|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 776-782
CHEMOKINES
Adaptor proteins CRK and CRKL associate with the serine/threonine
protein kinase GCKR promoting GCKR and SAPK activation
Chong-Shan Shi,
Joseph Tuscano, and
John H. Kehrl
From the B Cell Molecular Immunology Section, Laboratory of
Immunoregulation, NIAID, National Institutes of Health, Bethesda, MD,
and the Department of Medicine, UC Davis Cancer Center, University of
California-Davis, Sacramento, CA.
 |
Abstract |
STE20-related kinases play significant regulatory roles in a range
of cellular responses to environmental stimuli. GCKR (also referred to
as KHS1) is a serine/threonine protein kinase that has an STE20-like
protein kinase domain and that stimulates the stress-activated protein
kinase (SAPK, also referred to as Jun kinase or JNK) pathway. GCKR has
a large C-terminal regulatory domain that provides sites for
interactions with other proteins. Adaptor proteins mediate the
interactions between signaling molecules. In this study we showed that
the adaptor proteins Crk and CrkL associated with GCKR. When Crk-I,
Crk-II, or CrkL was transiently expressed in HEK 293T cells along with
GCKR, each coimmunoprecipitated with GCKR. Furthermore, in the Bcr-Abl
transformed cell line, K562 endogenous GCKR and CrkL
coimmunoprecipitated, indicating a constitutive association. Detection
of the CrkL-GCKR interaction required the SH3 domains of CrkL and 2 regions in GCKR 1 between amino acids 387 and 395 that contains a
consensus SH3 binding motif and the other between amino acids 599 and
696. Crk or CrkL overexpression increased GCKR catalytic activity. A
dominant negative form of Ras abolished Crk- or CrkL-induced GCKR
activation, suggesting a dependence on Ras activation for their
activation of GCKR. Finally, we showed impairment of the known ability
of CrkL to activate the SAPK pathway by a catalytically inactive form
of GCKR or by a GCKR antisense construct. Thus, GCKR associates with
other proteins through interactions mediated by SH2/SH3 adaptor
proteins, which can lead to GCKR and SAPK activation.
(Blood. 2000;95:776-782)
© 2000 by The American Society of Hematology.
| |
Introduction |
Mammalian cells have several serine/threonine protein
kinases related to the StE20 protein kinase in Saccharomyces
cerevisiae. Ste20 is of particular interest because it regulates a
mitogen-activated protein kinase (MAPK) pathway that controls the
mating response of haploid yeast cells.1 Genetic epistasis
analysis has positioned STE20 between the heterotrimeric G protein,
which is activated by pheromone exposure, and the MAPK cassette. Ste20
associates through its N-terminal regulatory domain with the small
GTPase Cdc42 and the scaffolding protein Ste5, interactions that result in the recruitment of STE20 to specific membrane sites.2
Among the mammalian proteins related to STE20, the PAK family of
protein kinases contains not only a related C-terminal kinase domain
but a similar overall protein structure with an N-terminal regulatory domain that also contains a binding site for Cdc42 and for
Rac.3 The PAK kinases have also been implicated in
activating a MAPK cassette; however, instead of the MAPK pathway, the
PAK kinases activate 2 related pathways, the stress-activated protein
kinase (SAPK), also referred to as Jun kinase (JNK), and the p38
pathways.3,4
The germinal center kinase (GCK) family is another subfamily of
serine/threonine protein kinases with a kinase domain related to that
of STE20. Eleven mammalian family members have been
identified.5 The GCK-related kinases can be subdivided into
2 broad groups based on their structural and functional properties. The
group 1 GCKs are closely related to GCK itself and include GCK,
GCK-related (GCKR, KHS), GCK-like kinase (GLK), hematopoietic
progenitor kinase-1 (HPK1), and Nck-interacting kinase
(NIK).6-12 These enzymes selectively activate SAPKs. The
group 1 GCK family members also have an approximately 350-A region that
can be divided into a leucine-rich domain and a 150-AA carboxyl
terminal (CT) region. Studies of GCK and GCKR indicate that the CT
region is required for binding to tumor necrosis factor (TNF)
receptor-associated factor-2 (TRAF2).13,14 GCK, GCKR, and
GLK are activated in vivo by TNF, a potent activator of the SAPK
pathway.7,9,13 Furthermore, TNF stimulation results in the
recruitment of GCKR to TRAF2.14
The carboxyl terminal region of the group 1 GCKs also includes at least
2 PEST motifs and a minimum of 2 polyproline-rich regions for binding
proteins that contain SH3 domains. The regulatory domain of HPK1 has 4 proline-rich motifs that have been designated P1 to P4. Three of these,
P1, P2, and P4, mediate Grb2 binding.10,15 GCKR shares the
P2 and P4 proline-rich motifs with HPK1. GCK also shares 2 proline-rich
motifs with HPK1, P3 and P4. Recently, HPK1 has been shown to interact
with the first SH3 domain of the adaptor proteins Crk and
CrkL.16 A peptide that spans the P2 region of HPK1
effectively blocks the binding of CrkL to a known CrkL SH3 ligand.
Similar to HPK1, GCKR also bound Crk and CrkL.16
The adaptor protein Crk was originally discovered as an avian
retrovirus encoding an oncogene product v-crk.17 The
mammalian homologs of v-crk were subsequently identified as Crk-I and
Crk-II, alternatively spliced forms of the same gene. Crk-II has an
N-terminal SH2 domain followed by 2 SH3 domains, whereas Crk-1 has a
single SH3 domain. In addition, a closely related gene, CrkL, was
isolated and found to be constitutively phosphorylated in
Bcr-Abl-transformed cells.18 The first SH3 domains of Crk
and CrkL have similar binding specificities and are known to bind the
guanine nucleotide exchange proteins C3G and SOS, the tryosine kinase
Bcr-Abl, and DOCK18 0.19,20 A conserved lysine present in
the defined binding motif PPxLPxK contributes significantly to the
binding affinity and specificity of the first Crk SH3 domain. No
binding specificities of the second SH3 domains of Crk-II and CrkL are
known. The SH2 domain of Crk binds tyrosine-phosphorylated YxxP motifs
present in such proteins as p130Cas, Cas-L, and
paxillin.17,18 Transient expression of v-Crk, Crk-I, or
Crk-II activated the SAPK pathway in several different cell
types.21-24 In this study, we have explored the potential
role of GCKR in Crk-induced SAPK activation.
| |
Material and methods |
Cell lines and cell culture
HEK 293 and HEK 293T cells were maintained in Dulbecco's minimal
essential medium plus 10% fetal calf serum, whereas K562 cells were
maintained in RPMI 1640 plus 10% fetal calf serum. HEK 293 and HEK
293T (SV40 T-antigen transformed) are human embryonic kidney cell lines
that are readily transfected.
Plasmids and antibodies
The pMT3-HA-SAPK-p46 plasmid was provided by Dr J. Kyriakis
(Harvard, Boston, MA). The pcDNA3-Ras-N17 was a kind gift of Dr S. Gutkind (National Institutes of Health, Bethesda, MD). Dr S. Gutkind
provided the Crk-I and Crk-II expression vectors after obtaining
permission from Dr M. Matsuda (National Institute of Health, Tokyo,
Japan). The CrkL expression vector was obtained from Dr J. Groffen,
(Children's Hospital, Los Angeles, CA), and the Bcr-Abl expression
vector was obtained from Owen Witte (UCLA, Los Angeles, CA). The
pcDNA-HA-GCKR, pCRIII-GCKR, pCRIII-GCKRT178A, and pCRIII-GCKR(AS) were
described previously.7 The pcDNA-HA-GCKR deletion
constructs 1-699, 1-599, 1-496, 1-396, 386-846, and 399-846 were
created by inserting the appropriate polymerase chain reaction (PCR)
product from pcDNA-HA-GCKR into pcDNA-H(A) The pEBG
GST-CrkL1-109 was created by insertion of the appropriate
PCR product in frame with the GST coding sequence in pEBG-GST (kindly
provided by U. Siebenlist, National Institutes of Health). The
orientation and veracity of each of the constructs were verified by
nucleotide sequencing. The anti-HA (12CA), anti-pY4G10, and
the anti-Crk and CrkL antibodies were purchased from Boehringer Mannheim (Indianapolis, IN), 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.7
In vitro kinase assays
Equal numbers of HEK 293 cells were plated on 10-cm plates at an
approximate density of 5 × 105 cells/plate the day
before transfection. The HEK 293T and HEK 293 cells were transiently
transfected using a calcium phosphate method, and the K562 cells were
transfected using Superfect (Qiagen, Valencia, CA).
Transfected DNA levels were normalized with control plasmids.
Forty-eight hours after the transfection,
HA-immunoprecipitates were subjected to in vitro kinase
assays using myelin basic protein or c-jun1-79 as
substrates for the GCKR and SAPK assays, respectively.7,13 Before the in vitro kinase assay, the HA-immunoprecipitates were washed
3 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), 3 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 3 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, Piscataway, NJ).
Coimmunoprecipitations
The coimmunoprecipitations were performed using lysates (20 mmol/L
Tris, pH 8, 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 or HA-GCKR truncation mutants (2 µg) and
Crk-I, Crk-II, or CrkL or prepared from K562 cells. Anti-HA, anti-Crk,
anti-CrkL, anti-Erk-1, anti-myc, anti-GCKR, or the
4G10 monoclonal antibody was added, and the immunoprecipitates were
collected with the appropriate secondary antibody-coupled magnetic
beads (Dynal, Glastrup, Denmark). They were washed 3 times
in lysis buffer, twice in lysis buffer with 0.5 mol/L NaCl, fractionated by sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE), and analyzed by immunoblotting with the
appropriate antibody. The Bcr-Abl and GCKR coimmunoprecipitations in
the presence or absence of CrkL were performed with lysates prepared
from HEK 293T cells transfected with the indicated expression vectors.
| |
Results |
Crk-I, Crk-II, and CrkL all associate with GCKR
GCKR has 1 proline-rich sequence that could serve as an interaction
site with Crk proteins, though a similar site in HPK1, P2, served as an
interaction site for the C-terminal SH3 domain of Grb2.15
In addition there are several other proline-rich sites in the
regulatory domain of GCKR, but none fit the consensus sequence for a
Crk SH3-domain-binding site. To test whether Crk or CrkL interacted
with GCKR, we first coexpressed Crk-I or Crk-II along with an HA-tagged
version of GCKR in HEK 293T cells. We examined Crk immunoprecipitates
for the presence of HA-GCKR and HA immunoprecipitates for the presence
of Crk proteins (Figure 1). The Crk
immunoprecipitates from the Crk-I- and Crk-II-transfected cells
contained HA-GCKR. In the converse experiment, the HA
immunoprecipitates from the Crk-I- and Crk-II-transfected cells
contained Crk-I and Crk-II, respectively. In addition some endogenous
Crk-II could be detected coimmunoprecipitating with HA-GCKR in the
Crk-I-transfected cells. An anti-Flag monoclonal antibody failed to
immunoprecipitate either HA-GCKR or Crk proteins. Similar experiments
revealed that CrkL and GCKR also coimmunoprecipitated (shown as a
control in Figure 3 below).
View larger version (47K):
[in this window]
[in a new window]
| Fig 1.
Crk-I and Crk-II coimmunoprecipitate with GCKR. HEK 293T
cells were transfected with constructs that directed the expression of
HA-GCKR and Crk-I or Crk-II.
Lysates prepared from the transfected cells were immunoprecipitated
with HA, FLAG (control antibody), or a Crk antibody as indicated. The
immunoprecipitates were fractionated on SDS-PAGE, transferred, and
blotted with the HA antibody (top) or the Crk antibody (bottom).
Locations of HA-GCKR, Crk-II, and Crk-I are indicated with arrows as
the immunoglobulin light chains. These experiments were performed 3 times with similar results.
|
|
Mapping the regions in GCKR and CrkL responsible for their
interaction and demonstration of an association between the endogenous
proteins
To analyze the site in GCKR that is important for interaction with
Crk proteins, we expressed truncated HA-tagged GCKR proteins in HEK
293T cells along with CrkL, and we examined HA immunoprecipitates for
the presence of CrkL using a CrkL-specific antibody. We found that CrkL
readily coimmunoprecipitated with wild-type GCKR and with
GCKR1-696 (Figure 2). However,
the next truncation of GCKR, GCKR1-599, resulted in a
significant reduction in the interaction. GCKR1-496 very
weakly coimmunoprecipitated CrkL, and an interaction between GCKR1-396 and CrkL could only be detected on long
exposures. This result was surprising because we had assumed that the
P2 proline-rich site in GCKR, which is located at the C-terminus of
GCKR1-396 and present in GCKR1-496 and
GCKR1-596, would mediate the interaction with GCKR. These
results suggested that the P2 site alone was insufficient to mediate a
high-affinity interaction with GCKR.
View larger version (50K):
[in this window]
[in a new window]
| Fig 2.
Coimmunoprecipitation of CrkL with GCKR depends on the
region in GCKR between amino acids 599 and 699.
HEK 293T cells were transfected with constructs that directed the
expression of CrkL and GCKR or truncated versions of GCKR. Cell lysates
and myc (control antibody) and HA antibody immunoprecipitates were
analyzed by immunoblotting. The blot was sequentially reacted with the
HA antibody (top) and the CrkL antibody (bottom). CrkL is indicated by
an arrow. The truncated GCKR proteins can be seen in the cell lysate
and in the HA immunoprecipitation lanes (arrows). Several nonspecific
proteins are also present (top), the most prominent just below 95 kd in
the cell lysate lanes.
|
|
To examine the importance of the N-terminal portion of GCKR in the CrkL
interaction, we truncated the first 385 amino acids of GCKR and created
GCKR386-846, which lacked the GCKR catalytic domain, but
retained the P2 site. The expressed truncated protein readily
coimmunoprecipitated with CrkL, indicating that the catalytic domain of
GCKR was not required for the interaction with CrkL (Figure
3A). Next, by making a short additional
truncation, we created a construct GCKR399-846, which
expressed a version of GCKR that lacked the P2 site. In contrast to the
results with GCKR386-846, GCKR399-846
coimmunoprecipitated poorly with CrkL (Figure 3B). Based on this result, we expected that the SH3 domains of CrkL would be indispensable for its interaction with GCKR. When we expressed a truncated version of
CrkL that lacked both SH3 domains as a GST-fusion protein in conjunction with either full-length GCKR (data not shown) or with GCKR386-846, we were unable to detect a significant
interaction between the transfected proteins (Figure 3C). Although the
truncated CrkL protein did not bind GCKR, it did extract several
tyrosine-phosphorylated proteins from the cell lysate (data not shown).
In sum, these experiments revealed that the amino acids 600-695 and
387-398 in GCKR and the SH3 domains of CrkL are required for an optimal association between the 2 proteins.
View larger version (33K):
[in this window]
[in a new window]
| Fig 3.
Further requirements for the interaction of GCKR and CrkL
and demonstration of an association of the endogenous proteins.
(A) The regulatory domain of GCKR interacts with CrkL. Constructs
directing the expression of HA-GCKR 386-846 and CrkL were
cotransfected into HEK 293T cells. The indicated immunoprecipitates and
a cell lysate were examined for either HA or CrkL expression.
Anti-myc antibody was used as a control. (B) The P2 motif in
GCKR was required to detect a strong GCKR/CrkL interaction. Constructs
directing the expression of HA-GCKR386-846 (lanes 1-3);
CrkL (lanes 1-6); and HA-GCKR399-846 (lanes 4-6) were
transfected into HEK 293T. The indicated immunoprecipitates or cell
lysates were analyzed for HA or CrkL expression by immunoblotting. The
designation HA-GCKRT indicated both truncated forms of
GCKR, whose mobilities on SDS-PAGE were indistinguishable. (C) The CrkL
SH2 domain did not contribute to the interaction with the GCKR
regulatory domain. Constructs directing the expression of
HAGCKR386-846, GST (lanes 2, 4) or
GST-CRKL1-109 (lanes 1, 3) were transfected into HEK
293T cells. Cell lysates extracted with glutathione-Sepharose 4B beads
(Pharmacia Biotech AB) were analyzed for GST and
HA-GCKR expression by immunoblotting. HA-GCKR386-846
expression was similar in the GST- and the
GST-CRKL1-109-transfected cells (lanes 3, 4). (D) GCKR and
CrkL are constitutively associated in K562 cells. CrkL
immunoprecipitates of cell lysates prepared from
10 × 106 million K562 cells or cell lysates from
0.5 × 106 cells were analyzed for GCKR expression
using a GCKR specific antiserum. A preimmune antiserum (P.I.) was used
as a control. Results from 2 different experiments (lanes 1, 2 and
lanes 3, 4) are shown. Each of these experiments was preformed at least
twice with similar results.
|
|
To examine whether an interaction occurred between endogenous Crk
protein and GCKR, we examined whether CrkL and GCKR
coimmunoprecipitated using cell lysates prepared from K562 cells. These
cells are derived from human erythroleukemia and contain significant
levels of CrkL and the Bcr-Abl oncogene. An antipeptide antibody raised
against an internal peptide in GCKR was used to immunoblot CrkL
immunoprecipitates. We found that they contained significant amounts of
GCKR, whereas immunoprecipitates prepared using a control antiserum did
not contain GCKR (Figure 3D). Thus, Crk-I, Crk-II, and CrkL can all interact with GCKR; in K562 cells, GCKR and CrkL associate
constitutively. However, the constitutive association of GCKR and CrkL
in K562 may not indicate that this is the case in other cell types
because the presence of Bcr-Abl may alter either GCKR or CrkL.
Because we showed an association between Bcr-Abl and
GCKR,25 we determined whether the presence of CrkL enhanced
the ability to detect an association between those 2 proteins. HEK 293T
cells were transfected with constructs expressing Bcr-Abl and GCKR in the presence or absence of CrkL. The coexpression of CrkL enhanced the
amount of HA-GCKR immunoprecipitating with Bcr-Abl (Figure 4). The levels of Bcr-Abl and HA-GCKR
expressed were unaltered by the concomitant expression of CrkL. Thus,
CrkL enhanced the association between Bcr-Abl and GCKR.
View larger version (46K):
[in this window]
[in a new window]
| Fig 4.
CrkL enhances the association of Bcr-Abl and GCKR.
HEK 293T cells were transfected with constructs that directed the
expression of Bcr-Abl and HA-GCKR in the presence or absence of CrkL
(lanes 1, 2 versus lanes 3, 4). Bcr-Abl (lanes 2, 4) or control (lanes
1, 3) immunoprecipitates were analyzed by immunoblotting for HA-GCKR.
Cell lysates from the same transfection were analyzed for Bcr-Abl and
HA-GCKR by immunoblotting with a Bcr-Abl or an HA antibody. This
experiment was preformed twice with similar results.
|
|
Overexpression of Crk-I, Crk-II, or CrkL increases GCKR
kinase activity
To examine the effects of Crk-I, Crk-II, and CrkL on GCKR kinase
activity, we cotransfected HEK 293T with expression constructs for the
Crk proteins and HA-GCKR. We subjected HA-GCKR immunoprecipitates to an
in vitro kinase assay using the substrate myelin basic protein. We
found that Crk-I, Crk-II, and CrkL increased HA-GCKR kinase activity
approximately 4.8-, 10.3-, and 11-fold, respectively, compared with
HA-GCKR immunoprecipitated from control cells (Figure 5A, top). Crk-I appeared not to express as
well as Crk-II, though the Crk antibody did not detect Crk-I as well as
Crk-II. The coexpression of the Crk proteins with GCKR resulted in
minor increases in the amount of GCKR tyrosine phosphorylation detected
by phosphotyrosine immunoblotting of HA-GCKR immunoprecipitates (Figure
5A, second panel). The expression of Crk proteins enhanced the amount
of GCKR phosphorylation in the in vitro kinase assay. Compared with control transfections, Crk-I, Crk-II, and CrkL increased the amount of
GCKR phosphorylation 5.1-, 6.2-, and 10.9-fold, respectively (Figure
5B, top). The identity of the coimmunoprecipitating phosphorylated band
with a more rapid mobility than GCKR is unknown.
View larger version (35K):
[in this window]
[in a new window]
| Fig 5.
Crk proteins activate GCKR kinase activity.
(A) Coexpression of Crk proteins enhances GCKR kinase activity. HEK
293T was transfected with a control or with constructs that direct the
expression of Crk-I, Crk-II, or CrkL and HA-GCKR. HA-immunoprecipitates
were subjected to an in vitro kinase assay using myelin basic protein
(MBP) as a substrate (autoradiograph, labeled as kinase assay). The
SDS-PAGE fractionated in vitro kinase assay was analyzed by
immunoblotting for phosphotyrosine (pY blot). In addition, cell lysates
prepared from the same transfections were analyzed by immunoblotting
for HA-GCKR (HA blot), CrkL (CrkL blot), and Crk-I and Crk-II (Crk
blot). (B) Coexpression of Crk proteins triggers GCKR phosphorylation.
HA-GCKR immunoprecipitates from cells transfected as in A were
subjected to an in vitro kinase assay in the absence of an exogenous
substrate (autoradiograph, labeled as kinase assay). Cell lysate
analyzed for HA-GCKR expression by immunoblotting. Each of these
experiments was performed at least twice.
|
|
Crk-I, Crk-II, and CrkL activate GCKR through a Ras-dependent
mechanism
The first SH3 domain of Crk or CrkL binds SOS and
C3G.19,20 SOS is a nucleotide exchange factor for Ras,
whereas C3G is a nucleotide exchange factor for Rap1. Several pieces of
evidence indicate that Ras is important in Crk-induced signaling. A
dominant negative form of Ras suppressed v-Crk-induced transformation
of NIH 3T3 cells and CrkL-induced transformation of Rat-1 fibroblasts, and it inhibited Crk-II-induced apoptosis of HEK 293 cell.21,26,27 To examine whether Ras was important in
Crk-induced GCKR activation, we made use of a dominant negative form of
Ras, Ras N-17. We coexpressed expression vectors for Crk-I, Crk-II, or
CrkL with HA-GCKR and with either Ras N-17 or a control plasmid. We
found that the presence of Ras N-17 resulted in a significant reduction
in Crk-induced GCKR activation (Figure 6).
The expression of Ras N-17 did not alter the expression of any of the
Crk proteins. These data are similar to those we have observed with
Bcr-Abl-induced GCKR activation, where Ras N-17 also
blocks.25 In contrast, Ras N-17 has no effect on
TNF-induced GCKR activation (Shi C-S, unpublished observation).
View larger version (45K):
[in this window]
[in a new window]
| Fig 6.
Ras N-17 blocks Crk-induced GCKR activation. HEK 293T
cells were cotransfected constructs directing the expression of HA-GCKR
in conjunction with Crk-I (lanes 2, 3), Crk-II (lanes 4, 5), CrkL
(lanes 6, 7), or a control plasmid (lane 1) in the presence (lanes 3, 5, 7) or absence of Ras N-17 (lanes 1, 2, 4, 6).
HA-GCKR immunoprecipitates were assayed for activity using an in vitro
kinase with MBP as a substrate. The fold induction compared to HA-GCKR
immunoprecipitate from the control transfection is indicated. Levels of
Crk-I, Crk-II, CrkL, Ras N-17, and HA-GCKR expression as detected by
immunoblotting are shown. These experiments were performed twice.
|
|
The activation of the SAPK pathway by Crk-I, Crk-II, or CrkL is
partially blocked by a GCKR antisense construct or a dominant negative
form of GCKR
Transient overexpression of v-Crk-, Crk-I-, or Crk-II-activated SAPK
in HEK 293 cells, whereas the overexpression of CrkL activated the SAPK
pathway in Rat-1 fibroblasts.21-24 Because GCKR is
expressed in HEK 293 cells,7 we sought to determine whether GCKR activation contributes to activation of the SAPK pathway after Crk
overexpression. For these experiments, we used an antisense GCKR
construct known to reduce the endogenous level of GCKR in HEK 293 cells
and a dominant negative form of GCKR that inhibits TNF-induced SAPK
activation and Bcr-Abl- induced SAPK activation.7,25 We
first examined whether the GCKR antisense construct inhibited Crk-I-
and Crk-II-induced SAPK activation. HEK 293 cells were cotransfected
with either Crk-I or Crk-II in the presence or absence of a construct
that expresses antisense GCKR RNA. The coexpression of GCKR antisense
partially inhibited both Crk-I- and Crk-II-induced SAPK activation
(Figure 7). The antisense construct did not
alter the expression of either Crk-I or Crk-II. Similarly, the GCKR antisense construct inhibited SAPK activation by CrkL. In addition, the
coexpression of the kinase-deficient form of GCKR partially blocked
CrkL-induced SAPK activation (Figure 8).
The coexpression of GCKR T178A did not alter the levels of HA-SAPK or
CrkL in the transfected cells. In both sets of experiments, we often
failed to see a significant inhibition of Crk- or CrkL-induced SAPK
activation until we reached a critical level of expression of the
inhibitor (antisense- or kinase-dead GCKR). Neither GCKR antisense nor
GCKRT178A significantly impaired MEKK1-induced SAPK activation (data
not shown).
View larger version (45K):
[in this window]
[in a new window]
| Fig 7.
Evidence for GCKR involvement in Crk-I- and
Crk-II-induced SAPK activation.
HEK 293 cells were cotransfected with constructs that direct the
expression of HA-SAPK (lanes 1 to 6), Crk-II (lanes 1, 2), Crk-I (lanes
3, 4), or GCKR (lane 5) in the presence (lanes 1, 3) or absence (lanes
2, 4 to 6) of a construct that expressed a GCKR antisense RNA. HA-SAPK
immunoprecipitates were subjected to an in vitro kinase assay using
GST-jun1-79 as a substrate. The fold induction compared to
HA-SAPK alone is indicated. The levels of HA-SAPK, Crk-II, and Crk-I as
detected by immunoblotting are shown below. These experiments were
performed twice with similar results.
|
|
View larger version (33K):
[in this window]
[in a new window]
| Fig 8.
Evidence that GCKR is required for CrkL-induced SAPK
activation.
(A) A GCKR antisense construct inhibits CrkL-induced SAPK activation.
HEK 293 cells were cotransfected with constructs that direct the
expression of HA-SAPK (lanes 1 to 5), CrkL (lanes 2 to 5), and a GCKR
antisense RNA (lanes 4, 5). HA-SAPK immunoprecipitates were subjected
to an in vitro kinase assay using GST-jun1-79 as a
substrate. The fold induction compared to the control is shown below
the in vitro kinase assay result. Levels of HA-SAPK and CrkL expression
are shown as detected by immunoblotting. (B) GCKRT178A inhibits
CrkL-induced SAPK activation. HEK 293 cells were cotransfected with
constructs that direct the expression of HA-SAPK (lanes 1 to 5), CrkL
(lanes 2 to 5), and increasing concentrations of GCKRT178A (1, 2, and 3 µg/mL, lanes 3 to 5, respectively). HA-SAPK immunoprecipitates were
subjected to an in vitro kinase assay using GST-jun1-79 as
a substrate. The fold induction compared to cells transfected only with
the construct directing HA-SAPK expression is indicated below the
autoradiograph. Levels of HA-SAPK, CrkL, and GCKRT178A as detected by
immunoblotting are shown. Low levels of endogenous GCKR are detected
(lanes 1, 2, bottom). These experiments were performed twice with
similar results.
|
|
To examine whether CrkL overexpression could enhance SAPK activity and
to define the role of endogenous GCKR in SAPK activation by CrkL, we
transfected K562 cells with CrkL and measured SAPK activation by
immunoblotting for phosphorylated SAPK (pSAPK). We found that the
transfection of either GCKR or CrkL enhanced the levels of pSAPK in
K562 cells and that the concomitant expression of either the GCKR
antisense construct or the kinase inactive form of GCKR inhibited this
increase (Figure 9, top). Endogenous SAPK
and endogenous and transfected levels of GCKR and CrkL were detected by
immunoblotting. The antisense form of GCKR reduced the level of
endogenous GCKR by approximately 40%, and the presence of the kinase
inactive form of GCKR accounted for the enhanced GCKR band (Figure 9,
lanes 1 and 2, fourth panel, respectively).
View larger version (58K):
[in this window]
[in a new window]
| Fig 9.
GCKR involvement in CrkL-induced SAPK activation in K562
cells.
K562 cells were transfected with contructs directing the expression of
CrkL (lanes 1 to 3), antisense GCKR (lane 1), kinase inactive GCKR
(lane 2), or wild-type GCKR (lane 4). Levels of phosphorylated SAPK
were detected with an antibody specific for pSAPK. Levels of SAPK,
GCKR, and CrkL in the cell lysates were detected by immunoblotting with
SAPK, GCKR, and CrkL-specific antibodies. This experiment was performed
twice with similar results.
|
|
| |
Discussion |
We have provided evidence that the Crk adapter proteins interact
with the serine/threonine kinase GCKR and likely regulate its activity.
In support of this conclusion, GCKR coimmunoprecipitated with Crk-I,
Crk-II, and CrkL after transient transfection in HEK 293T and was found
constitutively associated with CrkL in K562 cells. Two regions in GCKR
are important for its interaction with CrkL, which were localized to
amino acids 386-398 and 599-696. That CrkL-GCKR interaction may
modulate GCKR function was supported by experiments showing that
overexpression of Crk-I, Crk-II, or CrkL enhanced GCKR kinase activity.
Implicating Ras activation in this process, the concomitant expression
of a dominant negative form of Ras blocked Crk-triggered GCKR kinase
activity. That GCKR activation contributed to Crk-induced SAPK
activation was based on data generated with a GCKR antisense and the
GCKR T178A constructs.
In the course of these experiments, the GCKR-related kinase HPK1 was
shown to bind selectively to the first SH3 domain of c-Crk and
CrkL.16 Based on a series of binding experiments, the P2
site in HPK1referred to as M2appeared to be important in mediating
the binding. GCKR, designated as KHS, was also shown to bind to Crk
proteins.16 Based on the shared P2 site with HPK1, that
region in GCKR was implicated in mediating the GCKR-Crk interaction.
The similarity of the P2 site in GCKR to known Crk SH3-binding
sites19,20 suggested to us that this proline-rich site
would confer on GCKR the ability to interact with Crk proteins. Indeed,
the association of GCKR with Crk-I, Crk-II, and CrkL was readily
apparent on cotransfection of the appropriate expression vectors into
HEK 293T cells and endogenous CrkL, and GCKR could be
coimmunoprecipitated from K562 cells. Consistent with an SH3 domain-mediated interaction between GCKR and the Crk proteins, deletion of the CrkL SH3 domains abolished our ability to detect an
interaction between GCKR and CrkL. Although the P2 site in GCKR is
likely indispensable for a high-affinity interaction between GCKR and
CrkL, the region in GCKR between amino acids 599 and 699 also appears
important. This region contains 2 sites that are weakly homologous to
the consensus CrkL SH3-binding site, PDRILPRK609 and
PLPSPLN673. However, whether these regions contribute to
the Crk-GCKR interaction requires further study. An alternative
possibility is that the region in GCKR between amino acids 599 and 699 does not directly bind to Crk proteins but that it serves as a
homodimerization interface that stabilizes the Crk-GCKR interaction.
CrkL is a prominent substrate of the oncoprotein Bcr-Abl, and the
first SH3 domain of CrkL binds Bcr-Abl.20,21 We have previously shown that GCKR is constitutively associated with the Bcr-Abl in K562 cell.25 However, our ability to
coimmunoprecipitate the 2 proteins depended on the presence of the CT
region, which is distinct from those important for interaction with
CrkL. Thus, it may be possible to assemble a trimolecular complex that
contains GCKR, Bcr-Abl, and CrkL or other Crk proteins. Consistent with that possibility, CrkL overexpression enhanced the ability to detect an
association between Bcr-Abl and GCKR. Furthermore, Crk proteins should
link GCKR to other signaling or docking molecules by virtue of
their SH2 domains, which can bind tyrosine-phosphorylated proteins such as CBL, HEF1, members of the p130Cas family, or paxillin.
These proteins may modulate GCKR activity or provide a mechanism to
translocate GCKR to sites of action.
Several studies have reported that the overexpression of Crk proteins
leads to the activation of the SAPK pathway but not the MAPK
pathway.21-24 In HEK 293T cells, Crk has been reported to
activate the SAPK pathway by the guanine-nucleotide exchange protein
C3G through a Ras-independent mechanism.21,27
Kinase-negative28 forms of the mixed lineage kinase (MLK)
and the dual leucine zipper kinase (DLK) inhibited C3G-induced SAPK
activation, suggesting their involvement in Crk-induced SAPK
activation. In this model Crk recruits C3G, which acts as an exchange
factor for a small GTPase whose identity is unknown. The activated
GTPase results in MLK3 activation, which leads to MEKK1 and subsequent
SAPK activation or to DLK activation and entrance into the SAPK cascade
at the level of Sek1/MKK4.29-32 In contrast to these
studies in COS-7 cells, Crk-induced SAPK activation depended on the
activation of Ras and Rac because dominant negative forms of either
small GTPase ablated the Crk-induced SAPK activation.24 In
the same study, Crk-induced SAPK was shown to depend on both the SH3
and the SH2 domains of Crk, suggesting that a signaling complex must be
assembled and indicating the probable involvement of p130Cas. Consistent with that possibility, overexpression of p130Cas also activated the SAPK pathway, though weakly. In this model Crk recruits SOS, DOCK180, or both, which leads to Ras and Rac activation. Rac
activates a PAK kinase or MLK3, which leads to MEKK1 and subsequent SAPK activation.24
In this article, we provide several pieces of evidence for the
participation of GCKR in Crk-mediated SAPK activation in HEK 293 cells.
First, GCKR is present in COS-7 and HEK 293 cells, and it is a potent
SAPK activator in them.7 Second, the transient expression
of Crk results in Ras activation, which is either a direct or an
indirect GCKR activator,25 thus providing a mechanism by
which Crk could activate GCKR. Indeed a dominant negative form of Ras
blocked Crk-induced GCKR activation. Third, the interaction of Crk
proteins with GCKR links GCKR to other proteins that may regulate its
activity. Fourth, a reduction of GCKR protein levels using the GCKR
antisense construct impairs Crk-mediated SAPK activation, and a
kinase-dead form of GCKR inhibits the ability of Crk proteins to
activate SAPK.
How do we reconcile this information with the previously discussed
models of Crk-induced SAPK activation? GCKR could act upstream of MLK3,
providing a linear pathway initiated by Crk- or CrkL-binding GCKR. The
Crk-GCKR complex could recruit through the GCKR C-terminal SH3-binding
domain (amino acids 486-490) MLK3/DLK. A similar region in HPK1 has
been shown to mediate binding to MLK3.11 In a physiologic setting, tyrosine phosphorylation of a receptor could assemble a
complex of Crk, GCKR, and MLK3 (or DLK) at the membrane in which Rac1
or Cdc42 activates the complex by MLK3 dimerization through its leucine
zippers. Recently, MLK3 dimerization was shown to activate its kinase
activity.33 Alternatively, Crk-mediated Ras activation
could lead to GCKR and subsequent SAPK activation pathway through
MEKK1. There is evidence that several of the GCK family members signal
the SAPK pathway through MEKK1.9,12,34 GCK has been shown
to interact directly with MEKK1. This interaction is dependent on the
CT region and its third PEST region.34 The CT region is
conserved with GCKR, and arguing that GCKR can also activate the SAPK
pathway through MEKK1, a construct expressing a MEKK1 dominant
negative, impairs GKCR-induced SAPK activation.7
Delineating how GCK family members traffic within the cell and interact
with other molecules will be crucial for understanding how MAPK
signaling modules couple to their activators. Both direct- and
adaptor-mediated interactions between GCK family members and other
signaling molecules have been found, and their consequences for GCK
family member function is just emerging. Because some MAP3Ks such as
c-raf undergo stimulus-induced membrane translocation for their
activation, the colocalization of GCK family members with them provides
an attractive hypothesis, whereby specific GCK family members activate
distinct MAP3Ks. Crk proteins may serve such a role for GCKR.
| |
Acknowledgments |
We thank Mary Rust for her fine editorial assistance and Dr Anthony
Fauci for his continued support. We thank Dr John Kyriakis for his
constructive discussions.
| |
Footnotes |
Submitted February 4, 1999; accepted September 30, 1999.
Reprints: John H. Kehrl, Laboratory of Immunoregulation, NIAID,
National Institutes of Health, Building 10, Room 11B08, 10 Center
Drive, MSC 1876, Bethesda, MD 20892-1876; e-mail:
jkehrl{at}atlas.niaid.nih.gov.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Herskowitz I.
MAP kinase pathways in yeast-for mating and much more.
Cell.
1995;80:187[Medline]
[Order article via Infotrieve].
2.
Pryciak PM, Huntress FA.
Membrane recruitment of the kinase cascade scaffold protein Ste5 by the G beta gamma complex underlies activation of the yeast pheromone response pathway.
Genes Dev.
1998;12:2684[Abstract/Free Full Text].
3.
Sells M, Chernoff J.
Emerging from the PAK: the p21-activated protein kinase family.
Trends Cell Biol.
1997;7:162.
4.
Kyriakis JM, Avruch J.
Sounding the alarm: protein kinase cascades activated by stress and inflammation.
J Biol Chem.
1996;271:24,313[Free Full Text].
5.
Kyriakis J.
Signaling by the germinal center kinase family of protein kinase.
J Biol Chem.
1999;274:5259[Free Full Text].
6.
Katz P, Whalen G, Kehrl JH.
Differential expression of a novel protein kinase in human B lymphocytes.
J Biol Chem.
1994;269:16,802[Abstract/Free Full Text].
7.
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.
1997;51:32,102.
8.
Tung RM, Blenis J.
A novel human SPS1/STE20 homologue, KHS, activates Jun N-terminal kinase.
Oncogene.
1997;14:653[Medline]
[Order article via Infotrieve].
9.
Diener K, Wang XS, Chen C, et al.
Activation of the c-Jun N-terminal kinase pathway by a novel protein kinase related to human germinal center kinase.
Proc Natl Acad Sci U S A.
1997;94:9687[Abstract/Free Full Text].
10.
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.
1996;10:2251[Abstract/Free Full Text].
11.
Kiefer F, Tibbles LA, Anfafi M, et al.
HPK1, a hematopoietic protein kinase activating the SAPK/JNK pathway.
EMBO J.
1996;15:7013[Medline]
[Order article via Infotrieve].
12.
Su YC, Han J, Xu S, Cobb M, Skolnik EY.
NIK is a new Ste20-related kinase that binds NCK and MEKK1 and activates the SAPK/JNK cascade via a conserved regulatory domain.
EMBO J.
1997;16:1279[Medline]
[Order article via Infotrieve].
13.
Pombo C, Kehrl JH, Sanchez I, et al.
Activation of the SAPK pathway by the human STE20 homologue germinal centre kinase.
Nature.
1995;377:750[Medline]
[Order article via Infotrieve].
14.
Anafi M, Kiefer F, Gish GD, Mbamalu G, Iscove NN, Pawson T.
SH2/SH3 adaptor proteins can link tyrosine kinases to a Ste20-related protein kinase, HPK1.
J Biol Chem.
1997;272:27,804[Abstract/Free Full Text].
15.
Shi CS, Leonardi A, Kyriakis J, Siebenlist U, Kehrl JH.
TNF mediated activation of the SAPK pathway: TNF receptor-associated factor-2 recruits and activates GCKR.
J Immunol.
1999;163:3279[Abstract/Free Full Text].
16.
Oehrl W, Kardinal C, Ruf S, et al.
The germinal center kinase (GCK)-related protein kinases HPK1 and KHS are candidates for highly selective signal transducers of Crk family adapter proteins.
Oncogene.
1998;17:1893[Medline]
[Order article via Infotrieve].
17.
Kiyokawa E, Mochizuki N, Kurata T, Matsuda M.
Role of Crk oncogene product in physiologic signaling.
Crit Rev Oncog.
1997;8:329[Medline]
[Order article via Infotrieve].
18.
Sattler M, Salgia R.
Role of the adapter protein CRKL in signal transduction of normal hematopoietic and BCR/ABL-transformed cells.
Leukemia.
1998;12:637[Medline]
[Order article via Infotrieve].
19.
Matsuda M, Ota S, Tanimura R, et al.
Interaction between the aminoterminal SH3 domain of Crk and its natural target proteins.
J Biol Chem.
1996;271:14,468[Abstract/Free Full Text].
20.
Posern G, Zheng J, Knudsen BS, et al.
Development of highly selective SH3 binding peptides for Crk and CrkL which disrupt Crk-complexes with DOCK180, SOS, and C3G.
Oncogene.
1998;16:1903[Medline]
[Order article via Infotrieve].
21.
Senechal K, Halpern J, Sawyers CL.
The CRKL adaptor protein transforms fibroblasts and functions in transformation by the BCR-ABL oncogene.
J Biol Chem.
1996;271:23,255[Abstract/Free Full Text].
22.
Tanaka S, Ouchi T, Hanafusa H.
Downstream of Crk adaptor signaling pathway: activation of Jun kinase by v-Crk through the guanine nucleotide exchange protein C3G.
Proc Natl Acad Sci U S A.
1997;94:2356[Abstract/Free Full Text].
23.
Zhu T, Goh EL, LeRoith D, Lobie PE.
Growth hormone stimulates the formation of a multiprotein signaling complex involving p130(Cas) and CrkII: resultant activation of c-Jun N- terminal kinase/stress-activated protein kinase (JNK/SAPK).
J Biol Chem.
1998;273:33,864[Abstract/Free Full Text].
24.
Dolfi F, Garcia-Guzman M, Ojaniemi M, Nakamura H, Matsuda M, Vuori K.
The adaptor protein crk connects multiple cellular stimuli to the JNK signaling pathway.
Proc Natl Acad Sci U S A.
1998;95:15,394[Abstract/Free Full Text].
25.
Shi C-S, Tuscano J, Witte W, Kehrl JH.
GCKR links the Bcr-Abl oncogene and Ras to the stress activated protein kinase pathway.
Blood.
1999;93:1338[Abstract/Free Full Text].
26.
Greulich H, Hanafusa H.
A role for Ras in v-Crk transformation.
Cell Growth Differ.
1996;7:1443[Abstract].
27.
Parrizas M, Blakesley VA, Bietner-Johnson D, LeRoith D.
The protooncogene Crk-II enhances apoptosis by a ras-dependent, Raf-1/MAP kinase-independent pathway.
Biochem Biophys Res Comm.
1997;234:616[Medline]
[Order article via Infotrieve].
28.
Tanaka S, Hanafusa H.
Guanine-nucleotide exchange protein C3G activates JNK1 by a ras-independent mechanism: JNK1 activation inhibited by kinase negative forms of MLK3 and DLK mixed lineage kinases.
J Biol Chem.
1998;273:1281[Abstract/Free Full Text].
29.
Ezoe K, Lee ST, Strunk KM, Spritz RA.
PTK1, a novel protein kinase required for proliferation of human melanocytes.
Oncogene.
1994;9:935[Medline]
[Order article via Infotrieve].
30.
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.
1994;9:1745[Medline]
[Order article via Infotrieve].
31.
Tibbles LA, Ing YL, Kiefer F, et al.
MLK-3 activates the SAPK/JNK and p38/RK pathways via SEK1 and MKK3/6.
EMBO J.
1996;24:7026.
32.
Hirai S, Katoh M, Terada M, et al.
MST/MLK2, a member of the mixed lineage kinase family, directly phosphorylates and activates SEK1, an activator of c-Jun N-terminal kinase/stress-activated protein kinase.
J Biol Chem.
1997;272:15,167[Abstract/Free Full Text].
33.
Leung IWL, Lassan N.
Dimerization via tandem leucine zippers is essential for the activation of the mitogen-activated protein kinase kinase, MLK-3.
J Biol Chem.
1998;273:32,408[Abstract/Free Full Text].
34.
Yuasa T, Ohno S, Kehrl JH, Kyriakis JM.
Tumor necrosis factor signaling to stress-activated protein kinase (SAPK)/Jun NH2-terminal kinase (JNK) and p38: germinal center kinase couples TRAF2 to mitogen-activated protein kinase/ERK kinase kinase 1 and SAPK while receptor interacting protein associates with a mitogen-activated protein kinase upstream of MKK6 and p38.
J Biol Chem.
1998;273:22,681[Abstract/Free Full Text].
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
Q. Deng, J. Sun, and J. T. Barbieri
Uncoupling Crk Signal Transduction by Pseudomonas Exoenzyme T
J. Biol. Chem.,
October 28, 2005;
280(43):
35953 - 35960.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. C. Sitko, C. I. Guevara, and N. A. Cacalano
Tyrosine-phosphorylated SOCS3 Interacts with the Nck and Crk-L Adapter Proteins and Regulates Nck Activation
J. Biol. Chem.,
September 3, 2004;
279(36):
37662 - 37669.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. M. Felschow, M. L. McVeigh, G. T. Hoehn, C. I. Civin, and M. J. Fackler
The adapter protein CrkL associates with CD34
Blood,
June 15, 2001;
97(12):
3768 - 3775.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction