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The Journal of Neuroscience, October 15, 2000, 20(20):7602-7613
Dual Roles for c-Jun N-Terminal Kinase in Developmental and
Stress Responses in Cerebellar Granule Neurons
Eleanor T.
Coffey1, 2,
Vesa
Hongisto1, 2,
Martin
Dickens3,
Roger J.
Davis4, and
Michael J.
Courtney1, 2, 5
1 Turku Centre for Biotechnology, Åbo Akademi
University and University of Turku, BioCity, FIN-20521 Turku, Finland,
2 Department of Biochemistry and Pharmacy, Åbo Akademi
University, BioCity, FIN-20521 Turku, Finland, 3 Department
of Biochemistry, University of Leicester, Leicester LE17RH, United
Kingdom, 4 Howard Hughes Medical Institute, Department of
Biochemistry and Molecular Biology, University of Massachusetts,
Worcester, Massachusetts 01655, and 5 A. I. Virtanen
Institute, University of Kuopio, FIN-70211 Kuopio, Finland
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ABSTRACT |
c-Jun N-terminal kinases (JNKs) typically respond strongly to
stress, are implicated in brain development, and are believed to
mediate neuronal apoptosis. Surprisingly, however, JNK does not respond
characteristically to stress in cultured cerebellar granule (CBG)
neurons, a widely exploited CNS model for studies of death and
development, despite the regulation of its substrate c-Jun. To
understand this anomaly, we characterized JNK regulation in CBG
neurons. We find that the specific activity of CBG JNK is elevated
considerably above that from neuron-like cell lines (SH-SY5Y, PC12);
however, similar elevated activities are found in brain extracts. This
activity does not result from cellular stress because the
stress-activated protein kinase p38 is not activated. We identify a
minor stress-sensitive pool of JNK that translocates with
mitogen-activated protein kinase kinase-4 (MKK4) into the
nucleus. However, the major pool of total activity is cytoplasmic,
residing largely in the neurites, suggesting a non-nuclear role for JNK
in neurons. A third JNK pool is colocalized with MKK7 in the nucleus,
and specific activities of both increase during neuritogenesis, nuclear
JNK activity increasing 10-fold, whereas c-Jun expression and activity
decrease. A role for JNK during differentiation is supported by
modulation of neuritic architecture after expression of dominant
inhibitory regulators of the JNK pathway. Channeling of JNK signaling
away from c-Jun during differentiation is consistent with the presence
in the nucleus of the JNK/MKK7 scaffold protein JNK-interacting
protein, which inhibits JNK-c-Jun interaction. We propose a model in
which distinct pools of JNK serve different functions, providing a
basis for understanding multifunctional JNK signaling in
differentiating neurons.
Key words:
cerebellar granule neuron; stress-activated protein
kinase; JNK; JIP; MKK4; MKK7; p38; c-Jun; nuclear translocation; neuronal differentiation; dominant negative; neuronal morphology
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INTRODUCTION |
c-Jun N-terminal kinases (JNKs)
phosphorylate the transactivation domains of several transcription
factors including c-Jun, inducing transcriptional activation. JNKs have
been extensively investigated in non-neuronal tissues. Like the related
kinase p38, they are also known as stress-activated protein kinases, because their activities typically increase up to more than 10-fold in
response to a wide range of cellular stresses such as treatment with
ribosomal inhibitors, osmotic stress, and short wavelength irradiation
(Kyriakis et al., 1995
; Kyriakis and Avruch, 1996; Iordanov et al.,
1997). Ten isoforms of JNK1, JNK2, and the neural JNK3 genes are
expressed in brain (Mohit et al., 1995; Gupta et al., 1996). Two JNK
kinases, mitogen-activated protein kinase kinase-4 (MKK4) and
MKK7, have been cloned (Dérijard et al., 1995; Moriguchi
et al., 1997), and these are further diversified by alternative
splicing. These kinases activate JNK by phosphorylation on both
tyrosine and threonine residues, after which JNK is understood to
translocate to the nucleus, the location of the well characterized JNK
substrates transcription factors c-Jun, ATF2, Elk, and Sap1a (Pulverer
et al., 1991; Gupta et al., 1995; Janknecht and Hunter, 1997). JNK
signaling can be further regulated by JIP (JNK-interacting protein).
JIP binds MKK7 and JNK, enhancing JNK activation by MKK7; however, JIP
does not interact with MKK4 (Whitmarsh et al., 1998). JIP competes with
c-Jun, ATF2, and Elk for JNK binding via its JNK-binding domain (JBD)
(Dickens et al., 1997), suggesting that it may channel JNK activity
toward alternative substrates that interact with JNK via motifs
different from the JBD.
It is widely believed that JNK mediates cellular apoptosis in response
to stress (Kyriakis and Avruch, 1996; Ip and Davis, 1998), a role
particularly emphasized in neurons and neuron-like cells (Xia et al.,
1995) where the JNK phosphorylation sites of c-Jun are required for
trophic factor deprivation-induced death (Ham et al., 1995; Watson et
al., 1998). It might be expected, therefore, that neuronal JNK would
have low basal activity and respond sensitively to apopototic signals.
Surprisingly, however, it has been reported that cerebellar granule
neuron JNK is nonresponsive to apoptotic stresses (Watson et al.,
1998). The literature suggests that JNK activity from CNS neurons may
be high (Xu et al., 1997), but no comparison with the well studied JNKs
from cell lines has been reported.
In this study we analyze cerebellar granule neuron JNK by methods used
to characterize JNK signaling in cell lines. We find that JNK of
differentiating neurons displays high constitutive activity that
resides predominantly in neurites. This largely masks a minor
stress-activatable pool of JNK that exhibits stereotypical behavior,
i.e., nuclear translocation on activation, and appears to have
preferential access to c-Jun. The constitutively active JNK is further
activated during differentiation, as are MKK4 and MKK7 activities.
Inhibition of JNK activity leads to morphological changes suggesting a
role for JNK in regulating neuritic architecture during
differentiation. Finally, we present a model whereby distinct JNK pools
coordinate to transduce stress and developmental signals to different
targets in differentiating neurons.
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MATERIALS AND METHODS |
Materials. Polyclonal antibodies against MKK4 (MEK-4,
K-18), MKK7 (MEK-7, C-19), p38 (C-20), and monoclonal anti-P63-c-Jun (KM-1) were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antiserum to SAPK was a gift from John Kyriakis [Massachusetts General
Hospital (MGH), Boston, MA). Isoform-specific antibodies to JNK1
(G151-333) and JNK1/2 (G151-666) were from PharMingen (San Diego, CA)
and to JNK3/1 were from Upstate Biotechnology (Lake Placid, NY).
Phosphospecific antibodies to JNK and p38 were from New England Biolabs
(Beverly, MA). Monoclonal anti-c-Jun was from Transduction Labs
(Lexington, KY). Monoclonal antibodies to JIP-1 have been described
previously (Yasuda et al., 1999). 6-Cyano-7-nitroquinoxaline-2,3-dione
(CNQX), (RS)-
-Methyl-4-carboxyphenylglycine [(RS)-MCPG], and (5R,
10S)-(+)-5-methyl-10.11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine/dizocilpine [(+)-MK801] were from Tocris Cookson (Bristol, UK). All other materials were from Sigma (St. Louis, MO), Baker (Deventer, Holland), or Calbiochem (La Jolla, CA).
Cell culture. Cerebellar granule neurons were prepared from
7-d-old rats as described previously (Courtney et al., 1997). This
preparation is well characterized and is reported to contain 95% small
interneurons, predominantly granule neurons (Thangnipon et al., 1983).
Cells were cultured in minimal essential medium (Life Technologies,
Paisley, Scotland) supplemented with 10% (v/v) fetal calf serum (Life
Technologies), 33 mM glucose, 2 mM glutamine, 50 U/ml penicillin, 50 µM streptomycin, and 20 mM supplementary KCl (final 25.4 mM KCl),
except those in Figures 2 and 6 that were grown without additional KCl.
Cells were plated at 250,000/cm2 onto
culture surfaces coated with poly-L-lysine (100 µg/ml): 35 mm dishes or wells of 12- or 24-well plates (Costar, Corning, NY,
and Greiner GmbH, Solingen, Germany) for kinase assays and immunoblotting, and 10.5 mm × 10.5 mm coverslips for
immunofluorescent staining. Culture medium was replaced after 24 hr
with the inclusion of 10 µM cytosine arabinofuranoside
(Sigma) to reduce non-neuronal proliferation. After this time, fresh
culture medium was not re-added to the cells, to avoid serum
glutamate-associated toxicity. PC12 cells were cultured on
collagen-coated dishes in minimal essential medium supplemented with
12.5% (v/v) horse serum (Life Technologies), 2.5% (v/v) fetal calf
serum, 2 mM glutamine, 50 U/ml penicillin, and 50 µM streptomycin. SH-SY5Y cells and HeLa cells were
cultured in minimal essential medium containing 10% (v/v) fetal calf
serum, 2 mM glutamine, and penicillin and streptomycin as
above. U937 cells were cultured in RPMI (Life Technologies) containing
10% (v/v) fetal calf serum, 2 mM glutamine, and penicillin
and streptomycin as above. All cells were cultured in a humidified 5%
CO2 atmosphere at 37°C.
Transfections and reporter assays. Cerebellar granule
neurons on 10.5 × 10.5 mm coverslips for neurite analysis, in
24-well plates for reporter assays, and in 35-mm dishes for kinase
assays were transiently transfected as described previously (Xia et
al., 1995; Dudek et al., 1997). Cotransfection at a 1:1 ratio
with two visible marker plasmids with similar detection sensitivities, pDsRed (Clontech, Cambridge, UK) and pEGFP-F (Clontech), shows that all
transfected cells express both proteins (data not shown). Thus the
ratio of 1:3 marker plasmid (
-galactosidase) to remaining DNA was
used. For morphological studies, cells at 72 hr after plating were
transfected with 0.5 µg of pCMV--galactosidase as a marker for
transfection together with 1.5 µg of pEBG empty vector, pEBG-SEK1K129R [a kinase dead mutant of SEK1 (MKK4) (Sánchez et
al., 1994)], or pcDNA3-JIP-JBD (Dickens et al., 1997). Forty-eight hours after transfection, cells were fixed and stained as described below. For kinase assays, 3.5 cm dishes of cerebellar granule neurons
at 6 d in vitro (DIV) (an age when transfection
efficiency is optimal) were transfected with 1.25 µg JNK11, 7.5 µg SEK1KR or JIP-JBD as indicated, and 1.25 µg pEGFP-C1. The amount
of total DNA was equalized in each sample using pEBG empty vector. At
28 hr after transfection, cells were lysed as described for
immune-complex kinase assays, and the activity of pEBG-JNK11 was
measured. For reporter assays, 9 DIV cells were transfected with a
firefly luciferase reporter plasmid driven by five GAL4 elements
in tandem: pGL3-G5E4
38 (Griffiths et al., 1998), a plasmid
expressing a fusion protein of the p38-specific substrate MEF2A with
the DNA binding domain of GAL4 (Han and Prywes, 1995), and pRL-CMV
(Promega, Madison, WI), expressing sea pansy luciferase as an internal
standard against which signals were normalized, and either pcDNA3-MKK6E
(Rain-geaud et al., 1996) expressing a constitutively active MKK6
and pEBG-p38 (Meyer et al., 1999) or empty vector pCMV (van den
Heuvel and Harlow, 1993) as indicated. Empty vector pCMV was added to
equalize transfections to 2 µg total DNA/well. Twenty hours after
transfection, cells were lysed in passive lysis buffer (Promega).
Firefly (reporter) and Renilla (internal standard) luciferase
activities were assayed with the dual luciferase assay kit (Promega)
according to the manufacturer's instructions.
Plasmids. pEBG-JNK11 was constructed by inserting
JNK11, prepared by PCR-based procedures from pcDNA3-JNK11 (Gupta
et al., 1996), into the BamHI site of pEBG. All other
plasmids were generous gifts from John Kyriakis (MGH, Boston), Peter
Shaw (University of Nottingham, UK), Ron Prywes, (Columbia University,
New York), Joël Raingeaud (Centre National de la Recherche
Scientifique, Orsay, France), Bruce Mayer (Children's Hospital,
Boston), and Sander van den Heuvel, (MGH, Boston).
Tissue extract preparation. Tissues from Sprague Dawley rats
at postnatal days 8, 9, 10, 13, and 25 were rapidly extracted subsequent to decapitation and snap-frozen in liquid
N2. This method maintains the JNK activity status
of brain tissues; it has previously been used to show ischemia-induced
activation of JNK in brain (Herdegen et al., 1998). Frozen tissues were
homogenized in ice-cold lysis buffer (see below) and precleared by
centrifugation at 10,000 × g for 15 min. Samples for
immunoblotting were normalized for protein using the Bradford method
(Bradford, 1976) and resuspended in 4× Laemmli sample buffer [1×:
62.5 mM Tris-HCl, pH 6.8, 2% SDS (w/v), 5%
2-mercaptoethanol, 10% glycerol (v/v), and 0.001% bromophenol blue
(w/v)].
Immune-complex kinase assays. After treatment, cerebellar
granule neurons, PC12 cells, SHSY5Y cells, or U937 cells were washed twice in ice-cold PBS and lysed on ice in 500 µl of lysis buffer [20
mM HEPES, pH 7.4, 2 mM EGTA, 50 mM
-glycerophosphate, 1 mM dithiothreitol (DTT), 1 mM Na3VO4, 1%
Triton X-100, 10% glycerol, 1 mM benzamidine, 50 mM NaF, 1 µg/ml of leupeptin, pepstatin, and aprotinin
and 100 µg/ml PMSF]. After homogenization and centrifugation (10,000 × g) at 4°C for 15 min, supernatants were
normalized for total protein or for kinase expression in the case of
specific activity measurements. Normalized lysates were incubated with anti-SAPK serum for 2 hr followed by 1 hr incubation with 20 µl of
50% protein A Sepharose or, for transfected cells, with 10 µl
S-hexylglutathione agarose (Sigma). Immobilized kinase complexes were
washed three times with lysis buffer, three times with LiCl buffer
[500 mM LiCl, 100 mM Tris,
pH 7.6, 0.1% Triton X-100, and 1 mM DTT], and
three times with kinase buffer [20 mM MOPS, pH 7.2, 2 mM EGTA, 10 mM
MgCl2, 1 mM DTT, and 0.1%
(v/v) Triton X-100]. Kinase assays were performed in kinase buffer
supplemented with 50 µM ATP, 5 µCi
[
-32P]ATP (Amersham International),
and 6 µg of glutathione S-transferase-c-Jun(5-89)/sample for 30 min at 30°C. Reactions were stopped by addition of 4 × Laemmli sample buffer. Samples were resolved on SDS-polyacrylamide gels
and exposed to film, typically for 2-4 hr. Nonsaturated films were
digitized by flat-bed scanning followed by quantification using imaging
software developed by the authors. Recombinant GST-c-Jun(5-89) was
prepared as described previously (Berberich et al., 1996). To measure
MKK4/7 activities, two-step immune-complex assays were performed. Cells
were lysed as for JNK assays, and where specific activities were to be
measured, lysates were normalized for kinase expression.
Immunoprecipitation was essentially the same except that 1 µg of
anti-MKK4 or 2 µg of anti-MKK7 was used for 0.5 ml cell lysate. After
washing, immune complexes were incubated in kinase buffer supplemented
with 30 mM MgCl2, 10 mM MnCl2, 50 µM ATP, and recombinant SAPK-1 (prepared by
thrombin cleavage from a GST fusion protein) for 20 min at 30°C. The
second step of the reaction, initiated by addition of
32P--ATP and 5 µg GST-c-Jun(5-89),
was also for 20 min at 30°C. Phosphorylation of c-Jun(5-89) by
MKK4/7-activated recombinant SAPK-1 was detected by autoradiography.
Alkaline phosphatase treatment. Selected JNK immune
complexes were incubated with or without alkaline phosphatase (100 U) for 15 min at 37°C as described previously (Park et al., 1995). After
phosphatase treatment, immune complexes were collected and washed three
times in lysis buffer containing phosphatase inhibitors, three times in
LiCl buffer, and three times in kinase buffer. Samples were then
assayed for kinase activity as described above.
Nuclear isolation. Nuclei were prepared as described
previously (Park et al., 1995) with some modifications. After
stimulation, cells were washed three times in ice-cold PBS + 1 mM MgCl2, and lysed in 20 mM HEPES, pH 7.4, 10 mM NaCl, 3 mM
MgCl2, 2.5 mM EGTA, 0.1 mM DTT, 50 mM NaF, 1 mM
Na3VO4, 1 mM
PMSF, and 5 µg/ml of leupeptin, pepstatin, and aprotinin. Nonidet
P-40 (NP-40) was added to a final concentration of 0.05%, and cells
were incubated at 4°C for 10 min. Samples were centrifuged at
300 × g for 10 min at 4°C. Nuclear pellets were
washed in lysis buffer without NP-40. Samples were quantitated for
protein and solubilized in Laemmli buffer, and equal proportions
(unless indicated otherwise) of nuclear and cytosolic fractions were
resolved by SDS-PAGE.
Immunoblot analysis and quantification. Cells were
stimulated as indicated, washed in PBS, and lysed with Laemmli buffer. Samples were resolved by 10% SDS-PAGE and transferred by semidry transfer onto nitrocellulose. Blots were developed using the enhanced chemiluminescence detection method. Films were preflashed, exposed for
suitable times to ensure exposures in the linear range, digitized by
flatbed scanning, and quantified as above.
Immunostaining and morphological analysis of transfected
cells. Immunocytochemical staining was performed as follows.
Coverslips with neurons at 6-7 DIV or HeLa cells were fixed with 4%
paraformaldehyde for 20 min followed by permeabilization in PBS/Triton
X-100 (1%) for 3 min. After washing with PBS, cells were blocked with
10% serum/0.2% Tween-20/PBS. Incubation with primary antibodies was overnight at 4°C using 1:200 SAPK antiserum (generous gift of John M. Kyriakis, MGH), 2 µg/ml MKK4 (K-18), 2.5 µg/ml MKK7 (C-19), or 5 µg/ml p38 (C-20) (Santa Cruz Biotechnologies), or with 1:25 anti-phospho-JNK (New England Biolabs). Immunostaining for JIP1 was
with 1:100 anti-JIP1 monoclonal (Yasuda et al., 1999). The specificity
of SAPK staining was verified by preincubating anti-SAPK with 150 µg/ml recombinant GST-SAPK to neutralize specific staining. Incubation with primary antibodies was followed by incubation with
1:800 affinity-purified goat anti-rabbit or 1:80 rabbit anti-goat IgG
biotin conjugates (Sigma) and 1:200 ExtrAvidin fluorescein conjugate
(Sigma). JIP1 immunoreactivity was detected with 1:500 Alexa 488 -mouse. Before mounting, nuclei were stained with 0.2 µg/ml
propidium iodide for JNK, phospho-JNK, p38, and MKK4, and with 0.02 µg/ml for MKK7. Slides were examined under FITC and TRITC wavelengths
using a Leica confocal microscope (Heerbrugg, Switzerland) with
a 100× objective, and a zoom of 2. Multiple sections were scanned, and
sections through nuclei are shown, with sections closer to the
coverslip shown in insets. Cerebellar granule neurons transfected with
pCMV--galactosidase for the morphological studies were fixed 48 hr
after transfection and stained as outlined above. Incubation overnight
with primary antibody 1:2000 rabbit anti--galactosidase (5'
3'
Inc., Boulder, CO) was followed by 1 hr incubation with
biotin-conjugated goat anti-rabbit IgG and then 1:500 streptavidin
Alexa-488 (Molecular Probes, Leiden, The Netherlands). Nuclei
were counterstained with Hoechst. Analysis of neurite number was
performed within 1 week of immunostaining using a Leica DM IRB
with 100× objective. This sensitive staining procedure was necessary
to allow visualization of fine processes. No distinction was made
between dendritic or axonal processes during the counting. A projection
emanating directly from the cell body equal to or greater in length
than one-half the cell body diameter was measured as a process.
Additional branching beyond the cell body was not counted. Accurate
counting of processes required focusing in different planes of view,
and thus the number of projections visible in one focal plane (as shown
in Fig. 11C) does not reveal processes in higher and lower planes.
RT-PCR. Total RNA was isolated from cells cultured in
24-well plates with 300 µl per well Tri-reagent (Sigma) according to the manufacturer's instructions. The RNA was heat-denatured (5 min at
72°) and poly(A+)-RNA was reverse-transcribed in a 20 µl reaction
containing 200 U Moloney murine leukemia virus (MMLV)-RT, 1 × MMLV-RT buffer (Promega), 30 U RNase inhibitor (5'3' Inc.), 1.25 mM dNTPs (Finnzymes, Espoo, Finland), and oligo-dT
(Promega), and incubated for 90 min at 42°C, and the reaction was
terminated by incubating for 5 min at 95°C. An aliquot of cDNA was
used in a 20 µl PCR reaction containing 1 U Dynazyme, 1 × Dynazyme buffer (Finnzymes), 1 µM of each primer (Protein
and Nucleic Acid Chemistry Laboratory, University of Leicester,
Leicester, UK), and 250 µM dNTPs (Finnzymes).
Amplification was performed with the following primer sets: (1)
-actin: sense (2255-2274) 5'-TCC GGA GAC GGG GTC ACC
CA-3' (Miller and Johnson, 1996), antisense (3111-3132) 5'-CTA GAA GCA
TTT GCG GTG CAC G-3'; (2) c-jun: sense (1728-1747) 5'-GCT TCT CTA GTG CTC CGT AA-3' (Schafer et al., 1996), antisense (2483-2502) 5'-TCT AGG AGT CGT CAG AAT CC-3' (Schafer et al., 1996);
(3) JIP: sense (1628-1648) 5'-TCG GCA TGA AGA TGA
ACT TGA-3'; antisense (1929-1949) 5'-CTT GTT ATT CTT TGG ATG GTA-3'.
These oligos are expected to recognize the following JIP
forms: mouse JIP-1a, JIP-2b, and JIP-3
(perfect match; rat sequences are not currently available for these),
and rat JIP-1b, JIP-1c, and JIP-2a (only one base mismatch). The oligos do not correspond to the recently
reported human JIP2 (Yasuda et al., 1999) and
JIP3 (Kelkar et al., 2000), which probably represent
additional families of JIPs. PCR parameters were as follows: 95°C,
1.5 min; 50°C (for c-jun and JIP) or 58°C
(for -actin), 1.5 min; 72°C 1.5 min, for 25 (-actin) or 30 cycles (c-jun). Aliquots of the PCR reactions were run on
1.5% agarose gels containing ethidium bromide. RT-PCR data shown are
representative of three separate experiments. Reactions produced only a
single band (two in the case of JIP) of the expected size,
and PCR without template produced no detectable product.
| |
RESULTS |
Constitutive JNK activity in cerebellar granule neurons exceeds
that of anisomycin-stimulated kinase activity in U937 cells
JNKs are strongly regulated by stresses and are believed to play
an important role in neuronal apoptosis. Cerebellar granule neurons are
a widely used model for the study of neuronal death mechanisms.
Surprisingly, however, apoptotic stresses have been reported not to
induce substantial JNK activation in cerebellar granule neurons
(Kawasaki et al., 1997; Gunn-Moore and Tavare, 1998; Watson et al.,
1998). It has been speculated that these cells have a high basal
activity, possibly explaining the lack of JNK response to apoptotic
stimuli (Kawasaki et al., 1997; Gunn-Moore and Tavare, 1998). Thus we
assessed the level of basal activity of cerebellar granule
neuron-derived JNK compared with JNK from the U937 macrophage line, a
cell type in which JNK is well characterized (Dai et al., 1995).
Lysates containing equal protein from U937 cells and cerebellar granule
neurons were run on SDS-PAGE and immunoblotted with antiserum
recognizing all JNK isoforms. JNK expression (54 and 46 kDa bands) was
higher in cerebellar granule neurons than in U937 cells (Fig.
1A); therefore, lysates
from cerebellar granule neurons and U937 cells were normalized for this
difference in JNK expression, and kinase assays were performed. The
specific activity of cerebellar granule neuron JNK was eightfold
greater than JNK from U937 cells (Fig. 1B).
Anisomycin (50 µg/ml) induced an eightfold elevation in U937 cell JNK
activity as reported previously in these cells (Kyriakis et al., 1994;
Dai et al., 1995). General inhibition of phosphatases with excess
calyculin A (1 µM) moderately increased JNK
activity in both cell types. Interestingly, both the basal kinase
activity and the calyculin A-induced activity in U937 cells were
considerably lower than the basal activity in cerebellar granule
neurons. Thus, even anisomycin-treated U937 cells expressed lower
specific JNK activity than unstimulated cerebellar granule neurons.
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Figure 1.
Cerebellar granule neuron-derived JNK activity is
elevated above that of stressed U937 cells, whereas p38 activity from
neurons is low and responds to stress. A, Equal amounts
of protein (25 µg) from 1 DIV cerebellar granule neuron
(CBG) or U937 cell extracts were electrophoresed and
immunoblotted with anti-SAPK. JNK expression was on average 1.7-fold
higher in CBG extracts. B, Kinase assay lysates were
normalized for JNK expression, and specific JNK activity was measured
by immune-complex kinase assay. The specific activity of JNK from 1 DIV
CBG extracts was higher than that in anisomycin-treated U937 cells.
Treatments were with calyculin A (1 µM) for 30 min or
anisomycin (50 µg/ml) for 45 min. After stimulation, JNK was isolated
using polyclonal anti-SAPK, and immune-complex kinase assays were
performed using GST-c-Jun(5-89) as substrate. C,
Treatment of cerebellar granule neuron-derived JNK with alkaline
phosphatase indicated that the basal activity measured was
phosphorylation dependent. Representative images are shown, and
quantitated data from four experiments are in Figure 2.
D, p38 expression was compared in 1 DIV cerebellar
granule neurons and U937 cells. Forty micrograms of CBG and U937 cell
lysates were resolved by SDS-PAGE and immunoblotted using polyclonal
anti-p38 (n = 4). U937 cells expressed on average
sevenfold more p38 than cerebellar granule neurons. E,
As in B, lysates were normalized for kinase expression,
and specific activities of p38 from cerebellar granule neurons and U937
cells after treatment for 45 min with anisomycin (50 µg/ml) or
sorbitol (300 mM) are shown. Treatment with anisomycin lead
to a 399 ± 59% increase in p38 activity in neurons, comparable
with that in U937 cells of 302 ± 80%. Representative images from
four separate experiments are shown (A-E).
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Both threonine and tyrosine phosphorylation are required for activity
of JNK (Kyriakis et al., 1995). Because the basal activity in
cerebellar granule neurons was maintained by an unknown mechanism (Fig.
1B), we tested whether this activity was dependent on
its phosphorylation state. Samples were treated with alkaline
phosphatase, and kinase activity toward GST-c-Jun(5-89) was measured
after thorough washing of immunoprecipitates in buffers containing
phosphatase inhibitors (see Materials and Methods). Alkaline
phosphatase treatment strongly reduced the basal activity of
immunoprecipitated JNK (Fig. 1C), indicating that the high
basal activity measured from these cells was dependent on
phosphorylation of JNK.
Neuronal p38 shows typical responses to cellular stresses
Because the basal activity of neuronal JNK was elevated, we
decided to assess the activation state of p38, another
mitogen-activated protein kinase (MAPK) family member that is activated
by stress (Kyriakis and Avruch, 1996). Expression levels of p38 were
low in cerebellar granule neurons compared with the U937 cell line (Fig. 1D). As in Figure 1, lysates were normalized,
this time for p38 expression, and anti-phospho-p38 immunoreactivity was used as an indicator of the relative activities of p38 from both cell
types. Unlike neuronal JNK, p38 from neurons showed low basal specific
activity, comparable with that from U937 cells. Treatment with
anisomycin or osmotic shock for 45 min typically induces p38 activity
in cell lines (Kyriakis and Avruch, 1996; Hazzalin et al., 1997), and
p38 from neurons showed a classic stress response similar to that of
U937 cells (Fig. 1E). The low basal p38 activity in
these cells suggests that high JNK activity is not caused by stress
imposed by cell preparation or maintenance in culture.
Elevated JNK activity at 1 DIV is not mediated by glutamate
receptor activity
Neuronal JNK activity can be induced by extracellular glutamate
(Schwarzschild et al., 1997; Mukherjee et al., 1999). To assess whether
low levels of glutamate present in the culture medium (Aronica et al.,
1993) were responsible for the elevated JNK activity observed at 1 DIV,
cells were treated with glutamate receptor antagonists MK801, CNQX, and
MCPG, specific for NMDA, AMPA/kainate, and metabotropic-type receptors,
respectively. None of these treatments reduced basal JNK activity;
indeed, inhibition of metabotropic receptors with MCPG caused a small
but significant increase in JNK activity (Fig.
2). Furthermore, treatment of cells with
the L-type Ca2+ channel antagonist
nifedipine failed to reduce constitutively elevated JNK activity.
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Figure 2.
Glutamate receptor activity does not mediate
elevated basal JNK activity. To determine whether glutamate receptor
activity might contribute to basal JNK activity (~10-fold elevated
compared with cell lines), cerebellar granule neurons at 1 DIV were
treated for 1 hr with 50 µM CNQX, 25 µM
(RS)-MCPG, 2 µM (+)-MK801, or 1 µM nifedipine. JNK activity was assessed by phospho-JNK
immunoblotting and normalized to control samples. These treatments did
not reduce neuronal JNK activity; indeed, inhibition of mGluR-type
receptors caused a small but significant increase in JNK activity. To
investigate whether culture medium supplements might contribute to the
basal JNK activity, the effects of serum depletion ( FCS) and low KCl
were assessed by phospho-JNK immunoblotting. Total JNK activity was
measured from 7 DIV CBG neurons grown for 24 hr in the absence of FCS
but with supplementary KCl (25 mM) to prevent neuronal
death. Both activity and expression of CBG JNK decreased; thus specific
activities (s.a.) for JNK are shown for
these treatments. Serum depletion did not significantly alter specific
JNK activity. To assess whether KCl might contribute to the high basal
activity, we measured JNK activity from 1 DIV CBG neurons grown for 24 hr in low KCl (5 mM). The activity was not significantly
reduced. Quantitated data for JNK activation after treatment with
classic JNK stimuli as described in Figure 1 are shown. Cerebellar
granule neurons at 1 DIV were treated with anisomycin (50 µg/ml) or
sorbitol (300 mM) for 45 min, or with alkaline phosphatase
(see Materials and Methods); JNK activity was measured by
immune-complex kinase assay. Normalized, mean data ± SEM are
shown. Significance levels as assessed by Student's t
test are indicated (*p < 0.05 and
**p < 0.001).
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Elevated specific JNK activity in cerebellar granule neurons is not
reduced by removal of serum
To investigate whether high neuronal JNK activity resulted from
other components present in the culture medium, cerebellar granule
neurons were either (1) deprived of serum for 24 hr or (2) grown
in the absence of elevated KCl (Fig. 2). Expression of JNK protein
decreased after these treatments, but there was no decrease in specific
JNK activity. No decrease in specific JNK activity was detected with
shorter times of serum deprivation either (data not shown). Cerebellar
granule neurons are commonly grown in culture medium containing
elevated KCl (25 mM). This is thought to mimic the
innervation in vivo (Thangnipon et al., 1983) and protects
mature neurons in culture from a late phase of cell death (Gallo et
al., 1987; Courtney et al., 1997). To test whether the elevated JNK
activity observed by 1 DIV (Fig. 1) resulted from the excitation
induced by elevated KCl, freshly isolated cerebellar granule neurons
were plated in culture medium without supplementary KCl. Plating of
cells in nondepolarizing medium caused no significant decrease in JNK
activity and could not explain why this cell type had 8- to
10-fold higher activity than cell lines such as U937 cells, PC12 cells,
and SH-SY5Y neuroblastoma cells (Figs. 1, 4)
Cerebellar granule neuron JNK shows a moderate response to the
protein synthesis inhibitor anisomycin, a strong activator of JNK in
non-neuronal cells
c-Jun kinases typically undergo 10- to 20-fold activation when
treated with inhibitors of protein synthesis (Kyriakis et al., 1994;
Dai et al., 1995; Kyriakis et al., 1995; Iordanov et al., 1997). To
test the responsiveness of neuronal JNK to these compounds, immune-complex kinase assays were performed using GST-c-Jun(5-89) as
substrate. Differentiating cerebellar granule neurons were stimulated
with anisomycin (50 µg/ml, 40 min), an inhibitor of protein
synthesis. This treatment induced a moderate, twofold increase in total
JNK and MKK4 activity above basal levels (Figs. 2,
3). Treatment with anisomycin caused a
reproducible retardation in the mobility of c-Jun, with complete loss
of the fastest mobility band, consistent with c-Jun phosphorylation.
This result was corroborated by increased immunoreactivity of c-Jun
phosphoserine 63. A time course of JNK phosphorylation after treatment
with 10 µg/ml anisomycin (a concentration at which JNK activation is
maximal) (Iordanov et al., 1997) showed that p46 JNK phosphorylation
increased within minutes and remained elevated for up to 2 hr (Fig.
3B). The p54 JNK isoforms were also rapidly activated in
response to anisomycin (data not shown). The response of neuronal JNK
to osmotic stress, which typically activates JNK of non-neuronal cells
10-fold, was also measured by immune-complex kinase assay. Treatment of
cerebellar granule neurons with 0.3 mM sorbitol
for 45 min also resulted in a twofold activation above basal levels.
Quantitative data for these responses is in Figure 2.
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Figure 3.
Anisomycin induces moderate activation of MKK4/JNK
signaling and phosphorylation of c-Jun in cerebellar granule neurons.
A, Cerebellar granule neurons at 1 DIV were stimulated
with or without anisomycin (50 µg/ml) for 40 min. JNK and MKK4 were
immunoprecipitated from lysates, and immune-complex kinase assays were
performed. Representative images from multiple experiments
(n  4) are shown. Lysates of anisomycin-treated 1 DIV neurons were also blotted for c-Jun protein and Ser-63
phosphorylation. Although anisomycin only moderately activates JNK,
there is a mobility shift with a dramatic loss of the most mobile form
of c-Jun. B, A time course for anisomycin-induced (10 µg/ml) p-46 JNK activation is shown (mean ± SEM,
n = 3). C, Qualitative RT-PCR
analysis of c-jun mRNA from 5 DIV cerebellar granule
neurons after treatment for 3 hr with anisomycin (10 µg/ml) ± SB203580 (1 µM). The anisomycin-induced increase in
c-jun mRNA is not eliminated by treatment with the p38
inhibitor, suggesting that JNK activation might contribute to the
induction of c-jun. The bottom panel
shows actin levels from parallel samples, indicating that the changes
seen do not result from a general regulation of mRNA levels.
Lanes 1 and 2 on this panel show
reactions with equal (1×) or twice (2×) the
amount of input cDNA as in other samples, indicating that the reactions
were not saturating. D, SB203580 (1 µM)blocks p38 activity in these cells as
detected by MEF2A-GAL4 induction of a GAL4-driven luciferase reporter.
Quantitated data are shown (mean ± SEM).
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Contribution of JNK to elevated c-jun mRNA in response to
anisomycin-induced stress in cerebellar granule neurons
MEF2, a well known target of p38, regulates the c-Jun promoter
(Han and Prywes, 1995; Coso et al., 1997; Han et al., 1997), and c-Jun
can autoregulate its own promoter activity. Thus, it was feasible that
c-Jun promoter activity might be regulated in response to activation of
either p38 or JNK. Because c-Jun elevation is an important mediator of
apoptotic signaling in these neurons (Watson et al., 1998), we tested
whether c-jun levels were increased by p38 or JNK after
cellular stress with anisomycin (Fig. 3C). Qualitative
RT-PCR analysis using primers for c-jun showed an elevation
of c-jun mRNA after 3 hr treatment with 10 µg/ml
anisomycin. Cotreatment of cells with 1 µM of
the p38 and inhibitor SB203580 (IC50
values of 0.3 and 0.6 µM, respectively) (Li et
al., 1996; Kumar et al., 1997) did not eliminate the c-jun
elevation (Fig. 3C), although the drug blocked p38
activity (Fig. 3D). A broader spectrum inhibitor that blocks
p38, -, and -, SB202190 (3 µM), also
failed to block the c-jun elevation, even when including a 1 hr preincubation (data not shown), and p38
is reported not to be
expressed in brain (Jiang et al., 1997) or to activate the cJun
promoter (Marinissen et al., 1999). Thus, p38 activity alone cannot explain the c-jun induction in response to
anisomycin, suggesting that JNK signaling might contribute (Fig.
3A).
Activity of JNK from brain extracts and primary neurons in culture
exceeds that of the SH-SY5Y neuroblastoma and the PC12 neuronal cell
model
The previous figures showed that both the specific and the total
activity of JNK in cultured cerebellar neurons were higher than in U937
cells. To investigate whether this high activity was a general property
of neuronal systems, we determined the specific activity of JNK from
snap-frozen tissue samples, primary cultured neurons, and neuronal-like
cell lines. Developmental changes in brain tissues and primary neuronal
cultures were assessed by taking samples at different developmental
stages by a method demonstrated to retain JNK responsiveness in tissues
(see Materials and Methods). Equal protein was loaded onto gels, and
JNK was detected by immunoblotting. The relative expression of JNK
protein (per unit total protein) from cerebellar and forebrain extracts was high compared with liver (Fig.
4A, top
panel). Samples were normalized for this difference in JNK
expression, and equal amounts of JNK were used for kinase assays. It is
clear that brain-derived JNK also showed a high specific activity
(activity per unit JNK), whereas JNK from liver showed little or no
basal activity (Fig. 4A, bottom
panel). Although we cannot exclude a contribution from glial cells to the high JNK activity in brain, immunofluorescent studies failed to detect JNK activity in glia (data not shown). When
cultured cells were compared, similar expression levels of JNK protein
were found in primary cultured cerebellar granule (CBG) neurons and the
SH-SY5Y neuroblastoma line (Fig. 4A, top panel). However, the specific activity of JNK from primary
cerebellar granule neurons was clearly elevated compared with that
measured in cell lines (Fig. 4A, bottom
panel). Both expression and activity of PC12 cell JNK were
more similar to those of liver than brain, neurons, and neuroblastomas.
The levels of JNK expression (JNK per unit protein) were not
developmentally regulated from postnatal day 8-13 (Fig.
4A); however, there was a clear increase in the specific activity of JNK (activity per unit JNK) during development of
forebrain and the primary neuronal culture. As with liver, JNK
expression from heart, kidney, and spleen was relatively low (Fig.
4B, left panel).
When JNK levels were normalized between samples, the specific JNK
activity was clearly lower in non-neural tissues (Fig.
4B, right
panel).
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Figure 4.
Specific activities of JNK and JNK kinases from
brain tissues and primary neurons are elevated compared with those from
SH-SY5Y and PC12 cells. A, Rat brain tissues and
cerebellar granule neurons (CBG neurons) were compared
with non-neural tissue and neuron-like cell lines, SH-SY5Y and PC12
cells, for both JNK expression and specific activity. Top
panel, Equal protein (25 µg/lane) from tissues and cell
lysates was loaded and immunoblotted using anti-SAPK. Elevated levels
of JNK protein are seen in brain tissues and primary neurons. The
neuroblastoma cells also show elevated JNK expression compared with
PC12 cells. Bottom panel, Lysates for kinase assays were
normalized for JNK expression, and immune-complex kinase assays were
performed. Although JNK levels were normalized between samples, JNK
activities from brain tissues and primary neurons are still
considerably higher than from liver, SH-SY5Y, and PC12 cells. Data
shown for tissue samples and cells are representative of two and four
repeats, respectively. B, Specific JNK expression and
activity from forebrain was compared with other tissues as described in
A except that activities were this time measured with
phosphospecific JNK antibodies. Again, neural-derived JNK activity is
elevated above that of JNK from non-neuronal tissues. Data shown are
representative of tissues from three separate animals.
C, Specific expression and activity of JNK from rat
cerebellum of postnatal days 9 and 25 are shown. Normalization of
samples was performed exactly as described in A. The
specific activity of cerebellar JNK increases during this time. Data
shown represent results from three experiments. D, The
same procedure as described in A was used to compare
MKK4 and MKK7 expression in brain tissues and CBG neurons to cell
lines. Expression of both MKK4 and MKK7 is high in brain. In primary
cultured neurons and cell lines, levels of MKK4 and MKK7 are similar,
with the exception of MKK4 in neuroblastoma cells. Data from tissues
and cells are representative of two and four separate experiments,
respectively. E, Representative images from two-step
immune-complex kinase assays showing the developmental regulation
of MKK4 and MKK7 activities in cerebellar granule neurons are shown.
Representative images of four and three repeats are shown,
respectively. Quantitated data are shown in Figure 5.
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JNK activity from cerebellum and forebrain is
developmentally upregulated
The specific activity of JNK from forebrain increased from
postnatal day 8 to 13, whereas there was no clear upregulation of
cerebellar JNK activity during this period (Fig. 4A).
Because the cerebellum shows retarded maturation compared with
forebrain, we tested whether an upregulation of JNK activity occurred
at a later stage in the cerebellum (Fig. 4C). Interestingly,
there was an increase in specific activity of JNK from cerebella of postnatal day 25 compared with postnatal day 9.
JNK kinases MKK4 and MKK7 show high expression and activity in
brain extracts and neurons
JNK is activated by two known upstream kinases, MKK4 (also known
as SEK1, JNKK1, or SAPKK1) and MKK7 (also known as JNKK2 or SAPKK4),
dual specificity kinases that phosphorylate the JNK "TPY" motif and
are themselves activated by phosphorylation. We investigated the
expression of MKK4 and MKK7 from neural tissues, neurons in culture,
and neuron-like cell lines. Both MKK4 and MKK7 showed high expression
in brain tissues compared with liver; however, in the neuronal and
neuron-like cultured cells tested, levels of MKK4 and MKK7 expression
were comparable, with the exception of MKK4, which was very low in
neuroblastoma cells (Fig. 4D).
To investigate the developmental upregulation of MKK4 and MKK7
activity, immune-complex kinase assays of MKK4 and MKK7 isolated from
cells at 1, 3, and 6 DIV were performed (Fig. 4E).
This showed that total activities of both of these JNK kinases
increased during the neuronal differentiation. Quantitated data are
shown in Figure 5A.
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Figure 5.
JNK and MKK7 specific activities increase, whereas
expression and activity of c-Jun decrease during maturation of
cerebellar granule neurons. A, Lysates from
differentiating cerebellar granule neurons in culture were loaded
according to cell number and immunoblotted for JNK, p38, MKK4, MKK7,
and c-Jun expression ( ) or total phospho-JNK, phospho-p38, or
p63-c-Jun immunoreactivity, or MKK4 and MKK7 immune-complex kinase
activities were measured ( ). Quantitated data from multiple
experiments (mean ± SEM, n 3) are shown.
Error bars are shown for each data point; SEMs smaller than the symbols
are not visible. Although JNK and MKK7 expression are not significantly
upregulated during differentiation, their activities rise sharply.
Levels of c-Jun, p38, and c-Jun serine 63 phosphorylation, and
activation of p38, all decrease as cells mature in
vitro. B, Bright-field images of
cerebellar granule neurons differentiating in culture. By 6 DIV, a
dense network of processes has formed.
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Specific activities of MKK7/JNK increase during differentiation of
cerebellar granule neurons, whereas c-Jun activity decreases 5
Cerebellar granule neuron JNK activity was highly elevated 24 hr
after plating as shown in Figures 1 and 4. We assessed whether high
basal activity resulted from isolation-associated stress by measuring
activities of JNK, MKK4, and MKK7 at 1, 3, and 6 DIV (Figs. 4,
5A). During this period, neurons mature and form a dense
network of processes (Fig. 5B). Perhaps surprisingly, instead of a decline in JNK activity after isolation and plating of
neurons, total activities of JNK, MKK4, and MKK7 increased as neuronal
differentiation progressed. Because expression of many proteins
increases during differentiation of these neurons (Coffey et al.,
1997), increased protein expression could account for the regulation of
JNK, MKK4, and MKK7 activities measured. Although we found that MKK4
expression increased in parallel to its activity from 3 to 6 DIV, there
was no increase in JNK and MKK7 expression during this time (Fig.
5A). Thus a specific upregulation of MKK7/JNK signaling
occurred during the development of these neurons in culture.
Conversely, both expression and specific activity of p38 decreased
during development of these cells as did the expression and
phosphorylation of the nuclear JNK target, c-Jun (Fig.
5A).
Induction of JNK activity in maturing neurons is not dependent on
elevated KCl
Cerebellar granule neurons are typically grown in elevated
potassium, resulting in elevated cytoplasmic calcium levels (Courtney et al., 1990). This has been shown to facilitate their survival beyond
6 DIV (Thangnipon et al., 1983). Although culturing of these cells in
low KCl (5 mM) does not affect survival during the first 6 DIV (Courtney et al., 1997), some intracellular signaling responses are
altered during this period by culturing in low KCl (Courtney et al.,
1997). Thus, we assessed whether the maturation-associated increase in
JNK activity depended on elevated KCl (25 mM). As in
Figures 1 and 4, lysates from CBG neurons cultured in high or low KCl
were normalized for JNK expression, and specific JNK activities were
measured using phosphospecific JNK antibodies (Fig.
6A). The increase in
specific JNK activity was retained under these conditions of low KCl
(Fig. 6B).
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Figure 6.
Developmental upregulation of CBG JNK activity
occurs in cells grown with or without elevated KCl. A,
Specific JNK activities from differentiating cerebellar granule neurons
grown in high (25 mM) or low (5 mM) KCl
were measured as described in Figure 4B. Lysates
were normalized for expression of JNK, and kinase activity was assessed
using phosphospecific JNK. B, Quantitated data,
means ± SEM from four separate experiments, are shown.
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Bulk JNK activity resides in the cytoplasmic fraction in
resting cells
We have established that cerebellar granule neuron JNK activity
was highly elevated (Fig. 1) and that MKK7/JNK activities were
upregulated as these neurons differentiated (Fig. 5), suggesting they
may play a developmental role. To ascertain whether elevated JNK and
MKK activities were involved in nuclear or cytoplasmic signaling,
fractionation studies and immunofluorescent analysis were performed to
assess the subcellular localization of endogenous kinase expression and
activity in mature (6-7 DIV) neurons (Figs. 7, 8).
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Figure 7.
JNK activity is predominantly extranuclear in
cerebellar granule neurons in culture. A, Cytosolic and
nuclear fractions of cerebellar granule neurons at 7 DIV were analyzed
for expression of JNK and JNK kinases MKK4 and MKK7. Cultures were
lysed, cytosolic and nuclear extracts were separated, and amounts
corresponding to ~75,000 cytosols and nuclei were loaded on gels, and
expression levels were analyzed. Nuclear and cytosolic fractions were
then normalized for JNK expression, and specific phospho-JNK
immunoreactivity was measured (P-JNK). Nuclear
and cytosolic fractions were also blotted for c-Jun and I -B to
validate the fractionation procedure. B, Quantitated
data (mean ± SEM, n = 3) are shown.
C, Lysates from cerebellar granule neurons at 6 DIV were
immunoblotted with isoform-specific antibodies detecting JNK1, JNK2/1,
and JNK3/1, respectively. All three JNK isoforms are expressed
in cerebellar granule neurons in culture. D, Western
blots of CBG lysates show the relative specificity of the MAPK and
MAPKK antibodies used in the immunofluorescent analysis (Fig. 8).
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Figure 8.
JNK/MKK4 are cytosolic, whereas MKK7 is
exclusively nuclear in cerebellar granule neurons. Immunofluorescent
microscopy was used to investigate the subcellular localization of
endogenous JNK and JNK kinases in cerebellar granule neurons
in vitro. Shown are confocal scans of
immunofluorescent staining for JNK, P-JNK, MKK4, MKK7, and p38 in
cerebellar granule neurons at 6-7 DIV
(green). Nuclei were counterstained with
propidium iodide (red). Overlapping staining appears
yellow. Sections through the nuclei are shown with
insets of scans closer to the coverslip that highlights
staining in neuritic processes. Neuronal staining in the absence of
primary antibodies, using biotin-conjugated anti-rabbit
(1°r) and anti-goat
(1°gt) is shown. All
panels are scaled according to the scale bar depicted in
the p38 panel except for the HeLa staining, which has its own scale.
For comparison, endogenous MKK7 immunoreactivity in HeLa cells is
shown. Propidium iodide gives relatively higher background cytoplasmic
staining in HeLa cells, resulting in some yellow showing
in HeLa cytoplasm. Staining in HeLa cells in the absence of 1°
antibody is also shown (HeLa
1°gt).
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Amounts of cytosolic and nuclear fractions of mature (6-7 DIV)
cerebellar granule neurons corresponding to equal numbers of cytosols
and nuclei were loaded onto gels and immunoblotted for JNK, MKK4, and
MKK7 (Fig. 7A). The distribution of active JNK was assessed by normalizing cytosolic and nuclear fractions for JNK
expression and immunoblotting with phospho-JNK (Fig. 7A,
panel labeled P-JNK). This normalization for JNK
expression was necessary, because nuclear expression of JNK was
otherwise too low to directly compare phospho-JNK immunoreactivity in
cytosolic and nuclear fractions. The specific activity of cytosolic JNK
was sevenfold higher than JNK from the nuclear fractions (Fig.
7A,B). Blotting of the same
fractions for nuclear and cytosolic proteins c-Jun and I-B,
respectively, was performed to demonstrate fraction purity (Fig.
7A). Cumulative data from three experiments are shown in
Figure 7B. Not only is most of the JNK protein in the
cytoplasm in these cells, but the bulk of the total JNK activity
(>95%) also resides outside the nucleus. Our ability to successfully isolate the small nuclear pool of JNK activity from cells that contain
a much larger cytosolic activity was confirmed by the dramatically
different specific JNK activities retained in the two fractions (Fig.
7B, PJNK).
Cerebellar granule neurons differentiating in culture express all
three JNK isoforms
To identify which JNK isoforms were expressed in differentiating
cerebellar granule neurons, fractions were immunoblotted with
antibodies selective for JNK1, JNK1 and -2, and JNK3 and -1, respectively (Fig. 7C). The predominant JNK1 isoforms
expressed were in the 46 kDa molecular weight range, whereas the
antibody recognizing both JNK1 and JNK2 isoforms detected a 54 kDa
immunoreactive band, indicating that JNK2 isoforms were also expressed.
Similarly, a 54 kDa JNK3/1-specific immunoreactive band suggests the
presence of the JNK3 isoform in these cells.
Neuronal JNK and MKK4 are localized to both processes and cell
bodies; MKK7 resides exclusively in the nucleus
Cerebellar granule neurons are small, their cell bodies having
diameters of ~5-8 µm. The cell soma consists mainly of nucleus, with very little cytoplasmic space. Thus, we used confocal microscopy with a 100× objective to assess the subcellular distribution of endogenous stress-activated protein kinases. The antibodies that were
used detect virtually no nonspecific bands (Fig. 7D), and a
range of dilutions of antibodies were tested to avoid nonspecific staining. In addition, anti-SAPK was preadsorbed with recombinant SAPK,
and dilutions that did not produce background staining were used (data
not shown). Nonspecific nuclear staining with the SAPK and MKK4
antibodies was avoided by use of high dilutions of these antibodies.
Representative micrographs of confocal sections through nuclei
indicated primarily cytoplasmic staining for the MAPKs JNK and p38
(Fig. 8), with typical crescent-shaped perinuclear staining in cell
bodies (occasionally obscured by high staining in neurite bundles).
This perinuclear localization was the most predominant feature of p38
staining in these neurons. The confocal scans exaggerate the nuclear
distribution somewhat, because much of the dense network of neuronal
processes is below the plane of scans through the nucleus. Staining in
neuritic processes was most clearly seen in confocal sections closer to
the z-plane of the coverslip (Fig. 8, insets).
Thus, in the case of JNK and p38, intense staining of processes was
seen in scans closer to the coverslip. JNK showed low level nuclear
staining with more intense staining in neurites. JNK staining at 1 DIV
showed a similar distribution, with intense staining in the perinuclear
region and processes. To assess the location of active JNK in neurons,
cells were stained for phospho-JNK immunoreactivity. Phospho-JNK
staining localized to processes and cell bodies, with slight staining
in nuclei. Staining for MKK4 was typically punctate in the processes
and the cell body, again with slight nuclear staining. Conversely, MKK7
immunoreactivity was localized almost exclusively to the nucleus. This
distinct localization of MKK7 in the neurons contrasts sharply with its
localization in HeLa cells (Fig. 8), a standard cell line often used
for localization studies.
Nuclear levels of JNK and MKK4 increase after
anisomycin-induced stress
The subcellular localization of high, specific JNK activity to the
cytoplasm suggested a non-nuclear role for the elevated JNK activity
that increased further during differentiation. This diverges from the
classic stress-activated JNK response described in cell lines, which
results in activation and translocation of JNK to the nucleus where it
phosphorylates substrates such as c-Jun and ATF2 (Cavigelli et al.,
1995). Although we detected a small JNK activation by stress (40 min
anisomycin) at 1 DIV [percentage of control 192 ± 16 (Fig. 2)],
the fold activation falls during differentiation (percentage of control
137 ± 9 at 7 DIV), possibly because the stress-induced JNK
activation becomes progressively masked by the distinct,
developmentally regulated JNK activity. To examine whether a small pool
of neuronal JNK in these more mature neurons behaves like JNK in cell
lines, we investigated whether stress induced nuclear translocation and whether it increased nuclear activity of JNK. Upstream kinases were
also investigated (Fig. 9A).
In addition to the increase in nuclear JNK activity, we found a clear
nuclear translocation of JNK and MKK4 after 40 min stimulation with 50 µg/ml anisomycin, consistent with the classic JNK stress response of
non-neuronal cells.
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Figure 9.
Translocation of nuclear JNK occurs after stress
and during maturation of cerebellar granule neurons. A,
Nuclear levels of JNK, MKK4, MKK7, and P-JNK after treatment with
anisomycin were assessed by immunoblotting of nuclear fractions from 6 DIV cerebellar granule neurons treated ± anisomycin (50 µg/ml)
for 40 min. Levels of JNK, phospho-JNK, and MKK4 increase twofold in
nuclear fractions after stress, consistent with activation and nuclear
translocation of these kinases. B, The developmental
increase in JNK activity occurs in both cytoplasmic and nuclear
compartments. Nuclear and cytoplasmic fractions were prepared from CBG
neurons at 1, 3, and 6 DIV and immunoblotted for phospho-JNK. To enable
a direct comparison of nuclear and cytoplasmic fractions to be made,
10-fold more nuclei than cytoplasms were loaded (fraction of total).
C, Quantitated data (mean ± SEM,
n = 3) for multiple experiments as outlined in
B.
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To assess the subcellular localization of the developmentally regulated
JNK, we isolated nuclear and cytoplasmic fractions from cells at 1, 3, and 6 DIV and measured JNK activity (Fig. 9B). The larger
cytoplasmic pool of JNK activity increased 3.5-fold by 6 DIV, whereas
the nuclear pool activities increased by 10-fold (total phospho-JNK
immunoreactivity of 46 and 54 kDa bands) (Fig. 9C). Because
there was no upregulation in JNK protein levels in the nuclear
compartment or in total cell lysates from 3-6 DIV (data not shown),
these increases represent specific inductions of JNK activity in both
pools. Once again, the reliability of the nuclear-cytoplasmic
fractionation is confirmed by the dramatically different development
curves of P-JNK immunoreactivity despite the much larger levels of
P-JNK in the cytoplasm.
Cerebellar granule neurons express the long form of JIP
mRNA and protein
On the basis of the data in Figure 8 showing MKK7 localization in
the nucleus, we investigated the expression and location of the
JNK/MKK7 scaffold protein JIP (Dickens et al., 1997; Whitmarsh et al.,
1998; Yasuda et al., 1999). JIP isoforms contain putative nuclear
localization and DNA binding sequences, the latter of which is absent
in the shorter JIP-1a splice variant (Kim et al., 1999; Mooser et al.,
1999; Yasuda et al., 1999). Semiquantitative RT-PCR showed that
JIP mRNA was expressed in cerebellar granule neurons (Fig.
10A), the predominant
message being for the full-length JIP isoform. Antibody
raised against JIP-1 detected two bands that were preferentially
localized to the nuclear fraction (Fig. 10B). The
predominant form ran at 120 kDa, the molecular weight on SDS-PAGE of
JIP-1b [also known as IB1 (Bonny et al., 1998)], which possesses the
putative DNA binding domain. Immunofluorescence analysis of JIP-1 in 6 DIV CBG neurons showed that JIP-1 was concentrated in nuclei, the
location of MKK7. There is lighter punctate staining in the cytoplasmic
compartment (Fig. 10C) and also staining in growth cone-like
structures (data not shown). The later is consistent with a recent
report (Meyer et al., 1999). It may be that the predominant
localization to the nucleus of JIP, which binds MKK7 and not MKK4
(Whitmarsh et al., 1998), tethers MKK7 to the nuclear compartment in
CBG neurons.
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Figure 10.
JIP is expressed in nuclear fractions from
cerebellar granule cells. A, JIP mRNA was detected in 9 DIV cell extracts by RT-PCR with oligos complementary to both JIP-1a
and JIP-1b/1c/2/3, expected to produce fragments of size 324 (JIP ) and 465 (JIP+), respectively.
B, Equal proportions of cytosolic and nuclear fractions
from 6 DIV CBG neurons were loaded and immunoblotted with monoclonal
anti-JIP-1. JIP expression was predominantly nuclear, although lower
levels of JIP were detected in cytosolic fractions. C,
Confocal sections through nuclei of 6 DIV CBG neurons stained for JIP-1
and the corresponding 1° sample are shown; JNK staining is also
shown for comparison. The inset shows a section closer
to the coverslip.
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Inhibition of neuronal JNK activity leads to increased neurite
number, indicating a regulatory role for JNK during neurite
outgrowth
In the PC12 cell neuronal model, JNK activity increases during
neurite outgrowth induced by staurosporine (Yao et al., 1997), NGF
(Eilers et al., 1998), or protease inhibition (Giasson et al., 1999),
consistent with a role for JNK during neuronal differentiation. Our
observation of developmentally regulated JNK activity in cerebellar granule neurons (Figs. 4, 5, 9) supports such a hypothesis. To test
this, we used dominant inhibitory mutants to the JNK cascade, SEK1KR
(Sanchez et al., 1994) and JIP-JBD [JIP-JNK binding domain (Dickens et
al., 1997)], and assessed the effects on neurite outgrowth (Fig.
11). The efficiency of these mutants at
blocking JNK activity was assessed by cotransfecting cerebellar granule
neurons with JNK11 together with SEK1KR or JIP-JBD after which
immunopurified recombinant JNK11 activity was measured. Coexpression
of either SEK1KR or JIP-JBD blocked high basal JNK11 activity,
JIP-JBD being a more effective inhibitor (Fig. 11A).
The effect of inhibition of JNK activity on neuritic architecture was
measured by counting processes from neurons cotransfected with
-galactosidase, a marker for transfected cells that is expressed in
the cytoplasmic compartment and efficiently highlights neuritic
morphology. -Galactosidase expression was monitored using a
high-sensitivity staining procedure (see Materials and Methods) that
allows visualization of fine processes, and cells were examined using a
100× objective. Cells with projections at least one cell body radius
in length were counted; thus the number of projections and not process
length was examined. Counterstaining with Hoechst was used to measure cell viability. Dead cells, apparent by their shrunken nuclei, were not
included in the quantified data shown. There was no significant difference in viability of cells expressing pEBG empty vector, SEK1KR,
or JIP-JBD (data not shown). The percentage of total cells per
coverslip with one to five or more than five processes is shown in
Figure 11B. Inhibition of JNK activity with SEK1KR or JIP-JBD leads to an increased proportion of cells with four or five
projections from the cell body and fewer cells with only one or two
processes. Expression of the more effective JNK inhibitor, JIP-JBD,
leads to a twofold increase in the proportion of cells with more than
five neurites. Representative images are shown in Figure
11C. The increased number of processes per cell after inhibition of JNK activity may appear somewhat counterintuitive, given
that increased JNK activity correlates with a period of increased
neurite outgrowth. However, different neuronal subtypes in the brain
have their own stereotypic number of processes, which is determined by
a balance of positive and negative regulators (Tessier-Lavigne and
Goodman, 1996). Because both the preexisting arbor and the cell body
are potential sites for new process formation, the total negative
regulation of outgrowth may be higher in more mature cells with
increased arborization. Our data show that inhibition of the JNK
pathway, after the onset of differentiation, reduces process number and
may thus be one such negative regulator.
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Figure 11.
Inhibition of neuronal JNK activity results in
increased projections from the cell body. A, Five days
in vitro cerebellar granule neurons were transfected
with pEBG-JNK11, pEBG-SEK1KR, or pcDNA3-JIP-JBD as shown, and 24 hr
after expression, JNK11 was isolated on GSH beads and its activity
was measured. Recombinant JNK11 activity is elevated in unstimulated
neurons, thus mimicking endogenous JNK activity. Coexpression of
dominant inhibitory kinases SEK1KR or JIP-JBD effectively blocks
JNK11 activity. B, Three days in vitro
cerebellar granule neurons were transfected as above with the addition
of a -galactosidase transfection marker. Forty-eight hours after
transfection, cells were stained for -galactosidase expression, and
the number of processes emerging from living cells with lengths more
than or equal to the cell body radius were counted. The percentage of
cells with a given number of processes (1-5 or >5) were calculated
for randomly chosen fields from five to six coverslips per condition.
The number of cells counted under each condition was 233 for pEBG
(n = 5), 285 for SEK1KR (n = 5), and 305 for JIP-JBD (n = 6). Data are expressed
as means ± SEM. C, A representative image from
cells transfected as in A and B above is
shown.
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DISCUSSION |
A comparison of specific JNK activities from primary neurons, a
neuroblastoma cell line, and non-neuronal cell lines reveals that
cerebellar granule neuron-derived JNKs display considerable constitutive activity, exceeding that of stressed U937 cell JNK. Elevated JNK activity in primary neurons in culture was similar to the
high activity detected in brain extracts prepared at the equivalent
postnatal days (Fig. 4), implying that elevated JNK signaling may
represent the endogen
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ABSTRACT
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