two distinct PKC inhibitorsand utilizing phosphorylation of MARCKS, a bona-fidesubstrate of PKCa in SH-SY5Y (Goodall et al. 1997), as aproxy marker for PKC activity (Fig. 2b and Fig. S2). Ourexperimental design to apply a pharmacological pulse-chaseprotocol, where an initial burst of PKC activation wasfollowed by its acute pharmacological inhibition with the

Fig. 6 TPA-induced, PKC-dependent, C-tail phosphorylation of neu-

rofibromin correlates with cytoplasmic recruitment of the protein. (a)

Western blot-phosphatase assay of neurofibromin in supernatant and

nuclei of control and TPA-treated SH-SY5Y cells (upper panel) and

quantification of C-tail phosphorylation (lower panel). (b) Shift of

neurofibromin localization towards non-nuclear, cytoplasmic, struc-

tures upon treatment of SH-SY5Y cells with TPA. Cells were treated

with TPA and immunostained for neurofibromin and nuclear DNA.

Arrows in panel (i) point to strong nuclear localization of neurofibromin

in the majority of untreated cells; arrows in panels (ii) and (iii) point to

intense non-nuclear localization of neurofibromin after TPA treatment

in the majority of cells. Bar = 10 lm.

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580 | G. Leondaritis et al.

PKC inhibitors Ro-31-2280 and Go6976 (Fig. 4) revealedthe dynamics and significant level of coordination within thissignaling pathway. The rapid inactivation of PKC-dependentsignaling by this subsequent PKC inhibition stronglysuggests that PKC-dependent phosphorylation of c-Raf-1,SOS-1, MARCKS and neurofibromin, or the activation ofRas and ERK1/2, during the early phases of signaling,require constant PKC activation to ensue. Moreover, in long-term phase of TPA/PKC signaling, when cells have enteredthe differentiation program, several of these phosphorylation/activation events still persisted. Distinctively MARCKS thathas been directly implicated in cortical actin cytoskeletonremodeling during neurite outgrowth (Laux et al. 2000), wasconsistently phosphorylated (Fig. 1b), a modification alsoobserved for c-Raf-1 and, importantly, ERK1/2 (Fig. 1b).This implies that, as long-term treatment with TPA leadswithin hours to down-regulation of classic PKCs, particularlyof PKCa in neural cells (Mangoura et al. 2006a), othersignaling enzymes are recruited. Such mechanisms mayinvolve activation of both or either protein phosphatases(Strack 2002) and novel PKC isoforms. Amongst novelisoforms PKCe is prominently expressed in neural cells andits activity correlates well with neuronal induction inembryonic neurons and neuroblastomas, in particular inSH-SY5Y (Fagerstrom et al. 1996). These modulatorypathways should be in accordance with the necessity tostringently regulate ERK1/2 activity during both acute andlong-term signaling. ERK1/2 is the major output of thissignaling pathway and it is crucial for the expression ofneuron-specific genes neuropeptide Y, GAP43 and tyrosinehydroxylase in SH-SY5Y (Olsson and Nanberg 2001; Zhanget al. 2007) and its activity can be regulated at multiplelevels, particularly, at the level of Ras activation or at thelevel of inducible ERK1/2 phosphatases (Reffas and Schlegel2000).

Neurofibromin is a prominent neuronal RasGAP and aPKCa and e substrate and also it has a positive role onneuronal differentiation; thus it is an ideal PKC effectorlinking Ras activation to ERK activation during differenti-ation. By assessing its levels during both early and latephases of TPA/PKC signaling we have uncovered andcharacterized a PKC-dependent sustained phosphorylation atits C-tail and have tentatively identified the phosphorylatedresidue(s) as primarily Ser 2808. We first observed that thecommercially available GRP-D antibody, raised againstresidues 2798-2818 of the C-tail of the protein (Aaltonenet al. 1999), displayed a reduced affinity against phosphor-ylated neurofibromin under standard western blotting condi-tions (Fig. 1c and 2e). We then documented thatimmunoreactivity was reversed upon dephosphorylation ofdenatured or native neurofibromin (Fig. 3). Using a bioin-formatics analysis, we have found that serine 2808 of theSAGS*FKR stretch within the C-terminus is a PKCa/b-specific phosphorylation consensus site (Fig. 7); this serine

on neurofibromin was retrieved as phosphorylated in vivo inthe analysis of the phosphoproteome of adenocarcinoma cells(Kim et al. 2005). Moreover, when we used an antibodywhich recognizes the phosphorylated Ser on theTLASS*FKR peptide, a stretch highly homologues to Ser2808 on neurofibromin (Fig. 7), we found increased immu-noreactivity of neurofibromin with TPA treatment (data notshown). These results collectively support our conclusionthat Ser2808 is the primary target of PKC at the C-tail ofneurofibromin in vivo. Epitope masking effects have beenobserved with other antibodies against mTOR (Scott et al.1998), HDM2 (Khosravi et al. 1999), and p53 (Maya andOre 2000) and were similarly investigated with western blot-phosphatase assays. Therefore, the assay that we haveoptimized for neurofibromin (Fig. 3), may be used todistinguish whether reduced immunoreactivity in westernblotting analyses of the abundance of neurofibromin reflectsproteolysis of the protein (Cichowski et al. 2003) or C-tailphosphorylation.

A functionally important finding was that C-tail phos-phorylation of neurofibromin correlated with the subcellularshuttling of the protein. Neurofibromin has been reported tolocalize in many distinct subcellular compartments includingcytosol, and the actin and tubulin cytoskeleton systems(Gregory et al. 1993; Li et al. 2001), while its nuclearlocalization (Li et al. 2001; Vandenbroucke et al. 2004) hasbeen one of the most enigmatic features in neurofibrominbiology. Our experiments, utilizing both biochemical frac-tionation and immunocytochemistry experiments with amixture of antibodies to avoid any underestimation ofneurofibromin abundance, showed that a nuclear pool ofneurofibromin does exist (Figs 5 and 6). In addition, wefound that at basal conditions nuclear neurofibromin is C-tailSer2808 dephosphorylated whereas the cytosolic pool of theprotein is substantially C-tail Ser2808 phosphorylated(Fig. 5c). Most importantly, we showed that, upon activationof the Ras/ERK pathway by TPA, C-tail phosphorylation of

Fig. 7 Domain organization of neurofibromin. Functional regions of

neurofibromin include CSRD (cysteine/serine repeat domain, 543–

909aa), GRD (GAP-related domain, 1235–1451aa), Sec14 (Sec14-

homology domain, 1539–1698aa), PH (pleckstrin-homology domain,

1715–1816aa), and CTD (C-terminal domain, 2260–281aa). Shown in

detail are, the amino acid sequence of the GRP-D (sc-67) antibody

epitope within the C-tail of the protein (2798–2818aa) and the se-

quence of the epitope peptide recognized by the pS-PKC substrate

antibody (in bold); the asterisk indicates the PKC-phosphorylated

serine 2808 of neurofibromin.

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 109, 573–583

Neurofibromin C-tail phosphorylation | 581

neurofibromin rendered the protein primarily cytosolic(Fig. 6). Both C-tail phosphorylation and cytosolic recruit-ment persisted upon prolonged treatment with TPA and wereconcurrent to activation of ERK1/2 and the onset ofdifferentiation of SH-SY5Y. A plausible interpretation wouldbe that neurofibromin nucleocytoplasmic shuttling may beregulated or facilitated by C-tail phosphorylation. Recentstudies have shown that many tumor suppressor proteinsshuttle between the nucleus and cytosol often in a regulatedmanner (Fabbro and Henderson 2003). This type of dynamicmovement may also regulate the activity or stability of theprotein and it may operate in concert with protein–proteininteractions that serve to dock the protein in either compart-ment. Thus, C-tail phosphorylation of neurofibromin mayrecruit the protein to cytosol by stabilizing or facilitatinginteractions with cytosolic proteins, microtubules (Gregoryet al. 1993), actin cytoskeleton (Mangoura et al. 2006b), oractivated Ras itself. In this context, we propose that sustainedC-tail phosphorylation is a requirement for prolongedrecruitment of neurofibromin from the nucleus to the cytosolin order for a fine regulation of Ras/ERK pathway activity tobe achieved. This mechanism may also suggest a separate,Ras-independent role of neurofibromin in the nucleus,possibly as a scaffolding or structural protein, which remainsto be elucidated. Nevertheless, independent evidence fromthe expression pattern of the NF1-DE43 variant, which isunable to enter into the nucleus, suggests that nuclearexclusion, rather than nuclear localization, may play aprotective role against NF1-associated phenotypes (Van-denbroucke et al. 2002). A similar mode of regulation hasalso been described for another Ras-regulatory protein, themicrotubule-binding Ras effector RASSF1c, which primarilyresides in the nucleus. Upon induced DNA damage, how-ever, RASSF1c is released from the nucleus and targetsmicrotubule structures in the cytosol where, presumably byinteracting with activated Ras, facilitates activation of theMAPK JNK1/2 (Kitagawa et al. 2006).

In summary, we have identified for the first time a specificphosphorylation event at the C-tail Ser2808 of neurofibrominthat is associated with recruitment of the protein in thecytosol upon TPA and PKC-induced differentiation of SH-SY5Y cells. We are currently aiming at dissecting theindividual role(s) of C-tail phosphorylated serines on neuro-fibromin RasGAP activity and protein–protein interactionsduring differentiation. Importantly, the elucidation of thisregulatory pathway may have important clinical implications;new strategies aimed at regulating the nuclear export andcytoplasmic sequestration of neurofibromin may prove to bebeneficial therapeutically.

Acknowledgments

We thank Emmanouella Tsirimonaki, Sophia Karouzaki, Themis

Katsimihas and Theodora Kalpachidou for technical help, and Drs

Kostas Vekrellis and Leonidas Stefanis for the gift of the GFPu-SH-

SY5Y cells. G. L. was supported by a BRFAA postdoctoral

fellowship.

Supporting information

Additional Supporting information may be found in the online

version of this article:

Supplementary Materials and MethodsFigure S1 TPA-induced morphological differentiation of

SH-SY5Y cells is associated with up-regulation of neuronal genes

bIII-tubulin and a-adaptin.Figure S2 Effects of pharmacological inhibition of PKC, MEK1/

2 and EGFR on agonist-induced ERK1/2 and MARCKS phosphory-

lation in SH-SY5Y.

Figure S3 PKA activation by forskolin does not induce any

changes in neurofibromin immunoreactivity.

Figure S4 Neurofibromin is not degraded by the proteasome

upon activation of PKCa by TPA.

Figure S5 Sustained cytoplasmic retention of neurofibromin in

differentiated SH-SY5Y cells.

Please note: Wiley-Blackwell are not responsible for the content

or functionality of any supporting materials supplied by the authors.

Any queries (other than missing material) should be directed to the

corresponding author for the article.

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