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The Journal of Neuroscience, June 1, 2000, 20(11):4177-4188
A MAP Kinase-Signaling Pathway Mediates Neurite Outgrowth on L1
and Requires Src-Dependent Endocytosis
Ralf-Steffen
Schmid,
Wendy M.
Pruitt, and
Patricia F.
Maness
Department of Biochemistry, School of Medicine, University of North
Carolina, Chapel Hill, North Carolina 27599-7260
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ABSTRACT |
The neural cell adhesion molecule L1 mediates the axon
outgrowth, adhesion, and fasciculation necessary for proper development of synaptic connections. Mutations of human L1 cause an X-linked mental
retardation syndrome termed CRASH (corpus callosum hypoplasia, retardation, aphasia, spastic paraplegia, and hydrocephalus), and L1
knock-out mice display defects in neuronal process extension resembling
the CRASH phenotype. Little is known about the biochemical or
cellular mechanism by which L1 performs neuronal functions. Here it is
demonstrated that clustering of L1 with antibodies or L1 protein in
rodent B35 neuroblastoma and cerebellar neuron cultures induced the
phosphorylation/activation of the mitogen-activated protein kinases
(MAPKs) and extracellular signal-regulated kinases 1 and 2. MAPK activation was essential for L1-dependent neurite outgrowth,
because chemical inhibitors
[2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one and
1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] of
the MAPK kinase MEK strongly suppressed neurite outgrowth by cerebellar neurons on L1. The nonreceptor tyrosine kinase
pp60c-src was required for L1-triggered MAPK phosphorylation,
as shown in src-minus cerebellar neurons and by
expression of the kinase-inactive mutant Src(K295M) in B35
neuroblastoma cells. Phosphatidylinositol 3-kinase (PI3-kinase)
and the small GTPase p21rac were identified as
signaling intermediates to MAPK by phosphoinositide and Rac-GTP assays
and expression of inhibitory mutants. Antibody-induced endocytosis of
L1, visualized by immunofluorescence staining and confocal microscopy
of B35 cells, was blocked by expression of kinase-inactive Src(K295M)
and dominant-negative dynamin(K44A) but not by inhibitors of MEK or
PI3-kinase. Dynamin(K44A) also inhibited L1 antibody-triggered MAPK
phosphorylation. This study supports a model in which pp60c-src
regulates dynamin-mediated endocytosis of L1 as an essential step in
MAPK-dependent neurite outgrowth on an L1 substrate.
Key words:
neurite outgrowth; endocytosis; neural cell adhesion
molecule; signal transduction; Src; MAP kinase; PI3-kinase
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INTRODUCTION |
The cell adhesion molecule L1
functions broadly in regulating the growth of axons in developing
neurons and in fostering learning in the adult brain (Persohn and
Schachner, 1990 ; Scholey et al., 1993; Luthi et al., 1994). L1 is
localized in the plasmalemma of growth cones and processes of
developing neurons, on axons of mature nonmyelinated neurons, and in
Schwann cells, astrocytes, and some hematopoietic cells (for review,
see Schmid and Maness, 2000). L1 knock-out mice show axon
guidance errors in the corticospinal tract (Cohen et al., 1997) and
corpus callosum (Demyanenko et al., 1999), misoriented dendrites of
cortical pyramidal cells (Demyanenko et al., 1999), degeneration of
sensory axons (Haney et al., 1999), and enlarged ventricles (Dahme et
al., 1997; Demyanenko et al., 1999). Many of these features are present
in humans with the X-linked mental retardation syndrome termed CRASH
(corpus callosum hypoplasia, retardation, aphasia, spastic
paraplegia, and hydrocephalus), resulting from L1 gene mutations
(Fransen et al., 1997).
Little is known about the mechanism by which L1 directs axonal growth
or guidance, but the structural features of L1 suggest that it may
differ from that used by other classes of receptors to stimulate
neurite outgrowth. The L1 extracellular region mediates homophilic
(L1-L1) and heterophilic binding via its six immunoglobulin-like and
five fibronectin type III domains (Appel et al., 1993; Zhao et al.,
1998). Its short cytoplasmic region of 110 amino acids lacks a tyrosine
kinase domain but contains an actin-interaction domain (Dahlin-Huppe et
al., 1997), an ankyrin-binding region (Davis and Bennett, 1994), and a
neuronal-specific sequence, RSLE, which targets L1 to axons (Kamiguchi
and Lemmon, 1998). This motif is also involved in binding adaptor
protein 2, a clathrin adaptor, which enables L1 to be
internalized in growth cones via receptor-mediated endocytosis
(Kamiguchi et al., 1998). It is not known whether endocytosis of L1
plays any role in the biological functions of L1 or merely directs L1
to intracellular pathways for degradation.
A requirement for the nonreceptor tyrosine kinase pp60c-src in
L1-mediated neurite outgrowth has been identified in
src-minus neurons (Ignelzi et al., 1994), but how
pp60c-src regulates neurite growth on L1 is not known.
pp60c-src is tethered by N-terminal myristylation to the
cytoplasmic face of the plasma membrane and interacts functionally with
a variety of receptor types (Maness et al., 1996). On the basis of the
recent finding that pp60c-src is required for endocytosis and
signaling of the  -adrenergic receptor (Ahn et al., 1999), we have
investigated the hypothesis that pp60c-src regulates the
internalization of L1 as a critical determinant of L1-mediated neurite
outgrowth. Like the -adrenergic receptor, L1 clustering activates
mitogen-activated protein kinases (MAPKs) in neuronal cells (Schmid et
al., 1997; Schaefer et al., 1999), but it is not known whether MAPK has
a role in neurite outgrowth or other physiological functions of L1. L1
triggering also modulates intracellular levels of phosphoinositides,
pH, and Ca2+ (Schuch et al., 1989; von
Bohlen und Halbach et al., 1992) and activates tyrosine
phosphatases in growth cones (Atashi et al., 1992; Klinz et al., 1995),
suggesting that an array of signaling intermediates may coordinate the
complex intracellular program that allows growth cones to navigate
through the developing brain and form synaptic connections.
Here we demonstrate that MAPK activation triggered by L1 in neuronal
cells is required for neurite outgrowth on L1 and is mediated via
pp60c-src and the small GTPase p21rac
and phosphatidylinositol 3-kinase (PI3-kinase). Furthermore, it
is shown that pp60c-src functions by controlling
dynamin-mediated endocytosis of L1 as an essential step in MAPK
activation. This L1 pathway has the potential to regulate both actin
cytoskeletal dynamics in growth cones and gene expression in the
nucleus, events that may coordinate to regulate the growth and
navigation of neuronal processes.
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MATERIALS AND METHODS |
Antibodies and plasmids. Antibodies used in this
research were mouse monoclonal antibody Neuro4 against the L1
extracellular region (the gift of John Hemperly, Becton Dickinson);
monoclonal antibody HA.11 directed against the hemagglutinin
(HA)-epitope tag (Babco, Richmond, CA); anti-active MAPK polyclonal
antibody (Promega, Madison, WI) specific for dually phosphorylated,
activated MAPK; extracellular signal-regulated kinase 1 (ERK1)
polyclonal antibody K-23 and Raf-1 and B-Raf antibodies (Santa Cruz
Biotechnology, Santa Cruz, CA); and anti-active p38 antibody (New
England Biolabs, Beverly, MA). The following cDNAs were subcloned into
the vector pcDNA3 for expression in B35 neuroblastoma cells:
Fak-related nonkinase (FRNK) and Fak(397F) (Michael Schaller,
University of North Carolina, Chapel Hill, NC); Ras(15A), RhoA(19N),
Rac1(17N), and Cdc42(17N) (Channing Der, University of North Carolina,
Chapel Hill, NC); Src(K295M) (Sara Courtneidge, Sugen); PI3-kinase
 p85 (A. Baldwin and M. Mayo, University of North Carolina, Chapel Hill, NC); dynamin(K44A) (Marc Caron, Duke University, Durham, NC);
c-Raf-1(621A and R89L) and B-Raf (621A and R89L) (D. Morrison, National
Cancer Institute, Frederick, MD); and HA-tagged ERK2 (Melanie
Cobb, University of Texas Southwestern Medical School, Dallas, TX).
Other plasmids used encoded enhanced cyan fluorescence protein
(Invitrogen, San Diego, CA) and glutathione S-transferase (GST) plasmids encoding the c-Jun (1-79)-GST fusion protein (Shelton Earp, University of North Carolina, Chapel Hill, NC) and the
Rac-binding domain (RBD)-GST fusion protein (Richard Cerione,
Cornell University, Ithaca, NY).
Cell cultures and methods of L1 clustering. To produce a
cell line stably expressing human L1 (with RSLE), we transfected rat
B35 neuroblastoma cells (Schubert et al., 1974) with human neuronal L1
cDNA in the pcDNA3 vector (gift of John Hemperly, Becton Dickinson).
After selection in 0.5 mg/ml G418, 17 clones were assayed for L1
expression by Western blotting, and L1 localization on the cell surface
was verified by indirect immunofluorescence staining with Neuro4
antibodies. One clone (designated B35-L1) exhibiting intermediate
levels of L1 expression was chosen for the studies reported here.
B35-L1 cells were maintained in DMEM containing 10% fetal bovine serum
(FBS; HyClone, Logan, UT) and G418 (0.25 mg/ml). Primary cultures of
mouse cerebellar neurons were prepared from postnatal day 8 Sv129/C6B57
hybrid mice by the method of Schnitzer and Schachner (1981) and grown
in DMEM with 10% FBS (HyClone), 25 mM KCl,
and penicillin/streptomycin. The medium was replaced with OptiMEM (Life
Technologies, Gaithersburg, MD), and cells were incubated for 8 hr
before initiating signaling. For clustering of L1 on B35-L1 cells and
cerebellar neurons, L1 monoclonal antibody Neuro4 (30 µg) or L1-Fc
fusion proteins (50 µg) were preincubated with
F(ab')2 fragments of secondary antibodies raised
against Fc fragments of mouse or human IgG for 1 hr at 4°C in 50 µl
of OptiMEM. The complexes were added directly to cells in a 60 mm dish
containing 1 ml of medium at 37°C. Clustering of L1 into patches on
the cell surface was observed by indirect immunofluorescence staining
(data not shown).
MAPK phosphorylation assays. Cells were stimulated with the
indicated L1 antibodies or fusion proteins, rinsed once with cold HBSS, and extracted in Nonidet P-40 (NP-40) lysis buffer
containing 1% NP-40, 0.25% (w/v) Na-deoxycholate, 50 mM
HEPES, pH 7.4, 137 mM NaCl, 1 mM Na-EDTA, 10 mM NaF, 1 mM Na-orthovanadate, 10 mM p-nitrophenylphosphate, 1 mM phenylmethylsulfonyl fluoride, 10 mM -glycerophosphate, 10 µg/ml leupeptin,
0.1 TIU/ml aprotinin, 1 µg/ml pepstatin, 2 nM
calyculin A, and 10% (v/v) glycerol. Lysates were clarified by
centrifugation at 14,000 × g for 20 min at 4°C. Protein concentration was determined using the bicinchoninic acid assay
(Pierce, Rockford, IL). Proteins in cell extracts were separated by
SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was
blocked for 2 hr in 1% bovine serum albumin (BSA) in Tris-buffered
saline containing 0.05% Tween 20 (TTBS) and then incubated overnight
at 4°C either with anti-active MAPK antibodies or p38 MAPK antibodies
in 1% BSA in TTBS. After washing in TTBS, the membrane was incubated
with goat anti-rabbit IgG horseradish peroxidase conjugate in TTBS for
1 hr. The membrane was again washed in TTBS, and immune complexes were
detected using enhanced chemiluminescence (ECL; NEN) with Sterling
x-ray film (BioWorld). Membranes were stripped and reprobed in the same
manner with ERK antibodies to detect total ERK protein.
For assaying phosphorylation of HA-tagged ERK2, B35-L1 cells were
transfected for transient expression with an HA-tagged ERK2 plasmid
with or without plasmids expressing dominant-negative mutants.
Subconfluent cultures were transfected in 60 mm dishes containing
OptiMEM using Lipofectamine according to the manufacturer's instructions (Life Technologies). Signaling experiments were initiated 36-40 hr after transfection. Cells were lysed in NP-40 lysis buffer, and HA-tagged ERK2 was immunoprecipitated from cell extracts (500 µg)
with anti-HA antibody (Babco) for 1.5 hr, followed by Protein G-Sepharose for 0.5 hr at 4°C. Immune complexes were washed in lysis
buffer and subjected to SDS-PAGE, followed by immunoblotting with
anti-active MAPK antibody. For all assays, the exposed bands on x-ray
film were quantitated by densitometric scanning and analysis with
Image-Quant NT software. MEK inhibitors
[2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one (PD98059; 50 µM; New England Biolabs) and
1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126; 10 µM; Promega)] or a PI3-kinase
inhibitor [Ly294002 (10-20 µg; Promega)] dissolved in
dimethylsulfoxide (DMSO; <0.01%) was added to cells for 1 hr before
signal initiation. The Rho inhibitor C3 botulinum endotoxin (20 µg)
was added with Lipofectamine (5 µg) to cells for 4 hr before
stimulation. Control cultures received the same concentration of DMSO
or Lipofectamine.
For assaying the activity of c-Jun N-terminal kinase (JNK), cell
extracts (100 µg of protein) in NP-40 lysis buffer were incubated with c-Jun (1-79)-GST fusion protein for 4 hr at 4°C. Immune
complexes were washed twice with lysis buffer and twice with kinase
buffer (25 mM HEPES, pH 7.4, 25 mM
MgCl2, 25 mM -glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na-orthovanadate, and
2 mM p-nitrophenylphosphate) and then
incubated in kinase buffer with 50 µM ATP and
0.5 µCi of [ 32P]ATP (ICN) at 30°C
for 30 min. Proteins were separated on SDS-PAGE. The dried gel was
exposed to x-ray film, and exposed bands were quantitated by
densitometric scanning and analysis with Image-Quant NT software.
Assay of phosphoinositides. PI3-kinase activity was assayed
by immunoprecipitating phosphotyrosine-modified PI3-kinase from cell
extracts with phosphotyrosine antibodies and measuring the production
of phosphoinositides from phosphatidylinositol in an immune complex
kinase assay (Myers et al., 1993). Cell extracts (800 µg) were
incubated with phosphotyrosine antibody 4G10 for 8 hr at 4°C and then
for 1 hr with Protein G-Sepharose. Immunoprecipitates were collected
and washed twice with buffers I, II, and III (buffer I, 20 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, and
100 µM Na-orthovanadate; buffer II, 100 mM
Tris, pH 7.4, 500 mM LiCl, and 100 µM
Na-orthovanadate; and buffer III, 10 mM Tris, pH 7.4, 100 mM NaCl, and 100 µM Na-orthovanadate). Phosphatidylinositol (0.5 mg/ml; Avanti Polarlipids) was sonicated in
20 mM Tris, pH 7.4, and 1 mM Na-EGTA and
incubated with the immune complexes for 10 min at room temperature in
kinase reaction buffer (total volume of 50 µl) containing 20 µM ATP, 1 µCi of [32P]ATP (3000 Ci/mmol), 10 mM MgCl2, 20 mM Tris, pH
7.4, and 100 mM NaCl. The reaction was terminating by
adding 6N HCl (15 µl) and 1:1 (v/v) chloroform:methanol (120 µl).
Samples were vortexed for 30 sec and centrifuged at 10,000 × g for 1 min. Aliquots (30 µl) of the chloroform phase were
spotted on SilicaG60 plates (Whatman, Maidstone, UK) and subjected to
thin-layer chromatography in chloroform:methanol:25% (w/w)
NH4OH:water (60:47:2:11.3, v/v/v/v). Commercially
available phosphatidylinositol 4-phosphate (PIP; Sigma, St. Louis, MO)
was used to indicate the location of the product. The plate was dried and exposed to x-ray film. Phospholipids were visualized by incubating the thin-layer plate with iodine vapors, and radioactive spots were
scraped from the plate and quantitated for
32P incorporation in a scintillation counter.
Assays for GTP-bound
p21rac and Raf kinase
activity. For measurement of activated, GTP-bound Rac (Bagrodia et
al., 1998), B35-L1 cells were stimulated with L1 antibodies or normal
IgG as described; then lysates (500 µg) were incubated for 30 min at
4°C with 20 µg of a purified GST fusion protein (RBD-GST)
consisting of the Rac-binding domain of PAK1 together with glutathione
Sepharose. RBD-GST binds only to the activated form of Rac
(Rac-GTP) but not to inactive Rac-GDP.
RBD-GST/Rac-GTP complexes were collected by centrifugation and
analyzed by SDS-PAGE and Western blotting with anti-Rac antibodies
(Transduction Laboratories, Lexington, KY).
To assay Raf activation, we subjected lysates in NP-40 lysis buffer
(800 µg) to immunoprecipitation using polyclonal antibodies specific
for c-Raf or B-Raf (Santa Cruz Biotechnology) and Protein G-Sepharose.
In a coupled assay measuring myelin basic protein (MBP) phosphorylation
(Upstate Biotechnology, Lake Placid, NY), Raf immune complexes were
preincubated with inactive MEK and MAPK for 30 min at 37°C and then
with MBP and [32P]ATP for 10 min at
37°C. MBP was adsorbed to Whatman P81 paper and quantitated for
incorporation of radioactivity by scintillation counting. An activated
form of Raf provided by the manufacturer was used as a positive
control. The amounts of Raf immunoprecipitated were determined in
parallel by immunoblotting with Raf antibodies.
Measurement of neurite outgrowth. Cultures of mouse
cerebellar neurons consisting chiefly of granule cells were prepared
from postnatal day 7 or 8 pups by the method of Schnitzer and Schachner (1981). Immunoaffinity-purified L1 protein from adult mice brain was
applied in 10 µl spots onto nitrocellulose-coated plastic coverslips
(22 × 22 mm2) in 35 mm Petri dishes
as described (Ignelzi et al., 1994). After blocking with 1% bovine
serum albumin, 2 × 106 cerebellar
cells were added per coverslip in 2 ml of basal medium with Eagle's
salts (Life Technologies) with 10% heat-inactivated horse
serum, 2.5 gm/l glucose, and penicillin/streptomycin. No neurons
attached to uncoated areas of the nitrocellulose. The MEK inhibitors
PD98059 and U0126 were dissolved in DMSO and added at the time of
plating at 50 or 10 µM final concentration, respectively, resulting in a final concentration of 0.1% DMSO. Control cultures received the same concentration of DMSO. Cells were fixed after 24 hr
with 4% p-formaldehyde and then mounted on microscope
slides. The length of the longest neurite per cell was measured using a
microscope-mounted image processor with cursor overlay. Only neurites
longer than ~10 µm and not in contact with other cells were
measured. Neuron attachment to L1 was assayed by counting at least 100 cells from 30 or more randomly selected fields. Data were obtained from
three independent experiments.
Immunofluorescence staining for endocytosis of L1. For
experiments in which cell surface L1 and endocytosed L1 were
differentially labeled in living cells, a modification of the method of
Kamiguchi et al. (1998) was used. Rat B35-L1 neuroblastoma cells
(20,000 cells/well) were plated in LabTek II chamber slides (Nunc,
Naperville, IL) coated with poly-D-lysine. After 24 hr,
cells were transfected using Lipofectamine with a plasmid encoding
enhanced cyan fluorescence protein (0.15 µg of cDNA) together with a
plasmid expressing one of the following (0.25 µg of cDNA): wild-type
c-Src, kinase-inactive Src(K295M), dominant-negative dynamin(K44A), or
dominant-negative PI3-kinase (p85). At 24 hr after the start of
transfection, cells were incubated for 45 min at 37°C with 25 µg/ml
Neuro4 IgG or normal mouse IgG to induce endocytosis. Cells were washed
with cold DMEM and then incubated with goat anti-mouse IgG conjugated to rhodamine for 30 min at 4°C to label L1 on the cell surface. The
cells were rinsed with HBSS and fixed in 4% p-formaldehyde for 30 min. After washing cells once with 0.1 mM
glycine in PBS and twice with PBS, cells were blocked with goat
anti-mouse IgG for 1 hr at 4°C. After rinsing once in cold PBS, the
cells were again fixed in 4% p-formaldehyde for 10 min at
room temperature. Cells were washed once with 0.1 mM glycine in PBS and twice with PBS, and the
membrane was permeabilized with 0.1% Triton X-100 in PBS containing
10% goat serum for 1 hr at room temperature. Cells were washed once
with PBS and incubated with anti-mouse IgG conjugated to fluorescein
isothiocyanate (FITC) for 1 hr to label cytoplasmic L1. After
additional washes, slides were mounted with Vectashield. Cells were
viewed in a Zeiss LSM10 confocal laser microscope equipped with an
argon laser (excitation lines of 488 and 514 nm) resulting in an
optical thickness of 0.5 µm at the University of North Carolina
Microscopy Services Facility, Department of Pathology (Chapel Hill, NC;
Dr. Bob Bagnell, Director).
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RESULTS |
L1 clustering activates the MAPK pathway in cerebellar neurons
L1 signal transduction was investigated in primary cultures of
mouse cerebellar neurons, which express endogenous L1 and are comprised
of >90% granule cells (Schnitzer and Schachner, 1981). Signaling by
cell adhesion molecules of the Ig superfamily, such as neural cell
adhesion molecule (NCAM), integrins, and certain receptor
tyrosine kinases, is initiated by the clustering of receptors with
multivalent ligands or antibodies (Heldin, 1995). L1 molecules were
clustered on the surface of cerebellar neurons with antibodies against
the L1 extracellular domain; then cell lysates were analyzed by
immunoblotting with phospho-specific MAPK antibodies recognizing a
dually phosphorylated/activated form of the extracellular-regulated kinases ERK1 and ERK2. These MAP kinases are activated by dual phosphorylation of Thr 202 and Tyr 204 (Crews et al., 1992; Marshall, 1994). L1 clustering in cerebellar neurons stimulated ERK2 dual phosphorylation fivefold, increasing rapidly within 5-10 min and then
declining to nearly basal levels by 20 min (Fig.
1). The decline in phosphorylation could
be caused by activation of a dual specificity tyrosine/threonine
phosphatase such as MAPK phosphatase (MKP-1) (Fuller et al., 1997) or
L1-triggered phosphatase activities present in a subcellular fraction
enriched in growth cone membranes (Klinz et al., 1995). L1 clustering
did not induce phosphorylation of ERK1 in the cerebellar neuron
cultures even though similar levels of ERK1 and ERK2 were indicated by
immunoblotting with antibodies against ERK1 and ERK2 protein (Fig.
1A). Nonimmune IgG complexes had no effect on ERK
phosphorylation. Multivalent L1 protein, consisting of the L1
extracellular region fused to the Fc portion of human IgG, also
stimulated the phosphorylation of ERK2 approximately four- to fivefold
in cerebellar neurons with kinetics similar to that induced by L1
antibody complexes (Fig. 1). ERK2 phosphorylation in cerebellar neurons
induced by cross-linked L1 antibodies or L1-Fc protein was effectively
inhibited by the inhibitor PD98059, which is specific for MEK (MAP
kinase kinase) (Fig. 1). PD98059 binds the inactive form of MEK1 and to
a lesser extent MEK2, preventing the MEK activation required for MAPK
phosphorylation (Alessi et al., 1995).
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Figure 1.
Clustering of L1 in cerebellar neurons activates
ERK2. A, Top, Cerebellar cultures were incubated with
nonimmune mouse IgG (NIgG), monoclonal
antibodies against L1 (Neuro4) complexed with F(ab')2
fragments of anti-mouse IgG (L1 Ab), or L1-Fc fusion
protein complexed with F(ab')2 fragments of anti-human IgG
(L1 protein). Where indicated (+PD),
cells were treated with the MEK inhibitor PD98059 (PD;
25 µM). Cell extracts were subjected to SDS-PAGE and
immunoblotting with anti-active MAPK antibodies specific for
phosphorylated ERKs (pERK1 and
pERK2). Bottom, The same nitrocellulose
filter was stripped and reblotted with antibodies recognizing
phosphorylated and nonphosphorylated ERK proteins (ERK1 and ERK2). This
experiment was repeated twice with similar results. B,
Densitometric quantification of the ERK phosphorylation in
A is shown in arbitrary units of phosphorylation
relative to that of the nonimmune IgG control. Error bars indicate SEs
based on three experiments; an asterisk denotes
statistical significance (p < 0.05).
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An intact MAPK pathway is required for neurite outgrowth on L1
To determine whether the MAPK pathway participated in L1-directed
neurite outgrowth, primary cultures of mouse cerebellar neurons
(postnatal day 7-8) were analyzed for neurite outgrowth on purified
mouse L1 protein adsorbed to nitrocellulose-coated dishes as described
previously (Ignelzi et al., 1994). L1-stimulated neurite outgrowth was
inhibited by the MEK inhibitors PD98059 and U0126 (Fig.
2, Table
1). Mean neurite length decreased 56% in
the presence of PD98059 and 66% with U0126 (Fig. 2). U0126 was
slightly more effective than PD98059, probably because U0126 inhibits
active and inactive forms of MEK (Favata et al., 1998). The residual
neurite outgrowth observed in the presence of MEK inhibitors suggested
that there may be both MAPK-dependent and -independent pathways for
neurite growth on L1. Neither PD98059 nor U0126 affected cerebellar
neuron attachment to purified L1 protein (Table 1;
Cells/mm2), the ability of cerebellar
neurons to initiate neurite growth (Table 1; Cells with
neurites/mm2; % cells with neurites), or
the morphology of the neurons. Cells did not adhere to or extend
neurites on areas of the nitrocellulose-coated dishes without L1
protein. Effects on fasciculation could not be evaluated in these
sparsely plated cultures. These results reveal a functional role for
the MEK-MAPK pathway in L1-dependent neurite outgrowth by cerebellar
neurons.
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Figure 2.
Effect of MEK inhibitors on L1-dependent neurite
outgrowth by cerebellar neurons. Mouse cerebellar neurons were plated
on purified L1 protein adsorbed to nitrocellulose-coated coverslips and
allowed to extend neurites for 24 hr without or with MEK inhibitors (50 µM PD98059 or 10 µM U0126). Cells were
fixed, neurite lengths were measured, and results were plotted in a
neurite length distribution curve. Two hundred or more cells were
analyzed for each condition (see Table 1 for additional data). This
experiment was repeated twice with similar results.
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L1 activates ERK1 and ERK2 in B35-L1 neuroblastoma cells
The rat neuroblastoma cell line B35 (Schubert et al., 1974) is a
useful CNS model for investigating signaling pathways of neural
cell adhesion molecules (Schmid et al., 1999). The B35 cell line
displays differentiated neuronal properties such as membrane
excitability and expression of enzymes for neurotransmitter metabolism
(Schubert et al., 1974) and is more easily transfected than primary
neurons. B35 cells express little or no L1 as detected by
immunoblotting and immunofluorescence staining with L1 antibodies. To
define the L1-signaling pathway for MAPK activation further, a B35 cell
line was generated that stably expressed the RSLE-containing neuronal
isoform of L1 (B35-L1 cells).
L1 molecules were clustered on the surface of B35-L1 cells with L1
antibody complexes; then cell lysates were analyzed for dual
phosphorylation of endogenous MAPKs by immunoblotting with phospho-specific MAPK antibodies. Phosphorylation of both ERK1 and ERK2
increased four- to fivefold within 10 min of L1 clustering, whereas
nonimmune IgG had no effect (Fig.
3A,B). Phosphorylation was
rapid and transient with similar kinetics for ERK1 and ERK2. Clustering
of L1 on the cell surface was necessary for MAPK activation, because
monovalent Fab fragments of L1 antibodies did not induce ERK
phosphorylation (data not shown). The ability of L1 to stimulate phosphorylation of ERK1 and ERK2 in B35-L1 cells, in contrast to the
phosphorylation of only ERK1 in cerebellar neurons, suggested that
these pathways were differentially regulated. Similar results were
obtained during NCAM stimulation in cerebellar neurons and B35 cells
expressing NCAM140 (Schmid et al., 1999). Selective activation of ERK2
and not ERK1 in neuronal cells also occurs during stimulation of
protein kinase C or NMDA receptors (English and Sweatt, 1996).
Differential MAPK regulation was characterized previously in yeast in
which mating and pseudohyphal differentiation are independently
controlled by the MAPKs Fus3 and Kss1 (Madhani et al., 1997).
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Figure 3.
Clustering of L1 in B35 neuroblastoma cells
transiently activates MAPK but not JNK or p38. A, Top,
Rat B35 neuroblastoma cells stably expressing L1 (B35-L1) were
incubated with either nonimmune mouse IgG or L1 mAb Neuro4 complexed
with F(ab')2 fragments of anti-mouse IgG for various times
(5-20 min). Cell extracts were subjected to SDS-PAGE and
immunoblotting with anti-active MAPK antibodies
(pERK1 and pERK2).
Bottom, The same nitrocellulose filter was stripped and
reblotted with an ERK antibody recognizing ERK1 and ERK2. This
experiment was repeated three times with similar kinetics and extent of
MAPK activation. B, Densitometric quantification of MAPK
phosphorylation in the experiment shown in A is
expressed in arbitrary units of ERK1 and ERK2 phosphorylation relative
to that of the nonimmune IgG control. Error bars indicate SEs based on
three experiments; an asterisk denotes statistical
significance (p < 0.05). C,
The same cell extracts shown in A were assayed for JNK
activation by the JNK immune complex kinase assay. Autoradiography
showed no increase in the phosphorylation of c-Jun(1-79) by JNK from
L1-stimulated cells (p c-Jun). As a positive
control, cells were incubated with 300 mM sorbitol for 15 min. This experiment was performed three times with similar results.
D, The same cell extracts shown in A were
subjected to SDS-PAGE and immunoblotting with anti-phospho p38 MAPK
antibodies, which recognize phosphorylated/activated p38
(p p38). No increase in phosphorylation
levels was seen. As a positive control, cells were incubated with 10 mM anisomycin for 10 min. This experiment was performed
twice with similar results.
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To address the specificity of L1 activation of the MAPK-signaling
pathway, B35-L1 cells were assayed for L1-dependent phosphorylation of
the MAP kinases, JNK, and p38, which are activated in response to multiple cellular stressors (Waskiewicz and Cooper, 1995). No
increase of JNK phosphorylation was observed within 5-20 min of L1
clustering, whereas sorbitol-treated cells as a positive control showed
prominent JNK activity (Fig. 3C). Similarly, the phosphorylation of p38 MAPK was not increased over basal levels compared with anisomycin-treated cells as positive controls (Fig. 3D).
L1 activates the MAPK pathway via PI3-kinase and Rac
To investigate whether L1 clustering activated PI3-kinase in L1
antibody-triggered B35-L1 cells, the production of phosphoinositides by
PI3-kinase was quantitatively determined. During activation, PI3-kinase
becomes phosphorylated on tyrosine and can be immunoprecipitated with
phosphotyrosine antibodies. Incubation of immune complexes from L1
antibody-stimulated B35-L1 cells with phosphatidylinositol and
[32P]ATP produced radiolabeled
phosphatidylinositol 3-phosphate, which was separated from reactants by
thin-layer chromatography (Fig.
4A). The amount of
activated PI3-kinase increased two- to threefold within 3-8 min after
antibody triggering. A similar extent of increase in PI3-kinase
activity occurs in cells treated with epidermal growth factor (EGF),
platelet-derived growth factor, or phorbol esters and is
effective in mediating their physiological functions (Conricode, 1995;
Nave et al., 1996; Cross et al., 1997). To determine whether PI3-kinase
was involved in regulating L1-triggered ERK phosphorylation, a
dominant-negative PI3-kinase mutant (p85) was cotransfected with
HA-ERK2 for transient expression in B35-L1 cells. This dominant
inhibitory mutant contains a deletion in the PI3-kinase regulatory
subunit (p85) that abolishes its binding to the catalytic subunit
(p110) (Hara et al., 1994). Stimulation of B35-L1 cells expressing this
mutant with L1 antibodies resulted in complete inhibition of ERK2
phosphorylation (Fig. 4B). Treatment of B35-L1 cells
with 10 µM Ly294002, a selective chemical
inhibitor of PI3-kinase (Vlahos et al., 1994), prevented L1
antibody-induced phosphorylation of endogenous ERK1 and ERK2 (Fig.
4D).
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Figure 4.
Dominant-negative mutants of PI3-kinase and Rac1
inhibit MAPK activation induced by L1 clustering in B35 neuroblastoma
cells. A, B35-L1 cells were incubated with mouse IgG or
the L1 monoclonal antibody Neuro4 complexed with F(ab')2
fragments of anti-mouse IgG. Cell lysates (800 µg) were
immunoprecipitated with anti-phosphotyrosine mAb (4G10), and the
precipitates were incubated in an in vitro kinase
reaction with phosphoinositides. Phospholipid products were separated
by thin-layer chromatography (top). Error bars in the
graph (bottom) indicate SEs based on three experiments;
an asterisk denotes statistical significance
(p < 0.05). B, Top
row, B35-L1 cells transiently expressing HA-ERK2 and the
dominant-negative (DN) mutants PI3-kinase
(PI3K) p85, RhoA(19N), Rac1(17N),
Cdc42(17N), FRNK, Fak(397F), or Ras(15A) were incubated with mouse IgG
or the L1 monoclonal antibody Neuro4 complexed with F(ab')2
fragments of anti-mouse IgG. As a control, cells were exposed to 100 ng/ml EGF for 5 min. Cell extracts were immunoprecipitated with anti-HA
antibodies and then subjected to SDS-PAGE and immunoblotting with
anti-active MAPK antibody (p ERK2). Bottom
row, The same nitrocellulose filters were stripped and
reblotted with an antibody recognizing ERK2 protein. The experiments
were performed three times with similar results. C,
B35-L1 cells were incubated with normal mouse IgG or the L1 monoclonal
antibody Neuro4 complexed with F(ab')2 fragments of
anti-mouse IgG for 3 and 8 min. GTP-loaded Rac was pulled down from the
cell lysates (500 µg) by the addition of RBD-GST fusion
protein conjugated to Sepharose beads, and the relative levels of
Rac-GTP were evaluated by SDS-PAGE and immunoblotting with anti-Rac1
antibodies. D, Top, B35-L1 cells were
exposed to the C3 toxin Rho inhibitor or the Ly294002 PI3-kinase
inhibitor as described in Materials and Methods and then incubated with
normal mouse IgG or the L1 monoclonal antibody Neuro4 complexed with
F(ab')2 fragments of anti-mouse IgG for 7 min. Cell
extracts were subjected to SDS-PAGE and immunoblotting with anti-active
MAPK antibody (p ERK2). Bottom,
The same nitrocellulose filters were stripped and reblotted with an
antibody recognizing ERK2 protein. ori, Origin;
Rel, relative.
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Rac and Cdc42 are members of the Rho family of small GTPases that
regulate actin cytoskeletal dynamics (Ridley et al., 1992), signal to
JNK (Vojtek and Cooper, 1995), and cause cross-cascade activation of
the ERK pathway (Frost et al., 1997). PI3-kinase contributes to the
activation of Rac and Cdc42 by providing phosphoinositides that bind
the plextrin homology domain of specific guanine nucleotide exchange
factors (GEFs) (Quilliam et al., 1995). To investigate the role of
small GTPases in L1-stimulated MAPK activation, B35-L1 cells were
cotransfected with HA-tagged ERK2 and dominant-negative Rac1, Ccd42,
and RhoA plasmids, which act by sequestering respective GEFs. ERK2
phosphorylation in antibody-triggered B35-L1 cells was effectively
blocked by the expression of dominant-negative Rac1(17N) but not
RhoA(19N) or Cdc42(17N) mutants (Fig. 4B). C3 botulinum toxin, which inhibits RhoA, B, and C but not Rac or Cdc42
(Braun et al., 1989; Chrzanowska-Wodnicka and Burridge, 1996), also had
no effect on L1-dependent ERK phosphorylation (Fig.
4D). The participation of Rac in L1 signaling was
confirmed in an assay that directly measured the production of
activated, GDP-bound Rac (Bagrodia et al., 1998). Cell lysates from L1
antibody-treated B35-L1 cells were incubated with purified
RBD-GST fusion protein, which selectively binds Rac-GTP and
not Rac-GDP. The amount of Rac-GTP pulled down by the RBD-GST
complexes was significantly increased after L1 stimulation of B35-L1
cells (Fig. 4C). These results supported a role for Rac in
L1 signaling to MAPK.
L1-triggered MAPK activation in B35-L1 neuroblastoma cells differed
from NCAM140 signaling to MAPK in its independence from Ras and the
focal adhesion kinase p125fak. Cotransfection of
B35-L1 cells with HA-ERK2 and a dominant-negative Ras(15A) plasmid,
which sequesters Ras-GEFs (Quilliam et al., 1994), had no effect on L1
antibody-stimulated ERK phosphorylation, whereas it strongly inhibited
MAPK activation by EGF (Fig. 4B). Expression of a Ras
dominant-negative mutant (Ras17N) or a competitive N-terminal Raf
peptide that binds Ras (Brtva et al., 1995) also had no effect (data
not shown). Under the same conditions, Ras(15A) and Ras(17N) interfered
strongly with NCAM140-dependent MAPK activation in B35 cells (Schmid et
al., 1999). In a coupled immune complex kinase assay for Raf-induced
phosphorylation of MBP (Fucini et al., 1999), neither c-Raf nor
B-Raf became activated to a significant degree (>10% over the normal
IgG control) during L1 antibody stimulation of B35-L1 cells.
Dominant-negative Raf-1 or B-Raf mutants (S621A or R89L) also produced
insignificant inhibition of ERK2 phosphorylation (>1.2-fold).
Similarly, expression of FRNK (Schaller et al., 1993;
Richardson and Parsons, 1996) or the autophosphorylation site mutant Fak(Y397F) (Schaller et al., 1994), which are dominant interfering inhibitors of p125fak, did not affect
L1-triggered HA-ERK2 activation (Fig. 4B), whereas under the same conditions they inhibited NCAM140-triggered MAPK activation in B35 cells (Schmid et al., 1999). Tyrosine phosphorylation of p125fak was also not increased after L1
antibody triggering, as assessed by Western blotting with
phosphotyrosine antibodies in Fak immunoprecipitates (data not shown).
pp60c-src mediates ERK
phosphorylation induced by L1
Because neuronal process extension on purified L1 is regulated by
the nonreceptor tyrosine kinase pp60c-src (Ignelzi et al.,
1994), we investigated whether pp60c-src played a role in MAPK
activation triggered by L1. B35-L1 neuroblastoma cells were
cotransfected with HA-ERK2 and the dominant-negative c-Src(K295M)
plasmid, which expresses a kinase-inactive form of pp60c-src
because of mutation of an essential lysine residue (K295) in the ATP
binding site (Twamley-Stein et al., 1993). Expression of the
dominant-negative src plasmid inhibited ERK2 phosphorylation by ~70% in B35-L1 neuroblastoma cells treated with L1 antibodies (Fig. 5A). Moreover,
endogenous ERK2 phosphorylation in cerebellar neurons from
src-minus mice was 60% lower than that in wild-type neurons
(Fig. 5B). The residual ERK2 phosphorylation in
src-minus neurons and B35-L1 cells expressing
dominant-negative Src suggested that there may an additional L1 pathway
that is pp60c-src independent, in accord with the incomplete
inhibition of L1-dependent neurite outgrowth in MEK-inhibited cells and
by src-minus neurons (Ignelzi et al., 1994). L1 did not form
a stable complex with pp60c-src, because L1 and
pp60c-src did not coimmunoprecipitate from Brij97-detergent
extracts of B35-L1 cells with or without L1 clustering (data not shown)
under the same conditions in which NCAM140 coimmunoprecipitated with the Src family kinase p59fyn (Beggs et al.,
1997).
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Figure 5.
Dominant-negative mutants of pp60c-src and
dynamin inhibit MAPK activation induced by L1 clustering in B35
neuroblastoma cells. A, Top, B35-L1 cells
transiently expressing HA-ERK2 and the dominant-negative mutants
c-Src(K295M) or dynamin (K44A) were incubated for 7 min with
mouse IgG or the L1 monoclonal antibody Neuro4 complexed with
F(ab')2 fragments of anti-mouse IgG. Cell extracts in NP-40
lysis buffer were immunoprecipitated with anti-HA antibodies and then
subjected to SDS-PAGE and immunoblotting with anti-active MAPK antibody
(p ERK2). Bottom, The same
nitrocellulose filters were stripped and reblotted with an ERK antibody
recognizing ERK2 protein. The experiments were performed twice with
similar results. B, Top, Cerebellar neurons from
wild-type mice (wt) or src-minus mice
(src /) were incubated with mouse IgG
or the L1 monoclonal antibody Neuro4 complexed with F(ab')2
fragments of anti-mouse IgG. Buffers of the cell extracts were
subjected to SDS-PAGE and immunoblotting with anti-active MAPK Ab
(p ERK2). Bottom, The same
nitrocellulose filters were stripped and reblotted with an ERK antibody
recognizing ERK2. The experiments were performed twice with similar
results.
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pp60c-src is required for
dynamin-mediated endocytosis of L1
L1 has been shown to be endocytosed at the growth cone by a
clathrin-mediated mechanism (Kamiguchi et al., 1998). To determine whether L1 endocytosis was mediated by pp60c-src, B35-L1 cells
were analyzed for L1 endocytosis by double immunofluorescence staining
to visualize internalized and surface L1 with different fluorochromes.
Cells were transfected with plasmids encoding wild-type Src,
kinase-inactive Src(K295M), or dominant-negative dynamin(K44A) together
with a plasmid expressing cyan fluorescent protein to identify
transfected cells. To induce internalization of L1, we incubated living
cells for 30 min at 37°C with the monoclonal antibody Neuro4, which
is directed against an extracellular epitope of L1. Cells were fixed
and stained for L1 on the cell surface with rhodamine-conjugated
secondary antibodies. Cells were then permeabilized and stained for
internalized L1 with FITC-conjugated secondary antibodies. Confocal
microscopy revealed strong FITC labeling of internalized L1 antibody
complexes in a punctuate, vesicular pattern throughout the cytosol both
in cyan-positive cells (Fig. 6,
arrows, arrowheads) expressing wild-type Src from a
transfected plasmid (Fig. 6A,E) and in nontransfected
cells expressing endogenous Src (Fig. 6D,H).
This observation suggested that the amount of endogenous
pp60c-src was not rate-limiting for endocytosis of L1 antibody
complexes in B35-L1 cells. In striking contrast, cyan-positive cells
(Fig. 6, arrows, arrowheads) in cultures transfected with
dominant-negative Src(K95M) showed little L1 endocytosis (Fig.
6B,F,C,G), whereas cyan-negative cells serving as
nontransfected controls within the same cultures exhibited prominent
vesicular staining in the cytosol. Rhodamine staining for L1 on the
cell surface appeared relatively unaltered by endocytosis, suggesting
that the amount of internalized L1 was a relatively small portion of
the total L1 expressed in B35-L1 cells. L1 endocytosis was not
inhibited in B35-L1 cells treated with the MEK inhibitor PD98059 (50 µM) (Fig. 6J,N), in
cells expressing PI3-kinase p85 (Fig. 6K,O), or in
cells treated with the PI3-kinase inhibitor Ly294002 (20 µM), suggesting that PI3-kinase and MEK-MAPK
did not contribute to the mechanism of endocytosis.
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Figure 6.
Endocytosis of L1 antibody complexes requires
pp60c-src and dynamin. Endocytosis of L1 antibody complexes
induced with Neuro4 monoclonal antibodies against an extracellular
epitope of L1 was visualized in transfected B35-L1 neuroblastoma cells
by double immunofluorescence staining for L1 on the cell surface
(rhodamine) and internalized L1 (FITC) using confocal microscopy as
described in Materials and Methods. B35-L1 cells were cotransfected
with one of the pcDNA3 plasmids listed below together with equimolar
amounts of a plasmid expressing enhanced cyan fluorescent protein
(ECFP) as a marker for transfected cells. Arrows and
arrowheads denote ECFP-positive cells.
Images show representative cells from all experiments.
A, E, Wild-type c-Src. B,
F, C, G, Dominant-negative Src(K295M).
D, H, No transfection. I,
M, Dominant-negative dynamin(K44A).
J, N, Treatment with MEK inhibitor
PD98059 (50 µM). K, O,
Dominant-negative PI3-kinase (p85). L,
P, Treatment with PI3-kinase inhibitor Ly294002 (20 µM). A D and I-L show L1
on the cell surface (rhodamine); E-H and
M-P show internalized L1 (FITC). Scale bar, 10 µm.
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Dynamin GTPase plays a critical role in the formation of
clathrin-coated vesicles during endocytosis of many receptors (Hinshaw and Schmid, 1995), as shown by the ability of dominant-negative dynamin(K44A) to block effectively the formation of endocytotic-coated vesicles (Damke et al., 1994). Dynamin(K44A) also prevented L1 internalization in L1-B35 cells (Fig. 6M), confirming
its role in internalizing L1 by a clathrin-mediated mechanism
(Kamiguchi et al., 1998) in B35 cells. Although Rac has been shown to
modulate receptor-mediated endocytosis in some systems (Lamaze et al., 1996), expression of dominant-negative Rac1(17N) in B35 cells did not
affect L1 antibody-induced internalization (data not shown), suggesting
that this small GTPase did not regulate L1 endocytosis. Importantly,
the dynamin(K44A) mutant effectively inhibited L1 antibody-induced
phosphorylation of HA-ERK2 in transfected B35-L1 cells (Fig.
5A), thus indicating that an intact endocytotic mechanism was necessary for L1 triggering of the MAPK cascade.
Taken together these results demonstrated that the dynamin-mediated
internalization of L1 in B35-L1 cells depended on pp60c-src and
that L1 endocytosis was required for activation of an MAPK cascade that
regulated neurite outgrowth on L1.
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DISCUSSION |
Here we report that an MAPK-signaling pathway mediates neurite
outgrowth on L1 and requires pp60c-src-dependent endocytosis.
This is the first demonstration of a neuronal function regulated by L1
via MAPK and of the role of pp60c-src in regulating
dynamin-mediated endocytosis of L1 as an essential initial step in MAPK
activation. Key intermediates on the L1-MAPK-signaling pathway were
identified to be the small GTPase Rac and PI3-kinase. Because ERKs
activate gene expression via phosphorylation of the transcription
factors Elk-1 (Marais et al., 1993) and CRE-binding protein (Ginty et
al., 1994), these results raise the possibility that neurite outgrowth
may rely on signaling events in the growth cone and nucleus. The
proposed signaling pathway is outlined in Figure
7.
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Figure 7.
Proposed L1-signaling pathway. Cross-linking of L1
molecules on the growth cone membrane is proposed to induce
dynamin-mediated endocytosis of L1 via the Src tyrosine kinase, leading
to initiation of an intracellular signal transduction cascade involving
the sequential activation of PI3-kinase, Rac, MEK, and MAPK. Inhibitors
of PI3-kinase (Ly294002) and MEK (PD98059 and U0126) are shown. It is
suggested that Rac activation in the growth cone leads to cytoskeletal
changes resulting in lamellipodia and that MAPK may have nuclear
effects on gene expression, both of which may be needed for neurite
outgrowth.
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Results of this study demonstrate the requirement of an intact MEK-ERK
pathway in regulating neurite outgrowth by cerebellar neurons on an L1
substrate. ERK phosphorylation is widely used for neurite outgrowth in
response to pleiotropic axon growth/guidance cues, which include L1m
NCAM (Schmid et al., 1999), the extracellular matrix (Ihara et al.,
1997), and neurotrophins (Xia et al., 1995; Riccio et al., 1997). L1
signaling to MAPK may also function in synaptic plasticity as suggested
from antibody perturbation experiments (Luthi et al., 1994), because
ERKs are phosphorylated in neurons during -adrenergic and
serotonergic stimulation (Koch et al., 1994; Michael et al., 1998) and
in hippocampal long-term potentiation (LTP) (English and Sweatt, 1997).
The extent of ERK phosphorylation triggered by L1 (four- to fivefold)
approximated that induced by NCAM (Schmid et al., 1999), NGF (Riccio et
al., 1997), integrins (Chen et al., 1994), and LTP (English and Sweatt,
1997), underscoring its potential physiological relevance. By
transducing signals that converge within the MAPK pathway, adhesion
molecules, growth factors, and neurotransmitters may coordinately
regulate the growth or branching of axons and dendrites. While our work
was in progress MAPK was shown to be activated in L1-transfected NIH3T3
cells dependent on endocytosis (Schaefer et al., 1999). Our studies in
neural cells are in accord with this report but go further by
demonstrating a physiological role for MAPK in L1-dependent neurite
outgrowth and by identifying pp60c-src, Rac, and PI3-kinase as
key signaling intermediates in the pathway.
An important finding of our work is the requirement for
pp60c-src in dynamin-dependent endocytosis of L1 and activation
of MAPKs in neural cells. These results provide a mechanistic
understanding of the inhibition of L1-dependent neurite outgrowth
observed in src-minus neuronal cultures (Ignelzi et al.,
1994). Our studies suggest (1) that pp60c-src acts as a
gatekeeper of the MAPK cascade by regulating L1 endocytosis and (2)
that endocytosis and MAPK activation are needed for neurite growth on
L1. Dynamin-mediated endocytosis is also an early step in MAPK
activation induced by EGF (Vieira et al., 1996), serotonin 5-HT1A
(Della Rocca et al., 1999), insulin-like growth factor I (Chow et al.,
1998), lysophosphatidic acid (Luttrell et al., 1997), and
2-adrenergic agonists (Daaka et al., 1998).
Although only L1 and the 2-adrenergic receptor
have been shown to depend on pp60c-src for endocytosis and MAPK
activation (Ahn et al., 1999), it is conceivable that Src family
kinases may participate in endocytosis/signaling by other receptors. It
remains to be determined whether L1 clustering activates
pp60c-src by dephosphorylating Tyr 527 in the Src C-terminal
domain (Bjorge et al., 1996) possibly via tyrosine phosphatase
PTP- (Ponniah et al., 1999). A role for tyrosine
phosphatases in L1 signaling is consistent with the ability of L1
antibodies to induce dephosphorylation of tyrosine in growth cone
proteins (Atashi et al., 1992; Klinz et al., 1995). Because neurite
outgrowth by cerebellar neurons on L1 was not totally suppressed by MEK
inhibitors or in src-minus neurons (Ignelzi et al., 1994),
there may be an additional Src/MAPK-independent mechanism for
L1-dependent neurite growth. For example, neurite growth responses in
common to L1, NCAM, and N-cadherin have been shown to occur via the
basic fibroblast growth factor receptor, phospholipase C, and
production of arachidonic acid (Saffell et al., 1997). It should be
noted that because different cell types (cerebellar neurons and B35
neuroblastoma cells) were used to measure neurite outgrowth and
endocytosis, the results do not formally prove that endocytosis of L1
is required for the regulation of neurite outgrowth. Other than
pp60c-src it is not clear whether components of the L1-MAPK
cascade identified in B35 cells are also deployed for signal
transduction and neurite outgrowth in cerebellar neurons and other
neuronal cell types. Nonetheless, the finding that pp60c-src
was needed for ERK activation in both cell types and that deletion of
the src gene causes inhibition of neurite outgrowth
in cerebellar neurons (Ignelzi et al., 1994) supports the possibility
that Src-dependent endocytosis mediates neurite outgrowth on L1.
L1 signaling, as delineated in B35 cells, differed from the
prototypical Ras-MAPK cascade used by NCAM and integrins (Schlaepfer and Hunter, 1997; Schmid et al., 1999) in its independence from Ras and
Fak. However, Ras-independent (Howe and Juliano, 1998) and
FAK-independent signaling pathways are used for some forms of integrin
signaling (Lin et al., 1997; Miranti et al., 1998). FAK-independent
signaling in platelets shares with the L1 pathway the involvement of
pp60c-src, Rac, and PI3-kinase (Miranti et al., 1998). Although
we have not found 1-integrins stably associated with L1, a role for
specific -integrin subtypes in L1-MAPK signaling remains open,
because L1 has been shown to interact functionally with integrins for neurite outgrowth and cell migration on L1, mediated in part by an RGD
sequence in the sixth Ig domain (Montgomery et al., 1996; Yip et al.,
1998).
Neuronal growth cones display distinctive morphologies on L1, NCAM, and
laminin (Payne et al., 1992; Abosch and Lagenaur, 1993; Burden-Gulley
et al., 1995, 1997); yet each substrate can activate MAPKs, raising the
question of how specificity arises. Activation of different signaling
intermediates on the MAPK pathway may allow neurons to respond to
extracellular cues in distinct ways. In B35 cells, L1 signals to MAPK
via pp60c-src, PI3-kinase, and Rac, whereas NCAM140 signals to
MAPK via Fyn, FAK, Ras, and Rho (Schmid et al., 1999). Nonetheless, the
involvement of Rac in L1 signaling is consistent with the lamellipodial
morphology displayed by growth cones of retinal ganglion cells on L1
(Payne et al., 1992; Burden-Gulley et al., 1995). Activated Rho family GTPases differentially modulate actin cytoarchitecture (Nobes and Hall,
1995). Rac generates lamellipodia by inducing actin depolymerization
via LIM kinase-1 (Arber et al., 1998; Yang et al., 1998), Rho
induces actin stress fibers and growth cone collapse (Kozma et al.,
1997), and Cdc42 elicits filopodia via LIM kinase-2 (Sumi et al.,
1999). PI3-kinase may promote Rac activation via the binding of
phosphoinositide products to the plextrin homology domain of Rac-GEFs
(Han et al., 1998; Nimnual et al., 1998). PI3-kinase is an important
determinant of growth cone guidance, required for the turning of
Xenopus growth cones toward a netrin gradient (Hong et al.,
1999).
The ability of L1 to phosphorylate ERKs via Rac could occur by
cross-cascade stimulation of MEK. Rac typically activates the JNK
pathway (Crespo et al., 1996) but is an upstream regulator of
p21-activated kinase, which directly phosphorylates MEK and stimulates
ERK phosphorylation in a Ras-independent manner (Frost et al., 1997).
Such cross-talk allows pathways for Rac activation to cooperate with
prototypical Ras-Raf-MEK-MAPK pathways (Frost et al., 1997). In this
way, NCAM or neurotrophin signaling via Ras may cooperate with L1
signaling via Rac for MAPK activation and neurite outgrowth. Although
we found no evidence of c-Raf 1 or B-Raf in L1 signaling, basal levels
of a Raf isoform might participate.
The ability of L1 clustering to activate PI3-kinase also supports a
role for L1 in neuronal survival. PI3-kinase promotes neuronal survival
and blocks apoptosis in response to neurotrophins and insulin-like
growth factor I via the serine/threonine kinase Akt (Ashcroft et al.,
1999). Although Akt was not examined in our studies, a role for L1 in
neuronal survival is consistent with a 30% reduction in the number of
hippocampal neurons in L1 knock-out mice (Demyanenko et al., 1999) and
with the ability of L1 to enhance the survival of dopaminergic neurons
(Hulley et al., 1998) and to prevent apoptotic death of cerebellar and hippocampal neurons in culture (Chen et al., 1999).
Src-mediated endocytosis of L1 has the potential for not only local
actin cytoskeletal rearrangements in the growth cone but also nuclear
transcriptional control. Via Rac and PI3-kinase, L1 could cause actin
rearrangements to form lamellipodial extensions needed for neurite
elongation. Endocytosis of L1 might also serve to release the growth
cone from attachment to the substratum, facilitating its forward
migration. L1 in endocytic vesicles would be oriented with its C
terminal projecting toward the cytoplasm, enabling downstream signaling
molecules in the growth cone to be recruited and/or activated, a
scenario consistent with the observation that activated ERK colocalizes
with L1 in endocytosed vesicles of L1-transfected fibroblasts (Schaefer
et al., 1999). L1-signaling complexes might also be transported from
the growth cone to the cell body, as shown for internalized NGF-Trk
receptor complexes in pheochromocytoma 12 cells (Riccio et al., 1997). There dually phosphorylated ERKs would be in position to translocate into the nucleus, where they may induce transcription of the genes needed for neuronal process extension or cell survival.
| |
FOOTNOTES |
Received Feb. 1, 2000; revised March 15, 2000; accepted March 17, 2000.
This research was supported by National Institutes of Health Grant NS
26620 to P.F.M. and by a grant from the University of North Carolina
Medical Alumni Association to R.-S.S. We are grateful to Dr. Ron Graff
for producing the L1-B35 cell line and to Drs. John Hemperly, Keith
Burridge, Channing Der, and Michael Schaller for providing reagents and
advice. We thank Drs. Melanie Cobb, Sara Courtneidge, Richard Cerione,
Deborah Morrison, Al Baldwin, Marty Mayo, and Shelton Earp for plasmids.
Correspondence should be addressed to Dr. Patricia F. Maness,
University of North Carolina, Department of Biochemistry
CB#7260, Chapel Hill, NC 27599-7260. E-mail:
srclab{at}med.unc.edu.
| |
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ABSTRACT
INTRODUCTION
