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INTRODUCTION |
To date, five members of the mixed lineage kinase
(MLK)1 family have been
identified: MLK1 (1), MLK2 (MST) (2, 3), and MLK-3 (SPRK/PTK) (4-6)
and the less highly conserved DLK (MUK/ZPK) (7-11) and LZK (12). With
the exception of MLK1, all MLK family members have been shown to
function as mitogen-activated kinase kinase kinases (MAPKKKs), which
predominantly activate the SAPK (JNK) stress-signaling pathway (9,
12-15). Although MLK-3 and MLK2 can activate SAPK via both SEK1
(MKK4) and MKK7, MLK2 shows preferential association with MKK7 (16) and
DLK activates SAPK via MKK7 only (17). This MKK7 specific SAPK
activation can be mediated by the recently identified scaffold proteins
JNK-interacting proteins JIP1 and JIP2 (18, 19). These scaffold
proteins interact specifically with MLK2/MLK-3/DLK, SAPK, and MKK7 but
not MKK4. In addition to the SAPK signaling pathway, MLK-3 also
activates the p38 (RK/HOG) signaling pathway via MKK3 and MKK6 (13).
Moreover, MLK2 and DLK have been found to slightly activate p38 and
MLK2 also appears to activate ERK weakly (9, 15). Besides functioning as a MAPKKK, MLK-3 also acts as an I
B kinase kinase and mediates activation of the transcription factor NF-
B, which targets genes such as those involved in immune and inflammatory responses (20).
A number of upstream molecules utilize MLK-3 in SAPK activation.
Dominant negative forms of MLK-3 have been shown to block SAPK
activation mediated through the Ste20 homologues germinal center kinase
(13) and hematopoetic progenitor kinase 1 (HPK1) (21), the small
GTPases Cdc42 and Rac (22), and the guanine-nucleotide exchange
protein CRK SH3-binding GNFP (C3G) (23) and Lbc's first cousin
(Lfc) (24), suggesting that MLK-3 is downstream of these proteins in
the SAPK signaling pathway. In Jurkat T cells, MLK-3 is activated upon
costimulation with CD3 and CD28, and Cdc42 and Rac also activate
NF-
B via MLK-3 (20, 25). Among these proteins, only HPK1, Cdc42, and
Rac have been shown to directly interact with MLK-3. HPK1 binds to the
MLK-3 SH3 domain and phosphorylates MLK-3 (21), whereas Cdc42 binds to
the Cdc42 and Rac interactive binding region (26) and induces MLK-3
homodimerization (27). While these two events are crucial for MLK-3
activation, their underlying mechanisms are not fully understood.
Many kinases are phosphorylated in the kinase activation loop located
between the conserved sequence DFG of subdomain VII and APE of
subdomain VIII (28). The activation loop plays a crucial role in
substrate recognition, and in many cases; phosphorylation in this
segment is required to allow correct alignment of the substrates to the
catalytic site (28-30). This segment can be regulated either through
autophosphorylation or phosphorylation by other kinases. For example,
cyclic AMP-dependent protein kinase autophosphorylates the
Thr197 located in its activation loop (31), whereas ERK2 is
phosphorylated on Thr183 and Tyr185 by MEK in
the corresponding activation loop (32). In both cases, phosphorylation
in the activation loop is essential for the activation of these
kinases. The putative kinase activation loop of MLK-3 contains three
potential phosphorylation sites at residues Thr277,
Thr278, and Ser281 that may play a role in
MLK-3 activation.
It is clear that MLK-3 plays a central role in integrating and
processing inputs into SAPK, p38, and NF-
B signaling pathways, however, the activation mechanism of MLK-3 has not been elucidated fully. We have demonstrated previously that dimerization via the tandem
leucine zippers is required both for MLK-3 autophosphorylation and the
activation of SAPK, suggesting that autophosphorylation might be an
intermediate step of MLK-3 activation (27). In this report, we aimed to
further define the mechanism of MLK-3 activation and examine the role
of the kinase activation loop in such a mechanism.
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EXPERIMENTAL PROCEDURES |
Expression Constructs--
The pcDNA3 HA-SEK1 and HA-HPK1
eukaryotic expression constructs were obtained from Dr. J. Woodgett and
Dr. F. Kiefer, respectively. The pcDNA3 Flag-MLK-3 and pEBG
GST-HA-MLK-3 expression constructs have been described previously (27).
MLK-3 expression constructs bearing mutation in the activation loop
were generated using a megaprimer PCR based mutagenesis method. First,
3' primers containing single/double mutations (T277A,
GGCACAAAGCCACACAAATG; T278A, GGCACAAAACCGCACAAATG; S281A,
AAATGGCTGCCGCGGGCACC; T277D, GGCAAAGACACACAAATG; S281D, AAATGGATGCCGCGGGCACC; T227D/S281D, GGCACAAAGACACACAAATGG) were used
independently with the 5' primer (Mut1: TGAGGAAGGGTGACCGT) for
PCR amplification. The resulting DNA fragments containing the mutations
(5' megaprimer) together with the 3' primer D8AA (GATGGGCAGTGTGAGCTTGT)
were then used in the second round of PCR. The second PCR products were
restriction digested with HpaI and BsrGI and
replaced the same DNA fragment in the pcDNA-3 Flag MLK-3/pEBG GST-HA MLK-3 wild type constructs. To generate the K144R/S281A mutant,
the Pflm I-Pflm I DNA fragment of the pEBG GST-HA MLK-3 K144R was
replaced with the Pflm I-Pflm I DNA fragment containing the S281A
mutation. The prokaryotic expression constructs bearing the MLK-3
activation loop were generated as follows. The 5' primer (BamHI-DFG,
TTTGGATCCCTGGCCCGAGTGGCAC) and 3' primer (NotI-APE, TTTGCGGCCGCCAGGCGTAGG) bearing restriction sites were used to amplify
the DNA sequence corresponding to the MLK-3 activation loop region from
the wild type and the double mutants described above. The resulting DNA
fragments were digested with BamHI and NotI and
inserted into the NotI and BamHI sites of the
pGEX 4T-2 vector (Amersham Pharmacia Biotech).
Expression and Purification of GST Fusion Protein and
Peptide--
The pGEX bacterial expression constructs were transformed
into BL21 cells, a protease-deficient strain of Escherichia
coli. The bacterial cultures were incubated overnight at 37 °C,
diluted 50 times into a total volume of 200 ml, and incubated at
30 °C. When the O.D. reached 0.7 to 1.0, the cultures were induced
with 0.2 mM isopropyl-
-D-thiogalactoside at
30 °C for 2.5 h. The bacteria were then resuspended in 10 ml of
phosphate-buffered saline supplemented with 1% Triton, 10 mM dithiothreitol, 50 mM EDTA, and protease inhibitors (50 µg/ml aminoethylbenzenesulfonyl fluoride, 1 µg/ml antiPAIN, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml
aprotinin) (ICN Pharmaceuticals), lysed on ice with 10 µg/ml lysozyme
and centrifuged to remove the cellular debris. The fusion proteins were
then affinity purified by incubating the lysates with 200 µl of the
50% glutathione-Sepharose 4B for 1 h at 4 °C with gentle rotation. After washing 3 times with 10 ml of phosphate-buffered saline
supplemented with 1% Triton, 10 mM dithiothreitol, and 50 mM EDTA, the glutathione-Sepharose bounded fusion proteins were either subjected to elution or thrombin digestion.
To elute the fusion proteins, the Sepharose was treated 3 times with
150 µl of 10 mM reduced glutathione (dissolved in 50 mM Tris-HCl, pH 8.0) at 37 °C for 5 min. The elutions
were combined and spun through a Centriplus concentrator (Amicon), with
a molecular weight cut-off of 10,000 daltons, to remove the reduced
glutathione and concentrate the fusion proteins. The columns were
subsequently washed 3 times with 500 µl of phosphate-buffered saline
containing protease inhibitors and spun at 4 °C for 30 min and the
concentrated GST fusion proteins stored at
20 °C with 50%
glycerol at a concentration of 5 µg/µl.
To release the peptide, the Sepharose was further washed 4 times with
10 ml of 50 mM Tris, pH 7.4, and 10 mM
MgCl2, then incubated with 95 µl of 50 mM
Tris, pH 7.4, and 5 µl of 0.5 unit/µl thrombin at room temperature
for 75 min. The Sepharose was then centrifuged and the supernatants
containing the thrombin and the peptide were spun through a Microcon
centrifugal filter device (Millipore) with a molecular cut-off at
30,000 daltons to separate the thrombin and the peptide. The
flow-through containing the peptide was stored at
20 °C and
subjected to mass spectrometry analysis to check the purity and size.
Antibodies--
Anti-phospho-SAPK, anti-phospho-p38, and
anti-p38 antibodies were gifts from Dr. J. McGlade. Anti-SAPK and
anti-phospho-SEK1 antibodies were purchased from New England Biolabs.
Mouse monoclonal M2 anti-Flag antibody was obtained from Eastman Kodak,
Rochester, NY, and ascitic fluid containing anti-HA antibody was
prepared from 12CA5 hybridoma cells using standard methods.
Cell Culture and Transient Transfections--
HeLa cells were
cultured in Dulbecco's modified Eagle's medium supplemented with 8%
fetal bovine serum (Life Technologies, Inc.), 250 ng/ml Fungizone
(Bristol-Myers Squibb), 200 unit/ml penicillin, and 100 unit/ml
streptomycin (ICN Pharmaceuticals). In most of the experiments, HeLa
cells were transfected using a vaccinia virus-based transfection method
as described in Tibbles et al. (13). For the experiments
involving the endogenous SAPK and p38, HeLa cells were transfected
using Lipofectin (Life Technologies, Inc.) according to the
manufacturer's instructions. 293 cells were cultured and transfected
as described previously (27).
Immunoprecipitation and Affinity Purification--
Transfected
HeLa and 293 cells were harvested and purified as described previously
(27) with the following modifications. Pre-chilled Nonidet P-40 lysis
buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 10%
glycerol, and 1% Nonidet P-40) supplemented with proteinase inhibitors
together with phosphatase inhibitors (1 mM sodium
orthovanadate, 10 mM sodium fluoride, and 10 mM
-glycerophosphate) was used to lyse the transfected cells. For
immunoprecipitation, antibody together with 0.1 mg/ml protein
A-Sepharose (Amersham Pharmacia Biotech), was added simultaneously to
the lysates for 1 h at 4 °C. For affinity purification,
GST-tagged MLK-3 proteins were incubated with 25-50 µl of 50%
glutathione 4B-Sepharose (Amersham Pharmacia Biotech) for 1 h at
4 °C. Finally, the proteins bound either to protein A or
glutathione-Sepharose were then washed 3 times with the Nonidet P-40
lysis buffer.
Western Blotting--
Western blotting using anti-Flag, anti-HA,
and anti-MLK-3 SH3 antibodies were carried out as described previously
(27). Western blotting using anti-phospho-SAPK, anti-SAPK,
anti-phospho-p38, and anti-p38 antibodies was carried as according to
the manufacturer's instructions.
In Vitro Kinase Assays--
The immunopurified Flag-MLK-3
proteins were incubated with 30 µl of kinase buffer I (50 mM Tris, pH 7.4, 10 mM MgCl2, 1 mM EGTA) and 5 µCi of [
-32P]ATP for 30 min at 30 °C. The immunopurified HA-HPK1 proteins together with 2.5 µg of the purified GST fusion proteins were incubated with kinase
buffer II (25 mM Tris, pH 7.5, 5 mM
-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, and 10 mM
MgCl2) and 5 µCi of [
-32P]ATP for 30 min
at 30 °C. The kinase reactions were terminated by adding 2 × SDS sample buffer containing 20 mM EDTA. The samples were
then boiled, separated by SDS-polyacrylamide gel electrophoresis, the
polyacrylamide gels were dried, and exposed to x-ray film. For the
nonradioactive labeled in vitro kinase assay of the purified peptides, HA-HPK1 immunoprecipitates were incubated with the peptide in
20 µl of kinase buffer II and 2 mM ATP for 35 min at
37 °C. The sample was then centrifuged and the buffer containing the peptide was removed and subjected to mass spectrometry analysis.
Mass Spectrometry--
Matrix-assisted laser desorption
ionization mass spectrometry time of flight analysis was performed by
the Mass Spectrometry Laboratory of the Molecular Medicine Research
Center at the University of Toronto. In brief, 1 µl of the peptide
sample and 1 µl of the matrix (saturated
-cyano-4-hydroxycinnamic
acid in 50% acetonitrile, 0.1% trifluoroacetic acid) were spotted on
the matrix-assisted laser desorption ionization and air-dried. The
sample was then analyzed using the positive mode on a Voyager-DE STR
matrix-assisted laser desorption ionization-time of flight mass
spectrometer (Perseptive Biosystems, Inc.), equipped with a pulsed
nitrogen laser (337 nm, 3-ns pulse).
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RESULTS |
Mutations in the Kinase Activation Loop of MLK-3 Affect MLK-3
Autophosphorylation Activity--
Many kinases are phosphorylated in
the activation loop between catalytic subdomains VII and VIII. For
example, mitogen-activated kinases such as the ERK2 and SAPK are
phosphorylated and activated by a mitogen-activated kinase kinase in
the TXY motif (31, 33). The MAPKKK C-Raf is phosphorylated
by PKC, whereas MEKK1 is activated by autophosphorylation in the
activation loop (34-36) (Fig.
1A). Within the putative MLK-3
activation loop between the DGF (subdomain VII) and APE (subdomain
VIII) residues, there are 3 serine/threonine residues
(Thr277, Thr278, and Ser281) that
might serve as potential phosphorylation sites.

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Fig. 1.
Serine/threonine to alanine mutations in
MLK-3 putative kinase activation loop affect MLK-3
autophosphorylation. Panel A is a schematic diagram
that illustrates the amino acid sequence of the (putative) kinase
activation loop between the catalytic subdomains VII and VIII of the
MAPK (32), SAPK (33), c-Raf (35), Mekk1 (36), and MLK-3 (13). The
serine and threonine residues of MAPK, SAPK, c-Raf, and Mekk1 that are
phosphorylated are depicted in bold, as are the potential
phosphorylatable serine and threonine residues in MLK-3. Panel
B, GST-HA MLK-3 expression constructs bearing the
mutations T277A, T278A, or S281A were generated as described under
"Experimental Procedures." These expression constructs together
with the wild type and kinase-dead K144R constructs were transfected
alone into HeLa cells, and the resulting cell lysates were affinity
purified (AP) using glutathione-Sepharose 4B
(GS4B). Two-thirds of each of the purified samples were
subjected to in vitro kinase assay, and the resulting
autoradiograph is shown in the upper panel. The balance of
the samples were Western blotted using anti-MLK-3 SH3 Ab and are shown
in the lower panel. "p-GST-HA MLK-3" denotes
the phosphorylated MLK-3 protein. Similar results were obtained when
Flag-tagged MLK-3 expression constructs or 293 cells were used (data
not shown).
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To investigate whether any of these amino acids play a role in MLK-3
autophosphorylation, each one was independently mutated to alanine
(T277A, T278A, and S281A) using PCR-based site-directed mutagenesis.
The mutant kinases were assayed to determine whether they retained
autophosphorylation activity. In these experiments, GST-HA-tagged MLK-3
expression constructs containing the above mutations were transfected
into cells, purified using GS4B-Sepharose, and the autophosphorylation
compared with the wild type and kinase-dead (K144R) forms of MLK-3 via
in vitro kinase assays. As shown in Fig. 1B, when
the various forms of MLK-3 were expressed at equivalent levels,
mutant T277A displayed autophosphorylation activity comparable to that
of the kinase-dead form of MLK-3; mutant T278A retained close to normal
autophosphorylation activity, whereas mutant S281A exhibited a low
level of autophosphorylation activity. These results suggest that the
activation loop plays a key role in MLK-3 autophosphorylation and in
particular, that the Thr277 (and Ser281 to
lesser extent) residue is crucial for MLK-3 autophosphorylation.
Autophosphorylation Is Required for MLK-3 Activation--
We have
proposed that MLK-3 dimerization leads to its autophosphorylation, its
subsequent activation, and ultimately leads to the phosphorylation of
MLK-3 substrates such as SEK1 and MKK3/6 (13). To establish whether
autophosphorylation is required for MLK-3 activation, we first tested
the ability of the various MLK-3 activation loop mutants to
phosphorylate SEK1 using a gel mobility shift assay and Western
blotting using phospho-specific SEK1 (Thr223) antibody. If
autophosphorylation is required for MLK-3 activation, a mutant lacking
autophosphorylation activity will be inactive and will not
phosphorylate SEK1 (and thus will not induce an electrophoretic shift
in SEK1). For these studies, HA-SEK1 was transfected either alone or
together with the Flag-tagged wild type T277A, T278A, or S281A
forms of MLK-3. Half of the resulting cell lysates were immunoprecipitated and Western blotted using anti-HA Ab to detect the
HA-SEK1 proteins. The same blot was then probed with anti-phospho-SEK1 (Thr223) antibody to detect the phosphorylated form of
SEK1. To detect the various forms of MLK-3, the balance of the lysates
were subjected to immunoprecipitation and Western blotting using
anti-Flag Ab. As shown in Fig. 2,
panel A, in the presence of the wild type MLK-3, the
electrophoretic mobility of SEK1 was retarded; whereas mutation of
MLK-3 Thr277 to alanine completely abolished the SEK1
mobility shift. The MLK-3 T278A mutant induced a SEK1 mobility shift
similar to that of the wild type MLK-3, whereas the S281A mutant
induced an intermediate shift. Reprobing of panel A with
anti-phospho-SEK1 antibody revealed that T278A (which exhibited
relatively high level of autophosphorylation activity) phosphorylated
SEK1 to the highest level among the three mutants. Notably, however,
T278A activity was lower than wild type in this experiment. While this
may due to experimental variation, we cannot rule out that mutation of
this residue has an effect on MLK-3 activity toward SEK1. Mutant T277A
that had very low level of autophosphorylation activity only weakly
phosphorylated SEK1. Although S281A caused an intermediate shift of
SEK1, the level of phospho-SEK1 detected was lower than anticipated
suggesting that the SEK1 mobility shift was due to phosphorylation at a
site other than the Thr233 residue. These observations
suggest that autophosphorylation is required for MLK-3 function and a
correlation between the level of MLK-3 autophosphorylation activity and
its ability to phosphorylate SEK1.

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Fig. 2.
Serine/threonine to alanine mutations in
MLK-3 putative activation loop affect MLK-3 phosphorylation of its
substrate SEK1. HA-tagged SEK1 protein was expressed alone or
together with the Flag-tagged WT, T277A, T281A, or S281A proteins. Half
of the resulting cell lysates were subjected to immunoprecipitation
(Ip) and Wester blot (WB) using anti-HA Ab and
the electrophoretic mobility of the HA-SEK1 proteins are shown in the
panel A. The same blot was then Western blotted with
anti-phospho-SEK1 (Thr223) antibody to detect the
phosphorylated form of SEK1 (panel B). Panel C
shows the expression level of the various forms of MLK-3 as detected by
Ip and WB using anti-Flag Ab. (Note: the MLK-3 proteins were separated
on an 8% polyacrylamide gel to show the expression level but not to
resolve the mobility shift of MLK-3.)
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Autophosphorylation Is Required for MLK-3-mediated Activation of
the Stress Signaling Pathways--
To corroborate these findings, we
next asked if activation of endogenous SAPK and p38 is also depended on
MLK-3 autophosphorylation activity. For these experiments,
GST-HA-tagged WT, K144R, T277A, T278A, and S281A MLK-3 expression
constructs were transfected into HeLa cells. Part of the resulting cell
lysates were separated on SDS-polyacrylamide gels and Western blotted
with anti-phospho-SAPK, anti-SAPK, anti-phospho-p38, and anti-p38
antibodies to detect the endogenous phospho-SAPK, total SAPK,
phospho-p38, and total p38 proteins, respectively. The rest of the
lysates were affinity purified using glutathione-Sepharose 4B and
Western blotted with anti-MLK-3 Ab. As shown in Fig.
3, similar levels of phospho-SAPK were
detected following the expression of the wild type or T278A mutant. No
phosphorylation of SAPK was observed with either the kinase-dead or the
T277A mutant, whereas only low levels of phospho-SAPK were detected in
the presence of the S281A mutant. Very similar results were observed
with p38. We consistently observed the highest levels of phospho-p38 in
the presence of the wild type and T278A relative to the S281A and T277A
mutants (although a basal level of the phospho-p38 was detected in the
nontransfected HeLa cells). Notably, T278A mutant, which displayed a
reduced ability to phosphorylate SEK1 (Fig. 2), appeared to activate
SAPK as efficiently as wild type. The fact that MLK-3 can activate SAPK
via other signaling molecules, for example, SEK4, may account for this
difference. These data suggest that the amount of activated SAPK and
p38 is dependent on the level of MLK-3 autophosphorylation activity. Collectively these data indicate that autophosphorylation is crucial for MLK-3 activation and the subsequent phosphorylation and activation of two separate stress signaling pathways.

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Fig. 3.
Serine/threonine to alanine mutations in
MLK-3 putative activation loop affect MLK-3 mediated activation of SAPK
and p38. The various GST-HA-MLK-3 expression constructs including
the WT, K144R, T277A, T228A, and S281A were transfected into HeLa cells
and harvested after 2 days. Aliquots of each lysate were separated on
four polyacrylamide gels and Western blotted with anti-phospho-SAPK,
anti-SAPK, anti-phospho-p38, and anti-p38 antibodies, and the resulting
autoradiographs are shown in panels A-D as indicated.
One-third of each cell lysate was affinity purified using
glutathione-Sepharose 4B (GS4B) and Western blotted with
anti-MLK-3 SH3 Ab, and the expression level of the various MLK-3 forms
is shown in the bottom panel. Similar results were obtained
when 293 cells were used (data not shown).
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Both Thr277 and Ser281 Residues May Serve
as the MLK-3 Autophosphorylation Sites--
We demonstrated that
substitution of the activation loop residues Thr277 and
Ser281 with alanine either abolished or diminished the
autophosphorylation activity of MLK-3. Since phosphorylation of the
kinase activation loop is a prerequisite for substrate recognition of
some kinases, it seemed reasonable to propose that the
Thr277 and Ser281 residues would be essential
for MLK-3 substrate recognition. To examine this possibility, we
mutated these two residues into negatively charged glutamic acid
residues, either independently or together (so as to mimic the
phosphorylation state), and tested whether the resulting mutants were
capable of activating downstream signaling leading to SAPK
phosphorylation. Wild type, T277D, S281D, and T277D/S281D MLK-3
expression constructs were transfected into 293 cells and the resulting
lysates analyzed for the presence of the activated endogenous SAPK
using anti-phospho-SAPK antibody. As shown in Fig.
4, phospho-SAPK was detected in all of
the lysates (except for the nontransfected control), suggesting that
all the mutants were able to activate endogenous SAPK. These results
imply that these negatively charged residues could functionally replace the Thr277 and Ser281 residues and allow normal
substrate recognition and kinase activity. The Thr277 and
Ser281 residues may therefore act as the MLK-3
(auto)phosphorylation sites. If so, because the mutation of the
Thr277 residue to alanine almost completely abolished MLK-3
autophosphorylation activity, whereas the S281A mutation only reduced
MLK-3 autophosphorylation activity, Thr277 is likely to be
the major autophosphorylation site of MLK-3.

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Fig. 4.
Mutation of Thr277 and
Ser281 to glutamic acid in MLK-3 putative activation loop
does not affect MLK-3-mediated SAPK activation. The GST-HA T277D,
S281D, and T277D/S281D expression constructs were generated as
described under "Experimental Procedures" and transfected into 293 cells (The WT MLK-3 expression construct was included as a control.)
The cells were harvested after 2 days and aliquots of the resulting
cell lysates were subjected to Western blots (WB) using
anti-phospho-SAPK and anti-SAPK antibodies. The results of the WB
analyses are shown in the first and second panel
as indicated. Half of each cell lysate was also affinity purified using
glutathione-Sepharose 4B (GS4B) and Western blotted with
anti-MLK-3 SH3 Ab to detect the various forms of MLK-3 protein
(bottom panel). Similar experiments were done in HeLa cells
with the same results (data not shown).
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HPK1 Phosphorylates the Activation Loop of MLK-3--
As mentioned
above, some kinases such as the mitogen-activated kinases ERK and SAPK
are phosphorylated by other kinases in their activation loop thereby
leading to their activation. HPK1 has also been shown to activate MLK-3
via phosphorylation (21); however, the precise site of HPK1
phosphorylation has not yet been identified. Accordingly, we
investigated the possibility that HPK1 phosphorylates MLK-3 within the
activation loop. We first tested whether HPK1 could phosphorylate a
peptide containing the MLK-3 activation loop amino acid sequence. A DNA
fragment corresponding to the activation loop was amplified via PCR and subcloned into a bacterial expression construct bearing an N-terminal GST tag. The peptide was then cleaved specifically from the purified GST fusion protein using thrombin protease and further purified. Mass
spectrometry analysis indicated that the molecular mass of the
resulting peptide was 2167.55 daltons, which matched the amino acid
sequence "GSLAREWHKTTQMSAAGTYA," this corresponds to the expected
size of the MLK-3 activation loop, except for the first 2 amino acids
resulting from the thrombin cleavage site of the GST protein. HA-HPK1
was then expressed in HeLa cells, immunopurified using anti-HA Ab and
incubated with the purified peptide in the presence of ATP and
MgCl2. When the peptide was subjected to mass spectrometry
analysis to detect any change in molecular weight, we observed an
additional peak of 2247.71 daltons (Fig.
5). The additional 80 daltons corresponds
exactly to the anticipated replacement of a hydroxyl group with a
phosphate group. As a negative control, the same peptide was also
incubated with a kinase-dead form of HPK1, but no additional peak was
observed (data not shown). Taken together, these observations indicate
that HPK1 phosphorylated one residue in the MLK-3 activation loop
peptide in vitro.

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Fig. 5.
HPK1 phosphorylates a peptide containing the
MLK-3 activation loop amino acid sequence. A peptide of the amino
acid sequence "GSLAREWHKTTQMSAAGTYA" was produced as described
under "Experimental Procedures." The amino acid sequence of this
peptide (except the first two amino acids "GS")
corresponds to the putative MLK-3 activation loop amino acid sequence.
The purified peptide was incubated with HA immunoprecipitates that
contained the HA-HPK1 proteins, along with Mg2+ and ATP.
After incubation, the samples were analyzed by a mass spectrometer. An
additional peak of 2247.7 daltons was observed only after the
incubation with the wild type (as shown in the figure), but not the
kinase-dead form of HA-HPK1 (data not shown).
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HPK1 Phosphorylates the Ser281 Residue of the
MLK-3activation Loop--
To determine whether Thr277,
Thr278, or Ser281 residue(s) was phosphorylated
by HPK1, we next examined whether HPK1 could phosphorylate GST fusion
proteins containing the same region of the MLK-3 activation loop with
only one of the three potential residues unchanged. Accordingly, double
mutants T277A/T278A, T277A/S281A, and T278A/S281A were generated. (The
C-terminal sequences together with that of the wild type and the vector
are listed in Fig. 6A.)
Affinity purified GST fusion proteins were incubated with
immunopurified HA-HPK1 in an in vitro kinase. As shown in
Fig. 6B, in the presence of equivalent amounts of GST fusion
proteins, HPK1 specifically phosphorylated the GST fusion proteins
bearing the wild type or the T277A/T278A double mutant, but not the
T277A/S281A or T278A/S281A double mutants or the GST control.
When the GST wild type activation loop fusion protein was incubated
with purified activated SAPK and ERK2, it was not phosphorylated by
either kinase (data not shown) confirming that the HPK1 phosphorylation
is specific. These results indicate that Ser281 is the HPK1
phosphorylation site since HPK1 phosphorylated the fusion protein only
when the Ser281 residue was preserved, as in the case of
the wild type and the T277A/T278A double mutant. Therefore,
Ser281 not only serves as potential MLK-3
autophosphorylation site but is also a HPK1 phosphorylation site.

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Fig. 6.
HPK1 phosphorylates GST fusion proteins
containing the MLK-3 activation loop sequence only when the
Ser281 residue is conserved. Panel A, a
schematic diagram showing the carboxyl terminus sequences of the GST
fusion proteins used in this experiment. The box labeled
GST denotes the GST moiety; the capital letters
represent the amino acids encoded by the MLK-3 cDNA sequences that
correspond to the activation loop, and the lower case
letters represent the amino acids encoded by the vector. In
addition, the underlying letters represent the mutated amino acids.
Panel B, the GST fusion proteins listed in panel
A were purified and incubated with the HA-HPK1 immunoprecipitates
in the presence of Mg2+ and ATP. The samples were then
separated on a polyacrylamide gel, stained, dried, and exposed to x-ray
film. The autoradiograph in the upper panel shows the
phosphorylated GST fusion protein whereas the Coomassie staining in the
lower panel illustrates the total amount of the GST fusion
protein used in these experiments.
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To determine whether HPK1 phosphorylates only the Ser281
residue within the entire MLK-3 protein, a kinase-dead mutant
containing the S281A mutation was generated and the ability of HPK1 to
phosphorylate this mutant was tested using in vitro kinase
assay. GST-HA-tagged K144R or K144R S281A constructs were
transfected alone or together with HA-tagged HPK1. The expressed
proteins were immunoprecipitated using anti-HA antibody; one-third of
the precipitates were subjected to Western blotting and the balance of
the samples were evaluated via in vitro kinase assay. As
shown in Fig. 7, both K144R and K144R/S281A mutants exhibited undetectable levels of kinase activity. HPK1 was able to phosphorylate the K144R form of MLK-3, however, only a
faint band (upon prolonged exposure) of the phosphorylated K144R/S281A
form was observed. The Ser281 residue therefore acts as the
major HPK1 phosphorylation site. Notably, the autophosphorylation
activity of HPK1 was greatly reduced in the presence of the K144R/S281A
MLK-3 mutant and also slightly reduced in the presence of the K144R
mutant, suggesting the MLK-3 Ser281 phosphorylation site
may compete with the HPK1 autophosphorylation site(s).

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Fig. 7.
The Ser281 residue is the major
HPK1 phosphorylation site. A GST-HA-tagged kinase-dead MLK-3
mutant containing the S281A mutation was generated as described under
"Experimental Procedures." GST-HA tagged forms of MLK-3 K144R,
K144R/S281A, and HA-HPK1 expression constructs were transfected alone
or together into HeLa cells. The resulting cell lysates were subjected
to immunoprecipitated (Ip) using anti-HA Ab to isolate both
the MLK-3 and HPK1 proteins. The HA immunoprecipitates were then
subjected to either in vitro kinase assay or WB. The
upper panel shows the phosphorylated HPK1 and MLK-3, whereas
the lower panel shows the expression level of HPK1 and the
various MLK-3 proteins.
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DISCUSSION |
The kinase activation loop, defined as the region spanning between
the subdomains VII and VIII, has been shown in a number of kinases to
regulate catalytic activity (28). In this study, we examined the role
of kinase activation loop in MLK-3 activation. First, we mutated the
three potential phosphorylation residues in the kinase activation loop
to investigate whether these residues are required for MLK-3
autophosphorylation activity. We found that mutating residue
Thr277 to alanine significantly reduced MLK-3
autophosphorylation activity. This same MLK-3 mutant only had
negligible phosphorylation activity toward its substrate SEK1 and did
not activate either JNK/SAPK or p38/RK. The S281A mutant displayed a
reduced level of autophosphorylation activity, and reduced activation
of SEK1, JNK/SAPK, and p38/RK, whereas the T278A mutant, which
exhibited close to normal autophosphorylation activity, can almost
fully activate these proteins. These results clearly demonstrate that
the activation loop is crucial for MLK-3 autophosphorylation and its
subsequent activation and suggest that the MLK-3 autophosphorylation
level correlates with activity.
Mutation of Thr277 and Ser281 might result in
the loss of MLK-3 autophosphorylation activity in two different ways;
first, it may affect the MLK-3 substrate binding ability and second, it
may eliminate the autophosphorylation site. Our data show that when the
Thr277 and Ser281 residues were mutated to
acidic residues, so as to mimic negatively charged phosphorylated
serine or threonine residues, the ability of MLK-3 to activate the SAPK
pathway was retained. These findings suggest that MLK-3 is functional
when the Thr277 and Ser281 residues are
phosphorylated and support the idea that these two residues may, in
fact, serve as the MLK-3 (auto)phosphorylation sites. We found that
when Thr277 is mutated, MLK-3 autophosphorylation activity
is minimal whereas when Ser281 is mutated, there are still
some levels of autophosphorylation activity indicating that
Thr277 can still be phosphorylated independent of
Ser281. One explanation for these observations is that
Thr277 is required for the subsequent phosphorylation of
the Ser281. An alternative possibility is that the
Thr277 is the only autophosphorylation site within the
activation loop and mutation of Ser281 simply affects the
"self" recognition and subsequent autophosphorylation of
Thr277. One way to distinguish these possibilities will be
to use phospho-specific antibodies when they become available.
Our results also indicate that all three residues (Thr277,
Thr278, and Ser281) are important for efficient
phosphorylation of SEK1 and therefore, substitution of any of these
neutral hydrophilic residues to hydrophobic alanine residues may affect
substrate binding specificity. However, since mutation of
Thr277 and Ser281 to alanine have substantial
effects on SEK1 phosphorylation whereas mutation of Thr278
to alanine only resulted in relatively minor reduction of SEK1 phosphorylation seems likely that both Thr277 and
Ser281 but not Thr278 need to be phosphorylated
for normal MLK-3 function toward SEK1.
Because MLK-3 lies downstream of HPK1 in the stress signaling pathway
and is directly phosphorylated by HPK1 (21), we also asked whether HPK1
could activate MLK-3 by phosphorylating MLK-3 activation loop. We
identified the Ser281 residue as the HPK1-phosphorylation
site, indicating that both autophosphorylation and HPK1 phosphorylation
could activate MLK-3 via the kinase activation loop which underscores
the complex regulation of MLK-3 activation. This mode of regulation, by
autophosphorylation and transphosphorylation by another kinase(s)
within the activation loop has also been reported for the tyrosine
kinase Lck (37).
We have shown previously that MLK-3 homodimerization is required for
its autophosphorylation, critical for its activation, and can be
induced by Cdc42 (27). In this report, we in fact demonstrated that
autophosphorylation is essential for MLK-3 activation. Taken together,
these data indicate that MLK-3 employs an activation mechanism similar
to that of receptor tyrosine kinases; that is, induced dimerization
leads to autophosphorylation and the subsequent activation of MLK-3.
Based on the fact that all MLK family members (except MLK1 which has
not been reported) display autophosphorylation activity and bear tandem
leucine zippers (2-12), it is reasonable to speculate that all MLK
family members utilize a dimerization-based activation mechanism which
ultimately leads to autophosphorylation and activation. Within the MLK
family, DLK has also been shown to also employ a dimerization-based
activation mechanism (38). Moreover, it is noteworthy that the two
residues corresponding to the MLK-3 potential autophosphorylation sites
are conserved among other MLK family members. As shown in Fig.
8, the Thr277 residue is
conserved in MLK1 and MLK2 (but substituted with a serine in
DLK/MUK/ZPK and LZK), whereas the Ser281 residue is
conserved in all MLK family members. Hence it is temping to hypothesize
that these two residues may also be crucial for the
autophosphorylation, thus activation, of other MLK family members.

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Fig. 8.
Potential phosphorylation sites in MLK
activation loop. A schematic diagram showing the putative
activation loop amino acid sequences of the MLK family members. The
potential autophosphorylation sites and the HPK1 phosphorylation site
of MLK-3 are shown in bold and larger font size.
The putative phosphorylation sites of other MLK family members
corresponding to the MLK-3 Thr277 and Ser281
residues are also highlighted.
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