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Originally published In Press as doi:10.1074/jbc.M302830200 on August 6, 2003

J. Biol. Chem., Vol. 278, Issue 42, 41355-41366, October 17, 2003
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Estrogens Down-regulate p27Kip1 in Breast Cancer Cells through Skp2 and through Nuclear Export Mediated by the ERK Pathway*

James S. Foster {ddagger}, Romaine I. Fernando {ddagger}, Noriko Ishida §, Keiichi I. Nakayama § and Jay Wimalasena {ddagger} 

From the {ddagger}Department of Obstetrics and Gynecology, Graduate School of Medicine, Program in Comparative and Experimental Medicine, University of Tennessee Medical Center, Knoxville, Tennessee 37920 and the §Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan

Received for publication, March 19, 2003 , and in revised form, July 29, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cyclin-dependent kinase (CDK) inhibitor p27Kip1 plays a key role in growth and development of the mammary epithelium and in breast cancer. p27Kip1 levels are regulated through ubiquitin/proteasome-mediated proteolysis, promoted by CDK2 and the F box protein Skp2 at the G1/S transition, and independent of Skp2 in mid-G1. We investigated the respective roles of Skp2 and subcellular localization of p27Kip1 in down-regulation of p27Kip1 induced in MCF-7 cells by estrogens. 17{beta}-Estradiol treatment increased Skp2 expression in MCF-7 cells; however, this increase was prevented by G1 blockade mediated by p16Ink4a or the CDK inhibitor roscovitine, whereas down-regulation of p27Kip1 was maintained. Exogenous Skp2 prevented growth arrest of MCF-7 cells by antiestrogen, coinciding with decreased p27Kip1 expression. Under conditions of G1 blockade, p27Kip1 was stabilized by inhibition of CRM1-dependent nuclear export with leptomycin B or by mutation of p27Kip1 (Ser10 -> Ala; S10A) interfering with CRM1/p27Kip1 interaction. Antisense Skp2 oligonucleotides and a dominant-interfering Cul-1(1–452) mutant prevented down-regulation of p27Kip1S10A, whereas Skp2 overexpression elicited its destruction in mitogen-deprived cells. Active mediators of the extracellular signal-regulated kinase (ERK) pathway including Raf-1caax induced cytoplasmic localization of p27Kip1 in antiestrogen-treated cells and prevented accumulation of p27Kip1 in these cells independent of Skp2 expression and coinciding with ERK activation. Genetic or chemical blockade of the ERK pathway prevented down-regulation and cytoplasmic localization of p27Kip1 in response to estrogen. Our studies indicate that estrogens elicit down-regulation of p27Kip1 in MCF-7 cells through Skp2-dependent and -independent mechanisms that depend upon subcellular localization of p27Kip1 and require the participation of mediators of the Ras/Raf-1/ERK signaling pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogenic steroids are essential for normal development and function of female reproductive tissues yet play a pivotal, causative role in the initiation and progression of breast cancer (1, 2). Estrogens and antiestrogens exert growth regulatory actions in normal and malignant breast epithelial cells through regulation of events in the G1 phase of the cell cycle (for review, see Ref. 3).

p27Kip1 is a key element of the G1 phase regulatory apparatus. Removal of cyclin-dependent kinase (CDK)1 inhibitory activity associated with p21Cip1 and p27Kip1 in G1 phase is essential to cyclin E-CDK2 activation and S phase entry (for review, see Refs. 4 and 5). Along with other members of the kinase inhibitory protein (Kip) family (p21Cip1 and p57Kip2) p27Kip1 serves both as an inhibitor of CDK2 activity in G0 and early G1 and as an assembly factor for cyclin D-CDK4/6 complexes in early G1 (for review, see Ref. 5). Functional expression of p27Kip1 is required for morphogenesis and normal proliferative responses in the mammary epithelium (6). Mammary glands of p27Kip1(–/–) mice are underdeveloped compared with the wild-type, whereas the mammary epithelium of p27Kip1(+/–) mice is hyperproliferative and susceptible to oncogene-induced tumorigenesis (6, 7). Breast cancer isolates frequently exhibit decreased p27Kip1 expression in addition to elevated expression of cyclin E (8–12). High levels of cyclin E-CDK2 activity in these tumors correlate with a high cyclin E/low p27Kip1 phenotype (13) as does poor disease outcome. Expression of p27Kip1 is an independent prognostic indicator for breast carcinomas and for a range of other tumor types (for review, see Ref. 14).

Estrogens elicit active cyclin E-CDK2, and G1/S transition in estrogen receptor-positive MCF-7 breast cancer cells corresponding with decreasing levels of p27Kip1 protein (15, 16) and a decrease in CDK inhibitory activity associated with both p27Kip1 and p21Cip1 (15–17). Levels of p21Cip1 and p27Kip1 increase in MCF-7 cells after treatment of proliferating cells with antiestrogen, resulting in decreased CDK2 activity and growth arrest (18). Inhibition of expression of either p21Cip1 or p27Kip1 with antisense oligonucleotides maintains active CDK2 and prevents antiestrogen-mediated G1 blockade (19, 20).

p27Kip1 levels are regulated in distinct fashion at different points in the cell cycle. After mitogenic stimulation of quiescent cells p27Kip1 levels fall beginning in mid-G1, primarily mediated through regulation of protein stability by ubiquitin/proteasome-mediated proteolysis (Ref. 21; for review see Ref. 14). p27Kip1 protein expression is also regulated at the level of transcription (22), translation (23, 24), as well as through proteolytic processing independent of ubiquitination (25) and independent of the proteasome (26, 27). Early in G1 inhibitory activity of p21Cip1 and p27Kip1 toward CDK2 is removed by sequestration of the proteins into newly formed cyclin D-CDK4/6 complexes (for review, see Refs. 4 and 5). CDK2 activation in MCF-7 cells corresponds both with p21Cip1/p27Kip1 sequestration in early G1 (16, 17, 28, 29) and with decreased p21Cip1/p27Kip1 protein levels in mid- to late G1 (15, 16, 19), suggesting removal of CDK inhibitory activity in two phases after estrogen stimulation. Our recent studies indicate that the later phase of CDK inhibitor removal in MCF-7 cells requires the proteasome and proceeds in the absence of cyclin D-CDK4 complex formation or G1 transit (28). Further observations also indicated that down-regulation of p27Kip1 expression in MCF-7 cells also corresponds with increased expression of Skp2 (29), the F box component of the Skp1/Cul-1/F box protein (SCF) ubiquitin-protein ligase which binds p27Kip1 phosphorylated on Thr187 allowing its ubiquitination and destruction in the proteasome (30–33). Skp2 exhibits oncogenic potential in breast epithelial cells and is overexpressed in a subset of breast carcinomas (34). Recent studies also indicate that p27Kip1 ubiquitination can proceed in the absence of Skp2 (35).

In the studies described herein we examined p27Kip1 down-regulation after estrogen stimulation of MCF-7 cells in detail with respect to the role of Skp2, as well as Skp2-independent mechanisms contributing to p27Kip1 down-regulation. The results suggest that Skp2 participates in ubiquitination/degradation of p27Kip1 in the nucleus of estrogen-treated MCF-7 cells and further demonstrate that Skp2 overexpression prevents antiestrogen-mediated growth arrest of MCF-7 cells by down-regulating p27Kip1 expression and maintaining CDK2 activity. In contrast, estrogen-induced down-regulation of p27Kip1 proceeds in the absence of any increase in Skp2 expression in MCF-7 cells subjected to G1 blockade with p16Ink4a or roscovitine and appears to depend in this context upon export of p27Kip1 to the cytoplasm. Our studies also indicate that this process requires phosphorylation of p27Kip1 on Ser10 as well as participation of the Ras/Raf-1/extracellular signal-regulated kinase (ERK) signaling pathway at a stage beyond phosphorylation of p27Kip1 on this residue. In addition, enforced activation of the Ras/Raf-1/ERK pathway elicited cytoplasmic relocalization of p27Kip1 and prevented growth arrest by antiestrogen.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Antibodies—Cell culture media and antibiotics, 17{beta}-estradiol (E2), histones, fetal bovine serum (FBS), glutathione-agarose beads, RNase A, propidium iodide, monoclonal (M2) and rabbit polyclonal anti-FLAG antibodies, and other chemicals were from Sigma Chemical Co. ICI 182,780 was supplied by Dr. Alan Wakeling at Zeneca Pharmaceuticals (Alderly Park, Cheshire, UK). Protein A/G beads, antibodies to CDK2 (M2), p27Kip1 (C19), p21Cip1 (C19), Skp2 (H435), Cul-1 (H213), ERK2 (C14), Cks-1 (FL79), and Skp1 (H6) were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-actin, and anti-hemagglutinin epitope antibodies were from Roche Applied Science. Monoclonal antibody to p27Kip1 was from BD Biosciences, and antibodies to green fluorescent protein (GFP) and E2F-1 were from Labvision/Neomarkers (Freemont, CA). Antibodies to active mitogen-activated protein kinases (MAPK) were from Promega (Madison, WI). [{gamma}-32P]ATP, and Tran35S-label were from ICN (Irvine, CA). Horseradish peroxidase-conjugated secondary antibodies were from Jackson Immunoresearch (West Grove, PA). Fluorochrome-labeled secondary antibodies to mouse and rabbit IgG (Alexa Fluor 350 and 488) were from Molecular Probes (Eugene, OR). MG-132, Myc epitope antibodies (clone 9E10), PD98059, and roscovitine were from Calbiochem. Leptomycin B (LMB) was provided by M. Yoshida (36).

Cell Culture, Viral Vectors, and Transfections—MCF-7 cells were a gift from R. P. Shiu (37) and were maintained as described previously (28). MCF-7/tTA (Tet-off) cells were derived by transfection with pTet-off (Clontech). Cells were growth arrested by 2–3 days of culture in phenol red-free Dulbecco's modified Eagle's medium with 0.1% FBS and 20 nM ICI 182,780 as described previously (28) (16). Chemical stock solutions were prepared in ethanol (17{beta}-estradiol, ICI 182,780, LMB) or dimethyl sulfoxide (PD98059, roscovitine), and control cultures received equal amounts of solvents as vehicle controls where appropriate. Plasmid vectors for p16Ink4a (pBI-p16) and cyclin E (pMTcycE) were provided by J. Lukas (38), and vector for constitutively active ERK kinase (MEK, EE mutant) was provided by M. Cobb. Plasmids for Skp2 and FLAG-tagged p27Kip1 (wild-type, and S10A/S10E mutants) have been described previously (39–41). Plasmid vector for human Skp2 was provided by M. Pagano (31). The dominant-negative ERK2 and RasN17 plasmids were provided by G. L. Johnson (42), the Ras V12S35 plasmid was provided by C. M. Counter (43), and the Cul-1(1–452) plasmid was provided by Z. Q. Pan (44). The YFPp27Kip1 expression plasmid was provided by J. Slingerland (45). Transfections were carried out using LipofectAMINE PLUS transfection reagent (Invitrogen).

Control and antisense Skp2 oligonucleotides (Sigma/Genosys) based upon a previous publication (31) were prepared in partially phosphothiorated form to minimize destruction by cellular nucleases and were transfected into growth-arrested MCF-7 cells using Lipofectin transfection reagent (Invitrogen). The nucleotide sequences of the oligonucleotides were: antisense Skp2, 5'-CCTGGGGGATGTTCTCA-3'; control, 5'-CCGCTCATCGTATGACA-3'. Oligonucleotides were transfected for 3 h at a final concentration of 1 µM with 18 µl of liposome reagent.

Replication-defective adenoviral vectors for expression of p16Ink4a, p27Kip1, and control ({beta}-galactosidase) adenovirus have been described previously (28, 46, 47) as has the adenoviral Skp2 vector (murine Skp2) (39). Adenoviral vector for the constitutively active Raf-1caax mutant was provided by J. R. Nevins (48). Adenoviruses were propagated in human embryonic kidney 293 cells (ATCC), and viral lysates for use in experiments were titered by a standard plaque assay to determine plaque-forming units (pfu)/ml.

Flow Cytometric Analysis—For flow cytometric analysis MCF-7 cells were harvested in saline-EDTA, fixed in cold 70% ethanol, and stored at –20 °C. Fixed cells were subsequently washed, treated with 100 µg/ml RNase A, and stained with 50 µg/ml propidium iodide. Analysis of DNA content was performed in a BD Biosciences FACScan with a minimum of 15,000 events collected for analysis using BD Biosciences Cell Quest software. For analysis of DNA synthesis bromodeoxyuridine (BrdUrd) was added to the medium as described in the text and BrdUrd incorporation measured by flow cytometry using fluorescein-labeled antibodies to BrdUrd (BD/Pharmingen) according to the manufacturer's protocol.

Western Blot Analysis and Immune Complex Kinase Assays—Cells were lysed as described previously (15, 28) in ice-cold Nonidet P-40 lysis buffer (20 mM Tris, pH 7.5, 250 mM NaCl, 0.5% Nonidet P-40, 0.1 mM EDTA, 1 mM NaOV4, 10 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride), with brief sonication and centrifugation to remove cellular debris. Nuclear/cytoplasmic cell fractionation was carried out by lysis in hypotonic buffer with 0.02% digitonin as described previously (41). Western blots were performed as described in detail previously (28). Equal protein loading was verified for all lysate blots by Ponceau S staining and/or by reprobing for actin expression. In experiments measuring expression of ectopic FLAG-p27Kip1 by Western blotting transfection was verified by coexpression of GFP (pEGFPN1, Clontech) and analysis by fluorescent microscopy/Western blotting with anti-GFP antibodies. Histone kinase assays were performed with anti-CDK2 immune precipitates as described (15, 28) using equal amounts of lysate proteins.

Metabolic Labeling and Pulse-Chase Analysis of p27Kip1 Stability— MCF-7 cells in 6-well plates were growth arrested and, to facilitate detection of labeled p27Kip1, were infected with Adp27 at 5 pfu/cell with or without Adp16, AdSkp2, or control adenovirus as required. 12 h after infection cells were treated as described, and at appropriate times cultures were labeled for 1 h with [35S]Met/Cys as described previously (15, 28). For the chase, monolayers were washed twice with fresh, complete medium and incubated for the indicated times before lysis in ice-cold Nonidet P-40 lysis buffer as described above. Immunoprecipitations were performed with 1 µg of p27Kip1 (C19) antibodies and protein A/G-agarose beads and analyzed as described (15, 28).

Subcellular Localization of p27Kip1 by Microscopy—MCF-7 cells grown on glass coverslips were transfected with the YFPp27Kip1 plasmid vector (45) or FLAG-p27Kip1 vectors (40, 41) alone or in combination with appropriate vectors as indicated. After treatment the cells were fixed with 3% paraformaldehyde, mounted on slides, and cells with nuclear versus nuclear/cytoplasmic YFPp27Kip1 were enumerated visually on an Olympus IMT-2 fluorescent microscope. For immunofluorescent localization of FLAG-p27Kip1 or epitope-tagged cotransfectants, fixed cells were permeabilized with 0.25% Triton X-100 and stained with the appropriate antibodies and fluorochrome-labeled secondary antibodies. For quantification, a minimum of 400 cells were counted for each determination, and results were derived from at least three independent experiments. Statistical analysis was performed with Graph-Pad Prism 3.0 software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Skp2 Expression in Response to E2 Is Prevented by G1 Blockade, but p27Kip1 Down-regulation Is Maintained—Estrogen stimulation of G0/G1-arrested MCF-7 cells leads to down-regulation of p27Kip1 protein expression corresponding with cyclin E-CDK2 activation and preceding entry into S phase (15, 16, 28). As we reported previously, p27Kip1 down-regulation in MCF-7 cells is prevented by chemical inhibitors of the proteasome but is not affected in MCF-7 cells transduced with an adenoviral vector for p16Ink4a despite effective inhibition of cyclin D-CDK complex formation, CDK2 activation, and cell cycle transit (28). Degradation of p27Kip1 via the ubiquitin-proteasome pathway is facilitated by interaction with the F box protein Skp2, which in conjunction with CKS1 binds p27Kip1 phosphorylated on Thr187 by CDK2 and mediates interactions with the SCF ubiquitin-protein ligase (30–33, 49, 50). Skp2 levels increase in estrogen-stimulated MCF-7 cells (29). Similar to other studies (31), the amounts of Skp2 and CKS1/2 increased in MCF-7 cells after estrogen treatment, beginning ~9 h after release from growth arrest (Fig. 1A). Increasing Skp2 expression thus correlates with the decline in p27Kip1 levels which is evident in E2-treated MCF-7 cells by ~9–10hin most experiments and maintained throughout S phase (Fig. 1A, data not shown). As reported in earlier studies (31), levels of Skp1 and Cul-1 remain essentially constant in these cultures (data not shown).



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  		FIG. 1.
Expression of Skp2 is cell cycle-dependent and is not required for down-regulation of p27Kip1. A, Western blot analysis of expression of p27Kip1 and components of the SCF ubiquitin-protein ligase in lysates of growth-arrested MCF-7 cells treated with E2 for the indicated times (note: the CKS1 antibody cross-reacts with CKS2). B, cell cycle-dependent expression of Skp2 and CKS1. MCF-7 cells transduced with control ({beta}gal) and p16Ink4a adenoviral vectors as indicated were growth arrested, treated for 20 h with E2, and lysates were assayed for expression of Skp2, CKS1/2, p27Kip1, p16Ink4a, and actin by Western blotting (top five panels) and for CDK2-associated histone kinase activity (bottom panel), showing the blockade of CDK2 activation by p16Ink4a. C, growth-arrested MCF-7 cells were treated for 20 h with E2 as indicated or with E2 and 25 µM roscovitine (lane 3). Expression of p27Kip1, Skp2, and actin was assessed by Western blotting. The cell cycle status of these cultures based on DNA content analysis of the proliferative fraction (S phase plus G2/M) is given below the figure.

 

Earlier studies indicate that Skp2 expression is cell cycle-regulated, with the protein appearing in mid- to late G1 phase (31, 51, 52). In contrast to the lack of any effect of p16Ink4a-mediated cell cycle blockade on p27Kip1 down-regulation (28), increased expression of Skp2 and CKS1/2 elicited by E2 was prevented by G1 blockade enforced by overexpression of p16Ink4a (Fig. 1B). Cell cycle progression and increased Skp2 expression in response to E2 were also effectively inhibited by the chemical CDK inhibitor roscovitine (Fig. 1C), but down-regulation of p27Kip1 was unaffected. These results indicate that Skp2 expression in MCF-7 cells is cell cycle-dependent and that p27Kip1 down-regulation induced in MCF-7 cells by estrogen is not strictly linked to Skp2 expression. Thus, to the extent that p27Kip1 down-regulation in G1 is expected to rely upon increasing Skp2 expression in mid-G1, this would suggest that alternate mechanisms participate in p27Kip1 destruction in MCF-7 cells. Because Skp2/p27Kip1 interaction requires CDK2-dependent phosphorylation of p27Kip1 on Thr187 (33, 53–55) it is also of interest that CDK2 inhibition by p16Ink4a or roscovitine did not prevent p27Kip1 down-regulation.

Skp2 Overexpression Down-regulates p27Kip1and Maintains Proliferation of MCF-7 Cells under Conditions of Mitogen and Estrogen Withdrawal—Skp2 has been characterized as an oncogene (56, 57) and elicits p27Kip1 down-regulation, activation of CDK2, and cell cycle progression when overexpressed in quiescent cells (32). We studied the effects of Skp2 overexpression on MCF-7 cells undergoing antiestrogen-mediated growth arrest. Treatment of proliferating MCF-7 cells with the steroidal antiestrogen ICI 182,780 causes the cells to arrest in G0/G1 with repression of CDK4/CDK2 kinase activities and increased levels of both p21Cip1 and p27Kip1 (18). Our studies indicated that expression of endogenous Skp2 was reduced in antiestrogen-treated cultures (Fig. 2A). Transduction of MCF-7 cells with adenoviral Skp2 vector maintained CDK2 activity in ICI 182,780-treated cultures at levels comparable with those in proliferating cells and prevented growth arrest elicited by antiestrogen as measured by flow cytometry (Fig. 2A). Reversal of antiestrogen-induced growth arrest by Skp2 overexpression corresponded with decreased p27Kip1 expression, but levels of p21Cip1 in ICI 182,780-treated cultures were unaffected (Fig. 2A, lower panels). Measurements of p27Kip1 stability in growth-arrested control and Skp2-transduced cells by pulse-chase analysis and confirmed that p27Kip1 down-regulation elicited by Skp2 expression in antiestrogen-arrested cells were reflective of decreased stability of the protein (Fig. 2B). In addition, overexpression of Skp2 in growth-arrested MCF-7 cells elicited active CDK2 and S phase entry in Go/G1-arrested MCF-7 cells in the absence of E2 or other mitogenic stimulus (Fig. 2C), and this correlated with a reduction in p27Kip1 levels.



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  		FIG. 2.
Overexpression of Skp2 prevents antiestrogen-mediated growth arrest in MCF-7 cells and induces CDK2 activation/cell cycle progression in G0/G1-arrested cells. A, proliferating MCF-7 cells were transduced with control or Skp2 adenoviral vectors and treated with 20 nM ICI 182,780 for 40 h as indicated. Replicate cultures were assayed for CDK2-associated histone kinase activity (top panel and % kinase activity values), proliferative fraction (% S+G2/M), BrdUrd incorporation (%BrdUrd (+)), incorporation measured in a 4-h pulse), and by Western blotting for expression of Skp2, p27Kip1, p21Cip1, and actin (bottom panels). B, Skp2 overexpression destabilizes p27Kip1 in antiestrogen-arrested MCF-7 cells. MCF-7 cells were growth arrested by antiestrogen treatment (48 h) and transduced with p27Kip1 adenovirus at low multiplicity of infection (5 pfu/cell) and with control or Skp2 adenoviral vectors (40 pfu/cell). The in vivo stability of p27Kip1 was assessed by pulse-chase labeling and immunoprecipitation of labeled p27Kip1 for 12 h after E2 treatment as described under "Experimental Procedures." Quantitative analysis based on densitometry is shown as a graph. C, MCF-7 cells were growth arrested by treatment with antiestrogen (2 days total) and E2 treated or transduced with adenoviral vector for Skp2. Cultures were assayed 20 h later for BrdUrd uptake (6-h pulse), CDK2 activity, and by Western blotting for expression of Skp2 and p27Kip1.

 

Nuclear Export of p27Kip1 Is Required for Down-regulation under Conditions of G1 Blockade—Recent studies suggest that destruction of p27Kip1 is mediated by multiple proteolytic pathways (25, 58). These pathways are, in part, independent of Skp2 and instead rely either upon a constitutive ubiquitination apparatus in the cytoplasm (35, 58) or proteolytic degradation independent of ubiquitination (25–27). In addition, several studies indicate that p27Kip1 undergoes CRM1-dependent nuclear export in response to mitogenic stimuli, including estrogen (45, 59), as well as upon ectopic expression of Jab1/CSN5, a component of the COP9 signalosome (60). Studies of Jab1/CSN5-mediated export indicate that p27Kip1 does not contain nuclear export sequences and that direct binding interactions between p27Kip1 and Jab1/CSN5 may serve to "bridge" p27Kip1 and CRM1 (61). Recent studies in our laboratories and others have suggested that p27Kip1 and CRM1 may interact directly (41, 45). We examined the role of nuclear export in p27Kip1 down-regulation elicited by E2 in MCF-7 cells with interest in cells subjected to G1 blockade with p16Ink4a where Skp2 expression is impaired. Our earlier studies indicate that Skp2 is localized to the nucleus in MCF-7 cells (29). LMB disrupts the interaction between CRM1 and nuclear export sequences in exported proteins (36) and prevents export of p27Kip1 in mitogen-stimulated fibroblast (41, 59). Pulse-chase analysis of p27Kip1 stability in MCF-7 cells confirmed that p27Kip1 was destabilized in vivo by E2 treatment of p16Ink4a-transduced, antiestrogen-arrested cultures and was, in turn, stabilized by he addition of LMB along with E2 (Fig. 3A). Transduction of E2/LMB-treated cultures with Skp2 completely overcame the effects of LMB and destabilized p27Kip1 to a degree beyond that provided by E2 alone. Similar to our results in previous studies (29) LMB inhibited p27Kip1 down-regulation in measurements of steady-state expression levels (Fig. 3B, third lane), and transduction with Skp2 vector led to virtual elimination of p27Kip1 despite LMB treatment (Fig. 3B, fourth lane). This could lend support to a role for Skp2 in mediating p27Kip1 destruction in the nuclear compartment in MCF-7 cells, i.e. p27Kip1 destruction is facilitated when LMB maintains nuclear localization of the protein and Skp2 is overexpressed. The results thus may suggest that p27Kip1 destruction induced by estrogeninMCF-7cellstransducedwithp16Ink4arequires CRM1-dependent export of the molecule to the cytoplasm because of a lack of Skp2 expression.



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  		FIG. 3.
Stability of p27Kip1 is increased by LMB in MCF-7 cells overexpressing p16Ink4a. A, Skp2 reverses the stabilizing effects of LMB. MCF-7 cells were growth arrested and transduced with p16Ink4a adenoviral vector (80 pfu/cell) along with p27Kip1 adenoviral vector (5 pfu/cell). After 24 h, cultures were treated with E2,E2 plus LMB, or E2 plus LMB in combination with adenoviral Skp2 transduction (40 pfu/cell). After 12 h, the stability of de novo p27Kip1 was measured by pulse-chase analysis as described under "Experimental Procedures." The quantitative results based on densitometry are presented as a graph. B, MCF-7 cells were growth arrested and treated with E2, E2 plus LMB, or E2 plus LMB in combination with adenoviral Skp2. Lysates prepared after 24 h were assayed for p27Kip1, Skp2, and actin protein expression by Western blotting. C, MCF-7 cells were transfected with vectors for FLAG-tagged wild-type p27Kip1 or the p27Kip1S10A mutant along with pEGFPN1 as a transfection control and transduced with control or Skp2 adenoviral vectors (10 pfu/cell). After 12 h in complete medium, cells were cultured in medium with 0.2% FBS and 20 nM ICI 182,780 for 40 h. Protein expression levels of FLAG-tagged exogenous p27Kip1 (wild-type and S10A mutant) and FLAG-Skp2 were analyzed by Western blotting with rabbit anti-FLAG antibodies. The blot was reprobed for expression of actin and GFP as loading controls. D, MCF-7 cells were transfected with p27Kip1S10A along with control plasmid or plasmid vector for human Skp2 and cultured as in C. Cellular localization of p27Kip1S10A and Skp2 was visualized by immunostaining with anti-FLAG (M2) monoclonal antibodies and rabbit polyclonal antibodies to Skp2.

 

CRM1-dependent nuclear export of p27Kip1 at the G0/G1 transition requires phosphorylation of the molecule at Ser10 and subsequent mitogen stimulation (41, 59). p27Kip1 is phosphorylated on Ser10 in MCF-7 cells growth arrested with antiestrogen and is exported to the cytoplasm after estrogen treatment (59). Phosphorylation on this site represents a large portion of total p27Kip1 phosphorylation, and mutation of this residue to alanine (S10A mutant) prevents interaction with CRM1 and nuclear export of p27Kip1 in response to mitogen, favoring nuclear localization of the molecule (41, 59). To test further whether Skp2 action in MCF-7 cells is oriented to p27Kip1 in the nuclear compartment, cells were transfected with vectors for epitope-tagged wild-type p27Kip1 or the S10A mutant with and without adenoviral Skp2 transduction. Skp2 expression down-regulated both wild-type p27Kip1 and the S10A mutant in mitogen- and estrogen-deprived MCF-7 cells (Fig. 3C). Immunofluorescent microscopy confirmed that transfected, exogenous Skp2 and the S10A mutant of p27Kip1 were localized to the nucleus (Fig. 3D). Further experiments verified that exogenous Skp2, whether plasmid- or viral vector-derived, is localized to the nucleus and that enforced Skp2 expression did not affect the subcellular localization of wild-type p27Kip1 or p27Kip1S10A under a variety of conditions (data not shown). Our results thus support a role for Skp2 in destruction of p27Kip1 localized to the nucleus.

We further investigated the role of nuclear export and Ser10 phosphorylation using ectopic expression of the epitope-tagged p27Kip1 variants with mutations on Ser10 (40). Estrogen treatment of transfected MCF-7 cells down-regulated the levels of wild-type p27Kip1 and the S10E mutant of p27Kip1, which is efficiently exported from the nucleus (41) as well as those of p27Kip1S10A, which is not (Fig. 4A). To assess more directly the role of the SCF ubiquitin-ligase in p27Kip1 down-regulation elicited by E2 we cotransfected p27Kip1S10A and p27Kip1 S10E along with a Cul-1(1–452) truncation mutant that interferes with SCF function (44). The Cul-1(1–452) mutant prevented down-regulation of p27Kip1S10A in response to E2 (Fig. 4B) but had no effect upon removal of p27Kip1S10E. In addition, down-regulation of the nonexported S10A mutant was inhibited in MCF-7 cells transfected with antisense Skp2 oligonucleotides (Fig. 4C), but control oligonucleotides had no effect. Antisense Skp2 oligonucleotides reduced expression of the protein in these cells by ~50%. These results, along with those in Fig. 3, support a model in which E2-induced down-regulation of p27Kip1 in the nuclear compartment is dependent upon Skp2 and function of the SCF ubiquitin-ligase and further suggest that Skp2-independent degradation of p27Kip1 requires export of the protein.



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  		FIG. 4.
Down-regulation of the nonexported p27Kip1S10A mutant requires SCF function. A, MCF-7 cells were transfected with plasmid vectors for FLAG-tagged wild-type, S10E, or S10A mutants of p27Kip1 along with control vector followed by growth arrest and 24 h treatment with E2. Expression of the transfected forms of p27Kip1 was assayed by Western blotting of whole cell lysates with anti-FLAG antibodies. B, MCF-7 cells were transfected with control plasmid or the Cul-1(1–452) vector along with pEGFPN1 and the indicated p27Kip1 vectors. Expression of FLAG Cul-1(1–452) and the FLAG-tagged p27Kip1 mutants was assayed by Western blotting with anti-FLAG antibodies using lysates prepared 20 h after E2 treatment of G0/G1-arrested cells. Blots were subsequently reprobed for GFP expression. Panels reflect different exposures derived from the same gel caused by differences in overall expression levels. C, MCF-7 cells were transfected with p27Kip1S10A and growth arrested. After 2 days, cultures were transfected with control oligonucleotides, antisense Skp2 oligonucleotides, or with lipid alone as indicated and treated with E2. After 24 h, lysates were analyzed for expression of the p27Kip1S10A mutant and Skp2 by Western blotting.

 

Ras/Raf/MEK/ERK Activation Relocalizes p27Kip1 to the Cytoplasm and Is Required for Nuclear Export and Down-regulation of p27Kip1 Induced by Estrogen—Subcellular localization and degradation of p27Kip1 is regulated by MAPKs, i.e. ERKs 1 and 2, activated through the Ras/Raf-1/MEK/ERK pathway (62–66). With respect to growth regulation by estrogen our earlier studies indicated that dominant-negative RasN17 prevented down-regulation of ectopic p27Kip1 and S phase entry induced by estrogen treatment of MCF-7 cells (28, 67). Estrogens activate ERKs in MCF-7 cells (68–70), and chemical inhibitors of ERK activation inhibit estrogen-induced accumulation of cyclin D1 (67). We performed experiments to examine the role of active ERKs in p27Kip1 down-regulation elicited by estrogen in MCF-7 cells. Cotransfection of dominant-negative ERK2 prevented down-regulation of both the p27Kip1S10A and p27Kip1S10E mutants in response to estrogen (Fig. 5A), leading to accumulation of p27Kip1 in estrogen-treated cells (lanes 3 and 7) as well as in growth-arrested cells (data not shown). This suggests a role for ERKs in this process independent of the requirement for Ser10 phosphorylation of p27Kip1 and is in agreement with earlier studies, which indicated that Ser10 on p27Kip1 is not a likely target of ERK activity (40, 59).



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  		FIG. 5.
Active MAPK is required for E2 -induced p27Kip1 down-regulation. A, MCF-7 cells were transfected with plasmid vectors for p27Kip1S10A (lanes 1–4), p27Kip1S10E (lanes 5–8), along with control plasmid or plasmids expressing dominant-negative MAPK (DNErk2) or dominant-negative CDK2 as indicated. After growth arrest and 24-h E2 treatment, protein expression levels of the p27Kip1 mutants were measured by Western blotting with anti-FLAG antibodies. Overall transfection and expression of the (non-epitope-tagged) dominant-negative cotransfectants were monitored by reprobing the blots for ERK2, CDK2, and GFP. B, Chemical inhibition of CDK2 causes accumulation of the p27Kip1S10A mutant. MCF-7 cells were transfected with plasmids for p27Kip1 S10A (left panel)orp27Kip1S10E (right panel) along with control vector. After growth arrest, cells were treated with E2 (all cultures) and 25 µM roscovitine (Rosc.) as indicated. Expression of Skp2 and the p27Kip1 mutants was measured by Western blot analysis of lysates prepared after 20 h of treatment.

 

CDK2-dependent phosphorylation of p27Kip1 at Thr187 and interaction with SCFSkp2 are required for p27Kip1 degradation at G1/S but not in G1 phase (58). Consistent with these studies and with respect to our own observations in p16Ink4a-expressing or roscovitine-treated cells we found that down-regulation of the nonexported S10A mutant of p27Kip1 was prevented by coexpression of a dominant-negative CDK2 mutant leading to marked accumulation of this mutant in E2-treated cells (Fig. 5A, lane 4). In contrast, down-regulation of the exported, S10E mutant of p27Kip1 after E2 was largely unaffected by dominant-negative CDK2 with no accumulation of this mutant being evident (Fig. 5A, lane 8). Also, addition of roscovitine to estrogen-treated cultures caused accumulation of p27Kip1S10A but not p27Kip1S10E (Fig. 5B). Along with results given above this confirms that Skp2-dependent, nuclear destruction of p27Kip1 requires CDK2 activity and also suggests that active ERKs participate in p27Kip1 down-regulation through actions that lie outside of Ser10 phosphorylation of p27Kip1.

Constitutive MEK/MAPK activation renders MCF-7 cells resistant to growth arrest by antiestrogens through altered expression, localization, and function of p27Kip1 (66). We utilized transfection of YFPp27Kip1 fusion protein (45) to explore further the relationship between active signal transduction pathways and p27Kip1 localization in MCF-7 cells. Localization of YFPp27Kip1 was exclusively nuclear in 75–80% of cells under conditions of mitogen/estrogen withdrawal (Fig. 6A, graph and photomicrographs). Cotransfection of the constitutively active MEKEE mutant yielded readily observable cytoplasmic localization of YFPp27Kip1 in the majority of cells under these conditions as did expression of the RasV12S35 mutant that preferentially activates the Raf-1/MEK/ERK pathway (43). Coexpression of cyclin E, which readily overcomes antiestrogen- and p16Ink4a-mediated G1 arrest in MCF-7 cells (28), did not increase the extent of cytoplasmic localization of YFPp27Kip1. Cell cycle analysis determined, however, that MEKEE, RasV12S35, and cyclin E did not abrogate G1 arrest enforced in these cultures by the combination of antiestrogen and YFPp27Kip1 and would indicate that cytoplasmic localization of YFPp27Kip1 is independent of cell cycle transit (data not shown).



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  		FIG. 6.
Ras/Raf-1/MEK activation elicits cytoplasmic localization and down-regulation of p27Kip1. A, MCF-7 cells were transfected with the YFPp27Kip1 vector, along with the indicated vectors in 3:1 excess, and incubated 48 h in medium with reduced serum (0.2%) and 20 nM ICI 182,780. Localization of YFPp27Kip1 was determined microscopically and expressed as the percentage of cells exhibiting cytoplasmic YFPp27Kip1. Values in the graph represent the mean ± S.D. from multiple determinations over three separate experiments. Statistical analysis was based on a two-tailed t test. ##, significantly different from control at p < 0.01; **, not significantly different from control at p < 0.05. The photographs present example fields from a single experiment showing colocalization of YFPp27Kip1 and cotransfected MEKEE detected by immunostaining with anti-hemagglutinin epitope antibodies, and with cyclin E detected with anti-Myc epitope antibodies as indicated. B, MCF-7 cells were infected with {beta}-galactosidase or Raf-1caax adenoviral vectors (30 pfu/cell) and treated with 20 nM ICI 182,780 in reduced serum as indicated. The culture shown in the first lane was maintained in complete medium. After 48 h, cultures were analyzed for cell cycle status by DNA content and for expression of p27Kip1, Skp2, and actin. C, MCF-7 cells were infected with {beta}-galactosidase and Raf-1caax adenoviral vectors as above and treated 2 days with ICI 182,780 in medium with 0.2% FBS. Lysates were analyzed with phospho-specific antibodies to active MAPK and with antibodies to ERK2 (note: the ERK2 antibodies cross-react with ERK1). D, control and AdRaf-1caax-transduced cultures were treated for 24 h with ICI 182,780, and the cells were fractionated into nuclear (N) and cytoplasmic (C) compartments. The proportion of p27Kip1 in the subcellular fractions was determined by Western blotting and densitometry and is represented by the ratios given below the figure. As a control for fractionation, blots were reprobed for expression of E2F-1 as a nuclear marker. D, bottom graph, MCF-7 cultures transfected with YFPp27Kip1 and transduced with control, Skp2, and Raf-1caax adenoviral vectors (10 pfu/cell) were cultured as above in A were analyzed microscopically for subcellular localization of YFPp27Kip1. The enumerated percentage of cells with evident cytoplasmic YFPp27Kip1 is given as percent of cells with evident cytoplasmic p27Kip1 ± S.D. based on counts of 400–500 cells over three separate cultures. The indications for statistics are as above in A.

 

To examine the consequence of Raf-1/MEK/ERK activation on expression of endogenous p27Kip1 and proliferation of MCF-7 cells we transduced cultures with a constitutively active Raf-1caax mutant and examined the cell cycle profile along with the expression of p27Kip1 and Skp2 after treatment of the cultures with antiestrogen. Similar to results with Skp2 overexpression in Fig. 2, MCF-7 cells expressing Raf-1caax did not undergo G1 arrest after a 48-h treatment with ICI 182,780 and exhibited levels of p27Kip1 comparable with those in untreated, proliferating cells (Fig. 6B, top panel). Skp2 expression was minimal in antiestrogen-treated cells as before and was expressed at even lower levels in antiestrogen-treated cultures transduced with Raf-1caax (Fig. 6B, middle panel) despite continuing proliferation in these cultures. Analysis with antibodies to active ERKs confirmed that MCF-7 cells transduced with Raf-1caax exhibited higher activity of ERKs 1 and 2 relative to controls (Fig. 6C). Nuclear/cytoplasmic fractionation of control and Raf-1caax-transduced MCF-7 cells treated with antiestrogen indicated that cells transduced with Raf-1caax exhibit a higher proportion of cytoplasmic p27Kip1 than controls (Fig. 6D, top panel). Visualization of YFPp27Kip1 localization in mitogen- and estrogen-deprived cultures transduced with control or Raf-1caax vectors confirmed the increased cytoplasmic localization in Raf-1caax-transduced cultures (graph at bottom of Fig. 6D). The results in Fig. 6 indicate that down-regulation of endogenous p27Kip1 induced by Raf-1caax is sufficient for maintenance of proliferation in antiestrogen-treated cells and is associated with cytoplasmic relocalization of p27Kip1 as described in earlier studies (66). This proceeds independently of Skp2 expression and indicates that Skp2 expression is, in this case, disconnected from cell cycle progression. We examined the role of ERKs in nuclear export of p27Kip1 induced by E2. Assays of ERK activation under our conditions of growth arrest confirmed that E2 treatment elicits phosphorylated, active ERK within 15 min of stimulation, and this activation is maintained for up to 2 h (Fig. 7A). The extent of ERK activation elicited by E2 is consistently less, however, than that elicited by growth factors (data not shown). Two recent studies indicate that p27Kip1 undergoes nuclear export by 8–12 h after E2 treatment of MCF-7 cells (45, 59). In our studies estrogen treatment doubled the percentage of cells exhibiting cytoplasmic YFPp27Kip1 8 h after treatment of G0/G1-arrested cells (Fig. 7B, left panel). Inhibition of ERK activation with either the chemical MEK inhibitor PD98059 or by cotransfection with dominant-negative ERK2 decreased the number of cells exhibiting cytoplasmic YFPp27Kip1 (Fig. 7B, both panels). The dominant-negative RasN17 mutant also prevented cytoplasmic relocalization of YFPp27Kip1, but cotransfection with p16Ink4a had no effect. Fractionation of MCF-7 cells verified that PD98059 decreased the amount of endogenous p27Kip1 in the cytoplasm 8 h after E2 treatment (Fig. 7C). These results indicate that ERK activation is a requisite aspect of estrogen action in promoting p27Kip1 export. Further experiments shown in Fig. 7D demonstrated that inhibition of ERK activation by PD98059 or dominant-negative ERK2 coexpression prevents export of p27Kip1S10E as demonstrated above for the wild-type protein. Given that p27Kip1S10E mimics Ser10-phosphorylated p27Kip1, this would indicate that the role of ERK activation with respect to p27Kip1 export is separate from Ser10 phosphorylation per se. Our experiments also verified, however, that p27Kip1S10A is not exported in response to E2, thus confirming the requisite nature of phosphorylation on this residue (Fig. 7D, right panel).



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  		FIG. 7.
ERK activation is required for p27Kip1 export elicited by estrogen. A, MCF-7 cells were growth arrested in medium with 0.2% FBS and 20 nM ICI 182,780 and treated with 20 nM E2 for the indicated times. Whole cell lysates were analyzed by Western blotting with antibodies to active MAPK (ERKs 1 and 2). The blot was reprobed with antibodies to ERK2 (lower panel). B, in the left panel of the graph MCF-7 cells were transfected with YFPp27Kip1, growth arrested, and E2 treated with or without 50 µM chemical MEK inhibitor PD98059 as indicated for 8 h. The proportion of cells exhibiting cytoplasmic YFPp27Kip1 was enumerated. For experiments represented in the right panel MCF-7 cells were cotransfected with YFPp27Kip1 and the indicated vectors in 3:1 excess, and the percentage of cells exhibiting cytoplasmic YFPp27Kip1 was enumerated with all cultures receiving 20 nM E2. The values for both sets of experiments are derived from three independent experiments and are given as the mean ± S.D. Statistical analysis in B and D was based on a two-tailed t test. ##, significantly different from control at p < 0.01; **, not significantly different from control at p < 0.05; $$, different from E2 value at p < 0.01; &&, different from both control and DNRas values at p < 0.02. C,G0/G1-arrested MCF-7 cells were treated 8 h with E2 or E2 plus 50 µM PD98059 as indicated. Nuclear (N) and cytoplasmic (C) fractions were analyzed for expression of endogenous p27Kip1 and E2F-1 as indicated. The relative proportions of nuclear and cytoplasmic p27Kip1 based on densitometry are given below that panel as ratios. D, MCF-7 cells were transfected with vectors for FLAG-p27Kip1S10E (left panel) or p27Kip1S10A (right panel) along with the indicated cotransfectants. After culture and estrogen treatment as in B, the localization of the p27Kip1 mutants was analyzed by immunostaining with anti-FLAG antibodies. The graph is based on values derived as in B.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Regulation of CDK inhibitor expression, and particularly that of p27Kip1, has emerged as a critical aspect of growth control in normal mammalian cells and as a frequent point of dysregulation in a wide variety of tumors including breast carcinomas (for review, see Ref. 14). Intracellular concentrations of p27Kip1 are regulated primarily through the ubiquitin-proteasome pathway (Ref. 21; for review, see Ref. 14). Expression of p27Kip1 at low levels in human tumors is frequently the result of increased proteolytic degradation of the protein rather than allelic loss (10, 71).

Studies using MCF-7 cells as a model of growth regulation by estrogens have demonstrated the importance of p21Cip1/p27Kip1 removal in estrogen-stimulated proliferative responses (15–17, 28) and the participation of these CDK inhibitors in growth arrest by antiestrogens (for review, see Ref. 3). Prior to the onset of p27Kip1 destruction, the earliest phase of CDK inhibitor removal in E2-stimulated MCF-7 cells depends on de novo cyclin D1 synthesis and redistribution of p27Kip1/p21Cip1 from cyclin E-CDK2 complexes to newly formed cyclin D1-CDK4 complexes (17, 28, 29). Steady-state levels of both p27Kip1 and p21Cip1 decline in MCF-7 cells after E2 stimulation, demonstrable within 8–10 h of treatment (15, 16, 19, 28). One recent study has associated the early phase of E2-induced cyclin E-CDK2 activation to decreased synthesis of p21Cip1 coupled with increased synthesis of cyclin D1 (72). This same study indicated that p27Kip1 synthesis was increased 8–10 h after E2 treatment, but stability of the protein was decreased markedly (72).

Our studies now strongly indicate that down-regulation of p27Kip1 protein levels induced in MCF-7 cells by estrogen proceeds through at least two mechanisms separable by blockade of G1 transit and differing in their reliance on Skp2 and nuclear export of p27Kip1 (Ref. 28 and this work). Both modes of p27Kip1 down-regulation ultimately result in proteolytic destruction of the molecule similar to evolving concepts of p27Kip1 elimination arising from studies in other cell systems (26, 35, 58). Our evidence would suggest that estrogen promotes relocalization of p27Kip1 in early to mid-G1, dependent upon prior Ser10 phosphorylation, CRM1, and active ERK signaling kinases, delivering p27Kip1 to Skp2-independent systems in the cytoplasm for degradation (25–27, 35, 41, 45, 59). Later in G1, and in S phase, increasing expression of Skp2 and the appearance of CDK2 activity targets p27Kip1 remaining in the nucleus for destruction mediated through Thr187 phosphorylation and ubiquitination. Skp2 overexpression prevents antiestrogen-mediated growth arrest of MCF-7 cells and is sufficient for p27Kip1 down-regulation and cell cycle entry in growth-arrested cells.

Degradation of p27Kip1 is promoted by CDK2-dependent phosphorylation on Thr187 allowing interaction with the SCFSkp2 ubiquitin-protein ligase through binding interactions with Skp2 and CKS1 (30–33, 49, 50). Periodicity of p27Kip1 expression through the cell cycle has been explained by the cell cycle dependence of Skp2 expression (51, 52) and CDK2 activity (73, 74). We found previously that estrogen-induced down-regulation of p27Kip1 in MCF-7 cells was mediated through proteasome-dependent destruction beginning in mid-G1 yet was independent of CDK2 activation and cell cycle progression (28). Conversely, p27Kip1 destruction did correspond with increasing Skp2 expression (29). The studies described herein expand upon these observations and demonstrate that estrogens likely regulate expression of p27Kip1 in MCF-7 cells both through Skp2-dependent pathways as well as through mechanisms that do not rely upon interactions with SCFSkp2. Most importantly, p27Kip1 instability and down-regulation of steady-state levels of p27Kip1 were readily apparent under conditions of G1 arrest where Skp2 expression was not up-regulated by E2. The potential for growth promotion by Skp2 in MCF-7 cells is readily demonstrable, however, because Skp2 overexpression effectively destabilized p27Kip1 and promoted S phase entry in growth-arrested cells in the absence of mitogen. Overexpression of Skp2 thus represents a potential mechanism of antiestrogen resistance in breast cancer cells (34).

While these studies were under way one of our laboratories has published an independent study demonstrating that ubiquitin/proteasomedependent degradation of p27Kip1 at the G0-G1 transition is independent of Skp2 and occurs in the cytoplasm in mouse embryo fibroblasts (35). This activity has been attributed to a heterodimeric Kip1 ubiquitinylation-promoting complex located in the cytoplasm.2 Skp2-, and CDK2-dependent p27Kip1 destruction occurs in the nuclear compartment later in G1 and in S phase (35, 58). Similarly our studies have demonstrated abundant p27Kip1-ubiquitinating activity in lysates of MCF-7 cells irrespective of G1 blockade and decreased Skp2 abundance (data not shown). Recent studies have also demonstrated that proteasome- and calpain-mediated mechanisms contribute to p27Kip1 elimination without any requirement for ubiquitination (25–27).

The demonstration that overexpression of Jab1/CSN5 elicited nuclear export/degradation of p27Kip1 suggested a requirement for relocalization of the molecule prior to degradation (60), and recent studies have further characterized the mechanisms underlying p27Kip1 export. Jab1/CSN5 promotes export and degradation of p27Kip1 through a CRM1-dependent mechanism (60, 61), possibly involving interactions of p27Kip1 with the adaptor protein Grb2 in the cytoplasm (75). Export of p27Kip1 to the cytoplasm after growth factor stimulation may also inhibit Grb2/SOS interactions and serve to limit Ras effector functions (76). Nuclear export of p27Kip1 requires phosphorylation on Ser10 and mitogen stimulation (41, 59). Ser10 phosphorylation of p27Kip1 has been related to a growth factor-dependent, nuclear kinase, hKIS (77), and to Akt (78). The Ser10-phosphorylated form of p27Kip1 accumulates in MCF-7 cells undergoing growth arrest induced by antiestrogen and is exported to the cytoplasm after E2 stimulation (45, 59). In our studies blockade of CRM1-dependent export with LMB stabilized p27Kip1 (Ref. 29 and this work), particularly when MCF-7 cells are transduced with p16Ink4a, and the effects of LMB were reversed by Skp2. One recent study has suggested that stabilization of p27Kip1 by LMB in MCF-7 cells is not reflective of disrupted p27Kip1/CRM1 interactions and may result from more general effects of LMB on cell cycle progression (45). Our studies consistently indicate, however, that nuclear export and down-regulation of p27Kip1 are unaffected by inhibition of G1 transit (Ref. 28 and this work). Degradation of the nonexported S10A mutant of p27Kip1 was inhibited by antisense Skp2 oligonucleotides and by coexpression of the Cul-1(1–452) mutant that interferes with SCF function. Consistent with participation of SCFSkp2 in elimination of p27Kip1 in the nucleus, the S10A mutant of p27Kip1, which localizes to that cellular compartment, was down-regulated by enforced Skp2 expression in mitogen- and estrogen-deprived cells. We find no evidence that Skp2 expression alters the subcellular localization of p27Kip1.

The Ras/Raf-1/MEK/ERK pathway has been implicated in regulation of expression and subcellular localization of p27Kip1 (62–66, 79). Active Ras elicits p27Kip1 down-regulation in fibroblast through active ERK and shortened p27Kip1 half-life (62), and ERK activity induced by Erb2/neu overexpression promotes degradation of p27Kip1 and cytoplasmic localization of both p27Kip1 and Jab1/CSN5 (63, 64). Degradation of p27Kip1 induced by active ERK is independent of CDK2 and Thr187 phosphorylation (65). In addition, one recent study has shown that constitutive MEK/ERK activation contributes to antiestrogen resistance in breast cancer cell lines by altering phosphorylation, expression, and the CDK2 inhibitory function activity of p27Kip1 (66). We found that active MEK, and the selective MEK-activating RasV12S35 mutant, elicited cytoplasmic localization of p27Kip1 in antiestrogen-treated cultures. Furthermore, Raf-1caax prevented p27Kip1 accumulation and cell cycle arrest in antiestrogen-treated MCF-7 cells, corresponding with ERK activation and cytoplasmic localization of p27Kip1.

Estrogens activate the Ras/Raf/MEK/ERK signal transduction pathway in MCF-7 cells, and this pathway has been associated with promotion of growth and cell survival in MCF-7 cells and in estrogen-responsive tissues in vivo (63, 67–70, 80). In our previous studies dominant-negative Ras inhibited down-regulation of ectopic p27Kip1 in response to E2 (28). In the present study we found that dominant-negative ERK2 prevented E2-induced down-regulation of both the exported p27Kip1S10E mutant and the p27Kip1S10A mutant for which degradation appears to be restricted to the nucleus, leading to accumulation of both mutants in E2-treated cells. This may suggest a role for ERK in p27Kip1 destruction in either the cytoplasmic or nuclear compartments. Accordingly, dominant-negative ERK2 and the MEK inhibitor PD98059 inhibited nuclear export of p27Kip1, and this inhibition was independent of Ser10 phosphorylation based on blockade of the p27Kip1S10E mutant that mimics p27Kip1 phosphorylated on this residue. Our results thus suggest that ERKs participate in p27Kip1 export elicited by estrogen at a step subsequent to Ser10 phosphorylation and more broadly indicate an increasingly complex role for ERK activation in regulating the fate of p27Kip1 subsequent to estrogen/mitogen stimulation. A recent study indicates that calpain-mediated p27Kip1 degradation in choroid melanoma cells depends upon nuclear export and on MAPK activity, which appears to regulate p27Kip1 phosphorylation indirectly (26).

In conclusion, our studies indicate that p27Kip1 down-regulation in response to estrogen stimulation of MCF-7 cells is likely mediated both through Skp2-dependent mechanisms as well as through mechanisms that are independent of Skp2 and rely instead upon nuclear export of p27Kip1 to active proteolytic systems in the cytoplasm. Significantly, our studies also indicate a role for active ERKs in nuclear export and down-regulation of p27Kip1 elicited by E2. Removal of p27Kip1 CDK inhibitory activity in G1 thus appears to occur in at least three phases in this model system: sequestration in cyclin D-CDK complexes in early G1, export/cytoplasmic degradation in mid-G1, and Skp2-dependent degradation in the nuclear compartment beginning late in G1 phase. A simplified version of the interactions regulating p27Kip1 function in response to estrogen is shown in Fig. 8.



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  		FIG. 8.
Modes of p27Kip1 regulation by estrogens. A simplified version of the complex, interrelated events in p27Kip1 regulation in MCF-7 cells is shown. Estrogens regulate expression and function of p27Kip1 through Ras/Raf-1/ERK activation as well as through genomic targets of action such as Myc (for review, see Ref. 3). Phosphorylation of p27Kip1 at Ser10, Thr187, and as-yet undefined residues regulates subcellular localization, CDK inhibitory activity and stability of p27Kip1. Ras/Raf-1/ERK activation elicits CRM1-dependent export of p27Kip1 export, removing it from the inhibitory, nuclear pool and promoting its degradation. In the cytoplasm, p27Kip1 participates in formation of de novo cyclin D-CDK4 complexes induced by estrogen, and their import, leading to sequestration of p27Kip1 in this cellular compartment and possibly in the nucleus. Skp2 mediates ubiquitin/proteasome-mediated destruction of p27Kip1 localized to the nucleus, dependent on CDK2 activity and possibly ERKs. Undefined aspects of p27Kip1 regulation including the targets of ERK action, the site of p27Kip1 degradation mediated by Skp2, and the possible participation of exported p27Kip1 in interactions with cyclin D-CDK complexes or other effectors are indicated as (??). Given that most of the p27Kip1 in quiescent breast cancer cells is bound to cyclin E-CDK2 (83, 84), the equilibria existing between bound p27Kip1 and free, exportable p27Kip1 in the nucleus also remain unknown. Additional references for this figure have been cited in the Introduction and "Discussion."

 

In addition, our studies suggest modes of p27Kip1 down-regulation mediated by Skp2 or through oncogene-activated signaling pathways that may contribute to antiestrogen resistance in breast cancer cells. Our results demonstrate potent growth promotion by Skp2 with respect to antagonizing growth suppression by antiestrogens. A study published when this manuscript was in preparation (34) has demonstrated that Skp2 is overexpressed in a subset of estrogen receptor-negative/Her-2-negative breast cancers and that Skp2 overexpression abrogates antiestrogen-mediated growth arrest of MCF-7 cells. Similarly, our studies also indicate that cytoplasmic relocalization of p27Kip1 elicited by Ras/Raf-1/ERK activation can contribute to antiestrogen resistance in this model system. Previous studies have demonstrated a role for p27Kip1 in formation and nuclear localization of active cyclin D-CDK4 complexes as well as the particular importance of p27Kip1 and cyclin D-CDK4 to normal growth and function of the mammary epithelium (5, 6, 81, 82). Better understanding of the control exerted by estrogen over p27Kip1 export, degradation, and growth-promoting interactions with cyclin D-CDK4 complexes will clarify the complex interactions regulating growth in normal breast epithelium that ultimately contribute to tumor formation, antiestrogen resistance, and disease progression.


   FOOTNOTES
 
* This work was supported by Grant CA84048 from the NCI, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Obstetrics and Gynecology, Graduate School of Medicine, Program in Comparative and Experimental Medicine, University of Tennessee Medical Center, 1924 Alcoa Highway, Knoxville, TN 37920. Tel.: 865-544-8961; Fax: 865-544-6863; E-mail: jwimalas{at}utk.edu.

1 The abbreviations used are: CDK, cyclin-dependent kinase; BrdUrd, bromodeoxyuridine; E2, 17{beta}-estradiol; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; GFP, green fluorescent protein; Kip, kinase inhibitory protein; LMB, leptomycin B; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; pfu, plaque-forming units; SCF, Skp1/Cul-1/F box protein. Back

2 N. Ishida and K. Nakayama, submitted for publication. Back


   ACKNOWLEDGMENTS
 
We thank Dr. M. Cobb for providing the MEKEE construct and Dr. J. Slingerland for providing the YFPp27Kip1 construct. In addition, we are indebted to Richard Andrews for flow cytometric analysis performed for this and other studies.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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