(Received for publication, July 29, 1994; and in revised form, November 18, 1994)
From the
We report the first in vivo study demonstrating
tyrosine phosphorylation of mammary gland proteins including the
prolactin receptor, in response to the injection of prolactin.
Immunoblotting of mammary gland membrane extracts revealed that
subunits of 200, 130, 115, 100, 90, 70, and 45 kDa display increased
tyrosine phosphorylation within 5 min of prolactin administration. The
100-kDa component was identified as the full-length prolactin receptor
by a variety of means including immunoprecipitation and immunoblotting
with monoclonal (U5, 917, 110, and 82) and polyclonal (46) antibodies to the prolactin receptor. Maximal receptor
phosphorylation was seen within 1 min of hormone injection, and to
obtain a strong response it was necessary to deprive rabbits of their
endogenous prolactin for 36 h. Rapid tyrosine phosphorylation of the
full-length receptor was verified by its demonstration in Chinese
hamster ovary cells stably transfected with rabbit prolactin receptor
cDNA. Both in vivo and in vitro, the phosphorylation
signal was transient, being markedly reduced within 10 min of exposure
to prolactin. Tyrosine-phosphorylated receptor was shown to be
associated with JAK 2 by immunoblotting of receptor immunoprecipitated
from transfected Chinese hamster ovary cells with polyclonal 46. A
48-kDa ATP-binding protein was also shown to be associated with the
mammary gland receptor by U5 or polyclonal 46 immunoprecipitation of
receptor complexes following covalent labeling with
[-
P]azido-ATP.
Our demonstration of prolactin receptor tyrosine phosphorylation raises the possibility of signaling pathways regulated by receptor/SH2 protein interaction, which would facilitate prolactin specific responses. The fact that a period of hormone deprivation is needed for significant hormone triggered receptor phosphorylation indicates that the mammary gland receptor exists in a largely desensitized state in vivo, analogous to the related growth hormone receptor.
Despite its demonstrated role in a variety of important
functions such as reproduction, osmoregulation and immune
surveillance(1, 2) , the mechanism of action of
prolactin has remained an enigma until quite recently. Progress on this
problem required the development of a new paradigm, and this stemmed
from the realization that the prolactin receptor is a member of the
newly recognized cytokine receptor family, which includes the receptors
for growth hormone (GH), ()erythropoietin, several
interleukins, granulocyte colony-stimulating factor, and ciliary
neurotrophic factor(3, 4, 5, 6) .
These receptors possess common structural motifs externally, such as
two disulfide loops and the WSXWS homology box, and
internally, such as the proline-rich homology box 1. Based on the
crystal structure of the GH (receptor)
complex (7) and associated physicochemical and biological
evidence(8, 9) , it is believed that the initial event
in signal transduction by these receptors is hormone-induced
oligomerization of receptor subunits, and this brings into proximity
the proline-rich box 1 of the cytoplasmic domains, which is known to be
essential for signal
transduction(10, 11, 12) . This process is
associated with the very rapid activation of a member of the JAK (Janus
kinase) family of tyrosine kinases, which associate with these
receptors and become activated on hormone
binding(13, 14, 15, 16) . JAK
activation is followed by tyrosine phosphorylation of upstream members
of a variety of signaling pathways which include the cytoplasmic signal
transducer and activator of transcription factor
complex(16, 17, 18) , and the MAP kinase
pathway (19, 20, 21) .
In the case of the prolactin receptor itself, some of these details are inferred, but there is biological evidence for receptor dimerization (22, 23) , and rapid tyrosine phosphorylation of three proteins follows prolactin binding to the Nb2 lymphoma cell(24, 25) . These three proteins comprise 97-kDa and 40-kDa components, which may be themselves kinases(25, 26) , and a receptor-associated 121-kDa component, which appears to be JAK 2(27, 28) . In both reports examining prolactin-stimulated tyrosine phosphorylation in Nb2 cells, the 66-kDa mutant prolactin receptor was said to be only weakly phosphorylated (24) or not phosphorylated at all(25) .
We have recently found that herbimycin A, an inhibitor of JAK 2
kinase, is able to block a substantial portion of the prolactin signal
to the promoter of the milk protein gene, -lactoglobulin, when
expressed transiently with the prolactin receptor in CHO cells. (
)In order to define the role of tyrosine phosphorylation in
prolactin stimulation of mammary gland function, we have examined
hormone-stimulated tyrosine phosphorylation both in vivo,
using mid-lactating rabbit mammary gland, and in vitro, using
a stable CHO cell line expressing the full-length rabbit prolactin
receptor. We find that tyrosine phosphorylation of this receptor in
hormone-deprived cells is striking and very rapid, but transient, and
is associated with changes in tyrosine phosphorylation of a number of
cytoplasmic proteins.
Figure 1:
Total
tyrosine phosphorylation in solubilized mammary gland membranes
following prolactin injection. 15-day lactating rabbits pretreated with
bromocryptine for 36 h were injected with 1 mg of oPrl or vehicle, and
after 2 min, animals were killed and solubilized membrane extracts
prepared as described under ``Experimental Procedures.'' 0.5
ml of extracts were immunoprecipitated with the phosphotyrosine mAbs
UBI 4G10 (5 µg) and Frackleton PY (35 µg), or prolactin
receptor mAb U5 (5 µg). After running on an 8% reduced
SDS-polyacrylamide gel electrophoresis gel, proteins were transferred
to nitrocellulose and immunoblotted with 4G10 (UBI
PY) (panela) or anti-prolactin receptor polyclonal 46 (panelb). Immune complexes were revealed as described under
``Experimental Procedures.'' These data are representative of
three experiments.
Figure 2: Evidence that the 100-kDa tyrosine-phosphorylated protein is the prolactin receptor. Solubilized mammary gland extracts (0.5 ml) from a 15-day lactating rabbit injected with prolactin were immunoprecipitated with increasing quantities of receptor mAb U5, or with 5 µg of receptor mAbs 917, 110, or 82, as well as 10 µl of polyclonal 46. A control mAb to trophoblastin (C) showed no 100-kDa band. Panel a, blot revealed with anti-phosphotyrosine 4G10; panel b, blot revealed with anti-prolactin receptor 46. Data are representative of three experiments.
Figure 3: Co-localization of phosphotyrosine and prolactin receptor using enhanced chemiluminescence to reveal location of mAbs 4G10, U5, and 82 on Western blot. Immunoprecipitation of prolactin-injected mammary gland extracts was as for Fig. 1, using 5 µg of mAb U5.
In another experiment, mammary gland extracts of prolactin or saline-treated animals were immunoprecipitated with anti-ovine prolactin antiserum or with mAb U5, and the resulting immunoblot was probed for phosphotyrosine with mAb 4G10. This experiment was also designed to compare the conditions used by Argetsinger et al.(13) to obtain co-precipitation of JAK 2 with the GH receptor with the conditions routinely used in this study. It can be seen from Fig. 4that while tyrosine-phosphorylated receptor is immunoprecipitated by the prolactin antibody only after prolactin administration, there is no evidence for tyrosine-phosphorylated JAK 2 associated with the receptor using either extraction and immunoprecipitation condition.
Figure 4: Immunoprecipitation of tyrosine-phosphorylated receptor by antiserum to ovine prolactin. Mammary gland extracts were prepared from prolactin- or saline-injected animals either as described under ``Experimental Procedures'' (designated MW), or using the extraction buffer used by Argetsinger et al. ((13) , designated C-S) but using 0.5% Triton X-100 to ensure solubilization. Complexes were immunoprecipitated either with 5 µg of U5 or with anti-oPrl at 1:1000, then processed in the usual manner and immunoblotted with 4G10.
Figure 5: Prolonged hormone deprivation is required for maximum tyrosine phosphorylation response. 15-day lactating animals were treated with bromocryptine for either 7 or 36 h before killing. Figure shows extracts from 10 rabbits killed 5 min after prolactin or vehicle injection and processed as in Fig. 1.
Figure 6: In vivo tyrosine phosphorylation of receptor. Extracts from 15-day lactating or 20-day pregnant rabbits killed at different times after injection of vehicle or prolactin, with 8 or 36 h bromocryptine treatment. Rightside of panel shows result of probing immunoblot from maximal responding animals with anti-trophoblastin mAb.
The time course of receptor phosphorylation in response to hormone is illustrated in Fig. 7. The response is strong after only 1 min, is maintained for 5 min, but has declined markedly by 15 min after injection. A study of the time course of phosphorylation in vitro using isolated mammary gland acini from a 15-day lactating animal was unsuccessful because the receptor was fully phosphorylated before addition of hormone, presumably because the enzymatic digestion procedure had preactivated the tyrosine kinase (results not shown).
Figure 7: Time course of receptor phosphorylation in mammary tissue and comparison with liver. Rightpanel, rabbits were injected with vehicle or 1 mg of NIADDK oPrl S15 and killed at the given times after injection. Mammary glands were removed and processed for immunoprecipitation by U5 followed by immunoblotting with anti-phosphotyrosine 4G10. Leftpanel, a separate group of animals were injected with vehicle, oPrl as above, or with 0.8 mg of NIADDK oGH-15. Livers were removed 5 min after injections, and after preparing solubilized microsomes as for mammary tissue, were immunoprecipitated with either U5 (oPrl-injected animals) or anti-GH receptor mAb 263 (oGH- or vehicle-injected animals). Prolactin-treated animals showed a faint prolactin receptor band in only one of the hormone-treated animals, whereas livers from ovine GH-injected animals show a diffuse band around 125 kDa corresponding to GH receptor.
Fig. 7also shows that the livers taken from prolactin injected animals do not give a significant prolactin receptor signal in the 4G10 blots, although if the GH receptor mAb 263 was used, a diffuse band at 120-125 kDa is seen, but only in animals injected with 0.8 mg of ovine GH 5 min previously.
Figure 8: 4G10 phosphotyrosine immunoblots of total cell extracts (a) and prolactin receptor immunoprecipitates (Ab 46) (b and d) from CHO cells stably expressing the full-length rabbit prolactin receptor. Cells were hormone-deprived as described under ``Experimental Procedures,'' then oPrl was added to 400 ng/ml, and after the stated times, cells were washed and harvested. The cell pellet was then either boiled directly in SDS sample buffer (a) or immunoprecipitated with 46 (b), and then both were immunoblotted with 4G10 as set out in the experimental section. In panelc, the membrane was also probed with Ab 46 and developed for alkaline phosphatase localization of receptor. Panel d is a 4G10 immunoblot of prolactin receptor polyclonal 46 immunoprecipitates from another experiment with this cell line showing the transient phosphorylation response to hormone.
In Fig. 9, CHO cells expressing prolactin receptor were extracted with the phosphatase inhibitor buffer without EGTA and containing 10% glycerol. Extracts were immunoprecipitated with polyclonal 46 for 3 h at 4 °C. Probing the resulting immunoblot with JAK2 antibody revealed a 125-kDa band associated with the receptor after addition of prolactin. A variety of other maneuvers, including testing six different solubilizing detergents and use of anti-oPrl for capturing activated receptor complexes, were incapable of demonstrating JAK2 association in the presence of EGTA.
Figure 9: Association of JAK2 with prolactin receptor in CHO cells. CHO cells expressing rabbit prolactin receptor were treated with prolactin or vehicle, solubilized, and immunoprecipitated with prolactin receptor antiserum S46, and the immunoprecipitates were processed for Western blotting, all as described under ``Experimental Procedures.'' Blots were probed with 4G10, S46, or UBI JAK2 antibody and visualized by enhanced chemiluminescence.
Kinases possess an ATP
binding site, which can be affinity-labeled with
azido-ATP(26) , so in order to determine if other kinases were
associated with the EGTA extracted receptor, we incubated mammary
extracts with [P]azido-ATP, then UV
cross-linked, immunoprecipitated the receptor, and examined the labeled
component(s) autoradiographically after running the immunoprecipitate
on a reduced Laemmli gel. Fig. 10shows that the 100-kDa
receptor is not labeled, although a 48-kDa component with an ATP
binding site was found to be constitutively associated with the
receptor. Association is increased by hormone treatment, however. This
48-kDa component does not appear to be a tyrosine kinase, since we have
been unable to phosphorylate poly(Glu,Tyr) using protein G-bound
immunoprecipitates of rabbit mammary prolactin receptor and
[
-
P]ATP, even in the presence of 3 mM MnCl
. (
)
Figure 10:
[-
P]Azido-ATP
affinity labeling of receptor-associated proteins. Lots (0.5 ml) of
mammary tissue extracts from 15-day lactating animals injected with
vehicle or prolactin were incubated with Mn
and
Mg
, plus the photoaffinity label as described under
``Experimental Procedures.'' Extracts were set up with or
without 10 mM ATP to demonstrate specific binding. After photo
cross-linking, receptors were immunoprecipitated with antibodies 46 or
U5 and run on an 8% reduced SDS gel, dried, and autoradiographed as
described under ``Experimental
Procedures.''
This work presents what we believe to be the first analysis of in vivo tyrosine phosphorylation in response to administration of prolactin. It shows that the prolactin receptor is strongly tyrosine-phosphorylated within 1 min of prolactin injection, and this phosphorylation is transient, a finding confirmed with a rabbit prolactin receptor expressing CHO cell line. Moreover, there exists in vivo refractoriness to this phenomenon, and a prolonged period of hormone withdrawal is needed to observe the maximum extent of receptor phosphorylation. Finally, we demonstrate JAK 2 association with the prolactin receptor, as well as association with an ATP binding subunit of approximately 50 kDa.
Although the total
tyrosine phosphorylation analysis was done on detergent-solubilized
mammary gland membranes, it is likely that there were some residual
cytoplasmic proteins, so the weaker 45-kDa band seen to be
tyrosine-phosphorylated could be the MAP kinase, in agreement with the
MAP kinase activation of 150-175% we have observed in the stably
transfected E32 cells in response to prolactin. Activation
and tyrosine phosphorylation of the 42/44-kDa MAP kinase through the
homologous GH receptor has been reported by
others(19, 20, 37, 38) , and a
tyrosine-phosphorylated protein of mass around 40 kDa was reported in
the Nb2 line (24) to be prolactin-sensitive. Likewise, it is
plausible that the weaker band at 90 kDa is the prolactin-stimulated
equivalent of the p91 component of the interferon-stimulated gene
factor complex thought to be responsible for transactivation of JAK 2
responsive genes(18) . The prominent band at 125-130 kDa
presumably corresponds to JAK 2, as demonstrated by immunoblotting in
the transfected CHO cells. This kinase was recently reported to be
activated by prolactin in mouse mammary gland explants and in Nb2
cells(28) . The identity of the other tyrosine-phosphorylated
components at 70, 115, and 200 kDa remains to be determined.
We
believe that the evidence in support of hormone-dependent tyrosine
phosphorylation of the prolactin receptor is strong. First, the 100-kDa
phosphorylated band co-localizes with the receptor in Western blots
using different receptor antibodies, including mAbs. Second, this
phosphorylated band is seen only in CHO cells expressing the
full-length receptor. Third, the 100-kDa tyrosine-phosphorylated
protein is immunoprecipitable by anti-hormone antibody, but only if
ovine prolactin is bound to the receptor. Finally, if the 100-kDa band
were the 97-kDa cytoplasmic tyrosine kinase reported to be rapidly
tyrosine-phosphorylated in response to several
cytokines(26, 39) , it would have been
affinity-labeled by the azido-[P]ATP (24) . In any case, this kinase does not immunoprecipitate with
the prolactin receptor of Nb2 cells(24) . We have also found
that the prolactin receptor is rapidly tyrosine-phosphorylated in BAF-3
lymphoid cells stably transfected with full-length receptor
cDNA(40) . However, tyrosine phosphorylation of the Nb2
prolactin receptor was reported to be weak and inconsistent by Rui et al.(24) and was not seen by Rillema et
al.(25, 28) , yet such phosphorylation of closely
homologous cytokine receptors is now firmly established (e.g.(13) and (41) ). Since Rui et al.(24) also used prolactin receptor antibodies to
immunoprecipitate before immunoblotting for tyrosine phosphate, we need
to consider why the signal we observed with the full-length rabbit
receptor was so strong. Apart from the obvious differences of species
and cell type, the most likely possibility is that the Nb2 receptor
lacks the tyrosine residues targeted for phosphorylation because of the
198-residue deletion within its cytoplasmic domain(42) . In
this regard it is interesting to consider that the Nb2 line is
exquisitely sensitive to prolactin, presumably because its receptor
lacks this domain. This reasoning would imply that the missing domain
contains a negative regulatory region analogous to that of the
homologous erythropoietin receptor(41) . Tyrosine
phosphorylation within this domain could be the negative regulator to
limit the hormone signal to a pulse, so that conversion of receptor
cytoplasmic tyrosine residues to alanines, particularly tyrosines 407,
432, and 503, which lie within the Nb2 deleted 323-520 segment (43) may enhance the hormone response. Enhancement of receptor
responsiveness is indeed possible, since removal of the 322-333
sequence from the rabbit receptor increases prolactin transactivation
of the lactoglobulin promoter(12) . Alternatively, tyrosine
phosphorylation within this domain could be involved in transduction of
mammary specific functions through SH2 domain containing signaling
proteins such as phospholipase C
, Grb2, or the 85-kDa
phosphatidylinositol kinase(44, 45) . In support of
this view, milk protein gene response to prolactin in mammary gland
explants is blocked by a number of tyrosine kinase
inhibitors(46) . Moreover, deletion of residues
carboxyl-terminal to 320 markedly reduces the
lactoglobulin-chloramphenicol acetyltransferase response(12) .
The transient nature of the receptor tyrosine phosphorylation
illustrated in Fig. 7and Fig. 8is presumably the result
of tyrosine phosphatase action, for example PTP1C (47) or
PTP2(48) , which can associate with phosphorylated cytoplasmic
receptor domains through SH2 interactions(49, 50) .
The physiological reason for a brief phosphorylation signal is unclear.
Taking the argument set out above that tyrosine phosphorylation
represents a ``switch off'' signal, phosphatase action could
be seen as a means of returning the system quickly to the basal state,
ready for the next hormone pulse. Alternatively, it is known in the
case of the homologous interleukin 3 receptor that ligand-mediated
tyrosine phosphorylation of the subunit markedly accelerates its
proteolytic cleavage(51) , so that rapid dephosphorylation may
be a means of reducing receptor degradation. Of course, if tyrosine
phosphorylation is part of the signaling mechanism as for the epidermal
growth factor receptor(52) , then hormone-stimulated tyrosine
phosphatase activity as for the epidermal growth factor receptor (53) could serve as a desensitization signal. One could
speculate that the reason for reduced receptor phosphorylation in cells
previously exposed to hormone (``desensitized'') is an
increased phosphatase activity, or suppression of JAK 2 activity.
Indeed, the levels of these enzymes may be transcriptionally controlled
by prolactin.
Finally, although we found increased tyrosine phosphorylation of a 125-130-kDa band in solubilized mammary membranes from prolactin-treated animals, we were only able to show association of JAK 2 with the receptor when EGTA was omitted and glycerol was included in the extraction buffer. We have also observed a 48-kDa ATP binding subunit, which associates with the prolactin receptor, even in the absence of hormone. This protein does not appear to be a tyrosine kinase, based on our inability to obtain phosphorylation of poly(Glu,Tyr) in vitro with immunoprecipitated hormone-receptor complexes, and may represent a novel serine/threonine kinase. We are currently pursuing the identification of this interesting protein.