CPAP Is a Novel Stat5-Interacting Cofactor that Augments Stat5-Mediated Transcriptional Activity

Benjamin Peng, Kate D. Sutherland, Eleanor Y. M. Sum, Monilola Olayioye, Sergio Wittlin, Tang K. Tang, Geoffrey J. Lindeman and Jane E. Visvader

The Walter and Eliza Hall Institute of Medical Research & Rotary Bone Marrow Research Laboratories (B.P., K.D.S., E.Y.M.S., M.O., S.W., G.J.L., J.E.V.), Post Office Royal Melbourne Hospital, Victoria 3050, Australia; and Institute of Biomedical Sciences (T.K.T.), Academia Sinica, Taipei 115, Taiwan

Address all correspondence and requests for reprints to: Dr. Jane Visvader, Victorian Breast Cancer Research Consortium Laboratory, The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria 3050, Australia. E-mail: visvader{at}wehi.edu.au.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Stat5, a member of the signal transducer and activators of transcription (Stat) protein family, is a primary mediator of prolactin (PRL) signaling in the mammary gland. There are two distinct Stat5 genes, Stat5a and Stat5b. The Stat5a isoform has been demonstrated to have an essential role in mammary epithelial differentiation, whereas Stat5b is required for dimorphic sexual growth. To search for proteins that interact with the C terminus of Stat5a, a highly divergent region amongst Stat family members, we performed a yeast two-hybrid screen of HBL100 and primary breast adenocarcinoma libraries. This led to the identification of a protein that had previously been isolated as a centrosomal P4.1-associated protein (CPAP). CPAP was shown to specifically interact with Stat5a and Stat5b but not with Stat1 or Stat3. Both the tyrosine phosphorylated and unphosphorylated forms of Stat5, as well as Stat5a/Stat5b heterodimers, could associate with CPAP. CPAP was expressed in human breast cancer cell lines and the developing mammary gland as well as in other tissues. Indirect immunofluorescence and cellular fractionation studies revealed that CPAP was predominantly cytoplasmic, with low levels in the nucleus. Nuclear levels of CPAP increased substantially upon activation of the PRL pathway, most likely reflecting cotranslocation of this protein with activated Stat5. Furthermore, CPAP was found to augment Stat5-mediated transcription. Thus, we have identified CPAP as a novel coactivator of Stat5 proteins in the PRL (and probably other) pathways.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE SIGNAL TRANSDUCERS and activators of transcription (Stat) proteins play important roles in regulating fundamental cellular processes including cell growth, differentiation, and survival (reviewed in Refs. 1, 2, 3). Stats are activated in response to a wide variety of cytokines, growth factors, and hormones (4, 5). After ligand binding to its cognate receptor, cytoplasmic tyrosine Janus kinases (Jak) are recruited, triggering phosphorylation of the receptor and Stat proteins. This leads to Stat dimerization, which occurs via reciprocal interaction between the SH2 (Src Homology 2) domain of one Stat monomer and the phosphorylated tyrosine of another Stat monomer. Stat dimers then translocate to the nucleus, where they bind specific recognition sites and regulate transcription of target gene promoters (1, 5). Both the intensity and duration of the Stat response are tightly regulated, underscoring their importance in regulating proliferation and differentiation. Indeed, there is accumulating evidence that inappropriate Stat activation leads to oncogenesis (6).

Development of the adult mammary gland involves a complex cycle of morphogenetic changes, commencing with ductal elongation and branching in puberty, lobuloalveolar proliferation during pregnancy, followed by functional differentiation at parturition and the onset of lactation (7). Growth and differentiation during these stages is dictated by the coordinated action of steroid and peptide hormones. Prolactin (PRL), a pituitary polypeptide, is an important regulator of mammopoiesis (8, 9). During pregnancy, PRL is essential for expansion and differentiation of the mammary alveolar epithelium. After parturition, PRL acts in synergy with other hormones to induce terminal differentiation and milk production (7, 10). Binding of PRL to its cognate receptor (PRLR) triggers the Jak2-Stat5 pathway.

Stat5 was originally identified as a PRL-inducible transcription factor in the mammary gland (11). In addition to PRL, Stat5 proteins are activated by GH, erythropoietin, thrombopoietin, GM-CSF, IL-3, IL-2, IL-7, IL-9, IL-5, and IL-15 (reviewed in Ref. 12). Similar to the other family members, Stat5 comprises discrete structural domains: an N-terminal region that mediates oligomerization, a coiled-coiled domain, a DNA-binding region, an SH2 motif, a conserved tyrosine whose phosphorylation mediates dimerization between Stats, and a C-terminal transactivation domain (reviewed in Ref. 1). The C terminus has also been implicated in the degradation of activated Stat5 by the proteosome pathway (13). There are two closely related Stat5 genes (14, 15, 16, 17, 18), Stat5a and Stat5b, which colocalize and are highly conserved in both mouse and human. Upon activation, the encoded Stat5a and Stat5b proteins form either heterodimers or homodimers. In mammary epithelial cells, the Stat5a/Stat5b heterodimer and Stat5a homodimers are thought to have essential roles in milk protein gene regulation (19, 20, 21, 22).

Although Stat5a and Stat5b are widely expressed and respond to common stimuli, they have distinct biological functions. Targeted deletion of Stat5a results in impaired mammary gland development and failed lactogenesis, establishing an important role for this factor in mammary epithelial differentiation (23, 24). In contrast, Stat5b has been demonstrated to be essential for sexually dimorphic growth mediated by GH signaling (25). Female mice lacking both genes are infertile (24). Furthermore, mice deficient in either or both of these genes exhibit specific defects in hematopoietic development, including lymphopoiesis, erythropoiesis, and granulopoiesis (24, 26).

The specificity and diversity in Stat signaling pathways is dictated by multiple factors. The target genes activated by Stat proteins are dependent on both cell type and the activating stimulus (17, 27). In addition to Jaks, tyrosine kinases such as epidermal growth factor receptor (28) and nonreceptor tyrosine kinases such as Src (29) can induce Stat signaling. Moreover, phosphorylation of specific serine residues in some Stat proteins is required for maximal transcriptional activity (30, 31, 32). Serine phosphorylation may occur by ERK or other kinases, including JNK, p38MAPK, and protein kinase C (reviewed in Ref. 32). These observations emphasize the importance of cross-talk between different signaling pathways. Once latent Stat proteins become activated, specificity is likely to be determined by the context of the Stat-DNA recognition element, and by interactions with Stat-associated regulatory factors.

A number of proteins have been shown to interact with one or more members of the Stat family (33). Stat5-associated proteins include ERK, CBP/p300, Nmi, and the glucocorticoid receptor. The ubiquitous proteins ERK and p300 have been shown to bind the C-terminal transactivation domain of Stat5a (34, 35). The C-terminal regions of Stat proteins are the most divergent between the different family members. To further explore mechanisms contributing to the specificity of Stat5a function in the mammary gland, we searched for proteins that associated with the C-terminal domain. We report the identification of a novel Stat-interacting cofactor, which corresponds to the recently isolated protein termed centrosomal P4.1-associated protein (CPAP). Although CPAP can participate in centrosomal complexes, the majority (greater than 70%) of CPAP is found in soluble fractions (36). We demonstrate that nuclear levels of CPAP are elevated on activation of Stat5 and define a role for CPAP as a transcriptional coactivator in the Stat5 signaling pathway.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Carboxy Terminus of Stat5a Interacts with CPAP
To identify proteins that interact with the C terminus of Stat5a, we used a bait comprising the Gal4-DNA-binding domain fused to a cDNA encoding residues 618–793 of Stat5a, in a yeast two-hybrid screen of a breast epithelial HBL100 cDNA library. This region of Stat5a spans a portion of the SH2 domain, the invariant tyrosine residue at position 694, and the transactivation domain (Fig. 1AGo). Despite the presence of a transcriptional activation domain, the bait had only weak transactivating ability in yeast and did not yield any background colonies in the presence of 8 mM 3-aminotriazole (3-AT).



View larger version (14K):
[in this window]
[in a new window]
 
Figure 1. Schematic Diagram of Stat5a, Stat5b, and CPAP

A, Stat5a and 5b share 92% homology, with the majority of sequence differences located in the C-terminal transactivation (TA) domain (LDARLSPPAGLFTSARSSLS vs. QWIPHASQS). Furthermore, a PCEPA insertion occurs in Stat5b in the region proximal to the TA domain. The region corresponding to the Stat5a bait is shown. B, Schematic structure of CPAP. The cDNA clone encoding the Stat5-interacting protein (SIC) is identical with the published CPAP cDNA sequence (GenBank accession no. AF139625). CPAP contains three bipartite nuclear localization sequence motifs (K839RKIAPVKRGEDLSKSRR856, K1054RAEAIESSLEVEKKDK1070 and K1093KNYLPMQGNPPRRSK1108) represented as black bars. A region of homology with Tcp-10 (T-complex protein) at the C terminus is indicated by the shaded box. The original clone ({Delta}CPAP) isolated in the yeast two-hybrid screen is shown. C, Schematic diagram of deletion mutants of Stat5a. Wild-type Stat5a, the Y694F and deletion mutants used to determine the region of interaction with CPAP are shown.

 
A total of 20 x 106 transformants were screened, yielding 17ß-galactosidase-positive clones. One of these clones encoded a 1.2-kb open reading frame fused to the GAL4 activation domain and was also subsequently identified in a yeast two-hybrid screen of a primary breast adenocarcinoma library. Two full-length cDNAs of 4234 bp and 4358 bp were isolated by screening a {lambda} expression library derived from 184 human mammary epithelial cells. These encoded a potential protein of 1338 amino acids, corresponding to an EST isolated from female breast tissue (GenBank accession no. BE298403). More recently, the cDNA clones were found to be identical to the CPAP gene, which was isolated in a yeast two-hybrid screen using the head domain of the cytoskeletal protein 4.1R-135 (36) (Fig. 1BGo). Sequences located at the C terminus of this protein show weak homology to the human T-complex responder gene product 10 (Tcp-10). As reported, CPAP contains putative coiled-coiled domains, leucine heptad repeats, and a series of 21 nonamer repeats (G-box) at its C terminus (36). We have also noted three putative nuclear localization sequences KRKIAPVKRGEDLSKSRR (839–856), KRAEAIESSLEVEKKDK (1054–1070), and KKNYLPMQGNPPRRSK (1093–1108) (Fig. 1BGo) and two potential nuclear export signals LELDEFLFL (573–581) and LGNELKLNI (883–891) in CPAP.

A search of HTGS and other databases revealed that CPAP is located on the pericentromeric region of the long arm of chromosome 13 (13q11-12) spanning approximately 30 kb of genomic sequence (STS marker stSG46995), centromeric to the BRCA2 tumor suppressor gene located at 13q12.3.

Stat5a and CPAP Interact in Mammalian Cells and Form a Native Complex in Vivo
To establish whether CPAP could interact with Stat5 in vivo, human embryonal kidney epithelial cells (293T) were transfected with myc-tagged Stat5a or Stat5b and Flag-tagged CPAP expression plasmids and subjected to coimmunoprecipitation/Western blot assays. Initially, we tested the association using the CPAP clone originally identified in the yeast two-hybrid screen ({Delta}CPAP, Fig. 1BGo), encoding the C-terminal 354 residues. Interaction between {Delta}CPAP and Stat5a was found to be stronger than that between {Delta}CPAP and Stat5b (Fig. 2AGo, upper panels), as reproducibly less {Delta}CPAP was immunoprecipitated from cells expressing Stat5b, despite comparable levels of expression of these two Stat5 proteins (Fig. 2AGo, lower panels). To determine whether CPAP could interact with the activated form of Stat5, cells were cotransfected with a constitutively active form of Jak2 (Jak2-KL), which mediates phosphorylation of tyrosine 694 in Stat5a and Stat5b. Phosphorylation of Stat5, assessed using a monoclonal antibody specific for Y694, was only observed in the presence of Jak2-KL (Fig. 2AGo, lanes 1 and 3, middle panel). The association between {Delta}CPAP and Stat5a (Fig. 2AGo, lanes 1 and 2) or Stat5b (Fig. 2AGo, lanes 3 and 4) appeared equivalent in the presence or absence of Jak2-KL. This finding demonstrates that {Delta}CPAP can associate with both the latent and activated forms of Stat5. Furthermore, the Stat5a-Y694F mutant (Fig. 1CGo), in which the conserved tyrosine residue was mutated to phenylalanine, did not affect CPAP binding (data not shown). Thus, phosphorylation of tyrosine 694 is not necessary for mediating interaction with CPAP.



View larger version (33K):
[in this window]
[in a new window]
 
Figure 2. CPAP Forms a Complex with Stat5a in Vivo

A, CPAP interacts with both Stat5a and Stat5b, either phosphorylated or unphosphorylated, in mammalian cells. 293T cells were transiently transfected with myc-tagged Stat5a (lanes 1 and 2) or Stat5b (lanes 3 and 4) and HA-tagged {Delta}CPAP expression constructs, in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of a plasmid encoding a constitutively active form of Jak2 (Jak2-KL). Cell lysates were incubated with anti-myc antibody and immunoblotted with anti-HA antibody. C, Control isotype-matched monoclonal antibody. Western blot analysis confirmed expression of individual Stat5 and CPAP proteins (lower panels). Activation of Stat5 was confirmed using an antiphospho-Stat5 specific antibody (lanes 1 and 3). B, CPAP can interact with the Stat5a/Stat5b heterodimer. Plasmids encoding untagged Stat5a, myc-Stat5b and/or HA-tagged {Delta}CPAP were cotransfected into 293T cells, in the presence (lane 1) or absence (lane 2) of the Jak2-KL expression plasmid. Coimmunoprecipitation/Western assays in (B) and (C) were performed as in (A). C, Interaction between full-length CPAP and Stat5a. Full-length CPAP carrying an N-terminal Flag tag and myc-Stat5a encoding plasmids were cotransfected into human embryonic kidney epithelial 293T cells and analyzed by coimmunoprecipitation-Western blot assays. Anti-myc antibody could precipitate CPAP (myc, lanes 1 and 2), whereas control antibody, C, did not. Western analysis confirmed expression of Flag-CPAP and myc-Stat5a (lower panels, lanes 1 and 2). D, CPAP does not interact with Stat1 or Stat3. 293T cells were transiently transfected with HA-tagged CPAP and either myc-tagged Stat1 or Stat3 expression constructs, in the presence or absence of Jak2-KL-expressing plasmid. Anti-myc antibody was used for the immunoprecipitation, whereas anti-HA antibody revealed high level of expression of both Stat1 and Stat3 but no interaction with CPAP.

 
Stat5a and Stat5b form stable heterodimers upon phosphorylation of tyrosine 694. These dimers translocate to the nucleus, where they bind their cognate DNA sequence and regulate transcription (1, 5). To determine whether CPAP interacted with Stat5a/5b heterodimers, 293T cells transfected with {Delta}CPAP, Stat5a and myc-Stat5b, in the presence or absence of Jak2-KL, were analyzed by coimmunoprecipitation/Western blot assays (Fig. 2BGo). Immunoprecipitation with an anti-myc antibody revealed that CPAP did not disrupt formation of the heterodimer. Moreover, higher levels of {Delta}CPAP were associated with activated Stat5a/Stat5b (Fig. 2BGo, lane 1), compared with that associated with unphosphorylated Stat5 (Fig. 2BGo, lane 2). Thus, CPAP can form a heterotrimeric complex with Stat5a and Stat5b and preferentially associates with Stat5 dimers.

Full-length CPAP (carrying a Flag-tag) and Stat5 (with a myc-tag) were found to associate efficiently. CPAP, migrating as a 175-kDa protein, was immunoprecipitable with an anti-myc antibody (Fig. 2CGo, lane 1, myc), but not with an isotype-matched monoclonal antibody (Fig. 2CGo, lane 1, C). CPAP readily associated with the activated form of Stat5, in cells coexpressing Jak2 (Jak2-KL), consistent with the data shown in Fig. 2AGo. Stat5b was also found to interact with CPAP in transient transfection assays (data not shown). In contrast, no interaction was detected between {Delta}CPAP and either Stat1 (Fig. 2DGo, lanes 1 and 2) or Stat3 (Fig. 2DGo, lanes 3 and 4), either in the presence or absence of Jak-2. Thus, a specific interaction occurs between Stat5 and CPAP.

To test the interaction between endogenous CPAP and Stat5a proteins, whole cell lysates derived from normal mouse testes were analyzed by coimmunoprecipitation assays using anti-Stat5a or control monoclonal antibody. Testis has previously been shown to contain abundant CPAP mRNA (36). Immunoblotting of proteins precipitated with a polyclonal rabbit {alpha}-Stat5a antibody (Fig. 3Go, lane 2), but not with preimmune sera (Fig. 3Go, lane 1), revealed a specific interaction between Stat5a and CPAP. Comparison of immunoprecipitated proteins from testis extracts (lanes 5 and 8) with those from transfected 293T cell lysates verified the sizes of CPAP and Stat5a, respectively (Fig. 3Go, lanes 3, 6, and 9). Preimmune sera did not precipitate either CPAP or Stat5a (Fig. 3Go, lanes 1, 4, and 7). Thus, native Stat5a and CPAP proteins can associate in vivo.



View larger version (26K):
[in this window]
[in a new window]
 
Figure 3. Endogenous CPAP and Stat5a Proteins Interact in Vivo

Mouse testis lysate was immunoprecipitated with either preimmune serum (PI) (lane 1) or anti-Stat5a antiserum (lane 2) and blotted with affinity-purified anti-CPAP antiserum (36 ). Immunoblotting of lysates from 293T cells transfected with Flag-CPAP and myc-Stat5a provided size controls (lanes 3, 6, and 9). Endogenous CPAP and Stat5a proteins could be detected in testis lysates by immunoprecipitation/immunoblotting with the relevant antibodies (middle and lower panels).

 
Delineation of the Stat5a Region that Mediates Binding to CPAP
To further define the region within Stat5a that interacts with CPAP, a series of Stat5a deletion mutants carrying an N-terminal HA tag (see Fig. 1CGo) were cotransfected with Flag-tagged {Delta}CPAP into 293T cells and subjected to immunoprecipitation/Western blot analysis (Table 1Go). Mutants encompassing residues 618–793, 669–774, and 669–793 were immunoprecipitable by an anti-Flag antibody (Fig. 4Go, lanes 1 and 2), whereas Stat5a 677–793 did not associate with {Delta}CPAP (data not shown). Thus, portions of both the SH2 and the C-terminal transactivation domains are dispensable for association with {Delta}CPAP. The inability of the 677–793 mutant to bind CPAP indicates that residues 677–686 of the SH2 domain are necessary for interaction. The minimal interaction region within Stat5a comprises 106 residues, spanning the C-terminal portion of the SH2 domain, the conserved tyrosine residue, and the majority of the transactivation domain. This region shares 85% identity with Stat5b.


View this table:
[in this window]
[in a new window]
 
Table 1. Delineation of the Stat5a Region Required for Interaction with CPAP in Mammalian Cells

 


View larger version (30K):
[in this window]
[in a new window]
 
Figure 4. Delineation of a Minimal CPAP-Interacting Domain within Stat5a

Constructs encoding HA-tagged Stat5a deletion mutants (residues 669–774, lane 1; and residues 669–793, lane 2) were cotransfected with that encoding Flag-{Delta}CPAP into 293T cells. Lysates were immunoprecipitated with anti-Flag antibody, then immunoblotted with anti-HA antibody to reveal the Stat5a mutants. The 25-kDa band in the upper panel corresponds to light chain. The lower molecular mass bands correspond to Stat5 mutants encoding 106- and 125-amino- acid Stat5a-polypeptides (excluding the Flag epitope). Expression of HA-Stat5a mutants and Flag-{Delta}CPAP in cell extracts was confirmed by Western blotting, shown in the middle and lower panels.

 
CPAP Is Extensively Expressed in Breast Cancer Cell Lines and Normal Tissues
A panel of human breast cancer cell lines (37) was probed with a 1.2-kb CPAP cDNA, revealing a major transcript of 4.5 kb in all cell lines (Fig. 5AGo). RNA levels in these lines appeared greater than that in the nontransformed 184 human breast epithelial line. In human tissues, CPAP expression was variable, as previously noted (36). Highest levels were observed in haematopoietic organs, heart, brain, lung, and skeletal muscle (Fig. 5BGo).



View larger version (51K):
[in this window]
[in a new window]
 
Figure 5. Expression of CPAP in Breast Epithelial Cells and Human Tissues

A, CPAP is expressed in breast epithelial cells. Northern blot analysis of poly(A)+ RNA isolated from the indicated normal 184 and human breast cancer cell lines. The filter was hybridized with a human CPAP cDNA probe followed by a control GAPDH probe. B, CPAP is variably expressed in human tissues. Poly(A)+ RNA Northern blots (CLONTECH Laboratories, Inc.) were hybridized under high stringency with a human CPAP probe. C, CPAP expression in the developing mouse mammary gland. Semiquantitative RT-PCR analysis was performed on total RNA (1 µg) derived from mammary tissue isolated from virgin, pregnant, lactating, and involuting mice. PCR was carried out for 32 and 35 cycles before electrophoresis. Gels were blotted and hybridized with internal probes representing either CPAP or HPRT, the latter providing a control for RNA loading. D, CPAP is developmentally regulated in the mammary gland. Whole cell protein lysates (500 µg) derived from mouse mammary glands were immunoprecipitated with anti-CPAP antisera, then immunoblotted with the same antibody. Lysates from 293T cells transfected with CPAP provided a size control. dp, Days pregnant; dL, days lactation; dI, days involution.

 
To examine the expression of CPAP within the developing mouse mammary gland, we used semiquantitative RT-PCR to analyze RNA derived from virgin, pregnant, lactating, and involuting mouse mammary glands. Expression of CPAP mRNA was up-regulated during pregnancy but declined thereafter (Fig. 5CGo). Immunoprecipitation/Western blot analysis of protein lysates prepared from glands at different developmental stages, using an affinity-purified anti-CPAP antibody (Fig. 5DGo), confirmed higher CPAP expression in mammary glands derived from pregnant vs. those from virgin mice. CPAP expression was maintained during lactation but decreased in involuting mammary glands. Thus, CPAP appears to be developmentally regulated in the mouse mammary gland.

Colocalization of CPAP and Stat5a
CPAP has previously been reported to localize to the centrosome with the {gamma}-tubulin complex (36). In these studies, CPAP association with the centrosome was largely restricted to the insoluble cellular fraction. Greater than 70% of CPAP, however, is found in the soluble cellular fraction (36). To further examine the subcellular distribution of CPAP, breast epithelial MCF-7 and 293T cell lines were transfected with CPAP and immunofluorescence staining was performed using affinity purified anti-CPAP antiserum in conjunction with confocal laser scanning microscopy. CPAP primarily localized to the cytoplasm, but some nuclear staining was also detectable (Fig. 6AGo). Occasional perinuclear staining was visible, consistent with previous reports (36). Immunostaining of MCF-7 cells expressing Flag-tagged CPAP with an anti-Flag monoclonal antibody confirmed these results (Fig. 6AGo). Significantly, staining of HBL100 epithelial cells with anti-CPAP antiserum (36) revealed that endogenous CPAP was cytoplasmic as well as nuclear (Fig. 6BGo). Thus, CPAP appears to be predominantly cytoplasmic, with some nuclear staining, in the three cell lines tested. In transfected 293T cells, colocalization of CPAP and Stat5a was evident in the cytoplasm (Fig. 6CGo). The same observation was made in MCF-7 cells (data not shown).



View larger version (26K):
[in this window]
[in a new window]
 
Figure 6. Subcellular Localization of CPAP and Colocalization with Stat5

A, CPAP predominantly localizes to the cytoplasm. Indirect immunofluorescence, in conjunction with confocal microscopy, on MCF-7 and 293T cells transfected with Flag-CPAP using anti-CPAP antisera, preimmune (PI) sera, or anti-Flag antibody. The majority of CPAP was found in the cytosol, with low levels in the nucleus. B, Endogenous CPAP protein is primarily cytoplasmic. HBL100 epithelial cells were subjected to indirect immunofluorescence using rabbit polyclonal anti-CPAP antiserum. No staining was detectable with preimmune serum (PI). C, CPAP and Stat5a colocalize to the cytoplasm. 293T kidney epithelial cells were cotransfected with myc-Stat5a and Flag-CPAP. Cells were subjected to immunofluorescence using anti-myc and anti-CPAP antibodies. Bar, 10 µm.

 
CPAP Translocates to the Nucleus upon Activation of Stat5a
To explore whether the localization of CPAP was altered by activation of the Jak-Stat5 pathway, cellular fractionation studies were performed on 293T cells transfected with Flag-CPAP, myc-Stat5a, and/or Jak2-KL expression plasmids. The purity of fractionation was verified by immunoblotting with anti-{alpha}-tubulin and anti-Histone 1 (H1) monoclonal antibodies. Compatible with immunofluorescence studies, CPAP was identified in the cytoplasmic fractions (Fig. 7AGo) and at low levels in the nuclear fraction. Immunoblotting with anti-myc and antiphospho-Stat5 antibodies confirmed that Stat5 was primarily nuclear when activated by phosphorylation (Fig. 7AGo). Interestingly, nuclear levels of CPAP increased substantially when coexpressed with Stat5a and Jak2. Nuclear levels of CPAP remained low in cells expressing Stat5a in the absence of Jak2 (Fig. 7AGo). Coexpression of CPAP, Stat5b and Jak2, accompanied by phosphorylation and activation of Stat5, also resulted in nuclear translocation of CPAP (data not shown). Stat5a and CPAP were also shown to be associated in cytosolic fractions (Fig. 7CGo, lane 2), as demonstrated for whole cell extracts (Fig. 7CGo, lane 4), suggesting that the complex preexists in the cytoplasm.



View larger version (20K):
[in this window]
[in a new window]
 
Figure 7. CPAP Translocates to the Nucleus in the Presence of Activated Stat5a

239T cells transfected with CPAP, Stat5a and either (A) Jak2-KL, or (B) PRLR. Transfected cells were then fractionated into cytosolic and nuclear pools. Equal amounts (20 µg) of protein were loaded per lane. The antibodies used for immunoblotting were: anti-Flag, anti-myc, antiphospho-Stat5, antitubulin, and antihistone H1. More nuclear CPAP protein was detectable in cells containing phosphorylated myc-Stat5a, compared with those containing Stat5a but no Jak2. Furthermore, more CPAP was evident in the nuclear fraction of PRLR-expressing cells treated with PRL (for 2 h) than those untreated. Immunoblotting with {alpha}-tubulin or {alpha}-histone H1 confirmed the purity of the cytoplasmic and nuclear fractions, respectively. C, CPAP and Stat5a form a complex in the cytoplasm. Cytoplasmic extracts from transfected 293T cells were immunoprecipitated with anti-myc or control antibody, then immunoblotted with anti-Flag antibody (lanes 2 and 3). The upper band (denoted by an arrow) corresponds to CPAP. The upper band evident in lane 3 is at least 20 kDa smaller than CPAP and is a nonspecific product that is detected by the control isotype-matched antibody. Lane 1 represents a size control for CPAP. Whole cell extracts from transfected cells were also immunoprecipitated with Stat5-specific antibody, then blotted with anti-CPAP antiserum to provide a further control for CPAP (lane 4).

 
PRL mediates its effects through the Jak2-Stat5 pathway but also stimulates the MAPK and phosphoinositide 3-kinase pathways (10, 38). To confirm whether CPAP translocates to the nucleus upon activation of the PRLR, cells were transiently transfected with PRLR, Flag-CPAP, and myc-Stat5a, and subsequently treated with PRL (Fig. 7BGo). In parallel with the findings described above, cell fractionation studies revealed that CPAP was recruited to the nucleus by activation of the PRL receptor.

CPAP Enhances Stat5-Dependent Transcription
To determine whether CPAP affects Stat5 transcriptional activity, reporter assays were carried out in 293T cells using the lactogenic hormone response element (LHRE) reporter construct, which contains six copies of a Stat5 DNA-binding site linked to the luciferase gene. Activation of Stat5a (Fig. 8AGo) or Stat5b (Fig. 8BGo) by Jak2 resulted in a 2- or 5-fold increase in transcriptional activity, respectively. Cotransfection of full-length CPAP resulted in a further increase of 2- to 3-fold. CPAP augmented transactivation by Stat5b to a greater extent than that by Stat5a (Fig. 8Go, A and B) but itself had no effect on reporter gene activity (Fig. 8Go). Cotransfection of CPAP with both Stat5a and Stat5b did not lead to a further increase in activity relative to that obtained with Stat5b alone (data not shown). The expression levels of Jak2 and phosphorylated Stat5 were assessed in the same extracts as those assayed for luciferase reporter activity and were equivalent in the different transfections. As expected, CPAP was found to enhance PRL-mediated stimulation of the reporter construct, in cells carrying PRLR, Stat5a/5b and the reporter gene (data not shown). Thus, CPAP appears to be a positive cofactor for Stat5-mediated transcription.



View larger version (24K):
[in this window]
[in a new window]
 
Figure 8. CPAP Acts as a Coactivator in the Stat5 Signaling Pathway

Reporter gene assays were performed in 293T cells transfected with myc-Stat5a or Stat5b, Flag-Jak2, HA-CPAP expression plasmids and/or empty vector, together with the LHRE linked to the luciferase gene. Transfections with myc-Stat5a and myc-Stat5b are shown in A and B, respectively. The data represent an average of three independent experiments, performed in duplicate, with SEM indicated. Western blot analysis (shown for a representative experiment) using either antiphospho-Stat5 or {alpha}-Jak2 confirmed that the plasmids were equivalently expressed in the different transfectants.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Stat transcription factors are key mediators of cytokine signaling. These proteins are activated by a range of stimuli and different Stat members can be induced by the same stimulus. For example, Stat1, Stat3, and Stat5 are capable of activation by PRL (39). Stat5a, however, appears to be the most critical gene for PRL-induced milk protein gene expression and functional differentiation of the mammary gland. The mechanisms underlying the specificity of Stat action and target gene selection in different tissues are not well understood. Here we describe a novel interaction between Stat5 and CPAP, and a role for CPAP as a coactivator of Stat5-mediated transcription.

CPAP was previously identified by virtue of its interaction with the cytoskeletal protein 4.1R-135. Structurally, CPAP consists of multiple hydrophobic regions and a C-terminal domain that shares limited homology with Tcp-10, which has a role in sex transmission ratio distortion. The significance of this homology is not clear. CPAP was found to colocalize with {gamma}-tubulin in centrosomes, where it appears to be in the center of microtubule asters. The majority (

70%) of CPAP, however, occurs in the soluble cytosolic fraction rather than in the insoluble centrosomal fraction (36). Using confocal microscopy, we have demonstrated colocalization of Stat5 and CPAP in the cytoplasm of two different cell lines. Perinuclear staining of CPAP was evident in occasional cells, presumably corresponding to centrosomes. There is no evidence, however, to suggest the presence of Stat5 within centrosomes. Low levels of CPAP were also visible in the nucleus, consistent with the presence of three consensus nuclear localization signals in this protein. Thus, CPAP appears to be present in different subcellular compartments, including the cytoplasm, nucleus, and centrosomes. The precise localization of CPAP may vary with cell type or cell cycle status.

Both Stat5 and CPAP translocate from the cytoplasm to the nucleus in response to PRL-mediated activation of the Jak-Stat pathway. Association between Stat5a and CPAP could be demonstrated in cytosolic fractions, implying the presence of a preexisting cytoplasmic complex that translocates to the nucleus. Concomitant with PRL activation of its receptor or Jak2-mediated phosphorylation of Stat5a, nuclear levels of CPAP were found to increase significantly. Similarly, higher levels of CPAP were observed in the nucleus after activation of Stat5b (our unpublished data). The translocation most likely reflects recruitment of CPAP to the nucleus via its association with Stat5 dimers. In parallel with Stat proteins, CPAP appears to be phosphorylated on tyrosine residue(s) by Jak2 kinase (Peng, B., unpublished data). The effect of this phosphorylation on CPAP activity is not yet known.

CPAP specifically associated with Stat5 proteins but not with Stat1 or Stat3. The minimal interaction domain that was defined within Stat5a to mediate CPAP-binding contains 106 amino acids. This carboxy-terminal region encompasses the invariant tyrosine residue (position 694) that confers Stat dimerization, and part of the transactivation domain. The latter domain harbors the majority of differences between Stat5a and Stat5b, but it is the shared region within this domain that mediates association with CPAP. Binding of CPAP to Stat5 was independent of the phosphorylation status of Stat5. Moreover, both Stat5 homodimers and heterodimers could associate with CPAP (unpublished data). CPAP can also participate in a DNA-protein complex with the Stat5a/Stat5b heterodimer on a Stat5-binding site (unpublished data). Given that Stat5 proteins have the ability to form a tetrameric complex on DNA-target sites (40, 41), and that CPAP can dimerize (unpublished data), these proteins have the potential to form a high molecular weight multiprotein complex.

Stat5 proteins are likely to regulate the expression of common as well as distinct target genes. Only a few Stat5 target genes have been defined, including CIS (42), oncostatin M (43), ß-casein (19), ß-lactoglobulin (44), and the IL-2 receptor-{alpha} enhancer (45). The two Stat5 proteins appear to have distinct DNA-binding specificities (46). In mammary epithelial cells, Stat5a and Stat5b have different binding affinities for the various Stat5 target sites in the ß-lactoglobulin (44) and ß-casein gene promoters (19). As the two proteins differ primarily at their C terminus, this may in part reflect the proteins that associate with the C-terminal region of these Stats. CPAP was found to interact with both isoforms of Stat5. However, interaction between CPAP and Stat5a appeared to be of higher affinity than that between CPAP and Stat5b. Differences in the avidity of protein-protein interactions may affect the DNA-binding properties of Stat-protein complexes and contribute to target site selection.

Stat proteins associate with diverse proteins, including the transcription factors p48, c-Jun, Sp1, and the glucocorticoid receptor; the nuclear cofactors p300/CBP and Nmi; the inhibitory PIAS proteins; the DNA replication protein MCM5; NPI-1, which forms part of the nuclear translocation machinery; and the serine kinases ERK1/2 (reviewed in Ref. 33). The C-terminal transactivation domain of Stat5 has been demonstrated to bind CBP/p300 and ERK1/2. CBP and p300 serve as coactivators for several Stat proteins as well as a large number of other transcription factors, and are essential for mammalian proliferation, differentiation and development (47, 48). ERK1 and ERK2 are MAPKs that phosphorylate serine residues in response to diverse stimuli. Here, we have identified CPAP as a novel type of coactivator in Stat signaling. The mechanism by which CPAP augments Stat5-mediated transcription and the target genes influenced by CPAP’s association with Stat5 have yet to be determined.

Stat proteins 1, 3, and 5 have been implicated in oncogenesis (6, 49). Activated Stat signaling may contribute to transformation by stimulating cellular proliferation and preventing apoptosis. There is evidence suggesting that Stat5 is an important survival factor (26, 50). Stat5 is constitutively activated in multiple haematopoietic malignancies, including acute and chronic leukemias and lymphomas and in cell lines (6). CPAP localizes to chromosome 13q11-12, a region that has been linked to allelic loss in hematopoietic malignancies (51) and solid cancers but also appears to be in an area of allelic gain in prostate and ovarian cancers. The role of CPAP in normal cells and its influence on Stat5 activity in neoplastic cells remain to be established. The presence of CPAP in multiple subcellular compartments and its potential to shuttle between the nucleus and cytoplasm implies that this protein is multifunctional.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Constructs and Yeast Two-Hybrid Screen
The pGBT9-Stat5a bait was generated by PCR amplification of mouse Stat5a (residues 618–793) and subsequent cloning into the EcoRI-XhoI site of the plasmid pGBT9, using the following primers: forward 5'-GCGCGAATTCAGTGACTCGGAAATCGGGGC-3' and reverse 5'-GCGCCTCGAGTCAGGACAGGGAGCTTCTAGC-3'. The bait was used to screen HBL100 epithelial cell and primary breast adenocarcinoma libraries, generously provided by R. Baer and J. Byrne, respectively. Standard protocols, as detailed in the CLONTECH Laboratories, Inc. manual, were followed; 8 mM 3-AT (3-amino-1,2,4-triazole) was included in the selection plates, which lacked tryptophan, leucine, and histidine. Stat5a mutants were generated by PCR or restriction digest and cloned into either pGBT9 or HA-pcDNA3.1 expression vectors, as indicated in Table 1Go. Full-length mouse Stat5a was subcloned into the pEFrPGKpuro vector (52). Myc-Stat5a (mouse), Myc-Stat5b (mouse), and Flag-Jak2 were generously provided by W. Alexander and S. Nicholson. Jak2-KL, lacking residues 521–745 in the kinase-like domain, and HA-Stat5a(Y694F) were kindly provided by K. Harder and N. Hynes, respectively.

To isolate a full-length cDNA clone of CPAP, a {lambda}-Hybrizap expression library (Visvader, J., unpublished data) derived from 184 human mammary epithelial cell cDNA was screened with a partial-length CPAP clone. Full-length CPAP (residues 5–1338) or truncated {Delta}CPAP (residues 985-1338) cDNAs were cloned into HA-pcDNA3.0 or Flag-pEFPGKrpuro vectors for mammalian transfection studies.

Immunoprecipitation and Western Blot Analysis
For transient expression studies, 293T cells (10-cm plates) were transfected with 3 µg of each plasmid DNA using the calcium phosphate precipitation method (Life Technologies, Inc.). Forty-eight hours after transfection, cells were lysed in RIPA buffer (150 mM NaCl; 5 mM EDTA; 50 mM Tris, pH 8.0; 1% Nonidet P-40; 0.5% sodium deoxycholate; and 0.1% sodium dodecyl sulfate) containing protease inhibitors (Complete, Roche Molecular Biochemicals, Mannheim, Germany). Proteins were immunoprecipitated with the indicated antibody and protein G-Sepharose (Amersham Pharmacia Biotech, Arlington Heights, IL), then washed three times with cold buffer (as above). Samples were resolved by SDS-PAGE gradient (4–20%) gel electrophoresis (Novex) before transfer to Hybond-C extra membranes (Amersham Pharmacia Biotech). Filters were blocked, then incubated with primary antibody, followed by incubation with either antimouse or rabbit horseradish peroxidase-coupled secondary antibodies, and developed by enhanced chemiluminescence (Amersham Pharmacia Biotech). The immunoprecipitation and primary antibodies used for immunoblotting were as follows: mouse anti-myc monoclonal (a gift from S. Nicholson), mouse anti-Flag monoclonal antibody (Sigma, St. Louis, MO), mouse anti-HA monoclonal antibody (Sigma), rabbit anti-Stat5a polyclonal antiserum (Upstate Biotechnology, Inc.), mouse antiphospho-Stat5 monoclonal antibody (Upstate Biotechnology, Inc.), and affinity-purified anti-CPAP rabbit polyclonal antiserum (36).

For detection of endogenous proteins, murine testes were ground to a fine powder under liquid nitrogen, resuspended in TEB buffer (1% Triton X-100; 50 mM Tris, pH 7.5; 150 mM NaCl; 1 mM sodium orthovanadate; 20 mM ß-glycerophosphate; 10 mM sodium fluoride) plus protease inhibitors (Complete, Roche), sheared 10 times through a 21-gauge needle, incubated on ice for 10 min, and clarified by centrifugation. For immunoprecipitation of CPAP, 1.5 mg of protein extract was incubated on ice for 2 h with either anti-CPAP or anti-Stat5a antiserum. Complexes were immunoprecipitated with protein A-agarose, washed five times with TEB containing 0.1% deoxycholate, followed by one wash with TNE (50 mM Tris, pH 7.5; 150 mM NaCl; 5 mM EDTA). Samples were separated by SDS-PAGE (8%) and transferred onto polyvinylidene difluoride membrane for probing. For detection of CPAP in the mouse mammary gland, 500 µg of protein extract (at indicated time points) was immunoprecipitated with 1 µl of affinity-purified anti-CPAP antisera in RIPA buffer, before blotting with anti-CPAP antibody as described above.

RNA Expression and RT-PCR Analysis
Poly(A)+ RNA was isolated from breast cancer cell lines (37) and Northern analysis performed as described (53). Briefly, 3 µg of Poly(A)+ RNA was fractionated on a 1% agarose-formaldehyde gel, transferred to Hybond N+ membrane (Amersham Pharmacia Biotech), and hybridized with a CPAP (1.2 kb; residues 985-1338) or GAPDH cDNA probe. Human multiple tissue Northern blots (CLONTECH Laboratories, Inc.) containing 2 µg poly(A)+ RNA were hybridized with the CPAP probe. For RT-PCR analysis, RNA was prepared from mammary gland using RNAzol (Tel-Test, Friendswood, TX). PCR was performed on cDNA using the following primers: CPAP forward primer 5'-CTGGTTTCCCAATGGAACTCG-3', CPAP reverse primer 5'-GTGTCCATTAGCACATTACC-3', hypoxanthine guanine phosphoribosyl transferase (HPRT) forward primer 5'-CACAGGACTAGAACACCTGC-3', HPRT reverse primer 5'-GCTGGTGAAAAGGACCTCT-3', as described (54).

Experimental Animals
All animals used in the course of this study were treated within published guidelines of humane animal care.

Indirect Immunofluorescence and Confocal Microscopy
Cells were plated onto coverslips in six-well dishes before transient transfection using FuGENE (Roche). For immunostaining, cells were fixed after 48 h with PBS containing 4% paraformaldehyde and 2% glucose, permeablized by a short treatment with 0.2% Triton X-100, then blocked with PBS containing 5% goat serum and 0.1% Triton X-100. Slides were incubated with primary antibody at room temperature for 2 h or at 4 C overnight. Primary antibodies used for immunostaining were as follows: anti-Flag monoclonal Ab (Sigma), anti-HA monoclonal Ab (Sigma), anti-myc monoclonal antibody and affinity-purified anti-CPAP rabbit polyclonal antiserum (36). After washing, the slides were incubated with either antimouse secondary antibody conjugated to Texas Red (The Jackson Laboratory, Bar Harbor, ME) or antirabbit secondary antibody conjugated to fluorescein isothiocyanate (The Jackson Laboratory). Cells were counterstained with 1 µg/ml Bis-benzimide (Sigma) and the coverslips were mounted onto slides with Vectashield H-1000 (Vector Laboratories, Burlingame, CA) or with 1,4-diazabicyclo (2, 22) octane (Sigma) in fluorescent mounting medium (DAKO Corp., Carpinteria, CA). Cells were examined by confocal microscopy using a Leica Corp. TCS-4D confocal microscope.

Subcellular Fractionation
Nuclear and cytoplasmic extracts were prepared essentially as described (55). Transfected 293T cells, either treated with PRL (3 µg/ml) or not, were pelleted and resuspended in hypotonic buffer (10 mM HEPES, pH 7.9; 1.5 mM MgCl2; 10 mM KCl; 0.5 mM dithiothreitol; and 0.2 mM phenylmethylsulfonyl fluoride). Cells were homogenized by Douncing, centrifuged, and cytoplasmic fractions collected. Nuclear pellets were washed and lysed in 200 µl of high salt buffer (20 mM HEPES, pH 7.9; 25% glycerol; 1% Nonidet P-40; 420 mM NaCl; 1.5 mM MgCl2; 0.2 mM EDTA; 0.5 mM dithiothreitol; and 0.2 mM phenylmethylsulfonyl fluoride) to generate nuclear fractions. Protein concentrations were determined using the Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA). For Western blot analysis (see above), lysates containing 20 µg of protein were loaded in each lane.

Reporter Assays
For luciferase reporter assays, 293T cells grown in six-well culture dishes were transfected with 0.5 µg of each plasmid using the calcium phosphate method. The luciferase reporter plasmid contains six copies of a lactogenic hormone response element consisting of a high-affinity Stat5 binding site, upstream of a thymidine kinase minimal promoter sequence, kindly provided by V. Goffin (56). Rous sarcoma virus-ß-galactosidase plasmid (100 ng) was used in each transfection to normalize the transfection efficiency. Luciferase and ß-galactosidase activities were assayed as described (Promega Corp., Madison, WI).


    ACKNOWLEDGMENTS
 
We thank M. Santamaria and the confocal microscopy laboratory for excellent assistance. We are grateful to R. Baer and J. Byrne for cDNA libraries and to S. Jane for the CLONTECH Laboratories, Inc. blot. We also thank V. Goffins, N. Hynes, K. Harder, M. Crossley, S. Nicholson, and W. Alexander for cDNA constructs and J. Szer for invaluable help.


    FOOTNOTES
 
This work was supported by the Victorian Breast Cancer Research Consortium and the Rotary Bone Marrow Research Laboratories (Melbourne, Australia).

Abbreviations: CPAP, Centrosomal P4.1-associated protein; HA, hemagglutinin; HPRT, hypoxanthine guanine phosphoribosyl transferase; Jak, Janus kinase; PRL, prolactin; PRLR, PRL receptor; SH2, Src homology 2; Stat, signal transducer and activators of transcription.

Received for publication March 17, 2002. Accepted for publication June 5, 2002.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Darnell Jr JE 1997 STATs and gene regulation. Science 277:1630–1635[Abstract/Free Full Text]
  2. Horvath CM 2000 STAT proteins and transcriptional responses to extracellular signals. Trends Biochem Sci 25:496–502[CrossRef][Medline]
  3. Bromberg J, Darnell Jr JE 2000 The role of STATs in transcriptional control and their impact on cellular function. Oncogene 19:2468–2473[CrossRef][Medline]
  4. Darnell Jr JE, Kerr IM, Stark GR 1994 Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264:1415–1421[Medline]
  5. Ihle JN 1996 STATs: signal transducers and activators of transcription. Cell 84:331–334[Medline]
  6. Bowman T, Garcia R, Turkson J, Jove R 2000 STATs in oncogenesis. Oncogene 19:2474–2488[CrossRef][Medline]
  7. Hennighausen L, Robinson GW, Wagner KU, Liu W 1997 Prolactin signaling in mammary gland development. J Biol Chem 272:7567–7569[Free Full Text]
  8. Vonderhaar BK 1987 Prolactin: transport, function, and receptors in mammary gland development and differentiation. In: Neville MC, Daniel CW, eds. The mammary gland. New York: Plenum Press; 383–438
  9. Horseman ND 1999 Prolactin and mammary gland development. J Mammary Gland Biol Neoplasia 4:79–88[CrossRef][Medline]
  10. Bole-Feysot C, Goffin V, Edery M, Binart N, Kelly PA 1998 Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr Rev 19:225–268[Abstract/Free Full Text]
  11. Wakao H, Gouilleux F, Groner B 1994 Mammary gland factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response. EMBO J 13:2182–2191[Abstract]
  12. Leonard WJ, O’Shea JJ 1998 Jaks and STATs: biological implications. Annu Rev Immunol 16:293–322[CrossRef][Medline]
  13. Wang D, Moriggl R, Stravopodis D, Carpino N, Marine JC, Teglund S, Feng J, Ihle JN 2000 A small amphipathic {alpha}-helical region is required for transcriptional activities and proteasome-dependent turnover of the tyrosine-phosphorylated Stat5. EMBO J 19:392–399[Abstract/Free Full Text]
  14. Azam M, Erdjument-Bromage H, Kreider BL, Xia M, Quelle F, Basu R, Saris C, Tempst P, Ihle JN, Schindler C 1995 Interleukin-3 signals through multiple isoforms of Stat5. EMBO J 14:1402–1411[Abstract]
  15. Liu X, Robinson GW, Gouilleux F, Groner B, Hennighausen L 1995 Cloning and expression of Stat5 and an additional homologue (Stat5b) involved in prolactin signal transduction in mouse mammary tissue. Proc Natl Acad Sci USA 92:8831–8835[Abstract]
  16. Mui AL, Wakao H, O’Farrell AM, Harada N, Miyajima A 1995 Interleukin-3, granulocyte-macrophage colony stimulating factor and interleukin-5 transduce signals through two STAT5 homologs. EMBO J 14:1166–1175[Abstract]
  17. Ripperger JA, Fritz S, Richter K, Hocke GM, Lottspeich F, Fey GH 1995 Transcription factors Stat3 and Stat5b are present in rat liver nuclei late in an acute phase response and bind interleukin-6 response elements. J Biol Chem 270:29998–30006[Abstract/Free Full Text]
  18. Lin JX, Mietz J, Modi WS, John S, Leonard WJ 1996 Cloning of human Stat5B. Reconstitution of interleukin-2-induced Stat5A and Stat5B DNA binding activity in COS-7 cells. J Biol Chem 271:10738–10744[Abstract/Free Full Text]
  19. Schmitt-Ney M, Doppler W, Ball RK, Groner B 1991 ß-casein gene promoter activity is regulated by the hormone-mediated relief of transcriptional repression and a mammary-gland-specific nuclear factor. Mol Cell Biol 11:3745–55[Medline]
  20. Gouilleux F, Wakao H, Mundt M, Groner B 1994 Prolactin induces phosphorylation of Tyr694 of Stat5 (MGF), a prerequisite for DNA binding and induction of transcription. EMBO J 13:4361–4369[Abstract]
  21. Liu X, Robinson GW, Hennighausen L 1996 Activation of Stat5a and Stat5b by tyrosine phosphorylation is tightly linked to mammary gland differentiation. Mol Endocrinol 10:1496–506[Abstract]
  22. Cella N, Groner B, Hynes NE 1998 Characterization of Stat5a and Stat5b homodimers and heterodimers and their association with the glucocortiocoid receptor in mammary cells. Mol Cell Biol 18:1783–1792[Abstract/Free Full Text]
  23. Liu X, Robinson GW, Wagner KU, Garrett L, Wynshaw-Boris A, Hennighausen L 1997 Stat5a is mandatory for adult mammary gland development and lactogenesis. Genes Dev 11:179–186[Abstract]
  24. Teglund S, McKay C, Schuetz E, van Deursen JM, Stravopodis D, Wang D, Brown M, Bodner S, Grosveld G, Ihle JN 1998 Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses. Cell 93:841–850[Medline]
  25. Udy GB, Towers RP, Snell RG, Wilkins RJ, Park SH, Ram PA, Waxman DJ, Davey HW 1997 Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression. Proc Natl Acad Sci USA 94:7239–7244[Abstract/Free Full Text]
  26. Socolovsky M, Fallon AE, Wang S, Brugnara C, Lodish HF 1999 Fetal anemia and apoptosis of red cell progenitors in Stat5a-/-5b-/- mice: a direct role for Stat5 in Bcl-X(L) induction. Cell 98:181–191[Medline]
  27. Meinke A, Barahmand-Pour F, Wohrl S, Stoiber D, Decker T 1996 Activation of different Stat5 isoforms contributes to cell-type-restricted signaling in response to interferons. Mol Cell Biol 16:6937–6944[Abstract]
  28. Ruff-Jamison S, Chen K, Cohen S 1995 Epidermal growth factor induces the tyrosine phosphorylation and nuclear translocation of Stat 5 in mouse liver. Proc Natl Acad Sci USA 92:4215–4218[Abstract]
  29. Yu CL, Meyer DJ, Campbell GS, Larner AC, Carter-Su C, Schwartz J, Jove R 1995 Enhanced DNA-binding activity of a Stat3-related protein in cells transformed by the Src oncoprotein. Science 269:81–83[Medline]
  30. Wen Z, Zhong Z, Darnell Jr JE 1995 Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell 82:241–250[Medline]
  31. Bromberg JF, Horvath CM, Besser D, Latham WW, Darnell Jr JE, 1998 Stat3 Activation is required for cellular transformation by v-src. Mol Cell Biol 18:2553–2558[Abstract/Free Full Text]
  32. Decker T, Kovarik P 2000 Serine phosphorylation of STATs. Oncogene 19:2628–2637[CrossRef][Medline]
  33. Shuai K 2000 Modulation of STAT signaling by STAT-interacting proteins. Oncogene 19:2638–2644[CrossRef][Medline]
  34. Pfitzner E, Jahne R, Wissler M, Stoecklin E, Groner B 1998 p300/CREB-binding protein enhances the prolactin-mediated transcriptional induction through direct interaction with the transactivation domain of Stat5, but does not participate in the Stat5-mediated suppression of the glucocorticoid response. Mol Endocrinol 12:1582–1593[Abstract/Free Full Text]
  35. Pircher TJ, Petersen H, Gustafsson JA, Haldosen LA 1999 Extracellular signal-regulated kinase (ERK) interacts with signal transducer and activator of transcription (STAT) 5a. Mol Endocrinol 13:555–565[Abstract/Free Full Text]
  36. Hung LY, Tang CJ, Tang TK 2000 Protein 4.1 R-135 interacts with a novel centrosomal protein (CPAP) which is associated with the {gamma}-tubulin complex. Mol Cell Biol 20:7813–7825[Abstract/Free Full Text]
  37. Douglas AM, Goss GA, Sutherland RL, Hilton DJ, Berndt MC, Nicola NA, Begley CG 1997 Expression and function of members of the cytokine receptor superfamily on breast cancer cells. Oncogene 14:661–669[CrossRef][Medline]
  38. Kim DW, Cochran BH 2001 JAK2 activates TFII-I and regulates its interaction with extracellular signal-regulated kinase. Mol Cell Biol 21:3387–3397[Abstract/Free Full Text]
  39. DaSilva L, Rui H, Erwin RA, Howard OM, Kirken RA, Malabarba MG, Hackett RH, Larner AC, Farrar WL 1996 Prolactin recruits STAT1, STAT3 and STAT5 independent of conserved receptor tyrosines TYR402, TYR479, TYR515 and TYR580. Mol Cell Endocrinol 117:131–140[CrossRef][Medline]
  40. Meyer WK, Reichenbach P, Schindler U, Soldaini E, Nabholz M 1997 Interaction of STAT5 dimers on two low affinity binding sites mediates interleukin 2 (IL-2) stimulation of IL-2 receptor {alpha} gene transcription. J Biol Chem 272:31821–31828[Abstract/Free Full Text]
  41. John S, Vinkemeier U, Soldaini E, Darnell Jr JE, Leonard WJ 1999 The significance of tetramerization in promoter recruitment by Stat5. Mol Cell Biol 19:1910–1918[Abstract/Free Full Text]
  42. Matsumoto A, Masuhara M, Mitsui K, Yokouchi M, Ohtsubo M, Misawa H, Miyajima A, Yoshimura A 1997 CIS, a cytokine inducible SH2 protein, is a target of the JAK-STAT5 pathway and modulates STAT5 activation. Blood 89:3148–3154[Abstract/Free Full Text]
  43. Kamiya A, Kinoshita T, Ito Y, Matsui T, Morikawa Y, Senba E, Nakashima K, Taga T, Yoshida K, Kishimoto T, Miyajima A 1999 Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer. EMBO J 18:2127–2136[Abstract/Free Full Text]
  44. Philp JA, Burdon TG, Watson CJ 1996 Differential activation of STATs 3 and 5 during mammary gland development. FEBS Lett 396:77–80[CrossRef][Medline]
  45. Lecine P, Algarte M, Rameil P, Beadling C, Bucher P, Nabholz M, Imbert J 1996 Elf-1 and Stat5 bind to a critical element in a new enhancer of the human interleukin-2 receptor {alpha} gene. Mol Cell Biol 16:6829–6840[Abstract]
  46. Boucheron C, Dumon S, Santos SC, Moriggl R, Hennighausen L, Gisselbrecht S, Gouilleux F 1998 A single amino acid in the DNA binding regions of STAT5A and STAT5B confers distinct DNA binding specificities. J Biol Chem 273:33936–33941[Abstract/Free Full Text]
  47. Yao TP, Oh SP, Fuchs M, Zhou ND, Ch’ng LE, Newsome D, Bronson RT, Li E, Livingston DM, Eckner R 1998 Gene dosage-dependent embryonic development and proliferation defects in mice lacking the transcriptional integrator p300. Cell 93:361–372[Medline]
  48. Kung AL, Rebel VI, Bronson RT, Ch’ng LE, Sieff CA, Livingston DM, Yao TP 2000 Gene dose-dependent control of hematopoiesis and hematologic tumor suppression by CBP. Genes Dev 14:272–277[Abstract/Free Full Text]
  49. Danial NN, Rothman P 2000 JAK-STAT signaling activated by Abl oncogenes. Oncogene 19:2523–2531[CrossRef][Medline]
  50. Nosaka T, Kawashima T, Misawa K, Ikuta K, Mui AL, Kitamura T 1999 STAT5 as a molecular regulator of proliferation, differentiation and apoptosis in hematopoietic cells. EMBO J 18:4754–4765[Abstract/Free Full Text]
  51. Still IH, Roberts T, Cowell JK 1997 Fine structure physical mapping of a 1.9 Mb region of chromosome 13q12. Ann Hum Genet 61:15–24[CrossRef][Medline]
  52. Huang DC, Cory S, Strasser A 1997 Bcl-2, Bcl-XL and adenovirus protein E1B19kD are functionally equivalent in their ability to inhibit cell death. Oncogene 14:405–414[CrossRef][Medline]
  53. Visvader JE, Elefanty AG, Strasser A, Adams JM 1992 GATA-1 but not SCL induces megakaryocytic differentiation in an early myeloid line. EMBO J 11:4557–4564[Abstract]
  54. Lindeman GJ, Wittlin S, Lada H, Naylor MJ, Santamaria M, Zhang JG, Starr R, Hilton DJ, Alexander WS, Ormandy CJ, Visvader J 2001 SOCS1 deficiency results in accelerated mammary gland development and rescues lactation in prolactin receptor-deficient mice. Genes Dev 15:1631–1636[Abstract/Free Full Text]
  55. Lindeman GJ, Gaubatz S, Livingston DM, Ginsberg D 1997 The subcellular localization of E2F-4 is cell-cycle dependent. Proc Natl Acad Sci USA 94:5095–5100[Abstract/Free Full Text]
  56. Ferrag F, Chiarenza A, Goffin V, Kelly PA 1996 Convergence of signaling transduced by prolactin (PRL)/cytokine chimeric receptors on PRL-responsive gene transcription. Mol Endocrinol 10:451–460[Abstract]