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J. Biol. Chem., Vol. 278, Issue 35, 32929-32935, August 29, 2003
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From the
Walter and Eliza Hall Institute of
Medical Research and Bone Marrow Research Laboratories, 1G Royal Parade,
Victoria 3050, Australia and the ¶Hanson Centre
for Cancer Research, Frome Road, Adelaide, SA 5000, Australia
Received for publication, March 21, 2003 , and in revised form, June 20, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Several distinct PrlR isoforms are generated by alternative mRNA splicing
in a species-specific manner. The long form of the PrlR is the major
functional isoform. In humans, an intermediate and several short isoforms have
been characterized that share the same extracellular ligand binding and
transmembrane domains as the long PrlR but vary in the length of their
cytoplasmic tails
(14–16).
In addition, a 
S1 PrlR variant that lacks part of the extracellular
domain, resulting in a lower ligand binding affinity, has been described
(17). Functional analyses have
demonstrated a marked reduction in the ability of the intermediate PrlR to
transduce signals and mediate prolactin-induced proliferation compared with
the long form (14), whereas
the short isoforms appear to act in a dominant negative manner when
coexpressed with the full-length receptor
(15,
16).
Although tyrosine phosphorylation of the PrlR has been well studied, phosphorylation of this receptor on serine or threonine residues has not been described. Here we report a novel threonine phosphorylation site that is specific to the long form of the PrlR and mediates binding to 14-3-3 proteins. These proteins are small acidic, ubiquitous proteins that recognize serine/threonine-phosphorylated residues in a context-specific manner (18, 19). In mammals, seven highly homologous family members have been described that bind to many different types of proteins, including cell cycle regulators, transcription factors, and proteins involved in signaling and apoptosis (20, 21). In this study we provide evidence that threonine 391 phosphorylation of the human PrlR and accompanying 14-3-3 binding to this site may be involved in receptor desensitization, specifically affecting signaling to the MAPK and PI3K pathways. Furthermore, we demonstrate that okadaic acid stabilizes Thr391 phosphorylation, suggesting that phosphorylation of this site is regulated by a protein phosphatase 2A (PP2A)-like activity.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-Bos and HA-tagged PKB in pcDNA3 were gifts from W. Alexander and
J. Testa, respectively. HA-tagged Erk2 in pcDNA3, Gal4-Sap1a, and G5E4-lux
were kindly provided by Y. Nagamine. EE-tagged 14-3-3 
and 
cDNAs
in pEF1 were a gift from A. Villunger. Site-directed mutagenesis was
performed using the QuikChange kit (Stratagene) according to the
manufacturer's instructions. Antibodies—PrlR-specific polyclonal rabbit antiserum was kindly provided by J. Clevenger. Commercially available antibodies used were: V5-specific mouse monoclonal antibody (Invitrogen), Glu-Glu-specific mouse monoclonal antibody (Babco), 14-3-3-specific rabbit polyclonal antibody (Upstate Biotechnology), phosphotyrosine-specific mouse monoclonal antibody (clone 4G10) (Upstate Biotechnology), anti-phospho-Erk1/2 (T202/Y204) rabbit polyclonal antibody (New England Biolabs), anti-phospho-PKB (T308) rabbit polyclonal antibody (New England Biolabs), HA-specific rat monoclonal antibody (Roche Applied Science), anti-phospho-Stat5a/b (Y694/Y699) mouse monoclonal antibody (Upstate Biotechnology), anti-Stat5a rabbit antiserum (Upstate Biotechnology).
Generation of Anti-phospho-Thr391 PrlR Antibody—The anti-phospho-Thr391-PrlR antibody was raised by immunizing New Zealand White rabbits with the GSKCS(pT)WPLP peptide conjugated to keyhole limpet hemocyanin. The antibody was first affinity purified with the immunizing peptide conjugated to Sepharose, and then non-phospho-specific antibodies were removed by adsorption to the non-phosphorylated peptide conjugated to Sepharose. The specificity of the affinity-purified anti-phospho-Thr391-PrlR antibody was verified by dot immunoblots against the immunizing phosphorylated peptide, the corresponding non-phosphorylated peptide, and an irrelevant phosphopeptide (CLG-PPHSR(pS)LPDILG). The peptides were synthesized by Mimotopes Pty. Ltd. (Clayton, Victoria, Australia).
Cell Culture—SKBR3 and T47D cells were maintained in RPMI containing 10% fetal bovine serum (CSL) and 1 µg/ml insulin (Sigma). Cells were starved overnight in serum-free medium prior to stimulation with 1 µg/ml prolactin (kindly provided by G. Parlow). HC11 cells were grown in RPMI supplemented with 10% fetal bovine serum, 10 ng/ml EGF (Sigma), and 5 µg/ml insulin. For in vitro differentiation, cells were grown to confluence and maintained for 3 days, after which they were incubated in growth medium lacking EGF for 24 h prior to stimulation with 5 µg/ml prolactin and 1 µM dexamethasone (Sigma). 293T and NIH3T3 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. For transient transfections, 293T cells were transfected with FuGENE (Roche Applied Science) according to the manufacturer's instructions. NIH3T3 and HC11 pools stably expressing wild-type and T391A PrlR were generated by transfection followed by selection in 0.75 and 0.2 mg/ml neomycin, respectively. Treatment of cells with okadaic acid (ICN Biomedicals) was at 1 µM.
Protein Extraction of Cells—Whole cell extracts were
obtained by solubilizing cells in NEB (50 mM Tris, pH 7.5, 150
mM NaCl, 1% Nonidet P-40, 1 mM sodium orthovanadate, 10
mM sodium fluoride, 20 mM 
-glycerophosphate plus
complete (Roche Applied Science) protease inhibitors). Lysates were clarified
by centrifugation at 16,000 x g for 10 min.
Bacterial Expression of GST-14-3-3—An
EcoRI-BamH1-cDNA encompassing the coding region of
14-3-3 was cloned into pGEX-2T. E299 bacteria transformed with this
plasmid were used to produce a glutathione S-transferase 14-3-3
fusion protein (GST-14-3-3). An overnight culture was used to inoculate
a 500-ml culture that was grown to an optical density of 0.6–0.8 at 600
nm. GST-14-3-3 protein expression was then induced with 0.1 M
isopropyl--D-1-thiogalactopyranoside for 3 h. Following
induction, the bacterial cultures were harvested, and the pellets were washed
with ice-cold phosphate-buffered saline (150 mM NaCl, 16
mM Na2HPO4, 4 mM
NaH2PO4, pH 7.3) and resuspended in 10 ml of
phosphate-buffered saline containing 1% Triton X-100, 5 mM EDTA, 1%
aprotinin, 1 mM phenylmethanesulfonyl chloride, and 1 µg/ml
leupeptin. The suspension was then sonicated three times for 10 s on ice and
the lysate centrifuged for 10 min at 8,000 x g. Purification of
GST-14-3-3 was performed by mixing with glutathione resin (Sigma) for 1
h at 4 °C. The resin was washed three times with phosphate-buffered
saline. The purity and amount of bound GST-14-3-3 was then determined by
SDS-PAGE and Coomassie staining.
GST Pulldowns, Immunoprecipitations, and Western
Blotting—Pulldowns were performed by incubating protein lysates
with GST or GST-14-3-3 coupled to glutathione beads for 2 h on ice.
Beads were washed three times with NEB (see above). For immunoprecipitations,
equal amounts of protein were incubated with specific antibodies for 2 h on
ice. Immune complexes were collected with protein G-Sepharose (Amersham
Biosciences) and washed three times with NEB. Precipitated proteins were
released by boiling in sample buffer and were subjected to sodium dodecyl
sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) using 4–20%
gradient gels (Novex). The proteins were blotted onto polyvinylidine
difluoride membranes (Millipore). After blocking with 20% horse serum (Hunter)
in phosphate-buffered saline containing 0.1% Tween 20, filters were probed
with specific antibodies. Proteins were visualized with peroxidase-coupled
secondary antibody using the ECL detection system (Amersham Biosciences).
Stripping of membranes was performed in SDS buffer (62.5 mM Tris,
pH 6.8, 2% SDS, 100 mM -mercaptoethanol) for 30 min at 60
°C. Membranes were then reprobed with the indicated antibodies.
Luciferase Reporter Assays—293T cells were grown in 6-well dishes and transfected with 0.25 µg each of wild-type PrlR or T391A PrlR and Gal4-Sap1a expression plasmids and the G5E4-luciferase reporter construct (22). pRL-TK, a Renilla luciferase plasmid under the control of the thymidine kinase promoter (50 ng/well) was cotransfected as an internal control. 24 h post-transfection, cells were either treated with 1 µg/ml prolactin or left untreated for 2 and 4 h. Luciferase activities were measured with the dual-luciferase reporter assay system (Promega).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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isoform based on
its ability to interact with the granulocyte-macrophage colony-stimulating
factor receptor (23) and its
abundant expression in various tissues, including the mammary gland (data not
shown). Interestingly, the PrlR associated with GST-14-3-3 but not with
GST alone (Fig. 1B).
This interaction was further verified by coimmunoprecipitation of the PrlR
with both the 14-3-3 and isoforms
(Fig. 1C). The
14-3-3
isoform, whose expression is restricted to epithelial cells, was
also found to associate with the PrlR (data not shown). Mutation of the
central three amino acids within the putative 14-3-3 binding motif
(CST391 to AAA) of the PrlR disrupted its association with
GST-14-3-3, demonstrating that the interaction was mediated via this
consensus site (Fig.
1B).
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In most cases, 14-3-3 proteins associate with target proteins in a
phosphorylation-dependent manner. To explore whether Thr391
represented a novel phosphorylation site within the human PrlR and was
essential for interaction with 14-3-3 proteins, we carried out binding studies
with a mutant in which Thr391 was altered to alanine. The
interaction between this mutant with GST-14-3-3
(Fig. 1B) and its
coimmunoprecipitation with 14-3-3 and isoforms
(Fig. 1C) was markedly
reduced. Conversely, a S390A PrlR mutant still associated with 14-3-3 (data
not shown). These results demonstrate an absolute requirement for
Thr391 in the PrlR to create a binding site for 14-3-3
proteins.
In Vivo Phosphorylation of Thr391 and 14-3-3 Binding Are Regulated by a PP2A-like Activity—To confirm phosphorylation of Thr391 in the PrlR in vivo, we generated a polyclonal antibody that specifically recognized a phosphopeptide encompassing the 14-3-3 binding site in the human PrlR but did not cross-react with the corresponding unphosphorylated peptide. This antibody was used to demonstrate that the wild-type but not the T391A mutant receptor was phosphorylated in transiently transfected 293T cells (Fig. 2A). Phosphorylation of the wild-type receptor promoted association with endogenous 14-3-3 proteins, as determined by immunoblotting using an antibody that recognizes all 14-3-3 isoforms. Because of the lack of suitable antibodies for immunoprecipitation of 14-3-3 and PrlR proteins, we generated stable PrlR transfectants to address the interaction with endogenous 14-3-3 proteins as well as the phosphorylation of Thr391. Only the wild-type PrlR was found to be phosphorylated in NIH3T3 and EGF-dependent mouse mammary epithelial HC11 cells that stably expressed either the wild-type or the T391A mutant receptor (Fig. 2A). The presence of EGF had no effect on the level of Thr391 phosphorylation. To gain insight into potential kinases or phosphatases that modify this site, we treated cells with various stimuli or inhibitors and analyzed their effect on Thr391 phosphorylation. Activation of protein kinases A, B, and C was found to have little effect on phosphorylation of this site (data not shown). Interestingly, okadaic acid, a selective inhibitor of the serine/threonine phosphatase PP2A, significantly increased phosphorylation of Thr391 in NIH3T3 cells expressing the wild-type PrlR (Fig. 2B). This increase was associated with further recruitment of endogenous 14-3-3 proteins (Fig. 2B).
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Thr391 Phosphorylation in Breast Epithelial Cells Is
Independent of Prolactin Stimulation—We next investigated the
association between 14-3-3 and the endogenous PrlR in human breast carcinoma
cell lines, T47D and SKBR3, and in HC11 cells. GST-14-3-3 pulled down
the receptor in all cell lines (Fig. 3,
A and B), indicating that the PrlR is
phosphorylated on Thr391 in these cell lines. Phosphorylation of
this site appears to be constitutive and independent of ligand binding because
prolactin stimulation did not affect association of the PrlR with 14-3-3.
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Increased Tyrosine Phosphorylation of the T391A PrlR— Binding of prolactin to the PrlR activates Jak2 kinase, which in turn phosphorylates the receptor on tyrosine residues to generate docking sites for downstream signaling molecules. To investigate whether Thr391 phosphorylation influenced activation of the receptor, we transiently transfected vectors encoding wild-type or mutant receptor into 293T cells and then stimulated cells with prolactin for 10, 30, and 60 min. The receptor was immunoprecipitated from lysates and blotted with a phosphotyrosine-specific antibody (Fig. 4A). Interestingly, we observed increased basal as well as prolactin-induced tyrosine phosphorylation of the mutant receptor, suggesting that Thr391 phosphorylation may modulate PrlR activation. Increased tyrosine phosphorylation of the T391A mutant compared with the wild-type receptor was found to be highly reproducible as shown by densitometric quantification of three independent experiments (Fig. 4B).
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Enhanced Signaling of PrlR T391A to the MAPK and PI3K Signaling
Pathways—Stat transcription factors are one of the main targets of
prolactin signaling. We therefore analyzed how the phosphorylation site mutant
affects activation of Stat5 by cotransfecting 293T cells with expression
vectors encoding Stat5a and either wild-type or mutant PrlR, followed by
immunoblotting of lysates with a phospho-specific Stat5 antibody. Despite
increased tyrosine phosphorylation of the T391A mutant
(Fig. 4), no increase in the
level of activated Stat5a was observed in response to prolactin
(Fig. 5A). Even when
the concentration of prolactin was titrated to ensure that signaling to Stat5a
was not saturated, activation of Stat5a by the wild-type and T391A mutant PrlR
were comparable after 30 min of prolactin treatment
(Fig. 5B). However, a
small but reproducible increase in basal Stat5a phosphorylation was evident
prior to treatment with prolactin (Fig. 5,
A and B). Comparable findings were made in two
independent reporter assays using the Stat5-responsive -casein promoter
linked to a luciferase reporter. Although basal reporter activity was 1.5-fold
greater for the T391A PrlR compared with wild-type PrlR, Thr391
phosphorylation did not influence Stat5a signaling following prolactin
treatment (data not shown).
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The PrlR has been reported to stimulate the PI3K and MAPK signal transduction pathways in addition to Stat transcription factors. To explore the activation of these pathways, we cotransfected PKB (also known as Akt) or Erk2 expression plasmids together with those encoding the wild-type or mutant PrlR and analyzed lysates from prolactin-stimulated cells by immunoblotting with phospho-specific antibodies that recognize activated PKB or Erk. Interestingly, PKB activation appeared to be prolonged following treatment with prolactin (Fig. 5C), whereas enhanced activation of Erk2 was observed by the mutant PrlR relative to that of the wild-type receptor (Fig. 5D). To evaluate the increase in signaling at the transcriptional level, we employed a reporter assay dependent on endogenous Erk activity. Cells were transfected with a Gal4-Sap1a fusion protein in which the DNA-binding domain of Sap1a has been replaced by the DNA-binding domain of the yeast transcription factor Gal4. Upon phosphorylation of Sap1a by activated Erk, but not by related MAP kinases such as p38 or c-Jun NH2-terminal kinase (24), Gal4-Sap1 binds to the Gal4 binding sites in the promoter of G5E4-lux luciferase reporter (22). In agreement with the kinetics of Erk activation shown in Fig. 5D, the mutant PrlR was able to sustain higher basal and prolactin-induced Erk-dependent transcriptional activation than the wild-type receptor (Fig. 5E). This was observed after 2 and 4 h of prolactin stimulation. Taken together, these results suggest that Thr391 phosphorylation may negatively regulate signaling by the PrlR to specific downstream pathways such as the MAPK and PI3K pathways, thereby affecting the magnitude of transcriptional responses.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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chain of the interleukin 3, interleukin
5 and granulocyte-macrophage colony-stimulating factor receptors was found to
interact with 14-3-3. Ligand stimulation promoted chain
phosphorylation on Ser585 and consequent association with 14-3-3
(23), bridging the receptor to
the p85 subunit of PI3K, and providing a critical survival signal for
hemopoietic cells (25). Phosphorylation of Thr391 in the PrlR appears to provide a negative regulatory mechanism by which to down-regulate receptor activity. The Thr391 phosphorylation mutant displayed increased basal and ligand-induced tyrosine phosphorylation. Moreover, this was found to selectively enhance signaling along the MAPK and PI3K pathways but did not affect activation of the Stat5a transcription factor by prolactin. The intracellular domain of the long human PrlR contains 10 tyrosine residues whose extent of phosphorylation and function remain to be established. The most carboxyl terminal tyrosine (Tyr587) is known to be the primary site responsible for activation of Stat5 (26), although other tyrosines may be involved in engaging additional downstream effectors. The finding that the mutant PrlR induced Stat5 activation to a similar extent as the wild-type receptor suggests that tyrosine residues other than Tyr587 are affected by Thr391 phosphorylation. In contrast to Stat5a, increased Erk activation and Erk-dependent transcription were observed in the presence of the T391A receptor, while PKB activation was found to be prolonged. Thus, PrlR phosphorylation on the Thr391 site can influence both the intensity and duration of signaling of specific pathways activated by the PrlR.
Various mechanisms act in concert to control ligand-induced receptor
activation. The PrlR is known to be regulated by negative feedback involving
the induction of suppressor of cytokine signaling proteins that bind either
the receptor itself or Jak2
(27). In vitro
studies have implicated several members of the suppressor of cytokine
signaling family in the control of prolactin signaling
(28–30).
A physiological function for suppressor of cytokine signaling proteins in the
negative regulation of PrlR signaling in the mammary gland has been
established for suppressor of cytokine signaling 1
(30). Dephosphorylation by
site-specific phosphatases may also modulate the activation status of the
receptor, although such phosphatases have not yet been identified for the
PrlR. Nevertheless, it is possible that 14-3-3 proteins may recruit a
phosphatase to the PrlR that dephosphorylates specific tyrosine residues. For
example, the protein tyrosine phosphatase H1 was reported to associate with
14-3-3 (31). Finally,
serine/threonine phosphorylation has been implicated in attenuation of
receptor signaling. In the case of ErbB1 (also known as EGFR or HER1),
phosphorylation on Thr654 and Thr699, via protein
kinases C and D, results in down-regulation of EGF-induced c-Jun
NH2-terminal kinase activation, possibly involving receptor
internalization. The Erk pathway, however, remained unaffected by
phosphorylation of these sites
(32). These findings parallel
our observation that mutation of Thr391 in the PrlR only affects
selective signaling pathways.
Stability and internalization of the PrlR do not appear to be altered by Thr391 phosphorylation. Pulse-chase labeling experiments indicated that the basal turnover rates of the wild-type and mutant receptors were comparable (data not shown). The PrlR has been shown to be down-regulated by internalization through clathrin-coated pits upon ligand binding (33). However, this mechanism may not be affected through 14-3-3 binding, because the rate of prolactin-induced internalization of the T391A mutant PrlR was similar to that of the wild-type receptor (data not shown).
The stimuli that modulate phosphorylation of the PrlR on Thr391 are yet to be established. Phosphorylation of the PrlR on Thr391 appears to be independent of prolactin stimulation in different cellular systems (Fig. 3). Although EGF is known to block PrlR signaling (34), it does not accomplish this by inducing phosphorylation of Thr391, because EGF-starved mouse mammary epithelial cells showed no change in Thr391 PrlR phosphorylation status (Fig. 2A). We demonstrate here that okadaic acid treatment of cells leads to a marked increase in Thr391 phosphorylation, which was associated with increased 14-3-3 binding to the PrlR. Okadaic acid is an effective inhibitor of PP2A and can be used at concentrations up to 1 µM without any detectable inhibitory effect on protein phosphatase 1 or other major serine/threonine phosphatases (35). It is possible that Thr391 in the PrlR is dephosphorylated by PP2A. Alternatively, because the activity of many kinases is controlled by PP2A (36), the effect of okadaic acid on Thr391 phosphorylation could be indirect. Although the major cellular okadaic acid-sensitive phosphatase is PP2A, a role for other less abundant phosphatases such as protein phosphatases 4 and 5, which are also inhibited by okadaic acid (37, 38), cannot be excluded. Further understanding of the role of Thr391 phosphorylation in the PrlR will rely on examining its phosphorylation in different organs and at different developmental stages. Identification of the upstream kinase that modifies this site in vivo will provide insight into the pathways that cross-talk with PrlR signaling.
Prolactin and its receptor have been implicated in breast oncogenesis (39, 40). Higher prolactin levels have been associated with an increased risk of breast cancer (41), while the majority of human breast cancers are positive for the PrlR. Both normal and neoplastic breast tissues are sources of extrapituitary prolactin, implying an autocrine-paracrine role for prolactin in breast cancer. An alternative means of modulating PrlR signal transduction in cancer cells may involve deregulated phosphorylation of the Thr391 residue described here. It is tempting to speculate that decreased phosphorylation of this site may prolong prolactin signaling and contribute to breast neoplasia.
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FOOTNOTES |
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The on-line version of this article (available at
http://www.jbc.org)
contains two supplementary figures.
Supported by a long-term European Molecular Biology fellowship.
|| To whom correspondence should be addressed. Tel.: 61-3-9342-2165; Fax: 61-3-9347-0852; E-mail: lindeman{at}wehi.edu.au.
1 The abbreviations used are: PrlR, prolactin receptor; WT, wild-type; SH2,
Src homology 2; Stat, signal transducer and activator of transcription; EGF,
epidermal growth factor; HA, hemagglutinin; MAPK, mitogen-activated protein
kinase; PI3K, phosphatidylinositol 3-kinase; Erk, extracellular
signal-regulated kinase; Jak, Janus kinase; PKB, protein kinase B; PP2A,
protein phosphatase 2A.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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