From the Trescowthick Research Centre, Peter
MacCallum Cancer Institute, Melbourne 8006, Victoria, Australia and
¶ Department of Pharmaceutical Sciences, Northeastern University,
Boston, Massachusetts 02115
Received for publication, September 8, 2000, and in revised form, December 7, 2000
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ABSTRACT |
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MAP kinase signaling pathways are important
mediators of cellular responses to a wide variety of stimuli. Signals
pass along these pathways via kinase cascades in which three protein
kinases are sequentially phosphorylated and activated, initiating a
range of cellular programs including cellular proliferation, immune and
inflammatory responses, and apoptosis. One such cascade involves the
mixed lineage kinase, MLK2, signaling through MAP kinase kinase 4 and/or MAP kinase kinase 7 to the SAPK/JNK, resulting in
phosphorylation of transcription factors including the oncogene,
c-jun. Recently we showed that MLK2 causes apoptosis
in cultured neuronal cells and that this effect is dependent on
activation of the JNK pathway (Liu, Y. F., Dorow, D. S., and
Marshall, J. (2000) J. Biol. Chem. 275, 19035-19040).
Furthermore, dominant-negative MLK2 blocked apoptosis induced by
polyglutamine-expanded huntingtin protein, the product of the mutant
Huntington's disease gene. Here we show that as well as activating the
stress-signaling pathway, MLK2 is a target for phosphorylation by
activated JNK. Phosphopeptide mapping of MLK2 proteins revealed that
activated JNK2 phosphorylates multiple sites mainly within the
noncatalytic C-terminal region of MLK2 including the C-terminal 100 amino acid peptide. In addition, MLK2 is phosphorylated in
vivo within several of the same C-terminal peptides
phosphorylated by JNK2 in vitro, and this phosphorylation is increased by cotransfection of JNK2 and treatment with the JNK
activator, anisomycin. Cotransfection of dominant-negative JNK kinase
inhibits phosphorylation of kinase-negative MLK2 by anisomycin-activated JNK. Furthermore, we show that the N-terminal region of MLK2 is sufficient to activate JNK but that removal of the
C-terminal domain abrogates the apoptotic response. Taken together,
these data indicate that the apoptotic activity of MLK2 is dependent on
the C-terminal domain that is the main target for MLK2 phosphorylation
by activated JNK.
One mechanism by which cells respond to extracellular stimuli is
the recruitment of signaling networks that regulate changes in the
internal environment and control gene transcription (reviewed in Refs.
1 and 2). A group of signaling pathways that are highly conserved
through evolution are collectively known as mitogen-activated protein
kinase (MAPK)1 pathways (3,
4). The classical MAPK pathway is triggered by growth factors binding
to cell surface receptors leading to the activation of the GTPase, Ras.
This pathway results in activation of the extracellular
signal-regulated kinases (ERKs) leading to gene transcription and
cellular proliferation. A parallel MAPK pathway triggered by stress
factors such as osmotic shock, UV irradiation, cytotoxic drugs, and
inflammatory cytokines results in activation of the stress-activated
protein kinase/c-Jun N-terminal kinases (SAPK/JNKs) (2). A second
stress-activated pathway leads to activation of p38 MAPK. Effects of
stress activation vary from proliferation, differentiation, and gene
transcription to cell cycle arrest and apoptosis depending on cell type
and stimulus (2).
All MAPK pathways utilize a "three-kinase cascade" mechanism to
modulate incoming signals. Thus, each MAPK has upstream-activating kinases (MAPKKs) that are in turn regulated by MAPKK kinases (MAPKKKs) (reviewed in Ref. 5). Some of the upstream kinases appear to be highly
specific for a particular cascade, whereas others link to more than one
MAPK. The MAPKKs for the ERKs are the MAP/ERK kinases (MEKs) (6-8),
whereas activators for JNKs are the SAPK/ERK kinase/JNK kinase
(SEK1/JNKK), also known as MAPK kinase 4 (MKK4) (9-11), and the more
recently discovered MKK7 (12-14). In the case of p38, the main
upstream-activating kinases are MKK3 and MKK6 (11, 15, 16), but p38 can
also be activated by JNKK/MKK4 (11), providing crossover between the
JNK and the p38 pathways. MAPKKKs are a larger group of kinases that
have overlapping specificities for MAPKKs and are themselves activated
by an array of interactions in response to extracellular and internal
stimuli (reviewed in Ref. 17). As more upstream activators of the MAPKs
are identified, the picture of how these three parallel pathways are
regulated becomes more complex.
Recently it has become clear that MAPKs not only control downstream
signals to transcription factors but also exert effects upstream on
their activators. In budding yeast, the three components of the
pheromone-induced MAPK cascade are Ste11 The mixed lineage kinases (MLKs) (23, 24) are MAPKKKs that act upstream
of JNKK/MKK4 and MKK7 in the SAPK/JNK pathway (25, 26). MLK family
members MLK2, MLK3, and DLK activate the SAPK/JNKs, and to a lesser
extent p38 and ERKs, and MLK2 directly phosphorylates JNKK/MKK4
in vitro (26-30). Within their N-terminal regions, MLKs
1-3 contain several domains associated with protein interaction
including an Src homology (SH3) domain and two leucine zipper motifs
(24). In addition, each has a large C-terminal domain of unknown
function that is rich in serine, threonine, and proline amino acids.
Recently we reported that overexpression of active full-length MLK2
causes apoptosis in neuronal cells, and this is dependent on activation
of JNK (31). Transfection of kinase-negative MLK2 inhibits neuronal
cell apoptosis caused by expression of polyglutamine-expanded
huntingtin protein, the gene product associated with neuronal pathology
in Huntington's disease (32, 33). In addition, overexpression of the
N-terminal region of normal huntingtin protein partially rescues this
neuronal toxicity. A proline-rich peptide near the polyglutamine
insertion site in the N-terminal region of normal huntingtin protein
binds to the SH3 domain of MLK2, and polyglutamine expansion interferes with this interaction. As MLK2 is expressed at high levels in the
brain, these results suggest a role for MLK2 in the neuronal pathology
associated with polyglutamine expansion of huntingtin protein.
In the present report, we investigated signaling of MLK2 in the JNK
pathway and found that as well as being an activator of the SAPK/JNKs,
MLK2 is a substrate of activated MAPKs. JNK phosphorylation occurs at a
number of specific sites mainly within the C-terminal region of the
MLK2 protein, including the C-terminal 100 amino acid peptide.
Furthermore, the MLK2 C-terminal domain is phosphorylated in
vivo, and this phosphorylation increases after activation of JNK
by anisomycin treatment. In studies of MLK2 expression in epithelial
cells, wild-type MLK2 induced apoptosis, and this is dependent on both
its catalytic activity and the presence of the noncatalytic C-terminal
domain. Taken together, these studies demonstrate that the MLK2
C-terminal domain is a target for regulation by activated JNK and is
vital for MLK2-induced apoptosis.
Reagents--
Dulbecco's modified Eagle's medium (DMEM),
leupeptin, pepstatin, and aprotinin were purchased from ICN, Sydney,
Australia. Fetal calf serum (FCS) was from the Commonwealth Serum
Laboratories, Parkville, Victoria, Australia, and gentamicin was from
David Bull Laboratories, Melbourne, Australia. Rabbit anti-HA
antibodies were provided by Dr. D. Bowtell and monoclonal mouse anti-HA
(12CA5) by Dr. R. Pearson, both from the Peter MacCallum Cancer
Institute, Melbourne, Australia. Rabbit anti-JNK1 antibody (C-17) from
Santa Cruz Biotechnology and biotinylated sheep anti-mouse and goat anti-rabbit immunoglobulins, streptavidin-biotin-conjugated alkaline phosphatase, and Hybond C Super nitrocellulose membrane were from Amersham Pharmacia Biotech. Alkaline phosphatase substrate reagent, 5-bromo-4-chloro-3-indolylphosphate and FuGENETM 6 transfection reagent were from Roche Molecular Biochemicals, and nitro
blue tetrazolium was from Sigma. Protein A-Sepharose was from Amersham
Pharmacia Biotech, and [ Plasmids--
cDNAs encoding the complete human MLK2
polypeptide (24), the N-terminal region (nucleotides 289-1777), the
C-terminal region (nucleotides 1777-3454, including a stop codon
beginning at nucleotide 3151), and the C-terminal 300 nucleotides
(nucleotides 2851-3153) were each cloned into the vector pKH3,
immediately 3' to 3 copies of an oligonucleotide encoding the
hemagglutinin (HA) tag peptide. The pKH3 vector for mammalian
expression from the cytomegalovirus promoter was a kind gift of Dr. Ian
Macara, Department of Pathology, University of Vermont.
FLAG®-tagged versions of MLK2 cDNAs were made by replacing
the 3× HA tag coding sequence in pKH3 with a polymerase chain reaction
product encoding the FLAG® peptide epitope. GFP-tagged MLK2 cDNAs
were constructed by subcloning MLK2 cDNAs into the vector pEGFP-C1
(CLONTECH) that includes a gene for neomycin resistance. Expression vectors for human JNK1 (34), JNK2 (35), and JNKK
(10) were HA-tagged full-length cDNAs in the vector pSR Site-directed Mutagenesis--
The MLK2-K125A (KN-MLK2) mutant
was made using the Altered Sites® II in vitro mutagenesis
kit (Promega) essentially according to manufacturer's instructions,
with the following modifications. Taq polymerase (8 units/30
µl) was substituted for T4 polymerase in the second strand synthesis
reaction, and mutant plasmid DNA was transfected into Escherichia
coli ES1301 mutS by electroporation using a Bio-Rad
Gene Pulser. The mutagenesis template for KN-MLK2, a BamHI
fragment containing the first 1488 base pairs of the coding region of
human MLK2 cDNA (24), was subcloned into the pALTER-1 vector
(Promega). The oligonucleotide used to create the MLK2-K125A mutation
was
5'-146AGGAGGTGGCAGTCGCGGCCGCCCGGCTGG176
(altered bases underlined) based on the MLK2 nucleotide sequence. The
entire nucleotide sequence of the mutated insert was confirmed by
automated nucleotide sequence analysis on an ABI Prism model 373 DNA
Sequencer (Applied Biosystems) before re-inserting into HA-pKH3-HA-MLK2 vector.
Cell Culture, Transfection, and Treatment--
COS7 cells were
maintained in DMEM supplemented with 10% FCS and 20 µg/ml (20 IU)
gentamicin at 37 °C in 5% CO2 in air. Cells (0.5-1 × 106) were transiently transfected with 2-5
µg plasmid DNA using FuGENETM 6 transfection reagent,
electroporation, or CaPO4. For phosphopeptide mapping
experiments, 4 × 106 COS7 cells were transfected by
electroporation with 5 µg of HA-MLK2 pKH3, HA-KN-MLK2 pKH3, HA-C-MLK2
pKH3, and/or 3 µg of HA-JNK2 pSR3 Western Blotting and Immune Complex Kinase Assays--
Cells
were lysed in 1 ml of ice-cold lysis buffer (50 mM HEPES,
pH 7.2, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EGTA, and 1.5 mM MgCl2)
containing proteinase inhibitors (1 µg/ml each of aprotinin,
leupeptin, and pepstatin and 1 mM phenylmethylsulfonyl fluoride) and 50 mM NaPO4. Lysates were
incubated on ice for 10 min and then clarified by centrifugation in a
microcentrifuge at 4 °C. Lysate protein concentrations were
determined using the bicinchoninic acid assay (36). For Western
blotting, aliquots of cellular lysate containing 30 µg of protein
were subjected to 10% SDS-polyacrylamide gel electrophoresis and
proteins transferred to nitrocellulose. For immune complex kinase
assays (IP kinase assays), HA-tagged proteins were immunoprecipitated
from cell lysate volumes adjusted to contain equal amounts of protein
with anti-HA antibodies bound to protein A-Sepharose beads. Beads were resuspended in 30 µl of assay buffer (25 mM HEPES, pH
7.2, 100 mM NaCl, 10 mM MgCl2, 5 mM MnCl2, 100 µM
NaVO4, and 10% glycerol) containing 60 µM
ATP and 20 µCi of [ Phosphopeptide Mapping--
MLK2 tryptic phosphopeptides were
separated on two-dimensional thin layer cellulose essentially by the
method of Hunter and co-workers (37). Briefly, phosphorylated proteins
were excised from polyacrylamide gels and the gel slices suspended in
200 µl of 20 mM NH4HCO3.
L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (10 µg) was added, and the digest was allowed to proceed for
18 h at 37 °C. A total of 4 × 103 cpm/digest
were dried and resuspended in electrophoresis buffer, loaded onto thin
layer cellulose plates, and peptides resolved in two dimensions by
electrophoresis followed by ascending chromatography. The
electrophoresis buffer was 2% formic acid and 7.8% acetic acid in
H2O, pH 1.9, and chromatography was in 37.5%
n-butyl alcohol, 25% pyridine, and 7.5% acetic acid
in H2O. Labeled peptides were visualized by PhosphorImage
analysis of the thin layer plates.
In Vivo Labeling--
COS7 cells were transfected with
pKH3-HA-KN-MLK2 together with either pSR Phosphoamino Acid Analysis--
Following tryptic digestion of
phosphorylated MLK2 protein, extracted peptides were incubated with
constant boiling HCl at 95 °C for 120 min. After drying, amino acids
were dissolved in 7.5% acetic acid, 25% pyridine, 37.5%
n-butyl alcohol in H2O, resolved on a thin layer
cellulose plate at 1500 V for 2 h, and visualized by PhosphorImage analysis.
Staining of Cultured Cells for Microscopy--
Cells grown on
glass coverslips were incubated for 10 min in fixing solution (4%
paraformaldehyde in 100 mM Pipes, pH 6.9, 5 mM
MgSO4, 10 mM EGTA, and 2 mM
dithiothreitol), permeabilized in 0.1% Triton X-100 and 0.5% bovine
serum albumin in phosphate-buffered saline for 5 min, and treated with
RNase (400 µg/ml) for 30 min. Cells were stained with propidium
iodide (1 µg/ml, 20 min) before mounting coverslips onto glass
slides. Confocal images were captured using a Bio-Rad MRC-1000 Confocal
Imaging System.
MLK2 Activates JNKs 1 and 2--
Within MAPK cascades, preferences
for activation of particular components or downstream targets have been
reported in various cell types or activation conditions. In the case of
the JNK cascade, differential involvement of SAPK/JNK isoforms in
growth regulation and apoptosis induction has been demonstrated in
several different cell types (38, 39). To determine whether MLK2
activation of the SAPK pathway leads to preferential activation of JNK
isoforms, COS7 cells were cotransfected with expression plasmids for
HA-MLK2 (Fig. 1) together with either
HA-JNK1 or HA-JNK2. Lysates of the transfected cells were assayed for
JNK activation in IP kinase assays using GST-c-Jun as a substrate. As a
control for these experiments, a kinase-negative mutant in which
alanine replaces lysine at position 125 in the MLK2 kinase catalytic
domain (KN-MLK2) was prepared. Lysine 125 of MLK2 corresponds to lysine
72 of cyclic AMP-dependent kinase that is directly involved
in the phospho-transfer reaction (40) during catalysis. As can be seen
in Fig. 1B, there is very efficient phosphorylation of c-Jun
by both JNK1 and JNK2 when isolated from cells expressing wild-type
MLK2 (wt-MLK2), whereas both JNKs were essentially inactive toward
c-Jun when coexpressed with KN-MLK2. Although JNK1 appears to be more
highly activated than JNK2 in this assay, JNK1 has a higher basal
activity and thus the degree of activation by MLK2 is about 8-fold for both JNKs (Fig. 1C).
JNK Phosphorylates Its Upstream Activator MLK2--
In the course
of analyzing SAPK/JNK signaling, COS7 cells expressing KN-MLK2 were
treated with the protein synthesis inhibitor anisomycin, a potent
activator of JNK (2). In anti-HA immunoblots of recombinant MLK2
proteins from these cells, broadening of KN-MLK2 bands was evident
(data not shown). This suggested that MLK2 is phosphorylated in
vivo in response to anisomycin. However, due to the strong
constitutive autophosphorylation activity of overexpressed wt-MLK2,
this effect is only observable with the KN-MLK2 mutant. To determine
whether activated JNK has ability to phosphorylate MLK2, HA-JNK2 was
transfected into COS7 cells and immunoprecipitated after either serum
starvation or anisomycin treatment of the cells. Immune complex kinase
assays were performed with HA-KN-MLK2 that had been immunoprecipitated
from separately transfected untreated COS7 cells as the added substrate
(Fig. 2A). This revealed low level phosphorylation of added KN-MLK2 by JNK2 isolated from
serum-starved cells, and JNK2 isolated from anisomycin-treated cells
phosphorylated KN-MLK2 efficiently. Anisomycin activation of JNK in the
COS7 cells was confirmed by immunoblotting cellular lysates with
anti-JNK antibodies where a clear bandshift in JNK from
anisomycin-treated cells was observed (Fig. 2A, lower
panel).
As coexpression of active MLK2 has a significant effect on JNK
activity, we measured the ability of JNK2 to phosphorylate KN-MLK2
after activation by wt-MLK2. In this case, HA-JNK2 was coexpressed with
FLAG-tagged versions of either wt-MLK2 or KN-MLK2 and the JNK2
selectively immunoprecipitated with anti-HA antibodies. HA-KN-MLK2 was
separately transfected into COS7 cells and immunoprecipitated. HA-KN-MLK2 immunoprecipitate was then added to the HA-JNK2 for a kinase
reaction. As can be seen in Fig. 2B, HA-JNK2 that had been
coexpressed with active FLAG-MLK2 readily phosphorylated the added
KN-MLK2. Where JNK2 was expressed either alone or coexpressed with
FLAG-KN-MLK2, however, phosphorylation activity toward KN-MLK2 was
reduced, and no phosphorylation of KN-MLK2 was detected in anti-HA
immunoprecipitates from cells expressing FLAG-MLK2 alone (Fig.
2B, lane 3). This confirms that no active FLAG-MLK2 was present in the anti-HA immunoprecipitates and rules out the possibility that another kinase coimmunoprecipitating with KN-MLK2 may be responsible for the observed phosphorylation. Taken together, these
data demonstrate that JNK2 activated either by coexpression of wt-MLK2
or the classical JNK activator, anisomycin, readily phosphorylates
KN-MLK2 in vitro. The signaling pathway from MLK2 to JNK
leading to JNK phosphorylation of MLK2 is depicted in the diagram in
Fig. 2C. To demonstrate the involvement of the JNK cascade
further, we examined the requirement for JNKK activity in the ability
of activated JNK to phosphorylate KN-MLK2 in vitro (Fig.
2D). For this, HA-JNK2 was coexpressed with either wild-type or kinase-negative JNKK, and the cells were activated with anisomycin. The HA-JNK was immunoprecipitated and tested for the ability to phosphorylate added HA-KN-MLK2 immunoprecipitated from separately transfected cells. In this assay, phosphorylation of KN-MLK2 increased by 10-fold after anisomycin activation of the JNK pathway, and coexpression of kinase-negative JNKK inhibited this activation 50%.
It has been shown previously that JNK both binds to and phosphorylates
another MAPKKK, MEKK1 (20). To test the possibility that JNK may bind
directly to MLK2, HA-JNK2 was coexpressed with GFP-tagged wt-MLK2, and
anti-GFP immunoprecipitates were assayed for the presence of HA-JNK2 by
anti-HA immunoblotting. In addition, anti-GFP-MLK2 immune precipitates
were tested for c-Jun phosphorylation activity in an IP kinase assay.
In both cases, although GFP-wt-MLK2 was fully active, no HA-JNK2 band
or phosphorylation of c-Jun was detected (data not shown). Thus, by two
criteria, JNK was not detectable in the anti-GFP-MLK2 immune precipitates.
The C-terminal Region of MLK2 Is a Target for MAPK
Phosphorylation--
The 496-amino acid N-terminal region of the MLK2
protein (Fig. 1A) contains structural domains associated
with protein-protein interaction (24). The C-terminal 458-amino acid
region contains no known structural domains but has many potential
sites for proline-directed MAPK phosphorylation. To determine whether
sequences within the C-terminal region are necessary for MLK2 signaling
activity, HA-JNK2 was coexpressed with HA-MLK2, HA-KN-MLK2, HA-N-MLK2,
or HA-C-MLK2, and activation of JNK2 was measured in IP kinase assays
with GST-c-Jun as a substrate (Fig.
3A). This experiment revealed
that JNK2 immunoprecipitated from cells cotransfected with either MLK2
or N-MLK2 (lanes 2 and 4) phosphorylated c-Jun
efficiently, whereas JNK2 coexpressed with KN-MLK2 or C-MLK2 was
essentially inactive toward c-Jun.
It is also clear in this assay that there is a low level of
incorporation of phosphate into both KN-MLK2 and C-MLK2 (Fig. 3A,
lanes 3 and 5) when they are coexpressed and
coimmunoprecipitated with JNK2. Although C-MLK2 appears to be more
highly phosphorylated than KN-MLK2, the expression level of C-MLK2 is
consistently higher than that of full-length MLK2, as is evident in
anti-HA immunoblots of cell lysates from the assay (Fig. 3A,
lower panels). In addition, it is noteworthy that the C-MLK2 band
recognized by anti-HA antibodies in the immunoblot is somewhat broad,
and the in vitro incorporation of radioactive phosphate is
concentrated in the lower portion of the band (Fig. 3A, upper
panel, lane 5). As the bandshift is associated with
phosphorylation, this indicates that some phosphate is already
incorporated into C-MLK2 in vivo. The C-MLK2 protein generally runs as a broad band in immunoblots, and its bandwidth is
increased in lysates where JNK2 is coexpressed. The opposite situation
is observed for N-MLK2, which has a narrow band in the immunoblot but
shows a substantial shift after the in vitro kinase assay
(Fig. 3A, lane 4) indicating that much of the
phosphorylation has taken place in vitro.
To examine the effect of JNK activation on phosphorylation of
HA-KN-MLK2 and HA-C-MLK2, each was expressed in COS7 cells, immunoprecipitated, and used as substrates for JNK2 immunoprecipitated from either serum-starved or anisomycin-activated cells. IP kinase assays (Fig. 4A) confirm that
both KN-MLK2 and C-MLK2 are phosphorylated by JNK2 and that there is a
substantial increase in their phosphorylation (KN-MLK2
It has been reported that expression of MLK2 activates both p38-MAPK
and ERK (30, 42). JNK, ERK, and p38 all phosphorylate serine or
threonine residues followed by proline (5, 43), and such dipeptides are
prevalent in the MLK2 C-terminal domain (see Table
I). To test the possibility that
phosphorylation of MLK2 may be mediated by members of the ERK and p38
MAPK families, COS7 cells were transfected with HA-C-MLK2 together with
HA-MLK2, HA-JNK2, HA-p38, or HA-ERK2, and phosphorylation of C-MLK2 was measured in IP kinase assays (Fig.
5A). In this assay, C-MLK2 was
most efficiently phosphorylated by full-length MLK2 itself (Fig.
5A, lane 4) and by JNK2 (Fig. 5A, lane
2). Phosphorylation of C-MLK2 by ERK2 (Fig. 5B) was
observed; however, this was weak in comparison to that by JNK2, whereas
phosphorylation by p38 was barely detectable.
MLK2 Is Phosphorylated at Multiple Sites by Autophosphorylation and
by JNK2--
The catalytic domain of MLK2 has sequence motifs
associated with both the serine/threonine- and the tyrosine-specific
kinase families (24). To confirm the specificity of MLK2
autophosphorylation, the phosphorylated MLK2 band from an anti-HA IP
kinase reaction gel was excised and subjected to phosphoamino acid
analysis (Fig. 6A). The
migration pattern of phosphorylated amino acids was compared with that
of standard phosphoserine, phosphothreonine, and phosphotyrosine. This
analysis revealed that MLK2 autophosphorylates on both serine and
threonine but not tyrosine.
Due to the constitutive activity of overexpressed wt-MLK2, JNK2
phosphorylation of MLK2 can only be detected using KN-MLK2 as a
substrate, and this precludes measurement of MLK2 activation in
response to JNK phosphorylation. Therefore, we used phosphopeptide mapping to demonstrate that JNK2 phosphorylation of KN-MLK2 targets sites distinct form that of MLK2 autophosphorylation. For this analysis, phosphorylated HA-MLK2 was isolated from an IP kinase reaction gel, cleaved with trypsin, and resulting peptides resolved on
thin layer cellulose by electrophoresis in the first dimension followed
by ascending chromatography (37). The phosphopeptide mapping experiment
was repeated several times to ensure that the pattern was reproducible
and represented as complete a digest as possible. The final map (Fig.
6A) shows that 8 tryptic peptides are targets for MLK2
autophosphorylation with peptides 1, 4, and 5 being most highly
phosphorylated. In a time course in which MLK2 in vitro
autophosphorylated peptides were mapped at 2, 5, 10, and 20 min, all of
the same peptides were present, and only the intensity appeared to
change over time (data not shown).
Next, JNK2 immunoprecipitated from either serum-starved or
anisomycin-treated COS7 cells was used to phosphorylate HA-KN-MLK2 in vitro. Phosphopeptide mapping of the phosphorylated
HA-KN-MLK2 (Fig. 6B) revealed that JNK2 from serum-starved
cells targets a single MLK2 tryptic peptide. This peptide corresponds
to a major phosphopeptide present in the autophosphorylated wt-MLK2 map
(Fig. 6A, peptide 1). When activated
JNK2 from anisomycin-treated cells was used, a number of tryptic
phosphopeptides appear on the KN-MLK2 map (Fig. 6C), and
four of these (peptides 1, 4, 5, and 7) run at
similar positions to phosphopeptides in the wt-MLK2 autophosphorylation profile (Fig. 6A). Although it is possible that distinct
phosphopeptides may run at overlapping positions in the two maps, the
pattern created by these four phosphopeptides is strikingly similar in both maps, and it would be surprising to find four sets of distinct MLK2 phosphopeptides that comigrate in such a manner. It is
possible that another kinase coimmunoprecipitating with KN-MLK2 could
be responsible for the phosphorylation; however, when KN-MLK2 expressed alone was subjected to an IP kinase assay no in vitro
phosphorylation was detected (see Fig. 1).
In addition to the four peptides from the wt-MLK2 profile, there are
seven JNK2-specific phosphopeptides in the map of KN-MLK2 phosphorylated by activated JNK2 (Fig. 6C, peptides J1-J7).
Within the MLK2 protein, there are 40 predicted serine- and or
threonine-containing tryptic peptides (Table I) fairly evenly
distributed throughout the polypeptide chain. However, there are but
two hydroxy-amino acid-proline dipeptides in the N-terminal 496 amino
acids (peptides 1 and 20). In contrast, the
457-amino acid C-MLK2 polypeptide contains a total of 77 serine and
threonine residues of which 20 constitute potential sites for
proline-directed phosphorylation. These 20 sites are located within 12 predicted tryptic peptides.
To determine which JNK2 phosphorylation sites are located within the
C-terminal region of the MLK2 molecule, the JNK2 phosphorylation assay
was repeated with HA-C-MLK2 as the added substrate. Maps of HA-C-MLK2
tryptic peptides phosphorylated in vitro by JNK2 from either
serum-starved or anisomycin-treated cells are shown in Fig. 6,
D and E, with positions of phosphopeptides from
the KN-MLK2 map circled. These maps confirm that most of the
MLK2 sites phosphorylated by JNK2 are indeed located within the C-MLK2 protein. Only phosphopeptide J5, which is a prominent phosphopeptide in
the KN-MLK2 map, does not appear on the C-MLK2 map. However, signals
for several phosphopeptides, including numbers 4, J1, and J3, are
somewhat reduced in the C-MLK2 map compared with that of KN-MLK2, and
two new phosphopeptides (Fig. 6E, peptides C1 and
C2) that do not appear in the KN-MLK2 map are present in the C-MLK2 map. Taken together, the phosphopeptide maps clearly demonstrate specific phosphorylation of multiple MLK2 C-terminal domain sites by
activated JNK2.
JNK2 Phosphorylates the C-terminal 100-Amino Acid Peptide of
MLK2--
As can be seen in Table I, several regions of the MLK2
C-terminal domain sequence are rich in potential proline-directed phosphorylation sites. Peptide 38 in particular contains three Ser- or
Thr-Pro pairs as well as three other serine and four threonine residues. The C-terminal 100 amino acids of MLK2 includes peptide 38 as
well as 2 other tryptic peptides containing hydroxy-amino acids (Table
I, peptides 36 and 37). To begin to identify specific regions of the
MLK2 C-terminal domain that may be targets for JNK phosphorylation, we
cloned a cDNA fragment encoding the C-terminal 100 amino acids of
MLK2 (C100-MLK2) into a vector for mammalian expression with an HA tag.
The HA-C100-MLK2 vector was transfected into COS7 cells together with
either HA-MLK2 or HA-MLK2 plus HA-JNK2, and phosphorylation of
C100-MLK2 was determined by IP kinase assay (Fig.
7). In this assay, HA-C100-MLK2 was
phosphorylated by activated JNK2, while no phosphorylation of this
peptide by active MLK2 was detected. Phosphorylation of C100-MLK2 by
JNK2 but not MLK2 is consistent with data from the two-dimensional
phosphopeptide mapping experiments (Fig. 6) in which a number of C-MLK2
peptides are phosphorylated by JNK2 that are not present in the MLK2
autophosphorylation map.
JNK2 Phosphorylates MLK2 in Vivo--
To examine MLK2
phosphorylation in vivo, COS7 cells were transfected with
HA-KN-MLK2 together with either empty vector or HA-JNK2 and labeled
with [32P]orthophosphoric acid. Cells coexpressing JNK2
were first serum-starved and then incubated with or without anisomycin.
After immunoprecipitation and electrophoresis, phosphorylated KN-MLK2
was excised from the gel and subjected to two-dimensional tryptic
phosphopeptide mapping (Fig. 8). It is
evident from this analysis (Fig. 8A) that KN-MLK2 is
phosphorylated in vivo on six of the same peptides
phosphorylated by JNK2 in vitro. Furthermore, most of the
in vivo labeled peptides are also represented in the C-MLK2
map (Fig. 6E). In addition, a new phosphopeptide (V), that
is poorly labeled during in vitro JNK2 phosphorylation of
KN-MLK2 and C-MLK2 (Fig. 6, C and E) appears in
the in vivo maps. Surprisingly, however, peptide 1 that is present in all of the in vitro phosphopeptide maps is absent
from the in vivo maps, suggesting that phosphorylation of
this peptide is an artifact of the in vitro system.
When KN-MLK2 is coexpressed with JNK2 (Fig. 8B), the
in vivo labeled phosphopeptide pattern is almost identical
to that of KN-MLK2 expressed alone. Interestingly, however, two
phosphopeptides that are not present in the in vitro maps
appear very faintly when JNK2 is coexpressed with KN-MLK2
(arrowheads in Fig. 8B). When the cells are
treated with anisomycin (Fig. 8C), phosphorylation of these
two new peptides increases dramatically as does that of 5 other
phosphopeptides (2, 4, J2, J5, and V), indicating
that stress activation is involved in phosphorylation at these sites. Interestingly, however, the signals for two peptides (J4 and
J6, circled in Fig. 8C) decrease when the cells
are treated with anisomycin. It is not clear why these peptides, which
are prominent phosphopeptides in maps of KN-MLK2 phosphorylated
in vitro, decrease in signal while anisomycin causes an
~3.5-fold increase in KN-MLK2 phosphorylation (Fig. 4). The fact that
the position of peptides in the overall map does not change, however,
shows that the cleavage and migration conditions have not varied significantly.
Expression of wt-MLK2 causes apoptosis in neuronal cells, and this is
dependent on JNK activation (31). MLK2 expression also leads to
apoptosis in other cell types including melanoma and breast tumor lines
(data not shown) and Swiss 3T3 fibroblasts (42). To examine the roles
of the C-terminal domain and catalytic activity in MLK2-induced
apoptosis, we expressed GFP-tagged wt-MLK2, KN-MLK2, N-MLK2, and C-MLK2
into HEK 293 cells (Fig. 9). In transient transfection experiments, all of the GFP-wt-MLK2-expressing cells displayed classic hallmarks of apoptosis including cytoplasmic shrinkage, membrane blebbing, and nuclear distortion or fragmentation (Fig. 9A) within 24-48 h of transfection. No such
characteristics were observed with the expression of the
kinase-negative or truncated MLK2 proteins (Fig. 9, B-D),
and cells with stable expression of GFP-KN-MLK2, GFP-C-MLK2, or
GFP-N-MLK2 were selected. After these cells were expanded into stable
cell lines, the catalytic activity of the GFP-N-MLK2 protein was
compared with that of GFP-wt-MLK2 (Fig. 9E), and correct
sizes of expressed GFP-tagged MLK2, N-MLK2, and C-MLK2 proteins were
confirmed by anti-GFP immunoblotting. Wt-MLK2 and N-MLK2 are both fully
competent to signal to the JNK pathway as shown by immunoblotting of
coexpressed HA-JNKK, where a clear bandshift of phosphorylated JNKK was
evident (Fig. 9E, bottom panel). Although there are
morphological differences among the cell
lines,2 each has been
retrieved after storage in liquid nitrogen and cultured for periods of
several weeks without appreciable loss of viability or protein
expression. Furthermore, all of these cell lines appear to cycle
normally with only small differences in doubling times (data not
shown). These experiments clearly show that both kinase catalytic
activity and the C-terminal domain of MLK2 are required to induce
apoptosis.
Here we have shown that MLK2 activates both JNK1 and JNK2
and that all of the information required for JNK activation is
contained within the N-terminal 496 amino acids of the MLK2 protein
(Fig. 3). Furthermore, we show that MLK2 is phosphorylated by JNK
mainly within the C-terminal domain of the MLK2 protein. When JNK2 is activated by either anisomycin treatment or coexpression of wt-MLK2 incorporation of phosphate into the KN-MLK2 protein increases, and this
increase is significantly inhibited by coexpression of dominant-negative JNKK. Furthermore, MLK2 is also phosphorylated by
both p38 and ERK2 but somewhat weakly compared with JNK. In previous
studies, MLK2 was shown to activate p38 and ERK (30, 42); however, JNK
is activated to a much more significant extent than either of the other
MAPKs. In addition, MLK2 phosphorylates and activates JNKK and MKK7,
the direct upstream activators of JNK (25, 26), but is unable to
phosphorylate MEK, the MAPKK for ERK (30). Thus, in MAPK signaling
pathways, MLK2 activates MAPKs in a hierarchical manner, and this is
mirrored by the extent to which each MAPK reciprocally phosphorylates
MLK2 (Fig. 5).
Phosphopeptide mapping revealed that four MLK2 tryptic peptides
are phosphorylated by both autophosphorylation and by activated JNK2
(Fig. 6C, peptides 1, 4, 5, and 7),
whereas seven MLK2 peptides are phosphorylated specifically by
activated JNK. All but one of the JNK-specific peptides are located
within the MLK2 C-terminal domain (Fig. 6, C and
E). In cotransfection analysis, the C-terminal 100-amino
acid peptide of MLK2 is phosphorylated specifically by JNK2 but not by
MLK2 (Fig. 7). As all potential proline-directed sites within this
peptide are located within a 12-amino acid stretch near the C terminus
of MLK2 (Table I, tryptic peptide 38), this region of the MLK2 protein
may be important for regulation by MAPK phosphorylation.
Mapping of in vivo labeled KN-MLK2 phosphopeptides revealed
that all but one of the MLK2 peptides phosphorylated in vivo
have counterparts in the maps of both KN-MLK2 and C-MLK2 phosphorylated by JNK2 in vitro. This suggests that endogenous JNK, or
another kinase with very similar specificity, is responsible for a
large proportion of the in vivo phosphorylation of KN-MLK2
and that the MLK2 C-terminal domain is the main target for in
vivo phosphorylation. Furthermore, three KN-MLK2 peptides that are
not present in the in vitro maps (Fig. 8) are labeled
in vivo. It is possible that phosphorylation of these three
peptides within the cell requires the presence of other factors or
specific interactions that are missing in the in vitro
kinase reaction. It is also possible that kinases other than JNK2 are
involved. However, when JNK2 is coexpressed with KN-MLK2 and the cells
treated with anisomycin, incorporation of radiolabel into these three
peptides increases (Fig. 8C). In addition, anisomycin
treatment increases phosphorylation of four peptides that are also
present in the in vitro KN-MLK2 map. Thus, when MLK2 is
phosphorylated in vivo in conditions that activate JNK
signaling, sites are targeted that are also specifically phosphorylated by activated JNK2 in vitro. Taken together, these results
strongly support the conclusion that activated JNK is responsible for
the majority of phosphorylation in the in vivo labeling of
KN-MLK2.
Unexpectedly, two peptides observed during in vivo phosphate
labeling (Fig. 8, J5 and J6) decrease in signal
after anisomycin treatment. Although it is possible that phosphatases
activated by anisomycin treatment may be responsible for the reduction
in signal at specific MLK2 sites, to our knowledge, activation of phosphatases by anisomycin has not been reported. Therefore, it is
likely that activation of the JNK pathway by anisomycin influences either MLK2 conformation or interactions within the cell such that
certain sites become unavailable or unfavorable for phosphorylation. Recently, it has been shown that several MAPK pathways are regulated by
interactions of pathway components with scaffolding proteins (19,
44-49). These proteins may play roles in channeling MAPK signals to
maintain specificity or in localizing MAPK cascade components at
specific sites of activity within the cell. In budding yeast, a
scaffolding protein, Ste5, binds the three components of the pheromone
MAPK cascade, Ste11 (MAPKKK), Ste7 (MAPKK), and Fus3/KSS (MAPK), and
directs their specificity (44). Fus3 phosphorylates both Ste7 (19) and
Ste5 (45), and this "feedback phosphorylation" is thought to play a
role in complex formation among these proteins (19).
In the mammalian SAPK/JNK pathway, JNK binds to and phosphorylates
MEKK1 (20). As MEKK1 also binds JNKK (20), MEKK1 may act as a
scaffolding protein to regulate specificity of this JNK cascade (20).
In the present study, no direct binding of MLK2 to JNK was detected.
However, a pair of recently discovered mammalian scaffolding proteins,
JNK-interacting proteins 1 and 2, complex with MLKs as well as MKK7 and
JNK (46-49) and enhance JNK signaling in response to coexpression of
active MLK (49). Thus, JNK-interacting proteins play similar roles to
Ste5 for the kinase cascade from MLK to JNK in mammalian cells (47,
49). Therefore, phosphorylation of MLK2 by JNK may regulate such a
complex by a feedback mechanism, as has been suggested for Fus3
phosphorylation of Ste7 (19).
Expression of wt-MLK2 causes apoptosis in neuronal cells, and this is
dependent on its catalytic activity (Fig. 9) and on JNK activation
(31). However, the viability of cells stably expressing active N-MLK2,
which is fully competent to activate JNK, clearly demonstrates that
MLK2 signaling activity in itself is not sufficient to induce
apoptosis. Thus, sequences within the C-terminal region are required to
trigger a cell death response. As the C-terminal domain is a target for
phosphorylation by activated JNK, this phosphorylation has the
potential to regulate MLK2 interactions with proteins that may be
required for the apoptotic activity. It has previously been reported
that the MLK2 C-terminal domain binds to the signaling adapter 14.3.3
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ste7
Fus3/KSS (18).
Errede and co-workers (19) coined the term "feedback phosphorylation" to describe upstream phosphorylation of Ste7 by
Fus3. In mammalian cells, Xu and Cobb (20) report that JNK binds to and
phosphorylates MEKK1, a major MAPKKK for the JNK pathway (21, 22). In
addition, cotransfection of the noncatalytic domain of MEKK1
potentiates JNK activation
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (3000 Ci/mmol) and
[32P]orthophosphoric acid (9000 Ci/mmol) were from
PerkinElmer Life Sciences. L-1-Tosylamido-2-phenylethyl
chloromethyl ketone-treated trypsin (248 units/mg protein) was from
Worthington, and pre-coated thin layer cellulose plates were from
Merck. VectashieldTM mounting medium was from Vector
Laboratories (Burlingame, CA).
(10).
The JNK vectors and a cDNA encoding c-Jun fused to glutathione
transferase (GST-c-Jun) in pGEX were kind gifts from Dr. M. Karin,
University of California, San Diego.
and incubated in complete DMEM
at 37 °C in 5% CO2 in air for 24-36 h. For anisomycin
treatment, JNK2-expressing cells were washed after 12 h with
serum-free DMEM and incubated in serum-free conditions for 24 h.
In some cultures, JNK2 was activated by addition of 10 µg/ml
anisomycin to serum-starved cells for 30 min before harvest. For stable
transfectants, HEK 293 cells were electroporated with 5 µg of pEGFPC1
mutant MLK2 cDNA, and the cells were grown in the presence of 400 µg/ml G418 antibiotic. Clones of resistant cells were isolated and
assayed by microscopy for expression of the GFP fusion protein and by
immunoblotting with anti-GFP antibodies to confirm the size of the
expressed fusion protein.
-32P]ATP. Reactions were allowed
to proceed for 30 min at room temperature before proteins were
separated on 10% SDS-polyacrylamide gel electrophoresis, and
incorporated radioactivity was analyzed using a Molecular Dynamics
PhosphorImager (Sunnyvale, CA). Assays were repeated in triplicate, and
raw data were corrected for protein expression levels as determined by
anti-HA immunoblotting of an aliquot of each cell lysate.
-HA-JNK2 or empty vector and
incubated for 24 h in complete DMEM. For cells transfected with
JNK2, complete DMEM was replaced with serum-free DMEM, and cells were
incubated for a further 18 h. The medium of all cells was then
replaced with phosphate-free DMEM with or without 10% dialyzed FCS for
2 h. Cells were then incubated with
[32P]orthophosphoric acid (200 µCi per ml of medium)
alone or together with 10 µg/ml anisomycin for 30 min. Cells were
harvested and proteins immunoprecipitated with anti HA-antibodies.
Following electrophoresis, the KN-MLK2 protein was subjected to
two-dimensional phosphopeptide mapping as described above.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (28K):
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Fig. 1.
MLK2 activates both JNKs 1 and 2. A, schematic representation of MLK2 structural domains (24)
and constructs used for expression of epitope-tagged MLK2 proteins.
3×HA, 3 copies of the HA tag peptide; SH3, Src
homology 3 domain; kinase, catalytic domain;
LeuZip, dual leucine zipper domain; Basic, basic
motif; CRIB, cdc42/rac interactive binding motif (41);
K125A, catalytic domain inactivating mutation. B,
upper panels, IP kinase assays. COS7 cells were cotransfected with
5 µg of plasmid, pSR -HA-JNK1 (lanes 1-3), or
pSR
-HA-JNK2 (lanes 5-7) together with either pKH3 empty
vector (lanes 1 and 5), pKH3-MLK2 (lanes
2 and 6), or pKH3-KN-MLK2 (lanes 3 and
7), or pKH3-MLK2 with pKH3 empty vector (lane 4)
as indicated. After 24 h of incubation, cells were lysed and
anti-HA IP kinase assays performed with GST-c-Jun added as a substrate
(as described under "Experimental Procedures"). 32P
incorporation was visualized by PhosphorImage analysis of the kinase
assay gel. Lower panels, anti-HA immunoblots of cell lysate
proteins before immunoprecipitation. C, graphical
representation of 32P incorporation (PhosphorImager units)
into GST-c-Jun by JNK1 or JNK2, when coexpressed with HA tag (control),
HA-MLK2, or HA-KN-MLK2, as indicated. Each bar represents an
average of data from three separate experiments.
View larger version (34K):
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Fig. 2.
Kinase-negative MLK2 is phosphorylated by
activated JNK2. A, COS7 cells transfected with 5 µg
of pSR -HA-JNK2 were incubated for 24 h and then serum-starved
for 18 h before treatment with either medium alone or 10 µg/ml
anisomycin in medium, for 30 min, as indicated. Anti-HA kinase assays
were performed as described in the legend to Fig. 1, with HA-KN-MLK2
added as substrate. Upper panels, in vitro kinase
assay; lower panels, anti-HA and anti-JNK immunoblots of
expressed proteins before immunoprecipitation. B, COS7 cells
were transfected with 5 µg of pKH3-FLAG-MLK2, pKH3-FLAG-KN-MLK2,
and/or pSR
-HA-JNK2, as indicated. Cells were lysed, and HA-tagged
proteins were immunoprecipitated. Upper panels, IP kinase
assays performed as in A, lower panel, anti-HA
immunoblot of expressed proteins. C, diagram of the
phosphorylation cascade from MLK2 to JNK and the upstream
phosphorylation of MLK2 by JNK. D, COS7 cells transfected
with 3 µg of pSR
-HA-JNK2 together with 5 µg of either
pSR
-HA-wt-JNKK or pSR
-HA-KN-JNKK were incubated for 24 h and
then serum-starved for 18 h before treatment with either medium
alone or 10 µg/ml anisomycin in medium, for 30 min, as indicated.
Upper panels, IP kinase assays performed as in A,
lower panel, anti-HA immunoblot of expressed proteins.
View larger version (36K):
[in a new window]
Fig. 3.
The N-terminal 495 amino acids of MLK2 are
sufficient to activate JNK2. A, COS7 cells were
transfected with 5 µg of pSR -HA-JNK2 together with 5 µg of pKH3
empty vector, pHK3-HA-MLK2, pKH3-HA-KN-MLK2, pKH3-HA-N-MLK2, or
pKH3-HA-C-MLK2, as indicated, and cells were lysed 24-36 h later.
Upper panels, anti-HA IP kinase assays with added GST-c-Jun
substrate. Lower panels, anti-HA immunoblots of cell lysates
before immunoprecipitation.
3.5-fold and
C-MLK2
5-fold) by JNK2 following anisomycin treatment (Fig. 4A,
upper panel, lanes 2 and 4).
View larger version (50K):
[in a new window]
Fig. 4.
JNK2 phosphorylates KN-MLK2 and C-MLK2.
Upper panel, HA-JNK2 was transfected into 2 plates of COS7
cells, and after 24 h incubation, cells were serum-starved and
incubated with or without anisomycin (as described under
"Experimental Procedures"). Anti-HA-JNK2 IP kinase assays performed
as in the legend to Fig. 1 with substrates HA-CMLK2 (lanes 1 and 2) or HA-KN-MLK2 (lanes 3 and 4)
added as substrates. Lower panel, anti-HA immunoblots of
expressed proteins in cell lysates before immunoprecipitation.
SS, serum-starved cells; A, anisomycin treated
cells.
MLK2 tryptic peptides containing serine or
threonine
View larger version (19K):
[in a new window]
Fig. 5.
P38, ERK2, and wt-MLK2 phosphorylate the MLK2
C-terminal domain. COS7 cells were transfected with 5 µg of
plasmid pKH3-HA-C-MLK2 together with either 5 µg of pKH3 empty vector
(lane 1), pKH3-HA-MLK2, pSR -HA-JNK2, or pSR
-HA-p38
(A) as indicated or pSR
-HA-JNK2 or pSR
-HA-ERK2
(B). Cells were lysed 24-36 h later for anti-HA IP kinase
assays (upper panels) and anti-HA immunoblotting
(lower panels).
View larger version (37K):
[in a new window]
Fig. 6.
Activated JNK2 phosphorylates MLK2 at
multiple sites. Two-dimensional phosphopeptide maps of MLK2
proteins phosphorylated in vitro. COS7 cells were
electroporated with either 3 µg of pSR -HA-JNK2 or 5 µg of
pKH3-HA-MLK2, pKH3-HA-KN-MLK2, or pKH3-HA-C-MLK2. JNK2-expressing cells
were serum-starved and then incubated with or without anisomycin (as
described under "Experimental Procedures"). Proteins were
immunoprecipitated, and anti-HA IP kinase assays wer performed as
described in the legend to Fig. 1. Phosphorylated MLK2 proteins were
subjected to either phosphoamino acid analysis or two-dimensional
phosphopeptide mapping by electrophoresis (E) followed by
ascending chromatography (C) on thin layer cellulose as
described by Boyle et al. (37). A,
autophosphorylated HA-MLK2 and phosphoamino acid analysis (left
panel) and phosphopeptide map (right panel). Maps of
phosphopeptides from HA-KN-MLK2 phosphorylated by JNK2 from
serum-starved (B) or anisomycin-activated cells
(C). Maps of phosphopeptides from HA-C-MLK2 phosphorylated
by JNK2 from serum-starved (D) or anisomycin-activated cells
(E). Numbers 1, 4, 5, and 7 in
E refer to phosphopeptides that appear in the map of
autophosphorylated WT-MLK2 (A) and numbers J1-J7
refer to peptides that are specific to KN-MLK2 phosphorylated by JNK2
in vitro. Circles show positions of
phosphopeptides from the map of KN-MLK2 phosphorylated by activated
JNK2 (C).
View larger version (24K):
[in a new window]
Fig. 7.
JNK2 phosphorylates the C-terminal 100-amino
acid peptide of MLK2. COS7 cells were transfected with 3 µg of
plasmid pKH3-C-100-MLK2 together with 3 µg of pKH3-HA-MLK2 and
pSR -HA-JNK2 (lane 1) or pKH3-HA-MLK2 and pKH3 empty
vector (lane 2) and lysed 24 h later. Left
panel, anti-HA IP kinase assays; right panel, anti-HA
immunoblots of cell lysates before immunoprecipitation.
View larger version (42K):
[in a new window]
Fig. 8.
JNK2 phosphorylates MLK2 in
vivo. COS7 cells were cotransfected with 5 µg of
pKH3-KN-MLK2 and either 3 µg of pKH3 empty vector (A) or 3 µg of pSR -HA-JNK2 (B and C). Cells were
labeled with [32P]orthophosphoric acid (as described
under "Experimental Procedures") before immunoprecipitation and
two-dimensional phosphopeptide mapping. A, maps of KN-MLK2
peptides phosphorylated in vivo (phosphopeptides are
numbered according to their positions in Fig. 6 and
arrowheads refer to peptides absent from the maps of MLK2
proteins phosphorylated in vitro); B, KN-MLK2
phosphorylated in serum-starved cells coexpressing JNK2. C,
KN-MLK2 phosphorylated in anisomycin-treated cells coexpressing JNK2
(arrows indicate peptides with increased signal, and
circles indicate peptides with decreased signal, following
anisomycin treatment). D, autoradiograph of
immunoprecipitated JNK2 proteins from serum-starved (SS) or
anisomycin (Aniso)-treated cells.
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Fig. 9.
Catalytic activity and the presence of the
C-terminal domain are required for MLK2 induced apoptosis.
A-D, confocal fluorescence micrographs of HEK 293 cells.
Transiently transfected with pEGFPC1-MLK2 (A) or cell lines
stably expressing GFP-KN-MLK2 (B); GFP-C-MLK2
(C); or GFP-N-MLK2 (D). Cells cultured on glass
coverslips were fixed and stained with propidium iodide to highlight
nuclei (A-C) or left unstained (D).
E, analysis of signaling activity and sizes of expressed
GFP-tagged MLK2 proteins. HEK 293 cells were transiently transfected
with 5 µg of pEGFPC1-MLK2 and 3 µg of pSR -HA-JNKK or stable cell
lines expressing GFP, GFP-N-MLK2, or GFP-C-MLK2 were transiently
transfected with 3 µg of pSR
-JNKK. Upper panels, IP
kinase assays; lower panels, anti-GFP or anti-HA immunoblots
of cell lysates.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(42). 14.3.3 proteins are involved in both MAPK signaling and
regulation of apoptosis (50, 51), and they bind their targets through
motifs containing phosphorylated serine. The MLK2 C-terminal domain
14.3.3 motif is
Arg719-Gly-Leu-Ser-Pro-Pro724
(42) in which the serine is a proline-directed MAPK phosphorylation site. Thus, the 14.3.3/MLK2 interaction is a prime example of a complex
in which regulation of MLK2 activity and apoptosis may be influenced
through phosphorylation by activated JNK. Studies are now underway to
identify the specific sites of JNK phosphorylation of the MLK2
C-terminal domain and to determine the role of phosphorylation at these
sites in MLK2 induced apoptosis.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Michael Karin, University of California, San Diego, for providing plasmids. We are also grateful to Dr. Richard Simpson for advice on the manuscript and Drs. Richard Pearson and Nelly Marmy-Conus, Peter MacCallum Cancer Institute, for valuable discussion and assistance with phosphopeptide mapping.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed: Trescowthick
Research Centre, Peter MacCallum Cancer Institute, Locked Bag #1 A'Beckett St., Melbourne 8006, Victoria, Australia. Tel.:
61-3-9656-1249; Fax: 61-3-9656-1411; E-mail:
d.dorow@pmci.unimelb.edu.au.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M008237200
2 G. Price, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: MAPK, mitogen-activated protein kinase; SAPK/JNK, stress-activated protein kinase/c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; MLK, mixed lineage kinase; KN-MLK2, kinase-negative MLK2 mutant; C-MLK2, C-terminal 457 amino acids of MLK2; C100-MLK2, C-terminal 100 amino acids of MLK2; MEK, MAP/ERK kinases, MKK4 or MKK7, MAPK kinase 4 or 7; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GFP, green fluorescent protein; IP, immunoprecipitate; Pipes, 1,4-piperazinediethanesulfonic acid; GST, glutathione S-transferase.
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