(Received for publication, May 19, 1995)
From the
Chinese hamster ovary (CHO) cells, stably transfected with the
long form of the prolactin (PRL) receptor (PRL-R) cDNA, were used for
PRL-R signal transduction studies. Patch-clamp technique in whole cell
and cell-free configurations were employed. Exposure of transfected CHO
cells to 5 nM PRL led to the increase of Ca-
and voltage-dependent K
channel (K
)
activity. The effect was direct as it was observed also in excised
patch experiments. A series of tyrosine kinase inhibitors was studied
to investigate the possible involvement of protein tyrosine kinases in
K
functioning and its stimulation by PRL. Genistein,
lavendustin A, and herbimycin A decreased in a concentration and
time-dependent manner the amplitude of the K
current in
whole cell and the open probability of K
channels in
cell-free experiments. The subsequent application of PRL was
ineffective. The protein tyrosine phosphatase inhibitor orthovanadate
(1 mM) stimulated K
channel activity in excised
patches, indicating that channels can be modulated in opposite
directions by protein tyrosine kinase and protein tyrosine phosphatase.
Moreover, in whole cell experiments as well as in excised patch
recordings, anti-JAK2 tyrosine kinase antibody decreased the K
conductance and the open probability of the K
channels. Subsequent application of PRL was no longer able to
stimulate K
conductance. Immunoblotting studies using the
same anti-JAK2 antibody, revealed the constitutive association of JAK2
kinase with PRL-R. Preincubation of anti-JAK2 antibody with the JAK2
Immunizing Peptide abolished the effects observed using anti-JAK2
antibody alone in both electrophysiological and immunoblotting studies.
We conclude from these findings that these K channels
are regulated through tyrosine phosphorylation/dephosphorylation; JAK2
tyrosine kinase, constitutively associated with PRL-R, is implicated in
PRL stimulation of K
channels.
Prolactin (PRL) ()is a multifunctional pituitary
hormone involved in the control of a wide variety of physiological
processes in vertebrates, including lactation, reproduction, immune
responses, and osmoregulation, as well as cell
proliferation(1, 2, 3) . The PRL receptor
(PRL-R) belongs to the cytokine-growth factor receptor superfamily that
includes receptors for growth hormone, erythropoietin, numerous
hematopoietic interleukins (IL)-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-9,
granulocyte colony-stimulating factor, granulomacrophage
colony-stimulating factor, and ciliary neurotrophic
factor(4, 5) . This family of receptors possesses
common structural motifs, both external (two disulfide loops and the
WSXWS homology box) and internal (proline-rich homology box
1). Recent studies have been marked by considerable progress in
understanding the mechanisms of intracellular signaling for the
different members of this family, particularly for PRL-R. Most of the
data were obtained in the PRL-dependent rat T lymphoma cell line Nb2.
It has been shown by several groups that, following binding of PRL to
the PRL-R in these cells, dimerization of the receptor occurs (6, 7) prior to phosphorylation of an associated
tyrosine kinase (JAK2). This represents the first event in the process
of PRL-R signal transduction(8, 9) . Other studies
demonstrated that PRL stimulation of Nb2 cells induced a concentration-
and time-dependent activation of another protein tyrosine kinase,
p59
, from the Src protein tyrosine kinase family (10) . On the other hand, more and more studies demonstrate an
important role of ion channels in receptor signal
transduction(11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22) .
Several different mechanisms have been proposed for channel involvement
in signal transduction: direct agonist effect on the
channel(15, 16) , second messenger participation in
channel modulation(17, 18, 19) , and
regulation by kinases and phosphatases through channel
phosphorylation/dephosphorylation
processes(11, 20, 21, 22) . Moreover
channel regulation by phosphorylation has been shown to play a key role
in physiological processes such as proliferation and transformation (23, 24, 25) . Nothing, however, is known
about the putative role of channel phosphorylation in cytokine-growth
factor receptor superfamily signal transduction and, in particular, in
PRL-R signal transduction.
The signal transduction mechanism for the
full-length PRL receptor has been studied using a CHO line stably
transfected with the cDNA of the long form of rabbit mammary
PRL-R(26) . These CHO-transfected cells responded to PRL by
stimulating the cotransfected milk protein gene promoter(27) ,
proving that such cells are fully capable of transmitting the PRL
signal and that PRL-R is functional. In a series of studies using patch
clamp and microfluorimetric techniques, we analyzed the first steps of
the PRL-R signal transduction pathway: an increase in intracellular
Ca(28, 29) and direct stimulation
of calcium- and voltage-activated potassium channels (K
)
by PRL(29) . These observations suggested the existence of a
regulatory complex involving a protein kinase tightly associated with
K
channels and PRL-R. Furthermore, by immunoblotting
studies we presented evidence for the tyrosine phosphorylation of this
type of PRL-R, the association of JAK2 tyrosine kinase with the
receptor, as well as changes in tyrosine phosphorylation of a number of
cytoplasmic proteins(30) .
In this article, we report on the
very first steps in PRL-R signal transduction at the plasma membrane
level: we demonstrate an endogenous large conductance K channel as the primary ionic event triggered by PRL. These
K
channels are constitutively regulated through tyrosine
phosphorylation/dephosphorylation. We also show that JAK2 tyrosine
kinase, associated with PRL-R, is implicated in the stimulation of
K
channels by PRL.
Single
channel data analysis was performed after elimination of capacity
transients and leak current by subtraction of record averages without
channel activity from each current record. The openings and closings of
the channel were detected using a criterion of a 50% excursion between
fully open and fully closed states to determine the occurrence of an
opening or closing event such as crossings of the line at a
half-distance between zero current level and a level corresponding to
the average open channel amplitude. In this way, real current records
were put into ideal form by setting all intermediate amplitudes to the
level of zero current line or to the level of the average open channel
amplitude. The open probability (P) was calculated
as the open time integral divided by the number of channels in the
patch and the duration of the data segment analyzed. The number of
channels was estimated by examining the record for multiple openings
under conditions of high open probability (P
>
0.75).
Results are expressed as means ± S.D. where appropriate. Each experiment was repeated several times. Student's t test was used for statistical comparison among means and differences with p < 0.05 were considered significant.
As was demonstrated by our previous studies(35) , a
210-picosiemens K conductance, dependent on voltage
and intracellular Ca
, was revealed by patch-clamp
experiments in CHO cells. To check whether the K
channels
could be phosphorylated, we studied the effect of ATP, which serves as
a substrate for protein kinase, on the activity of the K
channels in CHO cells. Experiments using Mg-ATP (10-100
µM), applied to the cytoplasmic side of the membrane,
showed an increase in the open probability of the channel, proving that
a protein kinase is tightly associated with it. Fig. 1shows the
very low K
channel open probability values in the absence
of ATP in the internal solution and the much higher values in the
presence of ATP. The open probability values in the absence of ATP were
so low that it was impossible to carry out the statistical analysis
required to establish the effect of PRL on K
channel
activity under these conditions. In whole cell experiments without ATP
in the patch pipette, no effect of PRL on K
total
current was observed. ATP hydrolysis was required for channel
modulation, because the nonhydrolyzable ATP analog, AMPPNP, was
ineffective (not shown). As, on the one hand, tyrosine kinase was found
to be a primary target of PRL in PRL-R signal transduction(8, 9) and, on the other hand, the K
channels
stimulated by PRL were found to be associated with protein kinase, we
assumed the existence of a PRL-R-K
channel-tyrosine kinase
regulatory complex. To investigate whether the K
channels stimulated by PRL may be modulated by an endogenous
tyrosine kinase, we examined the effects of different tyrosine kinase
inhibitors on K
channel activity, using both whole cell
and cell-free modes of the patch-clamp technique. In these studies we
used two types of experiments differing in the duration of drug
application
Figure 1:
The time course of the activity of the
Ca-activated K
channel obtained from
excised (outside-out) patch recordings in the absence (
) or
presence (
) of 100 µM ATP in the patch pipette at a
membrane potential of +10 mV and 0.2 µM internal-free
Ca
. Representative experiments for such experimental
conditions are shown.
Bath application (3
min) of the protein tyrosine kinase inhibitor genistein (36, 37) caused a progressive reduction in the
K current (Fig. 2A). Further
application of PRL was ineffective. Genistein concentrations lower than
50 µM were ineffective under these experimental conditions (n = 6). After genistein was washed out, K
currents gradually recovered, indicating that the depression was
reversible. Genistin, an analog of genistein that lacks protein
tyrosine kinase inhibitory activity(36) , had no effect on
K
current (n = 4; Fig. 9A). We also tested two structurally distinct
protein tyrosine kinase inhibitors: herbimycin A (9, 38) and lavendustin A(36, 39) .
Herbimycin A (1.5 µM) and lavendustin A (10
µM) depressed K
currents to 57 ± 8% (n = 8) and to 54 ± 5% (n = 6)
of control, respectively. Fig. 2B shows an example of
K
current inhibition induced by 3-min application of 10
µM lavendustin A and current-voltage relationships for
this effect, where current amplitudes were plotted at different test
potentials. K
currents were evoked by 160-ms test pulses
from a holding potential of -40 to +10 mV.
Figure 2:
The effects of tyrosine kinase inhibitors
on K current in CHO-E32 cells. A illustrates
an example of the effect of 50 µM genistein on
K
current recorded before genistein application
(
), 8 min after the application (
), and after washout
(
). The membrane was depolarized from V
= -40 to +20 mV at 160-ms intervals. B illustrates an example of the effect of 10 µM of
lavendustin A on K
current recorded using a similar
protocol to the experiment in A. For both inhibitors the
original current traces are presented in the top panels and
current-voltage (I-V) relationships in the lower panels.
Calibration: 30 ms, 100 pA. C illustrates the effects of 6-h
treatment of CHO E32 cells with varying concentrations of protein
kinase inhibitors (
, genistein;
, lavendustin A,
,
herbimycin A) on normalized peak K
currents. Results
shown (mean ± S.E.) are representative of at least three
separate, independent experiments.
Figure 9:
Summary histograms of K
channel modulation in CHO-E32 cells. A illustrates the
histogram showing the effects of the various drugs studied on
normalized peak currents obtained by whole cell experiments. B illustrates the histogram showing the effects of these drugs on
the ratio of the channel open probability (P
)
obtained by single channel recordings.
Cell-free experiments demonstrated
that the effects of PRL and protein tyrosine kinase inhibitors were not
mediated by intracellular processes, as they could be also observed in
detached patches. Fig. 3shows K channel
activity stimulation by PRL and its inhibition by genistein (n = 7/9 patches). PRL (5 nM) caused an increase in
the open probability of the channels (Fig. 3B),
displaying the half-maximum increase in the open probability within 3.6
± 1.3 min. The open probability of the channel after the
addition of PRL was not constant, but oscillated between lower and
higher open probability values (Fig. 3B). Subsequent
addition of 100 µM genistein inhibited this K
channel activity almost completely within 7 ± 2 min (Fig. 3B). The amplitude histograms (Fig. 3C) for K
channels in control
(mean = 8.48 ± 0.12 pA) and in the presence of PRL (mean
= 8.34 ± 0.18 pA) demonstrate that PRL does not activate
additional conductances. Moreover, prolactin did not stimulate
K
channel activity in the presence of 30 nM CTX, a K
channel inhibitor in CHO cells(20) ,
indicating that PRL activated the CTX-sensitive K
channels
and no other type of outward channels (not shown). As in whole cell
experiments, genistin was ineffective (Fig. 9B).
Application of 1.5 µM herbimycin A also decreased channel
open probability without affecting single-channel conductance (n = 4/5 patches, Fig. 4). The subsequent addition of
PRL was ineffective.
Figure 3:
Modulation of K single
channel activity by PRL and its inhibition by genistein. A illustrates representative recordings of single K
currents in a cell-free patch in the outside-out configuration.
K
channel activity in this patch was obtained with 0.1
µM internal-free Ca
at a membrane
potential of +40 mV. Recordings are shown for the control
solution, 6 min after application of 5 nM PRL, and 2 and 8 min
after the subsequent application of 100 µM of genistein,
respectively. The time course of the open probability of the K
channels in the control and in the presence of PRL and genistein
is demonstrated in B. C illustrates the amplitude
histograms for single K
channel conductance for
control and in the presence of 5 nM PRL.
Figure 4:
The effect of herbimycin A on K channel activity. Representative records of single K
currents in excised (outside-out) membrane patch (top).
K
channel activity in this patch was obtained with 0.5
µM internal-free Ca
at a membrane
potential of +20 mV. Records are shown for the control solution,
10 min after 1.5 µM herbimycin application and 3 min after
the subsequent addition of 5 nM PRL. The open probability of
the K
channel versus time is presented in the
lower panel.
As protein tyrosine kinase inhibitors in high
concentrations are known to be able to inhibit not only protein
tyrosine kinase but also protein kinase C and protein kinase A kinases
in some cell types(36) , we checked the putative involvement of
protein kinase C and protein kinase A in the mechanisms studied. We
tested both activators and inhibitors of protein kinase C and protein
kinase A (10-8 M phorbol 12-myristate 13-acetate
application, as protein kinase C activator; 10-6 M phorbol 12-myristate 13-acetate 24-h incubation, as protein kinase
C inhibitor; 250 µM phloretin, as protein kinase C
inhibitor; 2 µM forskolin and 1 mM 8-bromo-cAMP,
as protein kinase A activators) on K channel activity
and on the stimulated effect of PRL. None of these drugs had any
effect. A protein kinase C biochemical assay (40) was also
carried out. 50-100 µM genistein had no effect in
these studies.
The possibility that these channels may be regulated by protein tyrosine phosphatase as well as by protein tyrosine kinase was investigated by application of the protein tyrosine phosphatase inhibitor, sodium orthovanadate(41) , to the cytoplasmic side of the cell-free patch. Orthovanadate (1 mM) increased the open probability of the channels in a time-dependent manner (Fig. 5). On average, activity increased by 198 ± 27% within 5 min (n = 6/9 patches). Fig. 5B shows the duration histograms for the channel prior to application of orthovanadate, then 2 and 8 min afterwards, respectively. In the presence of orthovanadate, channel openings were longer, as indicated by an increase in the relative number of events and the time constant to for the open state.
Figure 5:
The effect of orthovanadate on K channel activity. A illustrates representative records
of single K
currents in excised (inside-out) membrane
patches. K
channel activity in this patch was obtained
with 0.2 µM internal-free Ca
at a
membrane potential of +40 mV. Records are shown for the control
solution, and 2 and 8 min after 1 mM orthovanadate
application, respectively. B illustrates the corresponding
open time distributions with single exponential fit. Time constants: t = 1.9 ms (n = 604) for control, t = 2.3 ms (n = 668) after 2 min, and t = 6.8 ms (n = 1023) after 8 min of
orthovanadate addition, respectively.
The preceding results strongly support the
conclusion that the functioning of PRL-stimulated K channels is modulated by protein tyrosine kinases and protein
tyrosine phosphatases and thus regulated by constitutive tyrosine
phosphorylation/dephosphorylation. It has already been shown that
tyrosine-phosphorylated PRL-R is associated with JAK2
kinase(30) . Additional immunoblotting experiments were carried
out to find out if this association was constitutive. Solubilized
proteins from E32 cells incubated with or without 500 ng/ml of
prolactin were immunoprecipitated with anti-PRL-R antibody 46 and
analyzed for the presence of JAK2 in the complex. As shown in Fig. 6A, a protein of 130 kDa was detected in the blot
hybridized with anti-JAK2, corresponding to JAK2 kinase. This protein
was revealed in the presence or absence of stimulation by ovine PRL,
showing that this protein is constitutively associated with the PRL
receptor. A rehybridization of the same blot with S46 shows the
presence of the same amount of receptor in each line. In Fig. 6B, we demonstrate the specificity of the
recognition of JAK2 by the antibody. Cell extracts were
immunoprecipitated with anti-JAK2 antibody or anti-PRL-R antibody and
subjected to SDS-PAGE and Western blotting. Each blot was incubated
either with anti-JAK2 antibody or with anti-JAK2 antibody preincubated
with a peptide corresponding to the amino acid residues 758-776
of murine JAK2 (JAK2 immunizing peptide). In JAK2 immunoprecipitates we
observed that a protein of 130 kDa was specifically displaced by the
presence of the peptide. Nonspecific bands were always present
following incubation in the presence of the peptide. The same finding
was observed in 46 immunoprecipitates. The 130-kDa band was not
revealed in the presence of the peptide. Therefore, the 130-kDa protein
revealed on the blot is the tyrosine kinase JAK2, and this kinase is
constitutively associated with the prolactin receptor. To investigate
whether K
channels are also constitutively associated with
JAK2 kinase we studied the effect of the anti-JAK2 kinase antibody on
the of the K
channel activity. Anti-JAK2 antibody (diluted
1:1000 from the indicated antibody) was introduced into the internal
solution of the patch pipette. In whole cell experiments, the amplitude
of the K
current gradually decreased following rupture of
the seal. In control conditions of whole cell experiments, the K
current was stable under internal perfusion. No decrease in
current (or ``run-down'') was observed during recordings
lasting 20 min or more. Fig. 7shows the time dependence of the
decrease in K
current caused by anti-JAK2 antibody. Within
approximately 10 min the current was almost completely inhibited (n = 5). Subsequent application of PRL was ineffective (Fig. 7). Anti-JAK2 antibody applied to the cytoplasmic side of
the membrane in inside-out studies decreased the open probability of
the channel to 73 ± 18% (n = 4/4 patches) of
control (Fig. 8). Subsequent application of PRL did not
stimulate channel activity (Fig. 8). When anti-JAK2 antibody was
preincubated with a peptide corresponding to the amino acid residues
758-776 of murine JAK2 (JAK2 immunizing peptide) and this mixture
applied to the cytoplasmic side of the membrane, the decrease in
K
current in the whole cell experiments (Fig. 7) and in the open probability of the channel in
inside-out experiments (Fig. 8) were not observed. As the
anti-JAK2 antibody was obtained from Upstate Biotechnology, Inc. in
rabbit serum, we checked the effect of non-immune rabbit serum on
K
conductance as a control. Rabbit serum at the same
dilution (1:1000) was ineffective in both whole cell and inside-out
experiments (not shown). An immune serum antibody (anti-rat PRL
antibody) was used as an additional control. At the same dilution it
was also ineffective.
Figure 6: Association of JAK2 with the prolactin receptor in CHO cells. A illustrates JAK2 constitutive association with the PRL receptor. E32 cells were left untreated or treated with PRL (500 ng/ml), solubilized, and immunoprecipitated with prolactin receptor antiserum 46. All samples were analyzed by SDS-PAGE under reducing conditions, followed by immunoblotting with anti-PRL-R antibody 46 or anti-JAK2 antibody (Upstate Biotechnology, Inc.). Molecular weight standards are indicated on the left. B illustrates the blockage of anti-JAK2 reactivity with JAK2 peptide. E32 cell lysates were immunoprecipitated with anti-JAK2 antibody or anti-PRL-R antibody 46 and the immunoprecipitates were processed for Western blotting. Blots were probed with anti-JAK2 antibody or with anti-JAK2 antibody + peptide (corresponding to the amino acid acid residues 758-776 of murine JAK2).
Figure 7:
Time
course of the inhibition of the K current by anti-JAK2
antibody. Top, current traces obtained after seal rupture in
the presence of anti-JAK2 antibody in the patch pipette. Currents were
evoked by 160-ms test pulses from holding potential of -60 mV to
+50 mV test potential. Lower panel, the respective plot
of normalized current (I/I
) with
anti-JAK2 antibody or with anti-JAK2 antibody+ peptide versus time.
Figure 8:
Effect of anti-JAK2 antibody on K channel activity. Representative records of single K
currents from excised (inside-out) membrane patch (top).
K
channel activity in this patch was obtained with 0.2
µM internal-free Ca
at a membrane
potential of +50 mV. Records are shown for control solution, 2 min
after anti-JAK2-antibody application, following the subsequent addition
of PRL and during recovery. The open probability of the K
channel versus time is presented in the lower
panel.
Fig. 9presents a summary of the
effects of all the drugs studied on normalized peak currents, obtained
by whole cell experiments (Fig. 9A) and normalized P, obtained by excised patch experiments (Fig. 9B).
We conclude from these findings that the activation of
K channels by PRL in CHO cells, transfected with cDNA of
the long form of PRL-R, is the primary ionic event in PRL-R signal
transduction. K
channels are constitutively regulated
through tyrosine phosphorylation/dephosphorylation. Stimulation of the
channels by PRL possibly occurs through phosphorylation of protein
tyrosine residues of the channel or of one or more associated proteins.
The observation that K
channel stimulation by PRL does not
occur in the presence of anti-JAK2 kinase antibody suggests that at
least one of the kinases involved in the channel stimulation by PRL may
be JAK2 tyrosine kinase.
Although the association of PRL-R with
JAK2(8, 9, 30) , JAK1(42) , or Fyn (10) tyrosine kinases has already been clearly demonstrated,
the cascade of ionic events induced by PRL and the nature of ion
channels involved has not yet been studied. Our earlier studies have
characterized a membrane hyperpolarization, caused by K channel stimulation, and Ca
influx among the
first detectable responses to PRL-R activation(29) . The
underlying mechanisms are, however, not very clearly understood. In the
present study, we applied the patch-clamp recording technique in order
to unravel the mechanism of K
channel activation by PRL
and to identify the nature of the associated protein kinase.
In our
experiments ATP (10-100 µM) increased the open
probability of the K channel, therefore showing that a
protein kinase is involved in the regulation of channel activity.
Moreover PRL was unable to stimulate this activity when ATP was absent
from the internal solution, demonstrating that kinase phosphorylation
is needed for channel stimulation by PRL. Thus, we concluded that
protein kinase is closely associated with K
channel and
PRL-R in a regulatory complex.
It was recently demonstrated that in
murine fibroblast cell lines, transfected with Ras or Raf plasmids, the
K, CTX-sensitive channel is up-regulated by oncogenic
p21
and that this regulation appears to be due to raf kinase-dependent induction of channel expression(43) . The
application of either epidermal growth factor or platelet-derived
growth factor to nontransfected cells caused a time-dependent induction
of K
channels, obviously, through activation of endogenous
cellular p21
. Epidermal growth factor induction of the
K
channel was blocked by the tyrosine kinase inhibitors
lavendustin A (1 µM) or genistein (50 µM).
However, application of genistein to cells transfected by oncogenic ras had no effect on K
current density,
indicating that genistein had no direct inhibitory effect on the
K
channel. These results suggest that ras regulates the K
channels through serine/threonine
kinase and not through protein tyrosine kinase. Our experiments using
tyrosine kinase inhibitors show a distinct mechanism of K
channel regulation in CHO cells transfected with cDNA of the long
form of PRL-R: the direct regulation of K
channels by
tyrosine kinase (as this activity was inhibited directly in whole cell
and single channel experiments by three distinct tyrosine kinase
inhibitors) and modulation of this activity by PRL (as PRL was no
longer able to stimulate K
channel activity when the cells
were treated with protein tyrosine kinase inhibitors). Experiments
using protein kinase C and protein kinase A activators and inhibitors
showed that these kinases are not involved in the channel regulation
mechanisms.
For epidermal growth factor receptor, which has
intrinsic tyrosine kinase(44) , the first event is activation
of voltage-independent Ca channels defined as direct
receptor-operated channels (12) . This in turn causes the
activation of Ca
-dependent K
channels, sensitive to charybdotoxin(12, 13) ,
resulting in delayed membrane hyperpolarization and leading to the
activation of a second class of hyperpolarization-sensitive
Ca
channels(14) . We did not observe the
Ca
conductance activation prior to K
channel stimulation(29) . Conversely, our results
demonstrate that the first ionic event in PRL-R signal transduction is
K
channel activation, since this activation is observed in
excised patches. Based on the observed inhibitory effects of protein
tyrosine kinase inhibitors on the activity of PRL-stimulated K
channels, we propose that tyrosine kinase is involved in the
positive regulation of these channels. The direct tyrosine
phosphorylation of the delayed rectifier K
channel has
also been proposed for the m1 muscarinic acetylcholine
receptor(11) . This tyrosine kinase regulation is obviously an
essential link in PRL signal transduction as it was recently found that
the tyrosine kinase inhibitor herbimycin A was able to block a
substantial portion of the prolactin signal to the milk protein gene
promoter,
-lactoglobulin (30) . In Nb2 cells it was shown
that herbimycin A could also abolish the JAK2 kinase and receptor
phosphorylation(9) .
In our study the effects of PRL and
protein tyrosine kinase inhibitors were observed in excised-patch
experiments, indicating that the effects are not controlled by cellular
metabolism, but are direct and that protein tyrosine kinase remains
closely associated with K channel activity. In this
context it was of interest to check the effect of anti-JAK2 antibody on
K
channel activity. The effectiveness of using antibodies
in patch-clamp experiments was previously shown by Schweizer et
al.(45) . When anti-JAK2 antibody was introduced into the
patch pipette in whole cell experiments, the K
conductance
was almost completely inhibited within an average of 15 min, and PRL
was no longer able to stimulate K
conductance. Anti-JAK2
antibody also decreased the open probability of K
channels
when it was applied to the cytoplasmic side of the membrane in the
inside-out patch mode. In immunoblotting experiments JAK2 kinase was
revealed in the presence or absence of stimulation by PRL, showing that
it is constitutively associated with the PRL-R. On the other hand,
electrophysiological studies using anti-JAK2 antibody showed that this
kinase is also constitutively associated with the K
channel. The results with JAK2 immunizing peptide, demonstrating
the suppression of the effects observed using anti-JAK2 antibody alone
in both electrophysiological and immunoblotting studies, show that the
effects of the anti-JAK2 antibody are specific. The ability of the
constitutively active kinases to stimulate cellular responses has
previously been shown for MAP and phosphatidylinositol
3-kinase(46) . On the other hand, there is growing evidence for
the existence of a protein tyrosine kinase activation mechanism that
functions indirectly by second messengers. For example, in the brain,
membrane depolarization, which causes an increase in intracellular
Ca
levels, increases protein tyrosine kinase (47) and protein mitogen-activated protein kinase (48) activity. Extracellular signals (in our case PRL) appear
to stimulate the activity of protein tyrosine kinase, but it may also
be regulated by other factors (e.g. membrane potential and
intracellular Ca
). This constitutive activity of JAK2
kinase is the reason for K
channel inhibition by the
anti-JAK2 antibody in the absence of PRL stimulation. Therefore, our
results demonstrate the functional involvement of JAK2 kinase in
constitutive K
channel activity and the stimulation of
these channels by PRL. However, it was shown by immunoblotting
experiments that genistein (100-500 µM) was not able
to inhibit the JAK2 kinase phosphorylation induced by growth hormone
but it blocked the tyrosyl phosphorylation of other proteins
(intermediary tyrosine kinases)(49, 50) . This fact
may be explained by the various examples of interactions at the level
of the same protein; many phosphoproteins are phosphorylated at the
same or at distinct residues by more than one protein kinase: in the
case of tyrosine hydroxylase, the nicotinic acetylcholine receptor,
synapsin 1, or Ca
channel of the L type(47) .
This multisite protein phosphorylation appears to be the rule rather
than the exception(47) . Therefore, we cannot completely
exclude the possible involvement of other tyrosine kinases in K
channel functions.
The fact that the channel was activated by
the protein tyrosine phosphatase inhibitor orthovanadate in excised
patches suggests that the channel can be modulated in opposite
directions by protein tyrosine kinase and protein tyrosine phosphatase.
This modulation may be due to phosphorylation/dephosphorylation of the
channels themselves or of regulatory protein(s) associated with these
channels and the PRL receptor. The protein tyrosine phosphatase
endogenous to CHO cells was observed in most excised patches and thus
may be a member of the transmembrane class of protein tyrosine
phosphatase (51, 52) or of the cytoplasmic protein
tyrosine phosphatase which possess an SH2 domain or another means of
association with membrane-bound proteins(53, 54) .
These studies suggest that tyrosine phosphorylation/dephosphorylation
systems can modify K channels rapidly, producing flexible
changes in PRL-R signaling. Similar types of complexes that contain
regulatory protein kinases and phosphatases have previously been shown
for a variety of different ion channels(20, 21, 22, 55, 56, 57) and
for K
channels in
particular(20, 21, 56, 57) . But the
participation of such protein tyrosine kinase modulation of
K
channels in PRL signal transduction has never been
shown. To our knowledge this study provides the first example of the
regulation of K
channels by PRL and protein tyrosine
kinase in cell-free preparations. Our study is in accordance with a
hypothesis previously proposed (57) about such submembrane
complexing of ion channel proteins with modulatory enzymes like kinases
and phosphatases as a common means by which cells achieve highly
localized regulation of ion channel function by otherwise ubiquitous
biochemical processes. Of particular interest is the finding that the
modulation described here is due to a constitutive JAK2 tyrosine kinase
activity. These results take on added significance because the PRL
receptors were expressed in a heterologous system. However, remarkably,
the endogenous JAK2 tyrosine kinase and endogenous K
channels in the host cell associate closely with heterologously
expressed PRL-R and this complex retains its ability to be stimulated
by PRL in a detached membrane patch.
As, on the one hand, PRL is
known to stimulate the process of proliferation (58, 59, 60, 61) and, on the other
hand, K channels have also been shown to be involved in
the control of cell
proliferation(25, 43, 62) , the mechanism of
PRL stimulation of K
channels through tyrosine
phosphorylation, presented in this work, could provide a clue to
understanding the regulation of cell proliferation by PRL.