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J. Biol. Chem., Vol. 276, Issue 31, 29403-29409, August 3, 2001
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,,,,,¶
From the Department of Molecular and Cellular
Oncology, The University of Texas M. D. Anderson Cancer Center,
Houston, Texas 77030 and § Cancer Center, University of
California at Davis, Sacramento, California 95817
Received for publication, April 9, 2001, and in revised form, May 23, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Etk/Bmx, a member of the Tec family of
nonreceptor protein-tyrosine kinases, is characterized by an N-terminal
pleckstrin homology domain and has been shown to be a downstream
effector of phosphatidylinositol 3-kinase. P21-activated kinase 1 (Pak1), another well characterized effector of phosphatidylinositol
3-kinase, has been implicated in the progression of breast cancer
cells. In this study, we characterized the role of Etk in mammary
development and tumorigenesis and explored the functional interactions
between Etk and Pak1. We report that Etk expression is
developmentally regulated in the mammary gland. Using transient
transfection, coimmunoprecipitation and glutathione
S-transferase-pull down assays, we showed that Etk directly
associates with Pak1 via its N-terminal pleckstrin homology domain and
also phosphorylates Pak1 on tyrosine residues. The expression of
wild-type Etk in a non-invasive human breast cancer MCF-7 cells
significantly increased proliferation and anchorage-independent growth
of epithelial cancer cells. Conversely, expression of kinase-inactive
mutant Etk-KQ suppressed the proliferation, anchorage-independent
growth, and tumorigenicity of human breast cancer MDA-MB435 cells.
These results indicate that Pak1 is a target of Etk and that Etk
controls the proliferation as well as the anchorage-independent and
tumorigenic growth of mammary epithelial cancer cells.
Epithelial and endothelial tyrosine kinase (Etk, also called
Bmx)1 belongs to the Tec
family of nonreceptor protein-tyrosine kinases that are characterized
by an N-terminal Tec homology domain located downstream of a pleckstrin
homology (PH) domain (1-3). In addition, Etk contains Src homology-3
(SH3) and -2 (SH2) domains, and a catalytic kinase domain (4). The PH
domain protein module is commonly found in signal transduction proteins
and is believed to help mediate lipid-protein or protein-protein
interactions (5). The PH domains of Etk and Btk (a related Tec family
member) have been shown to interact with heterotrimeric G protein and protein kinase C (6, 7), and these interactions are believed to
regulate kinase activity. Recent studies suggest that activation of
PI3-kinase stimulate Etk, probably because of the direct interaction between the lipid product resulting from PI3-kinase reaction and the PH
domain of Etk (8). The PH domain is believed to be important because
germline mutation in the PH domain of Btk leads to human X-linked
agammaglobulinemia (1, 2, 9). In contrast, overexpression of
kinase-active Btk induces cellular transformation and protects cells
from apoptotic signals (10). Although most of the Tec family kinases
such as Btk, Itk, and Tec are of hematopoietic origin, Etk is found to
be expressed in a variety of tissues and cell types, including lung and
prostate tissues and salivary epithelial and endothelial cells (3, 4,
11).
Because Etk is a cytoplasmic kinase with several motifs characteristic
of signaling molecules, it has been implicated in signal transduction
networks. For example, Etk/Bmx was shown to mediate activation of Rho
and serum response factor in response to the heterotrimeric G proteins
G In addition to Etk, the p21-activated kinases (Paks) represent another
well characterized family of effectors of PI3-kinase. Pak1 is a direct
target of the small GTPases Cdc42 and Rac1, and binding of GTPases to
Pak1 stimulates its kinase activity via autophosphorylation (13).
Expression of kinase-active Pak1 mutant triggers the formation of
lamellipodia, dissolution of stress fibers, and dissolution of focal
adhesion complexes in fibroblast cells (14, 15). Expression of another
kinase-active Pak1 mutant with a mutation in GTPase binding sites
triggers the formation of actin ruffles (15-17). Pak1 kinase activity
is essential for the formation of polarized lamellipodia at the leading
edge (18) and for actin-myosin-mediated cytoskeletal changes (19).
Expression of the kinase-inactive Pak1 mutant blocks the ability of Ras
to induce transformation of Rat1 fibroblast (20), suggesting that Pak1
plays a role in this cell transformation. Furthermore, expression of
kinase-active Pak1 in breast cancer cells stimulates
anchorage-independent growth (21, 22).
Despite the recent reports of the involvement of Etk in signaling
cascades in human cancer cells and the fact both Etk and Pak1 are
downstream of PI3-kinase, the relationship between Etk and Pak1 kinase
and the role of the Etk pathway in the biology of human mammary
epithelial cancer cells remain unknown. We sought to determine the role
of Etk pathway in breast cancer cells. We present new evidence that Etk
is an upstream effector of Pak1 tyrosine phosphorylation and that it is
directly associates with Pak1. Furthermore, we found that Etk activity
is discovered to be required for the proliferation,
anchorage-independent growth, and tumorigenicity of mammary epithelial
cancer cells. These results indicate that Pak1 is a target of Etk and
that Etk regulates the anchorage-independent and tumorigenic growth of
mammary epithelial cancer cells.
Plasmids and Antibodies--
T7-tagged wild-type (Wt)
pcDNA3-T7-Etk (T7-Etk), kinase-inactive pcDNA-T7-Etk (Etk-KQ),
and N-terminal and C-terminal deletion mutants of Etk were previously
described (4, 12). N-Ter ETK contains amino acids 1-240 and C-Ter has
amino acid 243-674 (4, 12). Myc-tagged Pak1 Wt and Pak1 K299R
mutants were generously provided by Jonathan Chernoff and have been
earlier described (16, 18). To construct T7-tagged central inhibitory
fragment of Pak1 (Pak aa 83-149, Ref. 18) Pak1 aa 83-149 domain was
amplified by PCR and subcloned into pcDNA3.1 His (Invitrogen).
Antibodies directed against T7, phosphotyrosine, Pak1, and c-Myc
were purchased from Novagen, Upstate Biotechnology, Santa Cruz
Biotechnology, and Neomarkers, respectively. Dr Hsing-Jien Kung kindly
provided monoclonal antibody to Etk.
Cell Culture and Transfection--
MCF-7, MDA-MB435 human breast
cancer cell lines (18), were maintained in Dulbecco's modified
Eagle's medium (DMEM)/F12 (1:1) supplemented with 10% fetal bovine
serum. Cells were transfected with the desired vector using Fugene-6
reagent (Roche, Nutley). Clonal stable cell lines overexpressing
Wt-Etk or Etk-KQ or control pcDNA were selected in the
presence of G418 resistance (500 µg/ml).
Immunoprecipitation and Kinase Assay--
Cells were lysed in a
buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10% glycerol, 1% Nonidet P-40, 10 mM
NaF, 1 mM NaVO4, and a mixture of protease inhibitors. The
T7-tagged Etk or Myc-tagged Pak1 were immunoprecipitated from the cell
lysates with mAbs directly against T7 or Myc, respectively, as
described (23). When indicated, the immuncomplex was washed with
kinase buffer (20 mM HEPES, pH 7.4, 1 mM
dithiothreitol, 10 mM MnCl2, and 10 mM MgCl2). The kinase reaction was carried out
in buffer supplemented with 7.5 µg of enolase (Sigma) for the Etk
kinase assay and with myelin basic protein (MBP) for the Pak kinase assay.
GST Pull-down Assay--
Wt-Etk, N-Ter, and C-terminal Etk
cDNA (4, 12) were translated in vitro using the TNT
reaction kit (Promega) in the presence of
[35S]methionine. Subsequently, 10 µl of reaction volume
was diluted in 400 µl of protein-binding buffer (20 mM
Tris, pH 7.5, 50 mM NaCl, 10% glycerol, 10 mM
NaF, 1% Nonidet P-40, 1 mM NaV04, and protease inhibitors)
and incubated with 2 µg of Pak1-GST or GST protein beads at 4 °C
for 4 h. The beads were washed six times with 1 ml of each binding
buffer and eluted with 2× SDS-polyacrylamide gel electrophoresis
sample buffer. Elutes were resolved onto a 10% SDS-polyacrylamide gel
electrophoresis and visualized using a phosphorimager.
Cell Proliferation and Soft Agar Colony Formation
Assays--
MCF-7 and MDA-MB435 cells (2 × 104 cells
per well in a 24-well plate) and counted daily for six days.
Anchorage-independent colony assays were performed as described
previously (21, 23). Briefly 1 ml of solution of 0.6% DIFCO Agar in
DMEM supplemented with 10% fetal bovine serum was layered onto 60 × 15 mm tissue culture plates. MCF-7 or MDA-MB 435 cells (10,000 cells) were mixed with 1 ml of 0.36% Bactoagar solution in DMEM
prepared in a similar manner and layered on top of the 0.6% Bactoagar
layer. Plates were incubated at 37 °C in 5% CO2 for two
weeks. In dominant negative Pak1 experiment, MCF-7 cells or
Wt-ETK cells were transfected with 10 µg of GFP or GFP-K299R
Pak1 or GFP-Pak1 inhibitor and tested for anchorage-independent growth.
Tumorigenecity Studies--
Exponentially growing cells (3 × 106) were injected into mammary fat pad (two
sites/animal) of female athymic mice (Nu/Nu, 4 weeks old). Every fourth
day, tumor volumes were measured with calipers along two major axes.
Tumor volume was calculated as follows V = (4/3) Apoptosis--
Tunnel method was used to detect DNA
fragmentation as previously described by Gabriel et al.
(25). Briefly, paraffin-embedded sections pretreated with protease were
nicked and labeled with biotinylated poly(dU), introduced by terminal
deoxy-transferase, and then stained using avidin-conjugated peroxidase.
Reverse Transcription (RT)-PCR and Southern
Hybridization--
Total cytoplasmic RNA was isolated from different
stages of mice tissue using the Trizol Reagent (Life Technologies,
Inc.) and 500 ng of RNA analyzed by RT-PCR. The forward primer for
mEtk was 5'-CACACCACCTCAAAGATTTCATGG-3' and the reverse primer
was 5'-CATACTGCCCCTTCCACTTGC-3'. RT-PCR products were run onto 1% agarose gel, transferred to a blot, and probed with a 520-bp cDNA of mEtk.
In Situ Hybridization--
For in situ hybridization,
mouse mammary glands or 12-day-old embryos were dissected out and fixed
with 4% paraformaldehyde. The tissues were processed into 10 µm of
frozen sections, and in situ hybridization was performed as
described (24). To make the probe, the Etk cDNA fragment was cloned
to TOPO II vector, and RNA probe was labeled with digoxigenin and was
synthesized by in vitro transcription. Sense-probe
hybridization was used as background control.
Etk Expression during Embryogenesis and Mammary Gland
Development--
To explore the role of Etk during cell proliferation
and differentiation, we first explored the expression profile of Etk during mouse embryonic development using in situ
hybridization. As shown in Fig. 1, Etk
mRNA was expressed in most tissues, with highest levels in the
nervous and epithelial tissues, i.e. encephalon, dorsal root
ganglia, pancreas, lung, and the intestinal mucosa. Etk mRNA
signals in the vertebral column and hearts were significantly lower.
In situ hybridization analysis of mouse mammary gland
demonstrated that Etk mRNA signal was much stronger in the
lactating alveoli than in the pregnant mammary gland (Fig.
2A).
Etk/Bmx is usually the only Tec family kinase expressed in epithelial
cells, but the expression level is generally low. Despite the low
level, accumulating evidence suggests that it play an important role in
the growth, differentiation, and apoptosis of epithelial cell (26). To
understand the potential function of Etk in mammary gland, we
investigated Etk expression during various stages of the mammary gland
development by RT-PCR followed by Southern hybridization with a
fragment of human Etk cDNA. Results indicated that Etk expression
appears to undergo a cyclic change as the expression levels of Etk were
down-regulated during pregnancy, early lactation, and again during the
late stages of lactation (Fig. 2, B and C). Etk
expression was lowest during pregnancy, a stage of high proliferation
for mammary glands (Fig. 2, B and C), and highest
in non-proliferative weaning and virgin mammary glands, suggesting that
Etk may have an important role in the biology of mammary epithelial cells.
Expression and Activation of Etk in Breast Cancer
Cells--
Because the potential role of Etk in mammary epithelial
cancer cells is not known, we next examined the expression of Etk in a
panel of breast cancer cell lines by Southern hybridization. Among the
breast cancer cell lines used, highly tumorigenic and metastatic
MDA-MB435 cells exhibited the highest Etk content, whereas the Etk
content of MCF-7 and SKBR-3 was lower (Fig. 2D). Because
MCF-7 and SKBR-3 cells do not form tumors and metastasis in
vivo, it appears that Etk expression may correlate with the degree
of transformation of breast cancer cell lines used here.
To assess the physiological significance of Etk expression in breast
cancer cells, we sought to determine whether Etk kinase activity can be
stimulated by physiologically relevant molecules in mammary gland, as
heregulin- Pak1 Phosphorylation and Activation by Etk in Breast Cancer
Cells--
Because both Etk and Pak1 are downstream of PI3-kinase, we
sought to determine whether Etk could activate Pak1. Co-transfection of
MCF-7 cells with T7-tagged Etk and Myc-tagged Pak1 constructs significantly increased Pak1 kinase activity as determined by an
immune-complex kinase assay using MBP as a substrate (Fig. 3B, upper panel). Because Etk is a tyrosine
kinase, we hypothesized that Etk phosphorylates Pak1 on tyrosine
residues. Indeed, we discovered that co-transfection of Etk and Pak1
was accompanied by a substantial stimulation of tyrosine
phosphorylation of Pak1 (Fig. 3B, lower panel).
To verify that Pak1 is downstream of Etk, we next demonstrated that
coexpression of a kinase-defective K299R Pak1 mutant protein
(designated dominant-negative (DN)-Pak1) suppressed the ability of Etk
to activate Pak1 kinase activity (Fig. 3C). In the targeted
Pak1 K299R sequence, the lysine 299 ATP binding site was replaced by
arginine, rendering Pak1 catalytically defective as shown by us and
others in cell lines (16, 18). To rule out that the observed inhibitory
effect of Pak1 K299R mutant was not due to ineffective transduction of
Cdc42/Rac signals, we next generated stable MCF-7 clones expressing the
central inhibitory fragment of Pak1 aa 83-149 (Fig. 3D,
upper panel), which does not interfere with cdc42/Rac1
binding. These stable cell lines expressing control vector or the
central Pak inhibitor were transfected with T7-tagged Etk, and the
effect Etk on Pak1 kinase activity was measured by subjecting the
immunoprecipitated T7-Etk to in vitro kinase assay using MBP
as a substrate. The Etk was able to activate Pak1 activity in
vector-transfected cells but not in the cells that express Pak
inhibitor aa 83-149 (Fig. 3D). Together, these results
confirm that Etk regulates Pak kinase activity. While at present, we do
not know whether tyrosine phosphorylation of Pak1 induced by Etk
contributes to its elevated activity. To our knowledge, this is the
first report that shows that Pak1 is tyrosine-phosphorylated and serves
as a downstream substrate of Tec family of kinase. While interesting
and reproducible, at present we do not know the significance of the
tyrosine phosphorylation, and experiments are planned to explore the
significance of this finding.
Interaction of Etk with Pak1 in Vivo--
To determine whether the
observed activation of Pak1 by Etk is due to interactions between the
two proteins, we examined the association between T7-tagged Etk and
Myc-tagged Pak1 in vivo by reciprocal co-immunoprecipitation
and Western blot assays. Results of a representative experiment are
shown in Fig. 3E. Transient expression of T7-tagged Etk, but
not of control T7 vector, in MCF-7 cells was accompanied by the
association of T7-tagged Etk with Myc-tagged Pak1 in both
immunoprecipitation experiments. These results suggest that Etk
associates with Pak1 and stimulates tyrosine phosphorylation and kinase
activity of Pak1 (Fig. 3E).
To determine whether the observed association between Etk and Pak1 was
direct or mediated via other proteins, we examined the ability of
in vitro translated Wt-Etk or Etk-KQ protein to bind
GST-Pak1 in GST pull-down assays. As shown in Fig. 3F, Etk and its N-terminal domain (aa 1-240) strongly interact with GST-Pak1; very little interaction was seen between Etk C-terminal domain (aa
243-674) and GST-Pak1, and GST alone provided a proper negative control.
Etk in Proliferation and Anchorage-independent Growth of Breast
Cancer Cells--
To further delineate the contribution of Etk in the
biology of breast cancer cells, we established stable MCF-7 clones
expressing T7-tagged Wt-Elk or kinase-inactive Etk-KQ or control
vector. The results shown in Fig.
4A demonstrate the expression
of tagged Etk in several representative clones. The functionality of
Etk was assessed by performing in vitro kinase assays using
enolase as an exogenous substrate (Fig. 4B). To determine
how Etk affects the Pak1 pathway, MCF-7/Etk cells were transfected with
Myc-Pak1. Overexpression of Etk in MCF-7 cells was associated with
significant increases in the phosphorylation of Pak1 on tyrosine (Fig.
4C). MCF-7 cells expressing Wt-Etk were transfected with or
without Myc-tagged Pak1, and tagged Pak1 was immunoprecipitated by an anti-Myc mAb and subjected to in vitro kinase assay. The
upper and lower bands in Fig. 4D
represent the autophosphorylated T7-Etk (73 kDa) and Myc-Pak1 (64 kDa),
respectively. These protein bands were identified due to their
differential electrophoretic mobilities in the gel.
To examine the influence of Etk expression on the growth
characteristics of breast epithelial cancer cells, we measured the proliferation rate and the ability of cells to grow in an
anchorage-independent manner. Compared with vector-transfected control
cells, cells in which Wt-Etk was overexpressed demonstrated greater
ability to form larger colonies in soft agar, and expression of Etk-KQ mutant led to a reduction in the anchorage-independent growth (Fig.
5,A and B). In
addition, as shown in Fig. 5C, the growth rate of
cells expressing Wt-Etk and Etk-KQ was affected 35-40% more than that
of the control vector-transfected clone. Because Pak1 has been shown to
promote the anchorage-independent growth (21, 22), we next determined
whether dominant-negative Pak1 could modulate the ability of Wt-Etk
cells to form colonies in soft agar. Wt-Etk cells were transfected with
GFP-dominant-negative Pak1 and GFP-Pak1 inhibitor and used for
soft agar assay. As shown in Fig. 6,
A and B, Wt-Etk cells transfected with
dominant-negative Pak1 or Pak1 inhibitor form small colonies compared
with Wt-Etk control cells. Together, these findings suggested that Pak1
is a downstream effector of Etk pathway and may contribute to the observed phenotypic changes by Etk.
Effect of Overexpression of Kinase-inactive Etk of Breast Cancer
Cells--
We next sought to determine whether Etk activity is
required for the maintenance of the transformed phenotypes in breast
cancer cell lines. Highly tumorigenic and invasive MDA-MB435 cells were stably transfected with T7-tagged kinase-inactive Etk mutant (Etk-KQ) or with control pcDNA vector (Fig.
7A, upper panel).
As expected from the results shown in Fig. 4, overexpression of Etk-KQ
led to a significant reduction in Etk and Pak1 kinase activities (Fig. 7B). The expression of Etk-KQ was accompanied by a
significant inhibition of the growth rate of MDA-MB435 cell (Fig.
7C). In addition, overexpression of kinase-inactive Etk-KQ
reduced the ability of cells to grow in soft agar as compared
with vector-transfected control cells (Fig. 7, D and
E).
To investigate the significance of Etk expression in vivo,
we next examined the ability of MDA-MB435 clones expressing
kinase-inactive Etk-KQ or vector control in a xenograft model. In these
experiments, cells were implanted into the mammary fat pad of athymic
mice. Inactivation of Etk in MDA-MB435 cells severely affected the
ability of cells to form tumors (i.e. by 55-70% compared
with vector-transfected cells) (Fig.
8A). Histological examinations
of tumors with hematoxylin and eosin staining revealed the presence of
necrotic areas in tumors from Etk-KQ clones (data not shown).
Reevaluation of these tumors with TUNEL staining confirmed that
apoptosis was widespread in Etk-KQ tumors (Fig. 8B).
Together, these findings suggest that Etk expression may be required
for the maintenance of transformed phenotypes in breast cancer
cells.
Etk is a member of the Tec family of the nonreceptor
tyrosine kinases that are characterized by N-terminal PH domains. The PH domain is important in protein-protein interactions and is involved
often in cytoplasmic signaling cascades. Etk is one of the few Tec
family members, which are expressed in epithelial cells (10). Here we
sought to determine the role of Etk in regulating breast cancer growth
regulation. We report that: 1) Etk expression is developmentally
regulated during mammary gland development; 2) Etk is expressed in the
highly tumorigenic MDA-MB435 cell lines; 3) Etk is
tyrosine-phosphorylated and is activated by physiologically relevant
growth factor in breast cancer cells; 4) Etk phosphorylates Pak1 on
tyrosine residues, and kinase-inactive Pak1 mutant blocked Etk
activation of Pak1; 5) Etk directly interacts with Pak1 via the
N-terminal PH domain-containing region; 6) overexpression of Wt-Etk in
noninvasive breast cancer line enhanced the ability of the cells to
grow in an anchorage-independent manner; and 7) expression of
kinase-inactive Etk inhibits tumorigenic phenotypes in a highly
tumorigenic breast cancer cell line. Taken together, these observations
suggest that Etk play an important role in the regulation of mammary
epithelial cancer cells.
The finding that Pak1 kinase activity is stimulated following Etk
kinase activation is important, as it implies that Etk kinase constitutes an initial signal for Pak1 activation. This hypothesis is
supported by several additional observations: 1) inhibition of Etk
kinase by a kinase-defective mutant was accompanied by concurrent
inhibition of Pak1 activity; 2) expression of dominant-negative Pak1
mutant blocked the ability of Etk to activate the Pak1 kinase and did
not affect the Etk kinase; 3) Etk directly interacted with Pak1 via its
N-terminal PH domain; and 4) Etk-mediated stimulation of
anchorage-independent growth was blocked by dominant-negative Pak1.
These results suggest that Pak1 may be downstream of Etk kinase in
breast cancer cells. These findings are inconsistent with those in a
recent report (6) that showed activation of Rho rather than of Cdc42 or
Rac1 (upstream regulators of Pak1) by Tec family members in mouse 3T3
fibroblast cells. To reconcile these findings, we suggest that the Tec
family may utilize distinct members of the small GTPase family members
in epithelial cancer and fibroblast cells in humans and/or mice.
Another notable finding in this study was that Etk induced tyrosine
phosphorylation of Pak1 and that Etk activity is required for the
proliferation, anchorage-independent growth and tumorigenicity of
breast cancer cells. This finding strongly suggests that Etk utilize
Pak1 tyrosine phosphorylation to influence transformed phenotypes that
are generally believed to be driven by tyrosine phosphorylation. This
hypothesis is further supported by recent findings by us and by others
that overexpression of kinase-active Pak1 mutant (T423E, predominantly
serine phosphorylation) in breast cancer cells selectively enhanced the
anchorage-independent growth of breast cancer cells (21, 22).
Currently, we do not know the precise mechanism by which Etk exerts its
profound stimulatory effects in the transformation functions of cancer
cells. It is possible that in addition to Pak1, there are other
unidentified downstream effectors for Etk pathway that contribute the
observed phenotypic changes. Studies are in progress to investigate
these and other possibilities.
Our findings clearly established, for the first time, that Etk kinase
directly associates with Pak1 and stimulates Pak1 tyrosine phosphorylation and that Etk controls the anchorage-independent growth
rate and tumorigenic behavior of human mammary epithelial cancer cells.
These observations open a new avenue of investigation closely linking
the Tec and Pak families with breast cancer cell activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-12 and -13 that can be activated by hormones and
neurotransmitters (6). In addition, Etk was shown to be a substrate of
Src kinases and to be responsible for Src activation of signal
transducer and activator of transcription factor 3 (STAT3) and for
cellular transformation (12). Experiments using a kinase inactive
mutant Etk-KQ showed that Etk kinase activity was required for
interleukin 6-induced neuroendocrine differentiation of prostate cancer
cells (4). Furthermore, dominant-negative mutants of PI3-kinase blocked
interleukin 6-induced stimulation of Etk in this system, suggesting
that Etk is an effector of PI3-kinase (4).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R12 R2, where R1 is radius
1 and R2 is radius 2 and R1 < R2
(n = 10 per group) (24).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Etk expression during
embryogenesis. In situ hybridization using antisense
(AS) or sense (S) probe. High Etk expression in
high levels in the nervous and epithelial tissue, i.e. the
dorsal root ganglia (Drg), the mucosa of the intestine
(Int), the pancreas (Pan), and the lung
(Lu).
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Fig. 2.
Etk expression during the mammary gland
development. A, in situ hybridization, a
strong Etk signal was observed in the epithelial cells during lactation
(L, day 4) but not pregnancy (P, day 15) stage.
L/S, sense probe hybridization in the lactation mammary
gland. B, RNA (1 µg) at various stages of mammary gland
development (V, virgin weeks; L, lactation days)
was analyzed for the expression of Etk and GAPDH control by RT-PCR
followed by Southern hybridization. C, RNA at virgin
(V), pregnancy (P), lactation (L) and
post weaning (PW) stages (days) of mammary gland development
was analyzed. D, expression of Etk in breast cancer cells.
RNA (1 µg) from the indicated cell lines was analyzed for Etk
expression by RT-PCR followed by Southern hybridization. The results
are representative of two similar experiments.
1 (HRG), a polypeptide growth factor with a role in the
development and tumorigenesis of mammary epithelial cells (18). Indeed,
we found that HRG activated the autophosphorylation of Etk in MCF-7
breast cancer cells (Fig. 3A).
Because of the low level of expression and relatively low avidity of
the antibodies currently available, it has been difficult to visualize
endogenous Etk in cell biology study. Therefore, we resorted to express
T7-tagged Etk for this type of analysis.
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Fig. 3.
Etk regulation of Pak1 in MCF-7 breast cancer
cells. A, HRG stimulation of Etk kinase activity.
T7-tagged Etk was transfected into cells (lanes 2 and
3), and cells were treated with HRG (30 ng/ml) for 10 min
(lane 3). Immunoprecipitated (IP) Etk was used
for an in vitro kinase, and the phosphorylated Etk is shown
(upper panel). Immunoprecipitated Etk was immunoblotted with
anti-T7 antibody (lower panel). B, Etk stimulates
tyrosine phosphorylation and kinase activity of Pak1. Cells were
transfected with Myc-Pak1 with or without T7-Etk. Cell lysates were
immunoprecipitated with Myc antibody and subjected to in
vitro kinase assay using MBP. The reaction products were resolved
by 10% SDS-polyacrylamide gel electrophoresis and transferred to a
nitrocellulose and autoradiograph showing MBP phosphorylation
(first panel). The blot was reprobed with mAb 4G10 to show
Pak1 tyrosine phosphorylation (second panel) and then with
Myc antibody (fourth panel). Cell lysates were
immunoprecipitated with T7-antibody to show the expression of T7-Etk
(third panel). C, cells were co-transfected with
Myc-Pak1 and T7-Etk in the presence or absence of DN-Pak1. Cell lysates
were immunoprecipitated with Myc antibody and subjected to in
vitro kinase assay using MBP, and the blot was blotted with Myc
antibody. D, Etk interacts with Pak1 in vivo.
Cells were transfected with Myc-Pak1 or T7-Etk or both. Cell lysates
were immunoprecipitated with antibodies directed against T7 or Myc and
Western blotted with antibodies against T7 or Myc as indicated.
E, stable clones expressing Pak1 inhibitors and
pcDNA control cells were transfected with Wt-Etk,
immunoprecipitated with Pak1, and subjected to in vitro
kinase assay using MBP (upper panel). Cell lysates were
immunoprecipitated with T7 antibody to show the expression of T7-Etk
(middle panel). The expression of Pak1 inhibitors
were verified by PCR (lower panel). F, GST pull-down assays
to show the association of Pak1 with the in vitro translated
Wt-Etk, N-Ter-Etk, or C-Ter-Etk. The first three lanes show
the inputs. The results are representative of three similar
experiments.
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Fig. 4.
Overexpression of Etk stimulated Pak1
tyrosine phosphorylation. A, characterization of MCF-7
clones expressing T7-tagged Wt or kinase-inactive (KQ) Etk
by Western blotting by anti-T7 mAb or control vinculin Ab.
B, baseline Etk kinase activity in Wt-Etk clones. Lysates of
cells were immunoprecipitated with anti-Etk antibody, followed by
in vitro kinase assays. The autophosphorylated Etk and
enolase as an exogenous substrate of Etk are shown. C, the
indicated Wt-Etk clones were transfected with or without Myc-tagged
Pak1. The Myc-Pak1 was immunoprecipitated by an anti-Myc mAb,
immunoblotted with antiphosphotyrosine Ab, and then immunoblotted with
anti-T7 and Myc mAb. D, Wt clones were transfected with or
without MYC-tagged Pak1, and tagged Pak1 was immunoprecipitated by an
anti-Myc mAb subjected to in vitro kinase assay.
Autophosphorylated Etk and Pak1 are also shown. Results shown are
representative of three experiments.
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Fig. 5.
Etk regulation of cell growth and
anchorage-independent growth of breast cancer cells. The effects
of Wt-Etk and Etk-KQ expression on anchorage-independent growth
of MCF-7 cells. MCF-7 cells, Wt-Etk and Etk-KQ cells (104
cells/plate) were seeded in soft agar dishes (35-mm diameter) and
colonies with diameters larger than 1 mm were counted after 2 weeks of
incubation. The results are representative of three similar
experiments. B, the numbers given are mean values ± S.E. of three independent experiments performed in triplicate.
C, the effects of Wt-Etk or Etk-KQ expression on the growth
rate of MCF-7 cells. Cells were seeded at a density of 2 × 104 in 24-well tissue culture plates in 10% fetal bovine
serum/DMEM. After 4 and 6 days, cells were counted using a coulter
counter. Each point represents the mean ± S.E. of two
replicate wells. Asterisks indicate p < 0.05 by Student's t tests.
View larger version (78K):
[in a new window]
Fig. 6.
Dominant-negative inhibition of Pak1
reduces soft agar cloning efficiency of Wt-Etk cells.
A, MCF-7 cells and Wt-Etk cells were transfected with
GFP-K299R Pak1 or GFP-Pak1 inhibitor and tested for
anchorage-independent growth. Using a fluorescent lamp with excitation
for GFP visualization captured the images presented on the left
panel, and the right panel represents the phase
contrast image of the same microscopic field to show total cells.
B, the colony diameters of Wt-Etk cells transfected with
Pak1 K299R and Pak1 inhibitor aa 83-149. Representation results from
two independent experiments are shown here.
View larger version (73K):
[in a new window]
Fig. 7.
Kinase-defective Etk inhibits tumorigenicity
of breast cancer cells. A, characterization of
MDA-MB435 clones expressing T7-Etk by Western blotting with anti-T7 mAb
and with vinculin Ab. B, Etk and Pak1 kinase activities in
Etk-KQ cells. Etk-KQ cells were immunoprecipitated with anti-Etk
antibody or PAK1 antibody followed by in vitro kinase
assays. The autophosphorylates Etk (upper panel) and
enolase (substrate for Etk) or MBP (substrate for Pak1) (lower
panel) are shown. C, effect of Etk-KQ on the growth
rate of exponentially growing clones as determined by counting the
numbers of cells. D, effect of Etk-KQ expression on
anchorage-independent growth of MDA-MB435 cells over 14 days.
E, representative results from two independent experiments
are shown here.
View larger version (61K):
[in a new window]
Fig. 8.
Effect of kinase-defective Etk on breast
cancer tumorigenicity in vivo. A,
MDA-MB435 clones expressing control vector or Etk-KQ clones were
injected subcutaneously in the mammary gland fat pad, and tumor volume
was recorded (n = 10/group). B,
histological examination of tumors by TUNEL staining. Quantitation of
apoptotic cells using NIH image analysis program from 10 fields is
shown in the lower right panel.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
| |
FOOTNOTES |
|---|
* This study was supported in part by National Institutes of Health Grants CA80066 and CA65746 and by the Breast Research program of The University of Texas M. D. Anderson Cancer Center (to R. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: E-mail: rkumar@mdanderson.org.
Published, JBC Papers in Press, May 29, 2001, DOI 10.1074/jbc.M103129200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: Etk, epithelial and endothelial tyrosine kinase; PH, pleckstrin homology; Pak1, p21-activated kinase; Wt, wild type; aa, amino acid; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; mAb, monoclonal antibody; MBP, myelin basic protein; N-Ter, N-terminal; C-Ter, C-terminal; RT, reverse transcription; HRG, heregulin.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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