(Received for publication, June 8, 1995; and in revised form, August 17, 1995)
From the
In rat liver epithelial cell lines (WB or GN4), angiotensin II
(Ang II) stimulates cytosolic tyrosine kinase activity, in part,
through a calcium-dependent mechanism. In other cell types, selected
hormones that activate G- or G
-coupled
receptors stimulate the soluble tyrosine kinase, p125
.
Immunoprecipitation of p125
from Ang II-activated GN4
cells demonstrated a doubling of p125
kinase activity.
However, an additional Ang II-activated tyrosine kinase (or kinases)
representing the majority of the total activity was detected when the
remaining cell lysate, immunodepleted of p125
, was
reimmunoprecipitated with an anti-phosphotyrosine antibody.
Cytochalasin D pretreatment blocks G-protein receptor-dependent
tyrosine phosphorylation in Swiss 3T3 cells. While cytochalasin D
decreased the Tyr(P) content of 65-75-kDa substrates in Ang
II-treated GN4 cells, it did not diminish tyrosine phosphorylation of
115-130-kDa substrates, again suggesting activation of at least
two tyrosine kinase pathways in GN4 cells. To search for additional Ang
II-activated enzymes, we used molecular techniques to identify 20
tyrosine kinase sequences in these cell lines. None was the major
cytosolic enzyme activated by Ang II. Specifically, JAK2, which had
been shown by others to be stimulated by Ang II in smooth muscle cells,
was not activated by Ang II in GN4 cells. Finally, we purified
Tyr(P)-containing tyrosine kinases from Ang II-treated cells, using
anti-Tyr(P) and ATP affinity resins; 80% of the tyrosine kinase
activity migrated as a single 115-120-kDa tyrosine-phosphorylated
protein immunologically distinct from p125
. In summary,
Ang II activates at least two separate tyrosine kinases in rat liver
epithelial cells; p125
and a presumably novel, cytosolic
115-120-kDa protein referred to as the calcium-dependent tyrosine
kinase.
In analyzing EGF()-dependent tyrosine phosphorylation
in rat liver epithelial cell, two waves of Tyr(P) substrate
phosphorylation occurring at 5 and 60 s were noted (1) . The
latter group appeared to be phosphorylated in part due to activation of
a second process (e.g. a downstream tyrosine
kinase)(2) . Subsequently, we identified several of the same
substrates in Ang II, vasopressin, or epinephrine-treated cells, and
determined that these G-protein-coupled receptors stimulated tyrosine
phosphorylation in a calcium-dependent, protein kinase C-independent
manner(2, 3, 4) . Since virtually all
tyrosine kinases autophosphorylate on tyrosine residues, immune complex
tyrosine kinase activity could be assessed in anti-Tyr(P)
immunoprecipitates from control and Ang II-treated cells. Ang II
increased tyrosine kinase activity within 15-30 s; maximal
activation was seen within 1 to 2 min. Activation was abrogated by
intracellular chelators that blunt the Ang II-induced calcium
signal(3) . The mechanism by which calcium increases tyrosine
kinase activity remains unclear, but the schema is presumably indirect
because tyrosine kinase activity can not be stimulated by adding
calcium to cell-free extracts of these cells. Thus, our previous work
showed that G-protein-coupled receptors generating a calcium signal
rapidly, but indirectly, activate one or more tyrosine
kinase(s)(2, 3, 4) .
Other hormones
activating G-protein-coupled receptors also increase tyrosine
phosphorylation (e.g. bombesin(5, 6) ,
bradykinin (7) , thrombin(8, 9, 10) ,
carbachol(11) , endothelin(5, 6) ,
cholecystokinin(12) , lysophosphatidic acid(13) , and
fMet-Leu-Phe (14) ), and our demonstration of Ang II- and
vasopressin-dependent tyrosine phosphorylation has been confirmed by
several
groups(15, 16, 17, 18, 19, 20) .
While many of these hormone receptors (like Ang II) activate
G-proteins increasing phospholipase C activity and
intracellular calcium(21) , several activate
G
-coupled receptors, which would not be expected to raise
intracellular calcium(22) . Whatever the mechanism, several
investigators established that stimulation of G
- or
G
-coupled receptors activated a 125-kDa tyrosine kinase.
This kinase, p125
, localizes to focal
adhesions(23, 24, 25) and had been cloned as
a substrate of
p60
(26, 27) . In
concurrent work, several laboratories, including our own, showed that
signaling via integrins also stimulated p125
phosphorylation and
activity(28, 29, 30, 31) . This
analogy between G-protein stimulation and cell surface perturbation is
extended by the fact that both integrin (32) and hormone
stimulation (see below) activate MAP kinase.
Many of the hormones
noted above that activate tyrosine phosphorylation also stimulate cell
proliferation and the immediate-early gene expression that accompanies
growth factor action. The latter is likely to occur by activation of
the MAP kinase (ERK 1 and ERK 2) pathway(33, 34) , or
the newly characterized c-Jun N-terminal kinase (JNK) pathway (35) . In fact, both G-coupled receptors (in some
instances via a calcium-dependent mechanism) and G
-coupled
receptors have been shown to activate MAP
kinase(15, 16, 20, 36, 37, 38, 39) .
In addition, our laboratory has shown recently that Ang II treatment of
rat liver epithelial cells produces a 200-fold activation of JNK
through a calcium- and tyrosine kinase-dependent process.
In summary, data from several cell types suggest that the
selected G
- and G
-coupled receptors as well as
other agonists (e.g. integrin stimulation) can activate
p125
, MAPK, and JNK. The potential stimulation of several
pathways thought to be controlled by tyrosine phosphorylation led us to
ask whether Ang II stimulated p125
or another tyrosine
kinase(s) or both.
We report that Ang II stimulates at least two
cytosolic tyrosine kinases. The first, pp125, is
tyrosine-phosphorylated and activated in these cells in an Ang
II-dependent manner; however, greater than 80% of the tyrosine kinase
activity resides in another molecule that can be distinguished from
p125
. Molecular means were used to identify 20 tyrosine
kinases in the cells, but none appear to be the calcium-dependent
cytosolic tyrosine kinase. In contrast, we have partially purified an
autophosphorylating kinase from Ang II-treated cells by sequential
affinity chromatography. This activity migrates as a 115-120-kDa
Tyr(P) protein that can be separated electrophoretically and
distinguished from pp125
. The possibility is raised that
distinct Ang II-dependent intracellular signaling pathways are
activated through separate tyrosine kinases.
Figure 1:
Subcellular
fractionation of WB cell phosphotyrosine substrates. WB cells were
treated with EGF (1 µg/ml) or Ang II (1 µM) for 1 min
and homogenized in buffers described under ``Experimental
Procedures.'' Following ultracentrifugation at 105,000 g for 45 min, the particulate fraction was resuspended in an
equivalent volume of buffer and each fraction (homogenate, cytosol, and
membrane) was boiled in SDS sample buffer. Following SDS-PAGE and
transfer to nitrocellulose a phosphotyrosine immunoblot was performed,
as under ``Experimental Procedures.'' Ang II-dependent Tyr(P)
substrates were found predominantly in the cytosolic
fraction.
To determine the
localization of the tyrosine kinase(s) activated in Ang II-treated
cells, the experiment was repeated using GN4 cells, a chemically
transformed line derived from WB, that expresses 3-fold more Ang
II-stimulated tyrosine kinase activity. After separating homogenate,
cytosol, and membrane fractions, detergent was added, followed by
immunoprecipitation with anti-phosphotyrosine monoclonal antibody PT66.
Immune complex tyrosine kinase activity was performed as described
under ``Experimental Procedures'' assessing the transfer of
P from [
-
P]ATP to the
exogenous substrate poly(Glu
-Tyr
). In
immunoprecipitates from the homogenate fraction, Ang II treatment
increased tyrosine kinase activity by
2.5-fold (2,637 cpm for
control to 6,199 cpm for Ang II-treated). At least 75-80% of this
activity was found in the immunoprecipitates of cytosolic Tyr(P)
proteins (740 cpm for control, 4,596 cpm for Ang II-treated). In
contrast, little activity was found in the immunoprecipitates from the
membrane fraction (699 cpm for control, 313 cpm for Ang II-treated).
Thus, the majority of the Tyr(P) substrates and virtually all of the
increased tyrosine kinase activity were found in the cytosolic
fraction.
Figure 2:
Phosphotyrosine immunoblot of
immunoprecipitated proteins following Ang II treatment. Rat liver
epithelial (GN4) cells were treated with Ang II and lysed as under
``Experimental Procedures.'' Samples were then
immunoprecipitated with normal mouse IgG (mIg),
anti-phosphotyrosine monoclonal PT66 (p-tyr), or
anti-p125 (FAK) antibodies as indicated, boiled
in SDS sample buffer, subjected to SDS-PAGE, and immunoblotted for
phosphotyrosine (PT66). Data in the single immunoprecipitation (left panel) indicate that Ang II stimulates p125
tyrosine phosphorylation, but that PT66 immunoprecipitations
contain substantially more Tyr(P) substrate. In the double
immunoprecipitation (right panel), the supernatants of the
first immunoprecipitation were reimmunoprecipitated with PT66 or FAK
antibody as labeled, showing the lack of depletion of
phosphotyrosine-containing proteins following the immunodepletion of
FAK.
While the above
indicated that p125 is only a small fraction of the
Tyr(P) substrate, it did not determine whether p125
was
the major autophosphorylating kinase in immune complexes. Tyrosine
kinase activity assays were performed on 2A7 (p125
monoclonal) and PT66 (anti-Tyr(P) antibody) immunoprecipitates.
In five experiments, the immune complex p125
activity
measured by the ability of p125
immunoprecipitates to
phosphorylate poly(Glu
-Tyr
) nearly doubled
when isolated from cells after Ang II treatment (1 min), from an
average of 6,747 cpm for control to 11,730 cpm for Ang II. The immune
complex tyrosine kinase activity of the PT66 immunoprecipitate paired
with a representative p125
experiment rose from a control
of 15,656 cpm to 66,342 cpm after Ang II treatment (1 min).
Autophosphorylation activity was also assessed by repeating the single
and sequential immunoprecipitation protocol in control or Ang
II-treated GN4 cells and assessing incorporation from
[
-
P]ATP into precipitated protein in
vitro, followed by gel electrophoresis and autoradiography of
dried gels as described under ``Experimental Procedures.'' Fig. 3demonstrates that p125
autokinase activity
in 2A7 immunoprecipitates is slightly elevated (lanes 3 and 6) in Ang II-treated cells (1 min), corresponding to the
increase in immune complex poly(Glu
-Tyr
)
activity noted above. Ang II-dependent PT66 immune complex auto kinase
activity was much greater (Fig. 3, lanes 2 and 5). In the sequential protocol, immunoprecipitation removing
nearly all immunoreactive p125
from the supernatant did
little to diminish the subsequent PT66-immunoprecipitable autokinase
activity seen in the 115-125-kDa region (Fig. 3, lanes
10 and 11). Examination of lanes 5, 10,
and 11 in Fig. 3revealed a diffuse band above 120 kDa
of phosphorylated protein and one sharp band of in vitro phosphorylated protein at
115-120 kDa. On shorter
exposures of the autoradiograph, this single band is even more
prominent. This is observed either before (lane 10) or after (lane 11) the removal of p125
by
immunoprecipitation, suggesting that a major autophosphorylation
protein was not removed by p125
antibody.
P-Phosphoamino acid analysis by two dimensional thin layer
chromatography has shown that all [
P]phosphate
added in vitro was on tyrosine residues (data not shown). This
analysis suggested that an autophosphorylating kinase at 115-120
kDa may be the most prominent in vitro labeled protein, a
supposition substantiated by the purification discussed below.
Figure 3:
Autophosphorylation of immunoprecipitated
GN4 cell Tyr(P) substrates. Rat liver epithelial (GN4) cells were
treated with Ang II (1 min) and lysed as under ``Experimental
Procedures.'' Samples were then immunoprecipitated with normal
mouse IgG (mIg), anti-phosphotyrosine PT66 (p-tyr),
or anti-p125 (FAK) antibodies as indicated.
Immune complexes were incubated with
[
-
P]ATP in phosphorylation buffer as
described under ``Experimental Procedures.'' Following a
2-min incubation the samples were boiled in SDS sample buffer,
subjected to SDS-PAGE, and autoradiographed. The data in the single
immunoprecipitation (left panel) indicate that Ang II
stimulates p125
autophosphorylation, but that the Tyr(P)
immune complexes exhibit much greater total autokinase activity. In the
double immunoprecipitation (right panel), the supernatants of
the first immunoprecipitation were reimmunoprecipitated as labeled,
showing the lack of depletion of tyrosine kinase activity following
immunodepletion of FAK, suggesting the presence of other activated,
tyrosine-phosphorylated, tyrosine kinases after Ang II
treatment.
In
studies of p125 activation via G-protein-coupled
receptors, others tested whether the cytoskeletal was involved in the
signal transduction pathway(19, 24) . Preincubation of
cells with cytochalasin D, an agent that disrupts actin microfilaments
and cytoskeletal movement, prevented the tyrosine phosphorylation by
G-protein-coupled receptor agonists (e.g. bombesin) in Swiss
3T3 cells(19, 24) . GN4 cells were preincubated with 2
µM cytochalasin D for 2 h prior to stimulation with Ang II
(1 min). Fig. 4demonstrates that cytochalasin D had little or
no effect on Ang II-dependent tyrosine phosphorylation of substrates in
the 115-130-kDa region. We did not assess p125
tyrosine phosphorylation in this experiment, but Fig. 2demonstrated that depletion of Tyr(P) p125
from the 115-130-kDa substrates region would not alter the
Tyr(P) immunoblotting pattern in Ang II-treated cells. Cytochalasin D
pretreatment did, however, distinctly inhibit tyrosine phosphorylation
of 65-75-kDa substrates in Ang II-treated GN4 cells. In other
cell types, Tyr(P) substrates in this region have proved to be the
cytoskeletal protein paxillin, which is tyrosine-phosphorylated in
cells stimulated by hormones binding to G-protein-coupled
receptors(24) . In conclusion, Ang II stimulates rapid tyrosine
phosphorylation of two groups of substrates: one sensitive and one
insensitive to cytochalasin D. This may occur because Ang II activates
at least two tyrosine kinase pathways.
Figure 4: Lack of complete inhibition of Ang II-dependent tyrosine phosphorylation in cells preincubated with cytochalasin D. GN4 cells were pretreated with 2 µM cytochalasin D for 2 h and then treated with Ang II for 1 min. Cells were lysed, and lysates were subjected to 8% SDS-PAGE and transferred to nitrocellulose as described under ``Experimental Procedures.'' Phosphotyrosine immunoblotting (PT66) shows the presence of tyrosine phosphorylation in Ang II-treated cells that is resistant to cytochalasin inhibition (115-130-kDa region). The tyrosine phosphorylation in the 65-75-kDa region stimulated by Ang II is inhibited by cytochalasin.
Figure 5:
JAK2 is not significantly
tyrosine-phosphorylated in Ang II-treated GN4 cells. GN4 cells were
treated with Ang II (AII, 1 µM), growth hormone (GH, 100 ng/ml), or interferon (IFN
, 500
units/ml) for the indicated times. Cells were lysed and
immunoprecipitated with anti-phosphotyrosine (PT66) antibodies. Immune
complexes were subjected to SDS-PAGE, transfer to nitrocellulose, and
immunoblotting with anti-JAK2 antibody (generously provided by James
Ihle, St. Judes). Growth hormone stimulated JAK2 autophosphorylation,
while Ang II did not. The effect of IFN
was minimal. Lane 1 was immunoprecipitated with anti-JAK2, showing total
immunoprecipitable JAK2 from GN4 cells.
Figure 6:
Purification of a 120-kDa tyrosine kinase
activity in GN4 cells by phosphotyrosine and ATP affinity column
chromatography. Cells were treated with Ang II (1 µM) for
1 min, lysed in detergent buffer, and purified over
phosphotyrosine-agarose (PT66) as described under ``Experimental
Procedures.'' Proteins were eluted from the PT66-agarose with 10
mM phenyl phosphate and passed over a ATP-Sepharose column,
from which they were eluted with 1 mM ATP. The eluates were
immunoprecipitated with PT66-agarose at 0 °C and eluted by boiling
in SDS sample buffer. Aliquots were saved at each step for Tyr(P)
immunoblot analysis. A, phosphotyrosine immunoblot of a
standard 3%/8% SDS-PAGE gel showing proteins eluted from
phosphotyrosine (PT66)-agarose (P-tyr eluate), the proteins
not bound by ATP-agarose (ATP flow thru), the eluate from the
ATP-agarose (ATP eluate), and concentration of the ATP eluate
by a second phosphotyrosine immunoprecipitation (Final p-tyr
step). B, a second purification was analyzed by
phosphotyrosine immunoblotting as in A, but using a gel with a
low bisacrylamide ratio designed to better separate the 120-kDa region
phosphoproteins. This panel indicates the presence of two tyrosine
phosphorylated proteins just below the mass of Tyr(P) substrates that
are selectively retained by ATP-Sepharose, suggesting that they are
ATP-binding proteins. One of these Tyr(P) proteins migrates just below
the 120-kDa marker. C, in a third purification, the fraction
eluted from the ATP resin by ATP was aliquoted, precipitated with
either anti-Tyr(P) (PT66), EGF receptor (EGFR(1382)), or
p125 (Fak(2A7)) antibodies, and run on a gel
with low bisacrylamide ratio in preparation for Tyr(P) immunoblotting.
Twenty-five-fold more immune complex was loaded into the EGFR and
p125
lanes. The immunoprecipitates demonstrate that at
least two known kinases (EGFR and p125
) were purified by
sequential Tyr(P) and ATP affinity chromatography. These two known
kinases were present in lower amounts than the major Tyr(P)-containing
protein, which migrated just below the 120-kDa
marker.
Fig. 6(panel
A) shows an analysis at the end of a typical purification using a
standard gel polyacrylamide gel. At least five Tyr(P)-containing
proteins adsorbed to the ATP column (p170, p140, p125, p115-120,
and p75) were eluted with 1 mM ATP and were concentrated by
overnight reincubation with PT66-agarose. This last step was followed
by washing and extraction of the purified proteins in SDS sample buffer
(final Tyr(P) step). The lower band in the 115-120-kDa region
corresponded to the most heavily autophosphorylated protein seen in Fig. 3. A second purification was examined using a gel with a
low bisacrylamide concentration to enhance separation of
phosphoproteins in the 120-kDa region (Fig. 6B). The
gel allows visualization of the 115-120-kDa Tyr(P) protein below
the 120-kDa prestained marker separated from the preponderance of
Tyr(P) substrates above the 120-kDa marker. Most of these did not
adsorb to or elute from the ATP column. This can be seen even more
clearly in Fig. 6C, which shows the results from a
third purification in which the ATP eluate was concentrated
20-fold using a final Tyr(P) step. Again the low bisacrylamide gel
was used. This third purification was also used to confirm the
specificity of the ATP affinity column by identifying two of the trace
Tyr(P) proteins in final ATP eluate as the EGF receptor and
pp125
. The eluate from the ATP affinity resin was
aliquoted into three fractions, which were concentrated by
precipitation with either PT66-agarose (labeled the final Tyr(P) step),
rat EGF receptor polyclonal antiserum, 1382, or the p125
monoclonal 2A7. The PT66 lane was loaded with only 1/25 the
sample used in the EGF receptor or p125
lanes. p170
eluted from the ATP column was precipitated by the EGF receptor
antibody. The Tyr(P)-containing protein band that migrated just above
the 120-kDa molecular mass marker on low bisacrylamide gels was
immunoprecipitated by p125
monoclonal. Two separate
immunoblots of the concentrated final Tyr(P) step were performed using
the p125
monoclonal antibody 2A7 or the polyclonal
antibody BC-2 (raised against the p125
tyrosine kinase
domain, amino acids 311-701). These confirmed that the band
migrating above the 120-kDa marker on the low bisacrylamide gel was
p125
; the substrate migrating below the 120-kDa marker
did not react with either the p125
monoclonal or antisera
even though there was greater than 20 times the silver staining protein
in the 115-120-kDa band (data not shown).
Fig. 7shows
that the tyrosine autokinase activity from an aliquot of the same
immune complexes used for the immunoblots depicted in Fig. 6C. The auto kinase activity is almost totally
confined to the band running just below the 120-kDa marker, with an
apparent molecular mass of 115-120 kDa. The EGF receptor
immunoprecipitate shows little or no autokinase activity, and the
p125 2A7 immune complex exhibited barely detectable
kinase activity. A reimmunoprecipitation with PT66 after
immunodepleting EGF receptor or p125
, respectively,
showed that PT66 could still precipitate the 115-120-kDa
autokinase activity. While the purification yielded
tyrosine-phosphorylated EGF receptor and FAK that bind the ATP column,
their tyrosine kinase activity is minimal either by virtue of their low
abundance (compared to the 115-120-kDa protein) or due to
differential loss of activity. The p140 and p75 substrates were not
phosphorylated in vitro in this preparation under these
conditions. Thus, 115-120-kDa protein appears to be the major
kinase from the Ang II-treated cells.
Figure 7:
Autophosphorylation of Tyr(P) proteins
immunoprecipitated from fractions eluted from ATP-Sepharose with ATP. A
portion of the purification used for the Tyr(P) immunoblot in Fig. 6C was used for the following immunoprecipitations
and immune complex kinase assays. Proteins eluted from the ATP resin
were immunoprecipitated: (i) directly with PT66-agarose (Final
tyr-p step), (ii) with EGF receptor antibody, 1382 (EGFR
IP), (iii) following the EGF receptor precipitation the remaining
supernatant was reimmunopreciptated with PT66-agarose (Final Tyr(P)
step-EGFR), (iv) with anti-p125 antibody, 2A7 (FAK IP), or (v) following the 2A7 precipitation the remaining
supernatant was reimmunoprecipitated with PT66-agarose (Final p-tyr
step-FAK). All immune complexes were incubated with
[
-
P]ATP 60 µM (10 µCi) in
phosphorylation buffer as described under ``Experimental
Procedures.'' Following a 1-min incubation, the samples were
boiled in SDS sample buffer, subjected to SDS-PAGE, and
autoradiographed. The EGF receptor and p125
constitute a
small fraction of the autophosphorylating kinase activity in the final
Tyr(P) step, the majority of the activity was found in the
115-120-kDa protein.
Downstream signaling from G-protein-coupled receptors is
extraordinarily diverse as these receptors are involved in some manner
in most physiologic processes. In general, stimulation of
G-protein-coupled receptors results in activation of Ser/Thr kinases (21, 22) . However, in certain cell types, hormones or
agonist lipids that bind to G-protein-coupled receptors stimulate
tyrosine phophorylation, and sometimes even cell proliferation. In
addition, this subset of hormone G-protein-coupled receptors can
activate the MAP kinase pathway(15, 16, 20, 36, 37, 38, 39) and
can lead to immediate early gene expression(50) .
In rat liver epithelial cells, Ang II alone not only stimulates
proliferation, it also activates the MAP kinase and JNK kinase pathways
and modifies transcription factor activity as adjudged by increased
AP-1 binding. The Ang II actions on MAP kinase and JNK are protein
kinase C-independent, as well as calcium/calmodulin-independent, yet
both actions are inhibited by genistein, implicating the involvement of
a tyrosine kinase.
Activation of JNK in a
calcium-dependent, tyrosine kinase-dependent manner has not been
observed in other cells(51) . Thus, binding to the Ang II
receptor and the resultant
and calcium signals may have
differential consequences depending upon the downstream signaling
elements expressed in the cell types under study. The expression of a
novel tyrosine kinase activated in a G-protein- and/or
calcium-dependent manner may allow additional Ang II-dependent actions
in some cells, i.e. Ang II may stimulate multiple pathways in
cells in which it activates more than one tyrosine kinase.
Subcellular fractionation demonstrated that many of the Tyr(P)
substrates and virtually all of the activated tyrosine kinase in Ang
II-treated cells were soluble. Our catalogue of rat liver epithelial
cell tyrosine kinases yielded several intracellular candidates
(p125 and the JAKs) as potential calcium-regulated
tyrosine kinases in the 115-130-kDa range. p125
is
both tyrosine phosphorylated and minimally activated in Ang II-treated
cells, but it is a minor component of the Ang II-dependent response ( Fig. 2and Fig. 3). The JAK kinases, while often found in
the particulate fraction, presumably non-covalently bound to their
associated cytokine receptor (52) , could be released from the
membrane and activated by a calcium-dependent step. In addition,
another group reported that Ang II activated JAK2 in smooth muscle
cells(48) , and a second group showed Ang II stimulation of
serum-inducible element binding (53) , often regarded as
evidence of JAK pathway activation(52) . We have not seen
evidence of increased serum-inducible element binding in Ang II-treated
GN4 cells
, nor do we detect significant JAK2 tyrosine
phosphorylation in an Ang II-treated rat liver epithlelial cells (Fig. 7). Evidence for JAK2 activation in smooth muscle cells
and its absence in rat liver epithelial cells confirms the cell type
complexity of intracellular tyrosine kinase signaling.
The
p125 immunoprecipitation experiments ( Fig. 2and Fig. 3) suggested that another tyrosine kinase was activated in
Ang II-treated cells; the kinase purification supports this conclusion.
The sharp band of activity seen in immune complexes (Fig. 3)
appears to be the major autophosphorylating kinase activity isolated by
large scale Tyr(P) and ATP affinity chromatography ( Fig. 6and Fig. 7). After the ATP affinity step, the p115-120-kDa
Tyr(P) autophosphorylating protein is the dominant band on
silver-stained gels, and in some preparations, it is the only band
(data not shown). By Tyr(P) immunoblotting, a much more sensitive
technique than silver staining, most preparations eluted from the ATP
resin and concentrated with PT66-agarose contain varying proportions of
five Tyr(P) proteins: p170, p140, p125, p115-120, and p75. The
p115-120 band is always dominant. The selectivity of the ATP
affinity step was confirmed by the identity of p170 and p125 as known
tyrosine kinases. The presence of the EGF receptor (p170) was
surprising because even with immunoprecipitation of a 60-mm GN4 culture
dish with anti-EGF receptor antibody, we have not observed Ang
II-dependent EGF receptor phosphorylation. However, in purifications
using 50 150-mm culture dishes, enough tyrosine-phosphophorylated EGF
receptor is present to be specifically purified by the dual affinity
procedure. The contribution of the EGF receptor and p125
to the autokinase activity in the final preparation is dwarfed by
the activity in p115-120-kDa protein (Fig. 7). Since the
p115-120-kDa protein is much more abundant, we cannot speculate
on the specific activities of the putative calcium-regulated tyrosine
kinase (p115-120) and p125
. The p115-120-kDa
protein does not immunologically cross-react with either
p125
monoclonal 2A7, or a polyclonal raised against the
p125
tyrosine kinase domain. This does not preclude the
putative novel kinases's similarity to p125
, but
suggests that any relationship may be distant. Finally, there is little
P incorporation into p140 and p75 at this stage of the
purification. We do not know whether these are low specific activity
kinases, other ATP binding proteins, or Tyr(P) proteins specifically
bound to one of the ATP binding kinases.
In summary, Ang II
activates at least two tyrosine kinases in rat liver epithelial cells,
p125 and a second activity, which appears to migrate at
p115-120-kDa. In addition, Ang II activates at least two
downstream signaling pathways: the Jun N-terminal kinase, activated in
a calcium- and tyrosine kinase-dependent manner, and MAP kinases whose
activation by Ang II is less dependent on a calcium signal.
It will be of interest to determine whether the Ang II-dependent
activation of distinct tyrosine kinases regulates separate
intracellular signaling cascades.