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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1338-1345
GCKR Links the Bcr-Abl Oncogene and Ras to the
Stress-Activated Protein Kinase Pathway
By
Chong-Shan Shi,
Joseph M. Tuscano,
Owen N. Witte, and
John H. Kehrl
From the B-Cell Molecular Immunology Section, Laboratory of
Immunoregulation, National Institutes of Allergy and Infectious
Diseases, NIH, Bethesda, MD; UC Davis Cancer Center, Department of
Oncology, Sacramento, CA; University of California Los Angeles, Howard
Hughes Medical Institute, Los Angeles, CA.
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ABSTRACT |
The Bcr-Abl oncogene, found in Philadelphia
chromosome-positive myelogenous leukemia (CML), activates Ras and
triggers the stress-activated protein kinase (SAPK or Jun
NH2-terminal kinase [JNK]) pathway. Interruption of Ras
or SAPK activation dramatically reduces Bcr-Abl-mediated
transformation. Here, we report that Bcr-Abl through a Ras-dependent
pathway signals the serine/threonine protein kinase GCKR (Germinal
Center Kinase Related) leading to SAPK activation. Either an oncogenic
form of Ras or Bcr-Abl enhances GCKR catalytic activity and its
activation of SAPK, whereas inhibition of GCKR impairs Bcr-Abl-induced
SAPK activation. Bcr-Abl mutants that are impaired for GCKR activation
are also unable to activate SAPK. Consistent with GCKR being a
functional target in CML, GCKR is constitutively active in CML cell
lines and found in association with Bcr-Abl. Our results indicate that
GCKR is a downstream target of Bcr-Abl and strongly implicate GCKR as a
mediator of Bcr-Abl in its transformation of cells.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
CHRONIC MYELOGENOUS leukemia (CML) is a
human leukemia associated with the t(9,22) Philadelphia
translocation, which fuses Bcr gene sequence upstream of the
c-abl gene.1 Consistent with a pathogenic role in
this leukemia, Bcr-Abl transforms fibroblasts and hematopoietic
cells and can recapitulate a CML-like disease in
mice.2-5 Bcr-Abl encodes, depending on the
breakpoint, a constitutively active 210-kD or 185-kD cytoplasmic
tyrosine kinase. Although the biochemical mechanisms used by
Bcr-Abl to transform myeloid cells remain incompletely
understood, considerable evidence supports a requirement for the small
GTPase Ras. Moreover, multiple and redundant signaling pathways lead
from Bcr-Abl to Ras activation.6,7
One of the consequences of Bcr-Abl-induced Ras activation is increased
activity of stress-activated protein kinases (SAPK, also referred to as
c-Jun NH2-terminal Kinases or JNK) and this activation
appears crucial for Bcr-Abl-induced cellular
transformation.8,9 The SAPK are the terminal kinases in a
three-tiered protein kinase cascade, which also includes a
mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK or MEKK),
and a MAPK kinase (SEK1/MKK4 or MEK). Thus, a MEKK activates a MEK that
in turn activates SAPK. Among the MEKK that can activate the SAPK
pathway are MEKK1, MEKK2, and the mixed-lineage kinase termed
MLK-3.10-14 Substrates of the SAPK include the
transcription factors AP-1 and Elk-1. These transcription factors
regulate the expression of many of the genes involved in the
inflammatory response.10,11
As their name indicates, the SAPK are activated by cellular stresses
such as heat shock, ultraviolet irradiation, or osmotic shock, and by
the inflammatory cytokines tumor necrosis factor (TNF) and
interleukin-1.10,11 These cellular stresses trigger intracellular signal transducers that lead to activation of the SAPK
cascade. Undoubtedly among them are members of an interesting subfamily
of serine/threonine protein kinases. GCK (Germinal Center Kinase),15,16 HPK1 (Hematopoietic Progenitor
Kinase),17,18 GCKR (Germinal Center Kinase
Related),19 the identical kinase is also termed kinase
homologous to SSP1/STE20 (KHS),20 and GLK (germinal
center-like kinase)21 all activate the SAPK, but not the
related MAPK pathway. Based on the observation that Bcr-Abl preferentially activated the SAPK pathway8 and preliminary experiments that showed Ras to be a potent activator of GCKR, we
examined whether GCKR could link Bcr-Abl to SAPK activation.
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MATERIALS AND METHODS |
Cell lines.
K562, BV173, KCL22, and KY04 are Bcr-Abl+ cell lines
whereas U937 cells were derived from a patient with a histiocytic
lymphoma and Jurkat cells from a T-cell leukemia. The BV173, KCL22, and KY04 cell lines were provided by Dr John Barrett (Bethesda, MD) whereas
the K562, Jurkat, HEK 293, and U937 cells were obtained from the
American Type Culture Collection (Rockville, MD). The HEK
293T (embryonal kidney) cell line was obtained following permission from Dr D. Baltimore (Pasadena, CA).
Plasmids and antibodies.
The pMT3-HA-SAPK-p46 plasmid was provided by Dr J. Kyriakis (Boston,
MA). Bcr-Abl, Bcr-Abl mutants, and v-Abl expression vectors in pSR MSVtk Neo were previously
described.7,25,26 pcDNA3-Ras-V12, pcDNA3-Ras-N17,
pcDNA3-Rac-QL, and pcDNA3-Cdc42-QL were kind gifts of Dr S. Gutkind
(Bethesda, MD). The pcDNA-HA-GCKR, pCRIII-GCKR, and pCRIII-GCKR(AS)
were described previously.19 The pcDNA-HA-GCKR deletion
constructs 1-691, 1-599,1-493, and 1-396 were created by inserting the
appropriate polymerase chain reaction product from pcDNA-HA-GCKR into
pcDNA-HA. The anti-HA (12CA), anti-pY (4G10), and anti-Abl antibodies
were purchased from Boehringer Mannheim (Mannheim,
Germany), Upstate Biotechnology (Lake Placid, NY), and
Santa Cruz Biotechnology (Santa Cruz, CA), respectively.
The GCKR antiserum was generated in rabbits by immunizing with a
peptide (RKETEARDEMC) coupled to Keyhole limpet hemocyanin as
described.19
In vitro kinase assays, immunoblotting, and coimmunoprecipitations.
The HEK 293T and HEK 293 cells were transiently transfected using a
calcium phosphate method. Transfected DNA levels were normalized with
control plasmids. Forty-eight to 72 hours after the transfection,
HA-immunoprecipitates were subjected to in vitro kinase assays using
myelin basic protein or c-jun (79) as substrates for the GCKR and SAPK
assays, respectively.19 Before the in vitro kinase assay
the HA-immunoprecipitates were washed three times with kinase lysis
buffer (20 mmol/L Hepes pH 7.4, 2 mmol/L EGTA, 50 mmol/L
-glycerophosphate, 1% Triton X-100, 1 mmol/L Na3V04, and 10% glycerol) to which a protease
inhibitor cocktail tablet was added [Boehringer Mannheim]), three
times with a LiCl wash buffer (500 mmol/L LiCl, 100 mmol/L Tris pH 7.4, 0.1% Triton X-100, and 1 mmol/L dithiothreitol), and three times with
kinase buffer (20 mmol/L MOPS pH 7.2, 2 mmol/L EGTA, 10 mmol/L MgCl2, and 0.1% Triton X-100). The GCKR (1:300
dilution), HA, and phosphotyrosine immunoblots were performed using
standard methodology. The signals were detected by enhanced
chemiluminescence (ECL; Amersham, Arlington Heights, IL). The
coimmunoprecipitation was performed using lysates (1% digitonin, 50 mmol/L Tris, pH 8.0, 150 mmol/L NaCl, 0.5 mmol/L EDTA, plus protease
inhibitors or 20 mmol/L Tris, pH 8.0, 137 mmol/L NaCl, 2 mmol/L EDTA,
1% Triton X-100, 1 mmol/L sodium orthovanadate, plus protease
inhibitors) prepared from HEK 293T cells coexpressing HA-GCKR (2 µg)
and Bcr-Abl wild type or mutants (2 µg) or from K562 cells. Anti-HA,
anti-Abl, anti-GCKR, or the 4G10 monoclonal antibody (MoAb) were added
and the immunoprecipitates collected with the appropriate secondary
antibody-coupled magnetic beads (Dynal Corp, Oslo, Norway). They were
washed three times in lysis buffer, twice in lysis buffer with 1 mol/L
NaCl, fractionated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), and analyzed by immunoblotting with the
appropriate antibody.
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RESULTS |
Bcr-Abl activates GCKR and the SAPK pathway through a Ras-dependent
mechanism.
We first examined the effect of Bcr-Abl, v-Abl, or an oncogenic form of
Ras on GCKR kinase activity, which we assessed using an in vitro kinase
assay that measures the ability of immunoprecipitated GCKR (tagged with
a HA epitope) to phosphorylate myelin basic protein (MBP). We observed
that HA-GCKR immunoprecipitates prepared from HEK 293T cells
transfected with constructs that direct the expression of HA-GCKR and
either Bcr-Abl, v-Abl, or Ras-V12 contained elevated levels of GCKR
kinase activity (Fig 1A). In
contrast, an in vitro kinase assay performed with a HA-GCKR-T178A
immunoprecipitate prepared from HEK 293T cells coexpressing
HA-GCKR-T178A and Bcr-Abl did not result in a significant
phosphorylation of myelin basic protein (data not shown). GCKR-T178A
had been previously shown to lack protein kinase activity and to behave
as a dominant negative inhibitor of wild-type GCKR
activity.19 Expression of activated forms of two other
small GTPases (cdc42 and rac), which likely activate the SAPK pathway
via a PAK kinase,22,23 did not appreciably activate GCKR.
Inspection of the in vitro kinase assay showed a 97 kD phosphoprotein,
which HA-immunoblotting identified as GCKR (Fig 1A, top and middle
panels). Of note, HA-GCKR migrated slower on SDS-PAGE when
immunoprecipitated from the Ras-V12, Bcr-Abl, or v-Abl transfected
cells. This result is consistent with GCKR autophosphorylation induced
by the activating stimuli and/or phosphorylation by a
coimmunoprecipitating protein kinase. However, the wash conditions employed for the in vitro kinase are very stringent and we did not
observe any other bands on the autoradiograph besides MBP and GCKR.
This would suggest that the phosphorylations, which occurred during the
in vitro kinase assays, are predominantly GCKR-mediated MBP
phosphorylation and GCKR autophosphorylation. Reimmunoblotting with an
antiphosphotyrosine MoAb showed the presence of tyrosine
phosphorylation, which was most evident with Bcr-Abl although also
observed with v-Abl, but not with Ras-V12 (Fig 1A, bottom panel).
Because we did not observe any bands in the in vitro kinase assay that
might correspond to Bcr-Abl or v-Abl, the GCKR tyrosine phosphorylation
likely occurred in vivo.
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| Fig 1.
GCKR is a downstream target of Bcr-Abl and
v-Abl. (A) Bcr-Abl, v-Abl, and Ras-V12 activate GCKR kinase
activity. HA-GCKR immunoprecipitates from 1 × 106 HEK
293T cells transfected with control vector (2 µg), Bcr-Abl (2 µg), v-Abl (2 µg), Ras-V12 (2 µg), Cdc42-QL (2 µg), or
Rac-QL (2 µg) in the presence of HA-GCKR (1 µg) were assayed for
kinase activity using myelin basic protein (MBP) as a substrate. The
amount of 32P incorporated into MBP was determined by
excising the appropriate band and scintillation counting (results shown
in bar graph) and the fold increases compared with control
transfections. A portion of the gel was transferred to nitrocellulose
and autoradiographed (AR). The same blot was reacted with an anti-HA
MoAb (WB), stripped, and then with an antiphosphotyrosine MoAb (pY).
Immunoreactivity was detected by enhanced chemiluminescence (ECL).
Experiments were performed three times with similar results. (B)
Overexpression of GCKR augments the induction of SAPK activity
triggered by Bcr-Abl, v-Abl, or Ras-V12. HA-SAPK immunoprecipitates
from 1 × 106 HEK 293T cells transfected with Bcr-Abl (2 µg), v-Abl (2 µg), Ras-V12 (2 µg) in the presence of pCR3-GCKR (1 µg) or a control vector (1 µg) along with HA-SAPK (1 µg) were
assayed for kinase activity using GST-Jun (79) as a substrate. Bcr-Abl,
v-Abl, GCKR, and Ras levels were assessed by immunoblotting (second,
third, and fourth panels). The GCKR immunoblot was performed with the
GCKR-specific antiserum. HA immunoblotting verified equivalent levels
of HA-SAPK (bottom panel). These experiments were performed three times
with similar results.
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Supporting a role for GCKR in Bcr-Abl-mediated SAPK activation;
GCKR-enhanced SAPK activation by Bcr-Abl or v-Abl. HEK 293T cells
expressing Ras-V12, Bcr-Abl, or v-Abl had modest increases in SAPK
activity (3-, 2.5-, and 2-fold, respectively, in the experiment shown),
whereas the addition of GCKR further augmented them (7-, 7.5-, and
7.8-fold, respectively, Fig 1B). Low levels of GCKR were transfected in
this experiment to avoid significant SAPK activation by GCKR alone.
Coexpression of GCKR did not alter Bcr-Abl, v-Abl, Ras-V12, or SAPK
levels. To test whether Ras was involved in Bcr-Abl-induced GCKR
activation we used a dominant negative form of Ras, Ras-N17, to inhibit
endogenous Ras. Coexpression of Ras-N17 blocked GCKR activation by
either Bcr-Abl or v-Abl nearly reducing GCKR activity in the in vitro
assay to baseline (Fig 2A). The
coexpression Ras-N17 did not alter the expression levels of GCKR,
Bcr-Abl, or v-Abl.
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| Fig 2.
Bcr-Abl-induced GCKR activation requires Ras and
Bcr-Abl-triggered SAPK activation requires GCKR. (A) A dominant
negative Ras inhibits Bcr-Abl-induced GCKR activation. HEK 293T cells
(1 × 106) were transfected with HA-GCKR (1 µg) and
Ras-V12 (2 µg), v-Abl (4 µg), or Bcr-Abl a (4 µg). The v-Abl and Bcr-Abl transfections were
performed in the presence of a control vector (4 µg) or Ras-N17 (4 µg), indicated by bracket. HA-GCKR immunoprecipitates were assayed by
an in vitro kinase assay. HA-GCKR and Abl immunoblotting were performed
by ECL. These experiments were performed two times with similar
results. (B) GCKR antisense expression impairs Ras- and
Bcr-Abl-induced SAPK activation. HA-SAPK immunoprecipitates from HEK
293T cells (1 × 106) transfected with Bcr-Abl (4 µg) or
Ras-V12 (2 µg) in the presence of pCRIII-GCKR-AS (4 or 8 µg) and
HA-SAPK (1 µg) were assayed for kinase activity. A HA-immunoblot
verified equivalent levels of HA-SAPK. Ras and Bcr-Abl levels were
verified by immunoblotting. Data are reported as increase in SAPK
activity compared with basal levels in control transfections. These
experiments were performed four times with similar results. (C) A GCKR
mutant impairs Bcr-Abl-induced SAPK activation. HA-SAPK
immunoprecipitates from HEK 293 cells (1 × 106)
transfected with Bcr-Abl (4 µg) and HA-SAPK (1 µg) in the presence
or absence of GCKR-T178A (2, 4, or 6 µg) were assessed for kinase
activity. Data are reported as in part B. A HA-immunoblot verified
equivalent levels of HA-SAPK. Bcr-Abl levels are shown. GCKR
(endogenous and the transfected mutant) were detected by immunoblotting
with the GCKR-specific antiserum. This experiment was performed twice
with similar results.
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To provide direct evidence that Bcr-Abl signals the SAPK pathway
through GCKR, we examined the effects of a GCKR antisense construct and
a GCKR mutant on Bcr-Abl-induced SAPK activity. It is known that HEK
293 and HEK 293T cells express GCKR, and that a GCKR antisense
construct reduces GCKR levels and inhibits TNF-induced SAPK
activation.19 Here, we transfected HEK 293T cells with
HA-SAPK along with either Ras-V12 or Bcr-Abl in the presence or
absence of the antisense GCKR. The GCKR antisense construct inhibited
both Ras- and Bcr-Abl-induced SAPK activation (Fig 2B). Expression of
the antisense GCKR construct failed to alter the levels of Bcr-Abl or
v-Abl. Not only did the antisense GCKR construct inhibit
Bcr-Abl-mediated SAPK activation, but in addition the transfection of
a construct that directs expression of GCKR-T178A had a similar result
(Fig 2C). The expression of GCKR-T178A was monitored using the
GCKR-specific antibody. The low level of endogenous GCKR is observed in
the first two lanes of the third panel in the figure. Thus, using two
different approaches to target endogenous GCKR, we successfully
inhibited Bcr-Abl-induced SAPK activation.
Bcr-Abl and GCKR associate in vivo.
Because we had noted that the coexpression of Bcr-Abl and GCKR resulted
in GCKR tyrosine phosphorylation, we sought to determine if GCKR
physically associates with Bcr-Abl. Because we had failed to observe a
band in the in vitro kinase assay that would correspond to Bcr-Abl, we
initially used a digitonin-containing buffer to preserve low-affinity
interactions. We transfected HEK 293T cells with Bcr-Abl or v-Abl
expression vectors along with HA-GCKR. A HA-immunoblot of anti-Abl
immunoprecipitates prepared from cell lysates buffer showed the
presence of HA-GCKR (Fig 3A, lanes 1 to 4),
a result consistent with an interaction between GCKR and Bcr-Abl as
well as between GCKR and v-Abl. Next, we employed a more stringent
lysis buffer that contained Triton X-100 rather than digitonin. Again
GCKR and Bcr-Abl associated. Bcr-Abl immunoprecipitates contained GCKR
(Fig 3A, lanes 5 to 7) and HA-GCKR immunoprecipitates contained Bcr-Abl
(Fig 3A, lanes 8 to 10). Thus, Bcr-Abl and v-Abl can associate with
GCKR.
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| Fig 3.
GCKR and Bcr-Abl associate in vivo. (A) GCKR and Bcr-Abl
coimmunoprecipitate. HEK 293T cells were transfected with constructs
expressing HA-GCKR and either Bcr-Abl (lanes 1, 3 to 10), or v-Abl
(lane 2). Cell lysates (2 × 106 cells) prepared using a
digitonin lysis (lanes 1 to 4) or a Triton X-100 lysis buffer (lanes 5 to 10) were immunoprecipitated with anti-HA, anti-Abl, or a control
antibody as indicated underneath. The immunoprecipitates or cell
lysates were analyzed by immunoblotting with anti-HA (lanes 1 to 7) or
anti-Abl (lanes 8 to 10) antibodies. The signal was detected by ECL.
(B) Mapping of the region in GCKR required for interaction with
Bcr-Abl. Lysates from HEK 293T cells (2 × 106)
transfected with constructs expressing HA-tagged GCKR deletion
constructs along with Bcr-Abl were immunoprecipitated with anti-Abl or
control antibody. The immunoprecipitates and lysates were analyzed by
immunoblotting with an anti-HA antibody. The signal was detected by
ECL. A nonspecific doublet in the range of 65 kD is present in
each of the lysates. The GCKR truncation mutants migrated with their
expected molecular masses and were visualized in the cell lysates.
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To begin to map the region in GCKR important for this interaction,
constructs expressing wild-type GCKR or one of four GCKR truncation
mutants were transiently transfected into HEK 293T cells along with
Bcr-Abl. Only the wild-type GCKR coimmunoprecipitated with Bcr-Abl (Fig
3B). This analysis showed a requirement for the C-terminal 150 amino
acids of GCKR to observe the interaction. This region lacks remarkable
motifs except for two tyrosine residues, amino acids 836 and 847. Between amino acids 389 and 396 GCKR contains a proline-rich region, a
likely src homology (SH) type-3 binding site24; however,
whether this site contributes to the interaction with Bcr-Abl remains
to be determined.
To further characterize the GCKR Bcr-Abl interaction, we examined four
Bcr-Abl mutant proteins. We chose p185 Bcr-Abl Y177F, which does not
associate with Grb2; p185 Bcr-Abl R552L, which lacks c-Abl's SH2
domain-dependent interactions25; p185 Bcr-Abl Y813F, which
lacks an autophosphorylation site that creates a docking site for
interacting proteins26; and p185 Bcr-Abl KN, which is
catalytically inactive. Although there was some interexperiment variation in the expression levels of the Bcr-Abl mutants and in the
amount of coimmunoprecipitating GCKR, GCKR consistently coimmunoprecipitated with each of the Bcr-Abl mutant proteins (Fig 4). Furthermore, expression of each of
the Bcr-Abl mutants with the exception of the p185 KN mutant resulted
in the tyrosine phosphorylation of GCKR. This was based on the presence
of a 97 kD band detected by phosphotyrosine immunoblotting of GCKR
immunoprecipitates of lysates from cotransfected cells (C-S Shi, data
not shown). In addition, phosphotyrosine immunoprecipitates prepared
from HEK 293T cells coexpressing GCKR and either p185 Bcr-Abl Y177F, R552L, or Y813F contained significant amounts of GCKR
(Fig 5A). In contrast, p185 Bcr-Abl KN
failed to autophosphorylate and its coexpression with GCKR resulted in
a substantial reduction in the amount of GCKR in phosphotyrosine
immunoprecipitates. Not surprisingly, the KN mutant failed to activate
either GCKR or SAPK kinase activity (Fig 5B). However, the three other
mutant Bcr-Abl proteins were also markedly defective in their ability to activate both GCKR and SAPK. Thus, despite the ability of p185 Bcr-Abl Y177F, R552L, and Y813F to associate with GCKR and to result in
GCKR tyrosine phosphorylation, only the wild-type Bcr-Abl protein
strongly activated GCKR and the SAPK pathway.
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| Fig 4.
P185 Bcr-Abl point mutants all associate with GCKR. p185
Bcr-Abl wild-type and mutant protein expression vectors (4 µg each)
were transfected into HEK 293T cells (2 × 106) along with
the HA-GCKR expression vector (2 µg). The association of Bcr-Abl
point mutants with GCKR was examined by HA-immunoblotting anti-Abl
immunoprecipitates. The presence of the p185 Bcr-Abl mutants in the
immunoprecipitates was verified by immunoblotting with the
immunoprecipitating antibody. The levels of expression of the Bcr-Abl
mutants and HA-GCKR in the cell lysates are shown below. All the
immunoblots were detected by ECL.
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| Fig 5.
Analysis of the effects of p185 Bcr-Abl point mutations
on GCKR. (A) Effect of p185 Bcr-Abl point mutants on the
immunoprecipitation of GCKR by a phosphotyrosine MoAb. p185 Bcr-Abl
wild-type and mutant protein expression vectors (4 µg each) were
transfected into HEK 293T cells (2 × 106) along with the
HA-GCKR expression vector (2 µg). The presence of p185 Bcr-Abl, p185
Bcr-Abl mutant proteins, and HA-GCKR in antiphosphotyrosine
immunoprecipitates (MoAb 4G10) was assessed by immunoblotting with
either anti-Abl or anti-HA. The levels of the p185 Bcr-Abl point
mutants and HA-GCKR in the cell lysates were also assessed. (B) Effect
of p185 Bcr-Abl point mutants on GCKR and SAPK activity. The GCKR and
SAPK in vitro kinase assays were performed similar to Fig 1. The fold
inductions relative to GCKR alone (control) are shown and were assessed
by scanning the autoradiographs and quantified using NIH Image.
Immunoblotting showed similar levels of HA-GCKR. The kinase assays were
performed five times with similar results.
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GCKR is activated in CML cell lines.
K562, KCL22, BV173, and KY04 are Bcr-Abl+ cell lines
derived from CML patients in blast crisis. An immunoblot of cell
lysates prepared from these cell lines and two
non-Bcr-Abl+ cell lines showed similar levels of GCKR
expression. However, GCKR immunoprecipitates from the CML cell lines
had twofold to sixfold more kinase activity than did the controls
(Fig 6A). GCKR immunoblotting of
antiphosphotyrosine immunoprecipitates showed that GCKR is tyrosine
phosphorylation or associated with a tyrosine phosphorylated molecule
in K562 (Fig 6B), KY04, and KCL22 cells (C. Shi, data not
shown). To determine whether endogenous GCKR and Bcr-Abl associate in
vivo, we examined GCKR and Abl immunoprecipitates from K562 cells for
the presence of Abl or GCKR, respectively. This analysis also showed
that GCKR and Bcr-Abl coimmunoprecipitate (Fig 6B).
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| Fig 6.
GCKR is a downstream target of Bcr-Abl in
Bcr-Abl+ cell lines. (A) Increased GCKR kinase activity
in Bcr-Abl+ cell lines. GCKR immunoprecipitates from
lysates prepared from 5 × 106 cells of the indicated cell
line were subjected to an in vitro kinase assay. The data were
normalized to the results with either U937 cells or Jurkat cells. The
data shown summarize the results from four experiments and are
presented as mean +/ 2 SD. Above the results of the in vitro
kinase assay is an immunoblot showing the levels of GCKR in cell
lysates prepared from the different cell lines. (B) Bcr-Abl and GCKR
associate in K562 cells. Lysates prepared from 1 × 107
K562 cells were immunoprecipitated with an antiphosphotyrosine MoAb
(PY), a HA MoAb, the GCKR antiserum, preimmune (PI) antiserum, or an
Abl polyclonal antiserum as indicated. The immunoprecipitates were
subjected to immunoblotting with the indicated antibodies. The GCKR
immunoblot (lanes 1 to 3) was stripped and reblotted for Abl (lanes 4 to 6). The Abl immunoblot (lanes 7 to 9) was stripped and reblotted for
GCKR (lanes 10 to 12). Bcr-Abl and GCKR were detected in 50 µg of
cell lysate (lanes 7 and 10, respectively). Molecular weight markers
and the location of Bcr-Abl and GCKR are indicated.
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DISCUSSION |
Bcr-Abl activates the SAPK pathway via an ability to recruit and
activate GCKR. This conclusion is based on the following evidence.
First, Bcr-Abl activates GCKR kinase activity and together they
potently activate SAPK activity. Second, either a GCKR antisense construct or a catalytically inactive form of GCKR substantially reduced Bcr-Abl-induced SAPK activation. Third, GCKR and Bcr-Abl physically associate both in transfected cells and in
Bcr-Abl+ cells. Fourth, a dominant negative form of Ras
blocks both Bcr-Abl-mediated GCKR and SAPK activation. Finally,
Bcr-Abl mutant proteins that have an impaired ability to activate GCKR
fail to activate SAPK.
The case for Ras involvement in Bcr-Abl-mediated GCKR activation is
based on the use of a dominant negative inhibitor of Ras, the result
with the p185 Y177F point mutant, and the observation that Ras itself
is a potent activator of GCKR kinase activity. Because the
cotransfection of Ras-N17 blocked the ability of Bcr-Abl to activate
GCKR kinase activity, Bcr-Abl-mediated Ras activation is crucial for
the ability of Bcr-Abl to activate GCKR and is consistent with the
previously known requirement for Ras in Bcr-Abl-induced SAPK
activation.8 The impaired ability of Y177F p185 Bcr-Abl to
activate GCKR underscores the essential role for phosphorylated Y177 in
Bcr-Abl-mediated Ras activation in fibroblasts.27
Unfortunately, the precise signaling defects created in Bcr-Abl by
R552L and Y813F are not yet known. Nevertheless, both mutant proteins
are defective in fibroblast transformation and for GCKR and SAPK
activation in HEK 293 cells. This argues that they do not fully
activate Ras in these cells. Because all the Bcr-Abl mutant proteins
interacted with GCKR and all but the kinase-dead Bcr-Abl phosphorylated
GCKR, we can conclude that the recruitment and/or tyrosine
phosphorylation of GCKR is insufficient for its activation. Further
support for this hypothesis is provided by experiments in which GCKR
(1-699) and GCKR (1-599) were coexpressed with Bcr-Abl. Although these truncated versions of GCKR are not readily associated with GCKR, Bcr-Abl still increases their basal level of kinase activity (C. Shi,
unpublished observation). Thus, Bcr-Abl-mediated Ras activation emerges as the key element for Bcr-Abl-mediated GCKR activation and is
consistent with the ability of Ras-V12 to activate GCKR. Furthermore,
because Ras-activated SAPK activation was markedly impaired by
coexpression of a GCKR antisense construct, GCKR appears to be directly
involved in Ras-mediated SAPK activation in HEK 293 cells. Overall our
results are consistent with a model in which Bcr-Abl assembles a
complex of signaling molecules, thereby providing a platform for Ras
activation and protein-protein interactions. In myeloid cells the
colocalization of GCKR and Bcr-Abl would be expected to localize GCKR
to a microenvironment in which activated Ras is present.
How Ras activates GCKR remains enigmatic. Although GCKR lacks the
consensus binding site delineated for some direct Ras
effectors,28 an intriguing possibility is that GCKR is
directly activated by Ras perhaps by a mechanism similar to how Ras
activates Raf-1. In support of that possibility, when both proteins
were expressed in HEK 293T cells Ras-V12 could be detected in GCKR
immunoprecipitates and vice versa (C. Shi, unpublished observation).
However, whether this is a direct or an indirect interaction has not
been determined. A caveat in interpreting this association data is that
both proteins are expressed at high levels perhaps permitting
unphysiologic interactions. So far we have been unable to document an
interaction between the endogenous proteins.
Consistent with its multiple protein interaction domains, Bcr-Abl binds
and/or phosphorylates upwards of 20 proteins.29 GCKR joins this group although the mechanism by which the association occurs requires clarification. The C-terminal 150 amino acids of GCKR
are necessary for the interaction. Other regions in GCKR may also be
important and need to be mapped using N-terminal deletion constructs.
Of note, the C-terminal 150 amino acids of GCKR are highly conserved
with that of GLK and well conserved with that of GCK and HPK1
suggesting that they also may be recruited by Bcr-Abl. Interestingly,
the C-terminal 150 amino acids of GCKR are also required for its
interaction with TNF receptor-associated factor 2 (C-S. Shi et al,
manuscript submitted for publication).
The hypothesis that Ras activates GCKR, which leads to SAPK activation,
is consistent with observations that Ras not only signals the MAPK
cascade, but also SAPK. For example, CD3 and CD28 costimulation of T
lymphocytes induces Ras-dependent SAPK activation,30
insulin stimulates a Ras-dependent increase in SAPK activity in Rat1
fibroblasts,31 and the 1-adrenergic receptor activates
SAPK through a pathway that requires Ras and a MEKK.32 In
preliminary experiments CD3 and CD28 costimulation of T lymphocytes increased GCKR kinase activity implicating GCKR in T-cell SAPK activation (J. Tuscano, unpublished observations). Thus, Ras-mediated GCKR activation may be an important mechanism by which Ras activates the SAPK pathway in a variety of cell types.
Ras causes cellular transformation by activating multiple signaling
pathways. Several studies ascribe a role for SAPK activation. In one
study a catalytically inactive form of SEK1/MKK4 selectively inhibited
oncogenic Ras-stimulated SAPK activity and transformation, but not MAPK
activation.33 Particularly relevant to our findings, a
cytoplasmic inhibitor of SAPK signaling blocked the transformation of
pre-B lymphocytes by Bcr-Abl.9 Experiments to test whether the catalytically inactive form of GCKR may also impair
Bcr-Abl-mediated cellular transformation are in progress. Because of
the potential importance of the SAPK pathway in Ras-mediated cellular
transformation, GCKR provides an intriguing upstream target in this pathway.
| |
ACKNOWLEDGMENT |
The authors thank Mary Rust for her excellent editorial assistance, Dr
John Kryiakis for helpful discussions, and Dr Anthony Fauci for his
continued support.
| |
FOOTNOTES |
Submitted May 1, 1998; accepted October 7, 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 correspondence to John H. Kehrl, MD, Laboratory of
Immunoregulation, NIAID, NIH, Bldg 10, Room 11B08, 10 Center DR MSC
1876, Bethesda, MD 20892-1876; e-mail: jkehrl{at}atlas.niaid.nih.gov.
| |
REFERENCES |
1.
Kurzrock R, Gutterman J, Talpaz M:
The molecular genetics of Philadelphia chromosome-positive leukemias.
N Engl J Med
319:990, 1988[Medline]
[Order article via Infotrieve]
2.
Lugo TG, Pendergast A-M, Muller AJ, Witte ON:
Tyrosine kinase activity and transformation potency of bcr-abl oncogene products.
Science
247:1079, 1990[Abstract/Free Full Text]
3.
McLaughlin J, Chianese E, Witte ON:
Alternative forms of the BCR-ABL oncogene have quantitatively different potencies for stimulation of immature lymphoid cells.
Mol Cell Biol
9:1866, 1989[Abstract/Free Full Text]
4.
Daley GQ, VanEtten RA, Baltimore D:
Induction of chronic myelogenous leukemia in mice by the p210bcr/abl gene of the Philadelphia chromosome.
Science
247:824, 1990[Abstract/Free Full Text]
5.
Elefanty AG, Hariharan IK, Cory S:
Bcr-abl, the hallmark of chronic myeloid leukaemia in man, induces, multiple haemopoietic neoplasms in mice.
EMBO J
9:1069, 1990[Medline]
[Order article via Infotrieve]
6.
Puil L, Liu J, Gish G, Mbamalu G, Bowtell D, Pelicci PG, Arlinghaus R, Pawson T:
Bcr-Abl oncoproteins bind directly to activators of the Ras signaling pathway.
EMBO J
13:764, 1994[Medline]
[Order article via Infotrieve]
7.
Goga A, McLaughlin J, Afar DEH, Saffran DC, Witte ON:
Alternative signals to RAS for hematopoietic transformation by the BCR-ABL oncogene.
Cell
82:981, 1995[Medline]
[Order article via Infotrieve]
8.
Raitano AB, Halpern JR, Hambuch TM, Sawyers CL:
The Bcr-Abl leukemia oncogene activates Jun kinase and requires Jun for transformation.
Proc Natl Acad Sci USA
92:11746, 1995[Abstract/Free Full Text]
9.
Dickens M, Rogers JS, Cavanagh J, Raitano A, Xia Z, Halpern JR, Greenberg ME, Sawyers CL, Davis RJ:
A cytoplasmic inhibitor of the JNK signal transduction pathway.
Science
277:693, 1997[Abstract/Free Full Text]
10.
Whitmarsh AJ, Davis RJ:
Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways.
J Mol Med
74:589, 1996[Medline]
[Order article via Infotrieve]
11.
Woodgett JR, Avruch J, Kyriakis J:
The stress activated protein kinase pathway.
Cancer Surv
27:127, 1996[Medline]
[Order article via Infotrieve]
12.
Ezoe K, Lee ST, Strunk KM, Spritz RA:
PTK1, a novel protein kinase required for proliferation of human melanocytes.
Oncogene
9:935, 1994[Medline]
[Order article via Infotrieve]
13.
Ing YL, Leung IW, Heng HH, Tsui LC, Lassam NJ:
MLK-3: Identification of a widely-expressed protein kinase bearing an SH3 domain and a leucine zipper-basic region domain.
Oncogene
9:1745, 1994[Medline]
[Order article via Infotrieve]
14.
Tibbles LA, Ing YL, Kiefer F, Chan J, Iscove N, Woodgett JR, Lassam NJ:
MLK-3 activates the SAPK/JNK and p38/RK pathways via SEK1 and MKK3/6.
EMBO J
24:7026, 1996
15.
Katz P, Whalen G, Kehrl JH:
Differential expression of a novel protein kinase in human B lymphocytes.
J Biol Chem
269:16802, 1994[Abstract/Free Full Text]
16.
Pombo C, Kehrl JH, Sanchez I, Katz P, Avruch J, Zon LI, Woodgett JR, Force T, Kyriakis JM:
Activation of the SAPK pathway by the human STE20 homologue germinal centre kinase.
Nature
377:750, 1995[Medline]
[Order article via Infotrieve]
17.
Hu MC, Qiu WR, Wang X, Meyer CF, Tan TH:
Human HPK1, a novel human hematopoietic progenitor kinase that activates the JNK/SAPK kinase cascade.
Genes Dev
10:2251, 1996[Abstract/Free Full Text]
18.
Kiefer F, Tibbles LA, Anfafi M, Janssen A, Zanke BW, Lassam N, Pawson T, Woodgett JR, Iscove NN:
HPK1, a hematopoietic protein kinase activating the SAPK/JNK pathway.
EMBO J
15:7013, 1996[Medline]
[Order article via Infotrieve]
19.
Shi CS, Kehrl JH:
Activation of stress-activated protein kinase/c-Jun N-terminal kinase, but not NF- B, by the tumor necrosis factor (TNF) receptor 1 through a TNF receptor-associated factor 2- and germinal center kinase related-dependent pathway.
J Biol Chem
51:32102, 1997
20.
Tung RM, Blenis J:
A novel human SPS1/STE20 homologue, KHS, activates Jun N-terminal kinase.
Oncogene
14:653, 1997[Medline]
[Order article via Infotrieve]
21.
Diener K, Wang XS, Chen C, Meyer CF, Keesler G, Zukowski M, Tan TH, Yao Z:
Activation of the c-Jun N-terminal kinase pathway by a novel protein kinase related to human germinal center kinase.
Proc Natl Acad Sci USA
94:9687, 1997[Abstract/Free Full Text]
22.
Coso OA, Chiariello M, Yu JC, Teramoto H, Crespo P, Xu N, Miki T, Gutkind JS:
The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway.
Cell
81:1137, 1995[Medline]
[Order article via Infotrieve]
23.
Minden A, Lin A, Claret FX, Abo A, Karin M:
Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42.
Cell
81:1147, 1995[Medline]
[Order article via Infotrieve]
24.
Ren R, Mayer BJ, Cicchetti P, Baltimore D:
Identification of a ten-amino acid proline-rich SH3 binding site.
Science
259:1157, 1993[Abstract/Free Full Text]
25.
Afar DEH, Goga A, McLaughlin J, Witte O, Sawyers CL:
Differential rescue of BCR-ABL point mutants with c-MYC.
Science
264:424, 1994[Abstract/Free Full Text]
26.
Pendergast AM, Gishizky ML, Havlik MH, Witte ON:
SH1 domain autophosphorylation of p210 BCR/ABL is required for transformation but not growth factor-independence.
Mol Cell Biol
13:1728, 1993[Abstract/Free Full Text]
27.
Pendergast AM, Quilliam LA, Cripe Ld, Bassing CH, Dai Z, Li N, Batzer A, Rabun KM, Der CJ, Schlessinger J:
BCR-ABL-induced oncogenesis is mediated by direct interaction with the SH2 domain of the GRB-2 adaptor protein.
Cell
75:175, 1993[Medline]
[Order article via Infotrieve]
28.
Clark GJ, Drugan JK, Terrell RS, Bradham C, Der CJ, Bell RM, Campbell S:
Peptides containing a consensus Ras binding sequence from Raf-1 and the GTPase activating protein NF1 inhibit Ras function.
Proc Natl Acad Sci USA
93:1577, 1996[Abstract/Free Full Text]
29.
Sawyers CL:
Signal transduction pathways involved in BCR-ABL transformation.
Bailiere's Clin Haematol
10:223, 1997
30.
Su B, Jacinto E, Hibi M, Kallunki T, Karin M, Ben-Neriah Y:
JNK is involved in signal integration during costimulation of T lymphocytes.
Cell
77:727, 1994[Medline]
[Order article via Infotrieve]
31.
Miller BS, Shankavaram UT, Horney MJ, Gore AC, Kurtz DT, Rosenzweig SA:
Activation of c-Jun NH2-terminal kinase/stress-activated protein kinase by insulin.
Biochemistry
35:8769, 1996[Medline]
[Order article via Infotrieve]
32.
Ramirez MT, Sah VP, Znao XL, Hunter JJ, Chien KR, Brown JH:
The MEKK-JNK pathway is stimulated by alpha1-adrenergic receptor and ras activation and is associated with in vitro and in vivo cardiac.
J Biol Chem
272:14057, 1997[Abstract/Free Full Text]
33.
Clark GJ, Westwick JK, Der CJ:
p120 GAP modulates Ras activation of Jun kinases and transformation.
J Biol Chem
272:1677, 1997[Abstract/Free Full Text]
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Abstract
Introduction