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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3893-3899
Constitutive Activation of Extracellular Signal-Regulated Kinase in
Human Acute Leukemias: Combined Role of Activation of MEK,
Hyperexpression of Extracellular Signal-Regulated Kinase, and
Downregulation of a Phosphatase, PAC1
By
Seong-Cheol Kim,
Jee-Sook Hahn,
Yoo-Hong Min,
Nae-Choon Yoo,
Yun-Woong Ko, and
Won-Jae Lee
From the Department of Internal Medicine and Laboratory of
Immunology, Medical Research Center, Yonsei University College of
Medicine, Seoul, Korea.
 |
ABSTRACT |
Extracellular signal-regulated kinase (ERK) is an important
intermediate in signal transduction pathways that are initiated by many
types of cell surface receptors. It is thought to play a pivotal role
in integrating and transmitting transmembrane signals required for
growth and differentiation. Constitutive activation of ERK in
fibroblasts elicits oncogenic transformation, and recently, constitutive activation of ERK has been observed in some human malignancies, including acute leukemia. However, mechanisms underlying constitutive activation of ERK have not been well characterized. In
this study, we examined the activation of ERK in 79 human acute leukemia samples and attempted to find factors contributing to constitutive ERK activation. First, we showed that ERK and MEK were
constitutively activated in acute leukemias by in vitro kinase assay
and immunoblot analysis. However, in only one half of the studied
samples, the pattern of ERK activation was similar to that of MEK
activation. Next, by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis, we showed
hyperexpression of ERK in a majority of acute leukemias. In 17 of 26 cases (65.4%) analyzed by immunoblot, the pattern of ERK expression
was similar to that of ERK activation. The fact of constitutive
activation of ERK in acute leukemias suggested to us the possibility of
an abnormal downregulation mechanism of ERK. Therefore, we examined PAC1, a specific ERK phosphatase predominantly expressed in
hematopoietic tissue and known to be upregulated at the transcription
level in response to ERK activation. Interestingly, in our study, PAC1 gene expression in acute leukemias showing constitutive ERK activation was significantly lower than that in unstimulated, normal bone marrow
(BM) samples showing minimal or no ERK activation (P = .002).
Also, a significant correlation was observed between PAC1 downregulation and phosphorylation of ERK in acute leukemias (P = .002). Finally, by further analysis of 26 cases, we showed that a
complementary role of MEK activation, ERK hyperexpression, and PAC1
downregulation could contribute to determining the constitutive activation of ERK in acute leukemia. Our results suggest that ERK is
constitutively activated in a majority of acute leukemias, and in
addition to the activation of MEK, the hyperexpression of ERK and
downregulation of PAC1 also contribute to constitutive ERK activation
in acute leukemias.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
EXTRACELLULAR signal-regulated kinase
(ERK) is a signaling molecule common to pathways that regulate the
proliferation and differentiation in diverse cell types including
hematopoietic cells.1,2 In blood cells and their
precursors, the activation of ERK has been shown to be involved in the
proliferation and cellular response of various hematopoietic cytokines,
including steel factor, granulocyte-macrophage colony-stimulating
factor, interleukin-3 (IL-3), and IL-5.3-6 In response to a
wide range of cytokine and growth factor stimuli, activated ERK is
translocated into the nucleus and activates a number of nuclear
transcription factors7,8 that lead to a dramatic
recruitment and activation of a large group of cellular regulatory
processes. As far as the constitutive activation of ERK is concerned,
the importance of the activation of ERK by sequential upstream kinases,
referred to as the ERK cascade, has been generally accepted. On
stimulation by a variety of growth factors, the protein kinases, Raf
and MEK kinase (MEKK), MAPK/ERK kinase (MEK), and ERK, are successively activated by phosphorylation.9,10
Recently, a constitutively active mutant of MEK has been shown to
transform NIH3T3 cells,11,12 and some reports have shown constitutive activation of ERK in human malignancies, including acute
leukemia.13-15 Although previous studies showed
constitutive activation of ERK in human malignancies, detailed
mechanisms underlying such activation have not been well characterized.
A previous study with acute leukemia showed that samples with
constitutively activated ERK also showed elevated MEK
activity.13 This result supports the hypothesis that MEK
activation is necessary for ERK activation. However, Oka et
al14 reported that some discrepancy between ERK and MEK
activation was observed in renal cell carcinoma and suggested the
possibility of the presence of other mechanisms for constitutive
activation of ERK. Also, in previous studies with human
malignancies,13,14 no cases or only a small percent showed
mutation of ras, an upstream regulator of ERK cascade despite a high
frequency of constitutive activation of ERK. These results indicate the
possibility of other regulatory mechanisms, which may be critical in
the constitutive activation of ERK. In this respect, it is a notable
finding that hyperexpression of ERK, as a possible additional mechanism
of constitutive ERK activation, was found in breast
cancer.15
In general, the extent of protein phosphorylation is balanced by
antagonism of kinase and phosphatase. Therefore, recently cloned
dual-specificity protein-tyrosine phosphatases (PTPases) that
exhibited dual catalytic activity toward phosphotyrosine and
phosphothreonine in substrate proteins may play a pivotal role in the
regulation of the ERK signaling pathway.16-18 Phosphatase of activated cells (PAC1), a member of the ERK phosphatase family, predominantly expressed in hematopoietic tissues, exhibits stringent substrate specificity for ERK in vitro.18-21 The kinetics
of gene expression and nuclear localization and the ability of PAC1 to inactivate ERK are all consistent with a role of this phosphatase in
the compensatory inactivation of the stimulated ERK signaling pathway.
Based on these observations, we attempted to find molecular mechanisms
underlying constitutive ERK activation in acute leukemias. In this
study, we have examined the relationship between ERK activation and MEK
activation and also whether the level of ERK expression contributes to
the activation of ERK. Finally, we have studied the regulation of PAC1,
a phosphatase induced by the activation of ERK.
| |
MATERIALS AND METHODS |
Cells.
We used 79 programmed-frozen leukemia and 10 normal bone marrow (BM)
samples, which were separated by Ficoll-Hypaque. Acute leukemias
included 67 acute myelocytic leukemias (AMLs; one M0, 10 M1, 12 M2, 12 M3, 22 M4, 10 M5 in the French-American-British classification) and 12 acute lymphoblastic leukemias (ALLs).22
Purification of CD34+ cells.
To investigate the adequacy of unselected normal marrow cells for
comparison with immature leukemic cells, we also studied CD34+ cells, the early hematopoietic stem/progenitor cells,
from one normal marrow donor. CD34+ cells were purified by
immunomagnetic bead methods using anti-CD34 monoclonal antibody
(Miltenyl Biotech Inc, Bergisch Gladbach, Germany). The purity of
isolated CD34+ cells was more than 90% by flow cytometry
(FACScan; Becton Dickinson, Lincoln Park, NJ).
Cell cultures.
K562 cells (American Type Culture Collection Certified Cell Lines
[ATCC CCL] 243) were maintained in RPMI 1640 medium
containing 10% fetal bovine serum supplemented with 100 U/mL
penicillin, 100 µg/mL streptomycin, 2 mmol/L L-glutamine, 1 mmol/L
sodium pyruvate, and 1 mmol/L nonessential amino acids.
In vitro ERK assay.
For kinase assay and immunoblotting, samples were rapidly thawed and
washed twice with phosphate-buffered saline containing 1 mmol/L sodium
orthovanadate. The viability assay was performed by the trypan blue
exclusion test. All acute leukemia samples showed over 90% viable
blast cells. Whole cell lysates were extracted as reported
previously.23 Concisely, the cells were lysed in lysis
buffer (50 mmol/L  -glycerophosphate, 1 mmol/L sodium orthovanadate, 20 mmol/L HEPES [pH, 7.4], 2 mmol/L EDTA, 1 mmol/L dithiothreitol, 1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride [PMSF], 2 µg/mL leupeptin, 25 µg/mL aprotinin, 10% glycerol). After
incubating for 30 minutes at 4°C, lysates were centrifuged at
25,000g for 20 minutes. The protein concentration was
determined with aid of a Bio-Rad Protein Assay Kit (Bio-Rad
Laboratories, Richmond, CA). The activity of ERK was assayed by
measuring the kinase activity for the synthesized peptide containing
PLS/TP, a recognition sequence for ERK1/2 (Amersham,
Buckinghamshire, UK).24 Per manufacturer's information,
this peptide contains no other phosphorylation sites and is much more
specific for ERK1/2 than the commonly used substrate, myelin basic
protein. Reaction mixture containing 30 µg of protein was spotted
onto phosphocellulose paper (Amersham) and washed in 75 mmol/L
phosphoric acid. The radioactivity on the filter was measured by
densitometry scanning (BAS2500; FUJIX, Tokyo, Japan).
Immunoblot analysis.
The lysates containing the same amounts of protein were subjected to
10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically onto immobilon-P membrane (Millipore Corp, Bedford, MA). The transfer efficiency was
confirmed by Ponceau-S staining. Blots were blocked in 5% nonfat dried
milk in 20 mmol/L Tris-HCl (pH 7.6), 137 mmol/L NaCl, and 0.05% Tween
20 and then probed with a phospho-specific ERK1/2 antibody that
recognizes Thr202/Tyr204 phosphorylated ERK1/2,
a phospho-specific MEK1/2 antibody that recognizes
Ser217/221 phosphorylated MEK1/2 or a total
ERK1/2 antibody that recognizes total ERK1/2 (New England Biolabs,
Beverly, MA). Also, we examined the immunoblot analysis using
 -tubulin (Oncogene, Cambridge, MA) as a protein loading control to
adjust the difference in protein loading amounts for individual
samples. Immunoreactive bands were detected by horseradish peroxidase
(HRP)-conjugated secondary antibody using enhanced
chemiluminescence reagents (Amersham). The signal intensity of the
autoradiogram was quantified by using densitometry scanning and the
value of the phosphorylation or expression of each molecule divided by
the -tubulin expression was used for comparison with the
phosphorylation or expression of each molecule.
Semiquantitative RT-PCR.
Total RNA from leukemia and normal BM cells was extracted with
commercial kit (RNeasy Mini Kit; QIAGEN, Hilgen, Germany). First-strand cDNA was synthesized in 20 µL reaction mixture
containing 1 µg of total RNA, 1 mmol/L of each deoxynucleotide
triphosphate (dNTP), 20 U of avian myeloblastosis virus
(AMV) reverse transcriptase (Boehringer Mannnheim,
Mannheim, Germany), 1.6 µg of oligo-dT primer. The 50-µL PCR
reaction mixture contained cDNA derived from 100 ng of total RNA, 1.25 U Taq DNA polymerase, 0.2 mmol/L of each dNTP, 0.2 µmol/L of each
primer, 1.5 mmol/L MgCl2. The sequences of the primers
were: ERK-225: sense
5'-TCTGTAGGCTGCATTCTGGC-3'; antisense
5'-GGCTGGAATCTAGCAGTC-3'/PAC118: sense
5'-TTGCCCTACCTGTTCCTGGG-3'; antisense
5'-GTCTCAAACTGCAGCAGCTG-3'/-actin26: sense
5'-GTGGGGCGCCCCAGGCACCA-3'; antisense
5'-GTCCTTAATGTCACGCACGATTTC-3'. PCR was performed with a
DNA thermal cycler (Perkin Elmer-Cetus, Norwalk, CT) under the following conditions: denaturation at 94°C for 1 minute, primer annealing at 60°C for ERK2 and -actin or at 64°C for PAC1
for 1 minute and then chain elongation at 72°C for 2 minutes.
Optimal conditions for RT-PCR to quantitate the expression of each gene were determined as described previously,27 and we performed the amplification at 25 cycles for ERK2 and -actin or at 22 cycles for PAC1. Ten microliters of PCR products were separated on 1% agarose
gels containing 0.05 µg/mL of ethidium bromide and examined. We
analyzed the levels of ERK-2 and PAC1 gene expression using densitometry scanning.
Statistical analysis.
Student's t-test was used for comparison of in vitro ERK
activity and expression level of each gene by RT-PCR between leukemia and normal BM samples. Linear correlation between expression levels of
ERK from RT-PCR and those from immunoblot analysis was determined by
calculating Pearson's correlation coefficient. Fisher's exact probability test was used for all 2 × 2 tables. Data were
analyzed with SPSS statistical software package (SPSS Inc, Chicago,
IL). A P value less than .05 was considered to be statistically significant.
| |
RESULTS |
Constitutive activation of ERK in acute leukemias.
To determine whether ERK is activated in acute leukemia, we first
examined the activity of ERK by a commercial kit with synthesized peptide for ERK1/2. The ERK activity in 79 leukemia samples (16.6 densitometric unit ± 1.2 standard error [SE]) was
significantly greater than that in normal BM samples (5.2 densitometric
unit ± 1.2 SE) (P = .003). When the kinase activity was
analyzed according to the type of acute leukemia, AML samples (18.6 densitometric unit ± 1.3 SE) showed statistically higher activity
than ALL samples (10.2 densitometric unit ± 1.7 SE) (P = .03). For AML, the average activity of ERK was lower in the M3 subtype
than in other subtypes (P < .05). The increased ERK activity
was confirmed by immunoblot analysis using phospho-specific ERK
antibody in 26 leukemia samples. By immunoblot analysis, the
phosphorylation of the ERK2 was predominant than that of ERK1 in acute
leukemia samples (Fig 1), and thus we
defined ERK phosphorylation as the intensity of the phosphorylated ERK2
band. Human K562 cells, a chronic myeloid leukemia cell line in blast
crisis, showed high level of constitutive ERK activation (Fig 1).
Therefore, ERK was arbitrarily defined to be constitutively active when
the value of the ERK phosphorylation divided by the -tubulin
expression in the leukemia sample was greater than one third the value
measured in the K562 cells, because the phosphorylation of normal BM
samples was minimal or not detected. Activation of ERK in acute
leukemias was detected in 19 of the 26 leukemia samples analyzed
(73.1%) (Table 1) (Fig 1A, C,
and D). Unselected normal BM cells are heterogeneous and may not be
representative of acute leukemia because it is possible that ERK
activation observed in acute leukemia is simply due to enrichment of
immature progenitor cells, not to the leukemic process. Therefore, to
determine whether unselected normal BM cells are adequate for
comparison with immature leukemic cells and activation of ERK in acute
leukemias is not simply due to enrichment of immature progenitor cells,
we examined if the changes observed in acute leukemias occurred in
normal immature BM cells. From one normal BM donor, we isolated
CD34+ cells, the early hematopoietic stem/progenitor cells,
by immunomagnetic bead methods and examined immunoblot analysis. By
immunoblot analysis, the intensity of ERK phosphorylation in
CD34+ cells was not different from that in normal BM cells
(Fig 1A). Thus, these results suggest to us that unselected marrow
cells may be adequate for comparison with leukemic cells and the
results observed in acute leukemias are not simply associated with
enrichment of immature progenitor cells.
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| Fig 1.
Complementary role of MEK activation, ERK
hyperexpression, and PAC1 downregulation in determining the activation
of ERK in human acute leukemias. Normal unselected BM (N), purified
CD34+ (P), and acute leukemia samples were subjected to
immunoblot (IB) and RT-PCR analysis as described in Materials and
Methods. For immunoblot analysis, lysates containing the same amount of
protein were applied to each lane; 25 µg of protein for
phosphorylated ERK1/2 (ERK-P); 50 µg of protein for phosphorylated
MEK1/2 (MEK-P); 25 µg of protein for total ERK1/2 (ERK); 25 µg of
protein for  -tubulin. In all immunoblot analyses, K562 samples (K)
were loaded together as the evidence of same exposure time in
autoradiogram. Separated proteins were transferred to the immobilon-P
membrane and stained with phospho-specific ERK1/2 antibody or
phospho-specific MEK1/2 antibody or total ERK1/2 antibody or
-tubulin antibody, respectively. In immunoblot analysis of total ERK
using 25 µg protein, the ERK1 band was not visualized because ERK1
detected by total ERK1/2 antibody was much less abundant than ERK2. For
RT-PCR analysis, PCR was performed with PAC1 primer for 22 cycles, with
-actin primers for 25 cycles. PCR products derived from 20 ng of
total RNA were applied to each lane. Leukemia samples above the lanes
from (A) to (D) correspond with those in Table 1.
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Relationship between ERK activation and MEK activation in acute
leukemias.
To detect the activation of MEK in acute leukemia, we examined the
phosphorylation of MEK using phospho-specific MEK antibody and
determined whether the activation of ERK is accompanied by the
activation of MEK. Normal BM samples and purified CD34+
cells exhibited minimal MEK phosphorylation (Fig 1A). MEK was considered to be constitutively active when the value of the MEK phosphorylation divided by the -tubulin expression in the leukemia sample was greater than twofold the mean value measured in the normal
BM samples.14 Activation of MEK in acute leukemias was detected in 21 of the 26 cases analyzed (80.8%) (Table 1) (Fig 1A, B,
and D). In 14 of 26 cases (53.8%), the pattern of ERK activation was
similar to that of MEK activation. Although ERK activation seemed to be
associated with MEK activation, this relationship was not statistically
significant (P = .28) (Table 2).
Relationship between ERK activation and ERK expression in acute
leukemias.
To examine the expression level of ERK in acute leukemia, we examined
the ERK expression by semiquantitative RT-PCR and immunoblot analysis.
For RT-PCR analysis, we examined the expression of ERK2, the more
predominant ERK isoform in acute leukemia samples. Human K562 cells
constitutively expressed the ERK2 gene and showed 10-fold higher ERK2
gene expression than normal BM samples. Therefore, we used the K562
cells as a positive control, and the value of the ERK2 gene expression
divided by the -actin gene expression was used for comparison with
the expression of the ERK2 genes, with the value of gene expression in
K562 cells defined as 1.00. Figure 2 is a
schematic representation of the various expressions of the ERK2 gene in
79 acute leukemias. The average levels of ERK2 gene expression in acute
leukemia samples (0.43 ± 0.01 SE) were significantly higher than
those in normal BM samples (0.10 ± 0.01 SE) (P = .03). When
the expression levels of ERK2 gene were analyzed according to the type
of acute leukemia, there was no significant difference between AML and
ALL. For AML, the average levels of ERK2 gene expression were lower in
the M3 subtype than in other subtypes (P < .05). When
correlation was examined to confirm that the quantitation of ERK gene
expression by RT-PCR reflected the amount of ERK protein in the same
leukemia samples, a strong positive correlation was observed between
the levels of ERK2 gene expression quantified by RT-PCR and the levels
of total ERK protein by immunoblot analysis (Pearson's correlation coefficient, 0.437; P = .01).
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| Fig 2.
Relative expression of the ERK2 gene in acute leukemia
and normal BM samples. The value of ERK2 gene expression in individual
samples was obtained as described in the text (defined in K562 cells as
1.00).
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To examine whether the increased amount of total ERK protein
contributed to constitutive ERK activation in acute leukemias, we
studied the relationship between ERK expression and ERK activation in
26 cases by immunoblot analysis. The expression of ERK in normal BM
samples and purified CD34+ cells was minimal (Fig 1A).
Hyperexpression of ERK protein was considered to have occurred when the
value of the total ERK expression divided by the -tubulin expression
in the leukemia sample was greater than twofold the mean value measured
in the normal BM samples.14 Hyperexpression of ERK in acute
leukemias was detected in 14 of the 26 leukemia samples analyzed
(53.8%) (Table 1) (Fig 1A and C). In 17 of 26 cases (65.4%), the
pattern of ERK expression was similar to that of ERK activation.
Although ERK activation appeared to be associated with total ERK, this
relationship was not statistically significant (P = .19)
(Table 3).
Downregulation of ERK signaling pathway is compromised in acute
leukemias.
The constitutive activation of ERK suggested to us the possibility of
an abnormal downregulation mechanism of ERK. PAC1, a member of the ERK
phosphatase family, was seen as a maximally expressed PTPase in the
hematopoietic tissue and induced in response to ERK
activation.18-21 Therefore, to determine whether abnormal ERK downregulation mechanisms were present, we examined PAC1 gene expression by semiquantitative RT-PCR because PTPases, including PAC1,
are principally upregulated at the transcription level in response to
ERK activation.28 Interestingly, the average levels of PAC1
gene expression in acute leukemias (0.23 ± 0.01 SE) were significantly lower than those in normal BM samples (0.58 ± 0.01 SE) (P = .002). The CD34+ cells showed a level of
PAC1 expression similar to normal BM samples (Fig 1A). Then, to
determine whether the downregulation of PAC1 contributed to the
constitutive activation of ERK, we compared PAC1 expression with ERK
activation in each leukemia sample. PAC1 was considered to be
downregulated when the value of expression in the leukemia sample was
less than one half of the mean value measured in the normal BM samples.
Downregulation of PAC1 in acute leukemias was detected in 17 of the 26 cases analyzed (65.4%) (Table 1) (Fig 1A, C, and D). Furthermore, in 22 of 26 cases (84.6%), PAC1 downregulation was
significantly correlated with ERK activation (P = .002)
(Table 4).
| |
DISCUSSION |
The identification of constitutively activated signaling molecules
involved in transducing an oncogenic signal in leukemia cells is likely
to shed light on the mechanism of leukemogenesis. In this study, we
focused on the analysis of ERK, which is a key kinase in intracellular
signal transduction pathways for cell proliferation and
differentiation. Our study showed a high frequency of activation of ERK
in human acute leukemias, but we did not detect any, or only minimal,
phosphorylation of ERK in normal BM cells. This is in agreement with a
recent report that the constitutive activation of ERK occurs frequently
in acute leukemia cells.13 Also, when we examined the
activation of JNK (c-Jun NH2-terminal linases), a member of
MAPK family, on the seven cases (22, 49, 72, 75, 77, 78, and 79), which
showed constitutive ERK activation, none of these samples showed JNK
activation (data not shown). This result led us to the conclusion that
the ERK activation in acute leukemias is the ERK-specific event among
various MAPK pathways. Until now, the activated status of ERK has been
known to be predominantly determined by a highly conserved ERK
cascade.9,10 Actually, in a previous study examining
limited numbers of leukemia samples,13 ERK activation was
accompanied by MEK activation. However, in our study examining 26 leukemia samples, about one half of the leukemia samples showed a
relationship between ERK activation and MEK activation, but in the
remaining samples, any relationship of activation between both kinases
was not observed. Such a discrepancy was also observed in a previous
study with renal cell carcinoma.14 Therefore, although MEK
is the only activator, which is responsible for ERK
activation,29,30 our results suggest that the constitutive activation of ERK observed in acute leukemia cells is unlikely to
simply reflect phosphorylation of the protein by upstream kinases.
In our study, RT-PCR and immunoblot analysis showed a markedly
increased expression of ERK in acute leukemia, compared with normal BM
cells. Although there was no statistical significance, the pattern of
ERK expression was similar to that of ERK phosphorylation in 17 of 26 cases (65.4%) examined by immunoblot analysis. These findings are
consistent with a previous study of breast cancer, which demonstrated
that the activity and expression of ERK was elevated in both primary
and metastatic lesions.15 Whereas posttranslational ERK
modification has been extensively studied, relatively little is known
about altered ERK transcription. The variation of ERK mRNA levels in
different organs suggests that a tissue-specific transcription factor
may play an important role in the regulation of ERK
expression.31 Also, in the cloning study of a murine ERK1
gene, the promoter of ERK1 contains consensus binding sites for many
transcription factors, including AP-1, AP-2, Sp1, CTF-NF1, Myb, p53,
NF-IL-6, and Ets-1.32 Genetic strategies should be relatively straightforward in determining which transcription factors,
including tissue-specific factors, are important for ERK expression and
if there are mutations in the promoter of the ERK gene, which might
account for the hyperexpression of ERK in acute leukemia. Although the
molecular basis for ERK hyperexpression must be determined, the
increased amount of ERK protein available for phosphorylation by
activated upstream kinases in acute leukemia could increase the amount
of activated ERK and potentiate the proliferative capacity of leukemia cells.
ERK activation of acute leukemia samples in our study was considered to
be constitutive because the response of ERK to a wide variety of
extracellular stimuli showed transient phosphorylation and was followed
by rapid dephosphorylation.2,31 Furthermore, MEK activation
and ERK hyperexpression were not sufficient to explain the complete
mechanism of constitutive activation of ERK in acute leukemia. These
results raised the question of the abnormality of the downregulation
mechanism of ERK activation. In the cell, a dynamic balance exists
between phosphorylation and dephosphorylation, resulting from the
interplay between protein kinase and protein phosphatase. Therefore,
PTPases may play a distinct role in the regulation of the ERK signaling
pathway. PAC1, a member of the MAPK phosphatase family, was described
as a maximally expressed PTPase in the hematopoietic
tissue.18-21 Abundant data indicate that constitutive
activation of the ERK cascade increases the expression of PAC1,
providing a pivotal role for this phosphatase in compensatory
inactivation of the stimulated ERK signaling pathway because PAC1
exhibits stringent substrate specificity for ERK and constitutive PAC1
expression inactivates ERK.19,33-35 Furthermore, ERK
phosphorylation and activity are modulated by amounts of PAC1 in
transfection study using PAC1, thus highlighting a role for the MAPK
cascade.19 Because PAC1 is principally upregulated at the
transcription level in response to ERK activation,27 we
chose to study the PAC1 gene expression. Surprisingly, PAC1 gene
expression in acute leukemia samples showing constitutive ERK
activation was below one half that of the basal, unstimulated, normal
BM samples showing minimal or no ERK activation. Considering the
constitutively activated status of ERK in acute leukemia samples, downregulation of the PAC1 gene in acute leukemia samples is an attractive finding, and this result suggests that induction of the PAC1
gene is definitely downregulated in acute leukemia. In this respect,
our data provide the first evidence for downregulation of phosphatase
induction in response to activation of ERK in human malignancies. Taken
together, a significant correlation between PAC1 downregulation and ERK
activation suggests that no matter what is stimuli for activating ERK,
dysregulated switch-off mechanism for activated ERK contributes to the
constitutive activation of ERK in acute leukemia. Current research in
our laboratory is adopting a genetic strategy aimed at the introduction
of PAC1 into leukemic cells showing constitutive ERK activation and
downregulation of PAC1, which may allow us to determine whether the
inhibition of constitutively activated ERK by overexpression of PAC1
influences the growth of leukemic cells.
Furthermore, we found the complementary roles of MEK activation, ERK
hyperexpression, and PAC1 downregulation in determining the activation
of ERK by the analysis of individual cases. We attempted to classify 26 cases into four groups. In group 1 (Fig 1A), 6 cases showed strong ERK
phosphorylation by synergistic effect of MEK activation, ERK
hyperexpression, and PAC1 downregulation. In group 2 (Fig 1B), 7 cases
showed MEK activation, but not ERK activation, and 6 of 7 cases showed
PAC1 expression similar to normal BM samples. Considering the role of
MEK as a sole activator of ERK, inactivated ERK in these six cases may
be the result of the preferential dephosphorylation of ERK by normally
functional PAC1 under the affect of still-activated MEK. In group 3 (Fig 1C), 5 cases did not show MEK activation, although ERK was
activated. These findings suggest the possibility of the presence of
alternative ERK activators other than MEK1/2. However, another type of
MEK has been not identified in vivo. Also, certain leukemic oncogene products have been not reported to activate ERK independent of MEK.
Recently, a gain-of-function mutation of mammalian ERK2 (D319N ERK2)
was demonstrated.36 This mutant was shown to have an
increased sensitivity to lower levels of signaling in vivo as a result
of decreased sensitivity to phosphatase, but such mutant has not as yet
been found in human malignancy. However, on the basis of our results,
four of these five cases showed PAC1 downregulation. These findings
suggest that the downregulated status of PAC1 may be insufficient for
dephosphorylation of ERK, and therefore, the ERK was maintained in an
activated status despite dephosphorylation of MEK after withdrawal of
stimuli. Also, interestingly, K562 cells showed the mechanism of ERK
activation similar to this group. In group 4 (Fig 1D), seven cases
showed ERK activation, but not ERK hyperexpression. All cases in this
group showed PAC1 downregulation, and six cases showed MEK activation.
Therefore, in this group, ERK may be constitutively activated by a
combined effect of MEK activation and PAC1 downregulation despite
minimal ERK expression. Although this aspect calls for further
investigation, our present findings support the belief that the
complementary role of the three different regulatory
mechanisms could contribute to determining the constitutive
activation of ERK in acute leukemia.
In conclusion, ERK and MEK were constitutively activated in a majority
of human acute leukemias. Based on our data, in addition to activation
by sequential upstream kinases, the hyperexpression of ERK and the
downregulation of PAC1 are thought to contribute to constitutive ERK
activation. Furthermore, the complementary relationship of these
regulatory mechanisms may play an important role in constitutive ERK
activation in acute leukemia.
| |
FOOTNOTES |
Submitted February 17, 1998; accepted November 18, 1998.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests either to Seong-Cheol Kim, MD, Division of
Hematology-Oncology, Department of Internal Medicine, Yonsei University
College of Medicine, Seodaemun-Gu, Shinchon-Dong 134, Seoul 120-752, Korea; e-mail: seockim{at}chollian.net; or to Won-Jae Lee, PhD,
Laboratory of Immunology, Medical Research Center, Yonsei University
College of Medicine, CPO Box 8044, Seoul, Korea; e-mail:
wjlee1{at}yumc.yonsei.ac.kr.
| |
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