Cancer Research Translational Cancer Medicine 2008: Cancer Clinical Trials and Personalized Medicine Susan G. Komen for the Cure-AACR Outstanding Investigator Award for Breast Cancer Research
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Cancer Research 66, 9986-9994, October 15, 2006. doi: 10.1158/0008-5472.CAN-06-1596
© 2006 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Cyclin D1 Induction of Cellular Migration Requires p27KIP1

Zhiping Li, Xuanmao Jiao, Chenguang Wang, Xiaoming Ju, Yinan Lu, Liangping Yuan, Michael P. Lisanti, Sanjay Katiyar and Richard G. Pestell

Kimmel Cancer Center, Departments of Cancer Biology and Medical Oncology, Thomas Jefferson University, Philadelphia, Pennsylvania

Requests for reprints: Richard G. Pestell, Kimmel Cancer Center, Departments of Cancer Biology and Medical Oncology, Thomas Jefferson University, 233 South 10th Street, BLSB, Room 1050, Philadelphia, PA 19107. Phone: 215-503-5649; Fax: 215-503-9334; E-mail: Richard.Pestell{at}jefferson.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cyclin D1 gene is amplified and overexpressed in human breast cancer, functioning as a collaborative oncogene. As the regulatory subunit of a holoenzyme phosphorylating Rb, cyclin D1 promotes cell cycle progression and a noncatalytic function has been described to sequester the cyclin-dependent kinase inhibitor protein p27. Cyclin D1 overexpression correlates with tumor metastasis and cyclin D1–deficient fibroblasts are defective in migration. The genetic mechanism by which cyclin D1 promotes migration and movement is poorly understood. Herein, cyclin D1 promoted cellular migration and cytokinesis of mammary epithelial cells. Cyclin D1 enhanced cellular migratory velocity. The induction of migration by cyclin D1 was abolished by mutation of K112 or deletion of NH2-terminal residues 46 to 90. These mutations of cyclin D1 abrogated physical interaction with p27KIP1. Cyclin D1–/– cells were p27KIP1 deficient and the defect in migration was rescued by p27KIP1 reintroduction. Conversely, the cyclin D1 rescue of cyclin D1–/– cellular migration was reversed by p27KIP1 small interfering RNA. Cyclin D1 regulated p27KIP1 abundance at the posttranslational level, inhibiting the Skp2 promoter, Skp2 abundance, and induced p27KIP1 phosphorylation at Ser10. Together, these studies show cyclin D1 promotes mammary epithelial cell migration. p27KIP1 is required for cyclin D1–mediated cellular migration. (Cancer Res 2006; 66(20): 9986-94)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Several lines of evidence show a key role for cyclin D1 in human tumorigenesis. The cyclin D1 gene is the BCL1 oncogene, located at a breakpoint of the characteristic t(11;14)(q13;q32) clonal translocation in mantle cell lymphomas and a subset of multiple myeloma. Initially cloned by three independent groups, structural homology to eukaryotic cyclins led to the understanding that cyclin D1 functions as a regulatory subunit of a holoenzyme phosphorylating several downstream targets, including the retinoblastoma, pRb, protein. The finding that cyclin D1 is overexpressed in human tumors, the identification of cyclin D1 within driver amplicons in human cancer, the common coexpression of cyclin D1 with amplification, together with molecular genetic studies in murine models, have led to an understanding that cyclin D1 is an important collaborative oncogene in human cancer (1, 2).

As a regulatory subunit of the holoenzyme that phosphorylates Rb, cyclin D1 encodes the rate-limiting step in transitions from the G1 to S phase of the cell cycle in cultured cells. Molecular genetic analysis in transgenic animals has shown an essential role for cyclin D1 in the onset and progression of mammary tumorigenesis induced by Ras or ErbB2 (3) or gastrointestinal tumorigenesis induced by mutation of the Apc gene (4). In human tumors, cyclin D1 overexpression correlates with cellular metastases (5, 6), but does not correlate with indices reflecting cellular proliferation (7). Although cyclin D1 phosphorylation of Rb inactivates E2F repressor function in cultured cells, global gene expression of human tumors overexpression cyclin D1 suggested a lack of correlation between cyclin D1 overexpression and regulation of E2F target genes (8). Recent evidence suggests that cyclin D1 physically interacts with several proteins through distinct subdomains, including the p160 coactivator proteins, p300/CBP, and histone deacetylases (HDAC; ref. 1, 9, 10). Furthermore, a noncatalytic function of the cyclin D–cyclin-dependent kinase (CDK) complexes include sequestering the CDK-inhibitor proteins p21Cip1 and p27KIP1 (10).

The CDK inhibitor family p21 includes the members p21CIP1, p27KIP1, and p57KIP2. p27KIP1 inhibits most cyclin-CDK complexes, including cyclin E-CDK2, and induces cell cycle arrest in several cell types. p21 and p27KIP1 bind to CDK4/CDK6/cyclin D complexes but do not interfere with their kinase activity under stoichiometric conditions. p27KIP1 binding to cyclin D/CDK4,6 during the early phase of the cell cycle stabilizes the complex (11, 12). Curiously, CDK2 is dispensable for the cell cycle inhibition during the G1 phase of the cell cycle and is dispensable for their tumor-suppressor properties (13). Reduction in p27KIP1 abundance in tumors, however, correlates with tumor aggressiveness and dedifferentiation (14–16).

Recent evidence showed a role for p27KIP1 in cellular migration (ref. 17; reviewed in ref. 18). Intriguingly, although p27KIP1 inhibited migration of endothelial cells and vascular smooth muscle cells (19, 20), in a subset of cells, p27KIP1 stimulated cellular migration. In hepatocellular carcinoma cells and mouse embryonic fibroblasts (MEFs), p27KIP1 induced cellular migration (17, 21). The promigratory function of p27KIP1 was distinct from its cell cycle regulatory function. The NH2-terminal domain of p27KIP1 was considered to play an essential role in functional interactions regulating cell cycle progression. In contrast, the COOH-terminal 28 amino acids played a key role in migratory inhibitory activity (22).

In view of the previous studies demonstrating a correlation between cyclin D1 overexpression and cellular migration, analyses were recently conducted of cyclin D1–/– MEFs (23). These studies show cyclin D1–induced cellular migration, reducing cellular attachment. The induction of cellular migration was associated with the inhibition of Rho-GTPase activity and inhibition of thrombospondin secretion (23). As cyclin D1 is frequently overexpressed in human breast cancer, the current experiments were conducted to determine if cyclin D1 also regulated migration and transwell movement of mammary epithelial cells. Next, studies were conducted to determine the mechanism by which cyclin D1 induces cellular migration. Introduction of cyclin D1 promoted cellular migration and transmigration across a membrane. Mutational analysis identified a role for residues 47 to 90 in the NH2 terminus of cyclin D1 for cellular migration. These residues of cyclin D1 were required for enhancement of cellular migratory velocity. Molecular dissection of proteins binding to cyclin D1 mutants, defective in regulating cellular migration, identified a key requirement for these domains in physical association with p27KIP1 and CDK4, both in immunoprecipitation–Western blotting and mammalian two-hybrid analysis. Cyclin D1 inhibited Rho expression and coassociated with p27KIP1 in a complex with active Rho substrates. Depletion of p27KIP1, through small interfering RNA (siRNA), abrogated rescue by cyclin D1 of cellular migration. Conversely, p27KIP1 reintroduction obviated a defect of transmigration of cyclin D1–deficient cells. Cyclin D1–deficient cells show reduced levels of p27KIP1. Cyclin D1 induced p27KIP1 protein levels by inhibiting the Skp2 component of the SCF complex that degrades p27KIP1 and by inducing phosphorylation of p27KIP1 at Ser10. Together, these studies identify a novel molecular mechanism by which cyclin D1 promotes cellular migration through inducing the expression of p27KIP1 and by physically associating with p27KIP1 to promote cellular migration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice. All animal experiments were done in accordance with the guidelines for the care and use of laboratory animals at Georgetown University and Thomas Jefferson University. Cyclin D1–/– mice were maintained on a mixed C57Bl/6x129/SvJ background as described previously (4). Genotyping was done on tail DNA by PCR as described before (4).

Plasmids. The MSCV-IRES-GFP retroviral vector and cyclin D1 wild-type (23) or mutant constructs were cloned into the EcoRI site of the vector. pCMV5/human p27Kip1 full length, NH2 terminus (1-86 amino acids), and COOH terminus (77-198 amino acids) were provided by Dr. Joan Massagué (Memorial Sloan-Kettering Cancer Center, New York, NY). The full-length human p27Kip1 cDNA (KpnI to BamHI fragment from pCMV5/p27Kip1) was inserted into the MSCV-IRES-GFP vector at the EcoRI site upstream of the IRES driving expression of green fluorescent protein (GFP).

Cell culture. Cyclin D1+/+ and cyclin D1–/– primary MEF cultures were prepared as described previously (24). Human kidney 293, 293T, and MCF-7 cells were maintained in DMEM containing penicillin and streptomycin (100 mg of each/L) and supplemented with 10% fetal bovine serum (FBS).

For culture of murine mammary epithelial cells, cells were resuspended in DMEM/F12 medium supplemented with 10% FBS, 10 µg of insulin/mL, 20 ng cholera toxin/mL, 1 µg of hydrocortisone/mL, and 10 ng epidermal growth factor/mL.

Retroviral production and infection. Retroviral production and infection of cyclin D1–/– MEFs and MCF-7 cells were described in detail before (23). Fluorescence-activated cell sorting (FACS; FACStar Plus; BD Biosciences, San Jose, CA) sorted GFP+ cells were used for subsequent analysis.

Scanning electron microscopy. Cells were plated on fibronectin-coated glass coverslips and grown to ~80% confluence. The cells were prepared as described previously (25) for observation under scanning electron microscopy.

Immunofluorescence. Phalloidin staining was conducted as previously described (25). The samples were visualized on a Zeiss LSM 510 META Confocal Microscope with a 63x objective.

Immunoprecipitations and Western blotting. Cyclin D1–/– MEFs infected with GFP vector control or cyclin D1 wild type or a series of mutants were lysed in immunoprecipitation buffer (10 mmol/L Tris-HCL at pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 0.5% IGEPAL CA-630, 10% glycerol, 1 mmol/L sodium orthovanadate and protease inhibitor cocktail; Boehringer Mannheim, Mannheim, Germany). For each immunoprecipitation, 500 µL lysate (500 µg protein) and 10 µL anti-FLAG M2-agarose affinity gel (Sigma, St. Louis, MO) were incubated overnight at 4°C. Immunoprecipitates were washed five times in immunoprecipitation buffer, and 20 µL of 2x sample buffer was added to the bead pellet. The immunoprecipitates, as well as 50 µg proteins of the corresponding lysates were subjected to Western blotting as previously described (26). The following antibodies were used for Western blotting: mouse M2 anti-FLAG antibody, antivinculin antibody, and anti-{alpha}-tubulin antibody (Sigma), mouse DCS-6 anti-cyclin D1 antibody, rabbit polyclonal anti-p27Kip1 antibody, anti-p27Kip1 Ser10 phosphorylated antibody, anti-cdk4 antibody, anti-cdk5, anti-Skp2, anti-p57Kip2, anti-ß-tubulin antibody, and anti-CRM1 (Santa Cruz Biotechnology), rabbit anti-stathmin (Cell Signaling Technology, Danvers, MA).

Migration assay. Transwell migration assays were done as described before (27). Briefly, GFP+ cells were seeded on 8-µm-pore transwell filter insert (Corning Incorporated, Corning, NY) coated with 10 µg/mL fibronectin (Sigma). After 16-hour incubation at 37°C and 5% CO2, cells adherent to the upper surface of the filter were removed using a cotton applicator. Cells were fixed with 3.7% formaldehyde, stained with crystal violet, and the numbers of cells on the bottom were counted. Data are from three experiments done in triplicate (mean ± SE).

Time-lapse video. For time-lapse observation of cell movement, cells on 12-well plates were maintained in DMEM with 10% FCS and HEPES. Cells were placed in a temperature and CO2 controlled incubator to maintain the temperature at 37°C and CO2 at 5%. The cell movement videos were taken at 5-minute intervals for 2 hours by using a Nikon Eclipse TE-300 Inverted Microscope System. The cell movement velocity was determined by tracing the single cells at different time points using MetaMorph software.

Mammalian two-hybrid system. Mammalian two-hybrid was done by following the instruction of the manufacturer (Promega, Madison, WI). Human p27Kip1 cDNA was cloned into pBIND vector and human cyclin D1 cDNA (wild type or mutants) was cloned into pACT vector to generate fusion proteins with the DNA-binding domain of GAL4 (Gal4-p27Kip1) and the activation domain of VP16 (VP16-cyclin D1), respectively. Gal4-p27Kip1, VP16-cyclin D1, and pG5luc, which contains five Gal4 binding sites upstream of a minimal TATA box and the firefly luciferase gene (Promega) were cotransfected into MCF-7 cells with Superfect reagent. Two days after transfection, the cells were lysed and the amount of luciferase activity was quantitated as previously described (24).

Rho pull down assay. The activated Rho complex present in cyclin D1–/– MEFs infected with wild-type cyclin D1 or vector control was determined by using the Rho activation assay kit (Upstate Cell Signaling Solutions, Lake Placid, NY) according to the instruction of the manufacturer. The presence of cyclin D1 and p27Kip1 in the activated Rho complex was detected by immunoblotting.

siRNA. For suppression of cellular p27Kip1 expression, siRNA that specifically targets mouse p27Kip1 mRNA was purchased from Santa Cruz Biotechnology. MEFs were transfected with the p27Kip1 siRNA or control siRNA (Santa Cruz Biotechnology) using the Oligofectamine reagent (Invitrogen, Carlsbad, CA) according to the instructions of the manufacturer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Previous studies had shown defective migration of cyclin D1–deficient macrophages (25). To identify the molecular mechanisms by which cyclin D1 promoted cellular migration, a series of cyclin D1 expression plasmids were constructed. The CDK-binding defective mutant (K112E), the pRb-binding defective mutant [CC7,8GH(GH)], and the p160(SRC-1) coactivator-binding defective mutant of cyclin D1 [LL254, 255AA, (LLAA)] were previously described (24). Deletion constructs of the cyclin D1 carboxyl terminal 268 to 295 region (N1), deletion of the COOH-terminal region residues 179 to 295 (N4), deletion of the NH2-terminal 45 amino acids (C7) or NH2-terminal 90 amino acids (C6) were assessed (Fig. 1A ). These cDNAs were cloned into the MSCV-IRES-GFP expression vector and analyzed for their abundance in cultured cells through Western blotting to the NH2-terminal FLAG epitope. These studies showed levels of expression of these mutant proteins were similar to that of the cyclin D1 wild type when normalized to the loading control protein, GDI (data not shown). Scanning electron microscopy of MEF derived from cyclin D1–deficient cells, at passage 3, showed flattened spread morphology of cyclin D1–/– cells. Reintroduction of cyclin D1 and either the GH, LLAA, N1 or N4, or the C7 construct rescued the aberrant cellular morphology, returning the cyclin D1–/– MEFs to the rounded fibroblastoid morphology of wild-type cells (Fig. 1B). In contrast, the C6 mutant failed to rescue the aberrant cellular morphology (Fig. 1B).


Figure 1
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  		Figure 1. The cyclin D1 NH2 terminus and K112 regulate polar cellular morphology. A, schematic representation of wild-type and mutant human cyclin D1 expression vector structure. B, cyclin D1+/+, cyclin D1–/– MEFs, and cyclin D1–/– MEFs infected with mutant human cyclin D1 mutants N1, N4, C6, and C7 were plated on fibronectin (10 µg/mL)–coated coverslips and examined by scanning electron microscopy (x1,000). Bar, 20 µm. C, rhodamine-phalloidin staining of cyclin D1–/– MEFs transduced with control vector (GFP) or human cyclin D1 wild type (hD1wt) or KE mutant (KE), GH mutant (GH), LLAA mutant (LLAA), N1 mutant (N1), C6 mutant (C6), C7 mutant (C7). Nuclei were stained with 4',6-diamidino-2-phenylindole. Bar, 50 µm.

 
Filamentous actin (F-actin) staining was conducted using rhodamine-phalloidin staining; reintroduction of cyclin D1 wild type into cyclin D1–deficient cells induced the fibroblastoid distribution of F-actin (Fig. 1C). Similarly, the reintroduction of the expression vectors (GH, LLAA, N1, C7) rescued the spread morphology. However, the reintroduction of the expression vector C6 or the CDK binding defective mutant KE revealed the flattened spread morphology of the cyclin D1–/– cells, consistent with the failure of these two cDNAs to rescue the cellular morphologic appearance (Fig. 1C).

Cyclin D1 is overexpressed in ~50% of human breast cancers and is sufficient for the induction of mammary adenocarcinoma when targeted to the mammary gland in transgenic mice. We, therefore, sought to determine whether cyclin D1 is capable of promoting mammary epithelial cell migration. Primary murine epithelial cells (MEC) were prepared from cyclin D1+/+ and cyclin D1–/– mice. Cyclin D1–/– MEC showed a larger diameter when plated on fibronectin-coated plates. Cellular adhesion of primary murine mammary epithelial cells was assessed through crystal violet staining and measurement of absorbance (Fig. 2A ). Cellular attachment was increased >20% in cyclin D1–/– MECs. To examine the biological significance of the flattened cellular morphology of the cyclin D1–/– cells, migration assays were conducted. Transwell migration was done in which cells were plated on the superior aspect of a transwell membrane and the ability to transmigrate was assessed by cell counting of viable cells that have transversed the inferior membrane. The migration from the superior aspect of the membrane to the inferior aspect of the membrane was quantitated after 16 hours (Fig. 2B). A reduction in transwell migration was shown in cyclin D1–/– mammary epithelial cells (Fig. 2B). The ~3-fold reduction of transwell migration (P < 0.05) was observed in multiple separate MEC preparations from multiple mice of identical strain.


Figure 2
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  		Figure 2. Cyclin D1 promotes mammary epithelial cell migration. A, adhesion assay on fibronectin-coated, 96-well plates was conducted in either wild-type or cyclin D1–/– MECs. After 30 minutes, plates were washed, fixed, and stained with crystal violet. Adherent cells were observed under phase-contrast microscopy and the attached cells were counted as A550. B, transwell migration assay in wild-type and cyclin D1–/– murine mammary epithelial cells. * and **, significant difference between wild-type cells and cyclin D1–/– cells. C, single-cell analysis of cell movement velocity taken at 5-minute time intervals from cyclin D1–/– MEFs transduced with control vector (GFP) or mouse cyclin D1 (mD1) or human cyclin D1 wild type or KE mutant, GH mutant, LLAA mutant, N1 mutant, N4 mutant, C6 mutant, C7 mutant. D, the velocity of cell movement was determined by tracing the movement of single cells with time using MetaMorph software. Columns, mean cellular movement velocity for n ≥ 20 cells assessed at 5-minute intervals for 2 hours; bars, SE. **, P < 0.01.

 
The cyclin D1–/– MEFs were also defective in transwell migration (ref. 23; Supplementary Data 1), with ~4-fold less cells migrating compared with littermate control MEFs. Reintroduction of cyclin D1 restored transwell migration to that of wild-type cells. Deletion of amino acids 46 to 90 or point mutation of lysine 112 abrogated the ability of cyclin D1 to promote cellular migration (Supplementary Data 1). Persistence of migratory directionality was next assessed using videomicroscopy (Fig. 2D). Migratory velocity was enhanced by either murine or human cyclin D1, with a 3- or 4-fold increase in cellular movement velocity. Again, mutation of amino acid residues 46 to 90 or mutation of lysine 112 abrogated affects on cellular movement velocity (Fig. 2D).

Cyclin D1 and p27KIP1 function in trans during murine development (28). We therefore examined the physical association of cyclin D1 with p27KIP1 by transducing cyclin D1–/– MEFs with expression vectors for either wild-type or mutant cyclin D1, to determine whether the domains of cyclin D1 regulating cellular migration were required for physical association with p27KIP1. Western blot analysis showed similar levels of mutant cyclin D1 proteins compared with wild-type by FLAG antibody to the NH2-terminus of each cyclin D1 protein (Fig. 3A ). The relative abundance of p27KIP1 was induced by cyclin D1 transduction. The cells transduced with the cyclin D1 KE and C6 mutant did not induce p27KIP1 abundance by Western blotting. Using these cellular extracts, immunoprecipitation-Western blotting was conducted. An antibody to the FLAG epitope of the cyclin D1 proteins was used in immunoprecipitation. Western blot to FLAG showed the immunoprecipitation of cyclin D1 and the coassociation of CDK4 and p27KIP1. p27KIP1 associated with each of the cyclin D1 proteins except the cyclin D1 KE and cyclin D1 C6 mutant (Fig. 3B). Cdk4 was coprecipitated with each of the cyclin D1 mutants except the cyclin D1 KE mutant and the cyclin D1 C6 mutant. Collectively, these studies show a correlation between the ability of cyclin D1 to induce p27KIP1 abundance and the ability to bind p27KIP1 (Fig. 3C).


Figure 3
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  		Figure 3. The cyclin D1 NH2 terminus and K112 are required for p27Kip1 binding in cultured cells. A, Western blot for the amounts of p27Kip1, Cdk4, FLAG-tagged cyclin D1, and the loading control vinculin in the cell extracts used for immunoprecipitations. B, immunoprecipitation (IP) with a monoclonal FLAG antibody (M2) in cyclin D1–/– MEFs infected with control vector (GFP) or FLAG-tagged human cyclin D1 wild type or KE mutant, GH mutant, LLAA mutant, N1 mutant, N4 mutant, C6 mutant, C7 mutant. Immunoprecipitates were resolved on SDS-PAGE, and membranes were probed with a polyclonal p27Kip1 antibody (C19), Cdk4 antibody (C22), and FLAG antibody (M2). C, relative abundance of p27KIP1 in cyclin D1–/– MEFs transduced with cyclin D1 expression vectors.

 
To determine whether cyclin D1 directly interacted with p27KIP1, mammalian 2-hybrid experiments were conducted. The Gal4 DNA-binding domain was linked to the p27KIP1 protein. Cyclin D1 fragments were linked to the VP16 transactivation domain and the interaction between p27KIP1 and cyclin D1 was assessed using a multimeric Gal4 DNA binding site (pG5LUC; Fig. 4A ). The fusion of cyclin D1 to the VP16 activation domain reduced the transactivity of VP16 by 50%, consistent with the known binding of cyclin D1 to corepressors with HDAC activity (9). The association between p27KIP1 and cyclin D1 was shown by the induction of luciferase activity (Fig. 4A, lane 2 versus lane 3). The deletion of amino acid residues 46 to 90 abrogated the interaction between cyclin D1 and p27KIP1 in mammalian two-hybrid analysis (lane 4 versus lane 3). Similar analysis showed point mutation of cyclin D1 lysine 112 abrogated interaction between cyclin D1 and p27KIP1 (Fig. 4A). Cyclin D1 immunoprecipitation Western blot showed cyclin D1 coprecipitated p27KIP1 and this association was abolished by deletion of the p27KIP1 NH2 terminus (Fig. 4B). Together, these studies show the association with p27KIP1, requires cyclin D1 amino acids 46 to 90 and lysine 112.


Figure 4
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  		Figure 4. The cyclin D1 NH2 terminus and K112 are required for p27Kip1 binding in mammalian two-hybrid system. A, the mammalian two-hybrid system was used to detect the binding of cyclin D1 and p27Kip1. Left, schematic representation of the system. Middle and right, pG5luc activity in MCF-7 cells cotransfected with 0.3 µg pG5luc, Gal4-p27Kip1, and VP16-cyclinD1 wild type or mutants. B, left, schematic representation of full-length, NH2 terminus, and COOH terminus of p27Kip1; right, immunoprecipitation with a monoclonal FLAG antibody (M2) in HEK293T cells cotransfected with FLAG-tagged cyclin D1 and p27Kip1 full length, NH2 terminus, or COOH terminus. Immunoprecipitates were resolved on a SDS-PAGE gel, and membranes were blotted with a polyclonal p27Kip1 antibody (M197) and FLAG antibody (M2). The amounts of p27Kip1 and FLAG-tagged cyclin D1 in the cell extracts used for immunoprecipitations were also indicated as input. Cyclin D1 binds p27Kip1. Neither the p27Kip1 NH2 terminus or COOH terminus are sufficient to bind cyclin D1.

 
Previous studies have shown that p27KIP1 promotes fibroblast migration, although the mechanisms by which p27KIP1 promotes this activity remains controversial (29). Although the role of p27KIP1 in transwell migration had not previously been determined, we considered the possibility that cyclin D1–deficient cells may express less p27KIP1 protein, which, in turn, may contribute to the defect in migration of cyclin D1–/– MEFs. Western blot analysis revealed reduced p27KIP1 abundance in cyclin D1–deficient MEFs (Fig. 5A ). Recent studies have suggested an important role for p27KIP1 in promoting cellular migration through several distinct hypothesized mechanisms (18, 29). One mechanism by which p27KIP1 promotes cellular migration involves inhibition of Rho-GTPase activity. Reintroduction of cyclin D1 into cyclin D1–/– MEFs induced cyclin D1 and p27KIP1 levels with a commensurate reduction in Rho-GTPase activity (23) assessed by Rho-GTP pull down with p27KIP1 and cyclin D1 both detected in the Rho-GTP complex (ref. 23; Fig. 5B). We examined further the possible role for p27KIP1 in cyclin D1–mediated cell migration. To determine whether p27KIP1 rescued the cyclin D1–/– cell migration defect, cyclin D1–/– cells were transduced with a retroviral vector expression for p27KIP1 and transwell migration analyses were conducted. Reintroduction of p27KIP1 into cyclin D1–/– MEFs induced transwell migration (Fig. 5C) with a 2-fold increase in cell migration (Fig. 5C). The rescue function in transwell migration assays was similar to that of cyclin D1 in the number of migrating cells induced by p27KIP1 transduction (cyclin D1 transduction 72 ± 8 versus p27KIP1 transduction 61 ± 4, n = 3). Thus, p27KIP1 is sufficient to rescue the defect in migration of cyclin D1–deficient cells. To determine whether p27KIP1 functioned as a downstream target of cyclin D1–dependent rescue of cellular migration of cellular movement velocity, p27KIP1 siRNA was used (Fig. 5D-E). Cyclin D1 transduction of cyclin D1–/– cells induced cellular velocity 2.5-fold. Introduction of p27KIP1 siRNA abrogated p27KIP1 protein levels by Western blot and reduced cell movement velocity induced by cyclin D1 to that of cyclin D1–/– cells (Fig. 5D, E). Together, these studies show that cyclin D1 induction of cellular migration requires p27KIP1.


Figure 5
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  		Figure 5. Exogenous p27Kip1 rescues the migration defect of cyclin D1–/– MEFs. A, Western blot for p27Kip1, cyclin D1, and CRM1 protein expression in cyclin D1+/+ and cyclin D1–/– MEFs. B, glutathione S-transferase pull-down assay for activated Rho (Rho-GTP activity) in cyclin D1–/– MEFs infected with wild-type cyclin D1 (hD1wt) or vector control (GFP). The same membrane was blotted with FLAG antibody (mouse M2), p27Kip1 antibody (C19), and ß-tubulin antibody (H235) for cyclin D1, p27Kip1, and ß-tubulin expression in the activated Rho complex. p27Kip1 and cyclin D1 were detected in the activated Rho-GTP complex. ß-Tubulin is a negative control not associated with active Rho. C, transwell migration assay was conducted in cyclin D1–/– MEFs infected with either MSCV-GFP control vector or wild-type human p27Kip1. **, migrated cell number and the significant difference from GFP vector control. Bottom, exogenous p27Kip1 protein expression in cyclin D1–/– MEFs infected with vector control or p27Kip1-expressing virus. D and E, p27Kip1 siRNA blocked the promigratory effect of exogenous cyclin D1 in cyclin D1–/– MEFs. Phase-contrast, time-lapse video microscopy images of cyclin D1–/– MEFs infected with wild-type cyclin D1 without treatment or with control siRNA or p27Kip1 siRNA were traced by MetaMorph software (D). The cell movement velocity was determined by tracing the single cells at different time points. Columns, mean of at least 10 individual cells of the same cell type; bars, SE. Bottom, p27Kip1 protein expression was blocked after siRNA treatment.

 
The finding that the domains of cyclin D1–promoting cellular migration physically associated with p27KIP1 suggests that a physical interaction between these two proteins is important in cyclin D1–mediated migration. Thus, cyclin D1 promotes cellular migration through physical association with p27KIP1. These findings extend our previous understanding of the role of cyclin D1 in cellular biological activities. These studies also suggest the reduction in cyclin D1 abundance in cyclin D1–/– cells may have a secondary effect of reducing p27KIP1 levels, which, in turn, may contribute to the defect in cellular migration.

The finding that p27KIP1 protein levels were reduced in cyclin D1–/– MEFs led us to examine further the mechanism by which cyclin D1 regulated p27KIP1 abundance. p27KIP1 abundance is affected by transcriptional and translational pathways. However, the major mechanism is posttranslational phosphorylation of p27KIP1 on Thr187, which creates a recognition site for the Skp2-SCF-E3 ubiquitin ligase, thereby promoting ubiquitination and degradation by the proteasome (30, 31). An additional degradation pathway involves phosphorylation of p27KIP1 at Ser10. To determine the mechanism by which cyclin D1 induced p27KIP1 abundance, we examined the effect of cyclin D1 on p27KIP1 mRNA levels. Cyclin D1–/– MEFs were transduced with a cyclin D1 expression vector and p27KIP1 mRNA abundance was assessed by reverse transcription-PCR (RT-PCR). Cyclin D1 expression did not alter p27KIP1 mRNA levels (Fig. 6A ). We next assessed the effect of cyclin D1 on p27KIP1 protein levels. Cyclin D1–/– MEFs were stably transduced with either an expression vector encoding control (GFP) or cyclin D1/IRES-GFP. Cells transduced with cyclin D1 showed a 6-fold increase in p27KIP1 protein. Inhibition of new protein synthesis led to a reduction in both cyclin D1 and p27KIP1 levels, further suggesting the abundance of cyclin D1 protein maintains p27KIP1 protein levels (Fig. 6B). The abundance of the rate-limiting Skp2 component of the Skp2-SCF-E3 ligase that degrades p27KIP1 was next assessed. Skp2 abundance was inhibited by cyclin D1 expression (Fig. 6C), commensurate with the induction of p27KIP1 protein. Skp2 is regulated by transcriptional induction (32). We therefore assessed the regulation of the Skp2 promoter linked to a luciferase reporter gene. Cyclin D1 repressed the Skp2 promoter in a dose-dependent manner (Fig. 6D). Phosphorylation of p27KIP1 at Ser10 regulates p27KIP1 degradation by an ubiquitin ligase other than Skp2-SCF that remains to be defined. As p27KIP1 phosphorylation at Ser10 increases p27KIP1 stability, we conducted a Western blot with a phospho-Ser10-specific antibody. Cyclin D1–/– cells showed reduced p27KIP1 Ser10 phosphorylation. Cyclin D1 expression increased the relative abundance of p27KIP1 and associated Ser10 phosphorylation (Fig. 6E; ref. 33). p27KIP1 Ser10 is phosphorylated by cdk5, and cyclin D1 induced cdk5 abundance (Fig. 6E). As cyclin D1 induced p27 and cdk5 abundance, we investigated the possibility that cyclin D1 may induce additional cyclin D1–binding proteins. More than 30 cyclin D1 interactive proteins have been described (1). The well-characterized p21CIP1 protein was therefore assessed. Cyclin D1 induced expression of p21CIP1 but did not affect expression of p57KIP2 or cdk4 (Fig. 6F).


Figure 6
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  		Figure 6. Cyclin D1 induces p27KIP1 abundance, reducing the Skp2 component of Skp2-SCF-E3-ligase. A, p27KIP1 mRNA levels assessed by RT-PCR in cyclin D1–/– cells transduced with a cyclin D1 expression vector. Data are shown normalized to ß-actin. B, Western blot analysis for cyclin D1 and p27KIP1. Cyclin D1–/– cells, transduced with cyclin D1 expression vector, were subsequently treated with cyclohexamide for the time points indicated. Cyclin D1 and p27KIP1 protein abundance is shown graphically. C, Western blot for Skp2 and p27KIP1 in cyclin D1–/– cells, transduced with cyclin D1 expression vector encoding cyclin D1 with an amino terminal FLAG epitope. D, activity of the Skp2 promoter in HEK293T cells transfected with a cyclin D1 expression vector. Columns, mean of n > 5 separate transfections; bars, SE. E and F, Western blot analysis of cyclin D1–/– MEFs transduced with a cyclin D1 expression vector was done to assess abundance of p27KIP1 and cyclin D1 interacting proteins. G, TSP1 promoter activity was repressed by both cyclin D1 and p27KIP1 in a dose-dependent manner. H, schematic representation of model by which cyclin 1 regulates p27KIP1 abundance.

 
Recent studies (23) showed cyclin D1 promoted migration through reducing the expression and transcription of thrombospondin 1. Consistent with previous studies, cyclin D1 repressed TSP-1 (Fig. 6G). p27KIP1 also repressed TSP-1, consistent with our model in which cyclin D1 and p27KIP1 function cooperatively to promote cellular migration. Collectively, these studies suggest that cyclin D1 promotes migration through p27KIP1. Cyclin D1 associates with p27KIP1 and induces p27KIP1 abundance, through reducing p27KIP1 degradation by repressing Skp2 and inducing p27KIP1 Ser10 phosphorylation (Fig. 6H).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cyclin D1 is overexpressed in human breast cancer and the current studies identify a novel function for cyclin D1 in mammary epithelial cells. The studies herein show that cyclin D1 reduces attachment and induces mammary epithelial cell migration across a membrane. This new function of cyclin D1 may contribute to the clinical observation that cyclin D1 overexpression correlates with tumor metastasis. The current studies are consistent with and extend recent findings that cyclin D1 promotes motility of fibroblasts (23). These prior studies show that cyclin D1 induces MEF motility by inhibiting Rho GTPase activity. The current studies identify several key molecular mechanisms by which cyclin D1 promotes migration. First, using siRNA to p27KIP1, we showed that induction of cellular migration by cyclin D1 requires p27KIP1. Second, the domains of cyclin D1 required for the induction of cellular migration are shown to be required for p27KIP1 binding and for the induction of p27KIP1 abundance. Third, these studies show that cyclin D1 induces p27KIP1 abundance through two mechanisms: cyclin D1 represses the Skp2 component of the Skp2-SCF-E3 ligase that degrades p27KIP1 and cyclin D1 induces phosphorylation of p27KIP1 at Ser10, a modification known to enhance p27KIP1 stability (33, 34). Collectively, these studies show a novel function for cyclin D1 in mammary epithelial cells, and a new mechanism by which cyclin D1 regulates cellular migration and a new mechanism by which cyclin D1 regulates p27KIP1 abundance.

The domains of cyclin D1 required for the induction of cellular migration included amino acid residues in the NH2 terminus and Lys112. Both regions of cyclin D1 were shown to be required for association with p27KIP1 in mammalian two-hybrid. The requirement for p27KIP1 in cyclin D1–mediated migration suggests that the physical association of cyclin D1 with p27KIP1 plays an important role in the formation of a promigratory complex. These studies extend previous observations in which cyclin D1 association with p27KIP1 functioned to promote cell cycle function and DNA synthesis (35). In prior studies, a noncatalytic function of cyclin D–CDK4 included sequestration of p27KIP1 from the cyclin E–CDK2 complex (reviewed in ref. 35). These studies extend the prior observations, demonstrating a role for cyclin D1 association with p27KIP1 in promoting cellular migration.

These studies also extend prior observations that cyclin D1–/– murine bone marrow macrophages show reduced migration in response to CSF1 and increased cellular adhesion (25). This abnormality of cellular migration in cyclin D1–deficient cells was observed herein in fibroblasts and in mammary epithelial cells. Cyclin D1–deficient mammary epithelial cells showed a reduction in migration and reduced cellular velocity. The morphologic appearance of cyclin D1–/– MEFs were more spread and flattened as assessed by scanning electron microscopy. This spread morphology was rescued by murine or human cyclin D1 requiring the cyclin D1 NH2-terminal residues (46-90). The domains of cyclin D1 required for inducing the fibroblastoid polarized morphology were also required for the induction of cellular migratory velocity and transwell migration. The domains of cyclin D1 promoting cellular migration were also required for association with p27KIP1. Cyclin D1–deficient cells express reduced levels of p27KIP1 and p27KIP1 siRNA abrogated cyclin D1–mediated cellular migration. Collectively, these observations show that the cyclin D1–p27KIP1 complex is mechanistically involved in promoting cellular migration. Reintroduction of p27KIP1 rescued the cellular migratory defect of cyclin D1–/–, suggesting p27KIP1 is a key distal mediator of cyclin D1–mediated migration.

The mechanism by which p27KIP1 regulates cellular migration remains controversial. p27KIP1 can induce rearrangements of the actin cytoskeleton in either a Rac-dependent manner (21) or RhoA-dependent manner through inhibition of RhoA (17). In endothelial and vascular smooth muscle cells, p27KIP1 blocks cellular migration (19, 20). The subcellular distribution of p27KIP1 is important in promigratory function and cytoplasmic p27KIP1, rather than nuclear p27KIP1 promotes cellular migration (36). In addition to inhibiting RhoA activity, p27KIP1 binds stathmin, which may counteract the promigratory effects of stathmin, and cyclin D1 may antagonize this effect of p27KIP1 (22). Cyclin D1 is known to regulate E2F activity (37) and E2F transcription factors activate stathmin (38). However, cyclin D1 did not affect either stathmin abundance or stathmin promoter activity (Supplementary Data 2).

The current studies show that cyclin D1 regulates p27KIP1 abundance. Cyclin D1–/– cells were p27KIP1 deficient and reintroduction of cyclin D1 induced p27KIP1 abundance. Mutants of cyclin D1 that rescued cellular migration also induced p27KIP1 abundance, whereas mutants defective in rescue were also defective in regulating p27KIP1 abundance (Fig. 3C). Together, these studies suggest an additional mechanism by which cyclin D1 may promote migration indirectly through the induction of p27KIP1 abundance. p27KIP1 abundance can be regulated at both the transcriptional and posttranslational level. Phosphorylation of p27KIP1 at Thr187 creates a recognition site for a ubiquitin ligase that degrades p27KIP1. The Skp2 SCF ubiquitin ligase (31, 39–41) regulates p27KIP1 abundance by degradation. Herein, cyclin D1 repressed the Skp2 promoter and reduced Skp2 abundance, thereby inducing p27KIP1 protein abundance. Cyclin D1 is known to regulate the activity of a subset of transcription factors (1) through mechanisms that involve association with HDACs (9), providing a possible mechanism by which cyclin D1 may regulate the Skp2 promoter.

Herein, phosphorylation of p27KIP1 at Ser10, known to stabilize p27KIP1 (33), was increased by cyclin D1. Several different kinases have been linked to p27KIP1 Ser10 phosphorylation [Mirk/dyrk1B (42), hKIS (43), AKT (44), cdk5 (45), and extracellular signal-regulated kinase 2 (44)]. Phosphorylation at Ser10 is important in the inactivation of p27KIP1 tumor-suppressor function (17). In addition, p27KIP1 is exported from the nucleus to the cytoplasm at the G0-G1 phase of the cell cycle (34, 46, 47). Phosphorylation of Ser10 is required for the binding of p27KIP1 to CRM1, a carrier protein for nuclear export. KPC1 and KPC2-E3 ubiquitin-promoting complexes ubiquitinate and degrade p27KIP1 subsequent to CRM1-dependent nuclear export. Although the mechanism by which cyclin D1 regulates p27KIP1 Ser10 phosphorylation will require further analysis, AKT kinase that phosphorylates Ser10 in vitro was unaltered in these cells, suggesting that an alternate kinase, perhaps cdk5, is involved.

The current demonstration of a role for the physical interaction between cyclin D1 and p27KIP1 in the induction of cellular motility provides an important new link between the cyclins and the cyclin-dependent kinase inhibitor proteins. The biological significance of these studies is evidenced by the alteration of transwell migration, cellular adhesion, and cellular velocity. The demonstration that a single amino acid residue of cyclin D1 is required for p27KIP1 association and the induction of cellular migratory capability lends credence to the possibility that specific inhibitors may be designed to inhibit cyclin D1–dependent cellular migration in tumors.


   Acknowledgments
 
Grant support: NIH grants R01CA70896, R01CA75503, and R01CA107382, and NIH Cancer Center Core grant P30CA56036 for work conducted at the Kimmel Cancer Center (R.G. Pestell), a generous grant from the Dr. Ralph and Marian C. Falk Medical Research Trust, and the Susan Komen Breast Cancer Foundation grant BCTR0504227 (C. Wang). This project is funded in part under a grant from the Pennsylvania Department of Health. The Department disclaims responsibility for any analysis, interpretations, or conclusions.

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.

We thank Dawn Scardino and Almeta Mathis for assistance in preparing the manuscript, and Dr. Paul Bornstein (University of Washington, Seattle), Hui Zhang (Yale University, New Haven), and Michael J. Birrer (NCI) for providing TSP-1 luciferase, Skp2 luciferase, and stathmin luciferase plasmids.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 5/ 2/06. Revised 7/20/06. Accepted 8/18/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Fu M, Wang C, Li Z, Sakamaki T, Pestell RG. Minireview: cyclin D1: normal and abnormal functions. Endocrinology 2004;145:5439–47.[Abstract/Free Full Text]
  2. Arnold A, Papanikolaou A. Cyclin d1 in breast cancer pathogenesis. J Clin Oncol 2005;23:4215–24.[Abstract/Free Full Text]
  3. Yu Q, Geng Y, Sicinski P. Specific protection against breast cancers by cyclin D1 ablation. Nature 2001;411:1017–21.[CrossRef][Medline]
  4. Hulit J, Wang C, Li Z, et al. Cyclin D1 genetic heterozygosity regulates colonic epithelial cell differentiation and tumor number in ApcMin mice. Mol Cell Biol 2004;24:7598–611.[Abstract/Free Full Text]
  5. Drobnjak M, Osman I, Scher HI, Fazzari M, Cordon-cardo C. Overexpression of cyclin D1 is associated with metastatic prostate cancer to bone. Clin Cancer Res 2000;6:1891–5.[Abstract/Free Full Text]
  6. Jares P, Fernandez PL, Campo E, et al. PRAD-1/cyclin D1 gene amplification correlates with messenger RNA overexpression and tumor progression in human laryngeal carcinomas. Cancer Res 1994;79:4813–7.
  7. Oyama T, Kashiwabara K, Yoshimoto K, Arnold A, Koerner F. Frequent overexpression of the cyclin D1 oncogene in invasive lobular carcinoma of the breast. Cancer Res 1998;58:2876–80.[Abstract/Free Full Text]
  8. Ewen ME, Sluss HK, Sherr CJ, Matsushime H, Kato J-Y, Livingston DM. Functional interactions of the retinoblastoma protein with mammalian D-type cyclins. Cell 1993;73:487–97.[CrossRef][Medline]
  9. Fu M, Rao M, Bouras T, et al. Cyclin D1 inhibits PPAR{gamma}-mediated adipogenesis through HDAC recruitment. J Biol Chem 2005;280:16934–41.[Abstract/Free Full Text]
  10. Roberts BJ, Whitelaw ML. Degradation of the basic helix-loop-helix/Per-ARNT-Sim homology domain dioxin receptor via the ubiquitin/proteasome pathway. J Biol Chem 1999;274:36351–6.[Abstract/Free Full Text]
  11. Blain SW, Montalvo E, Massague J. Differential interaction of the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 with cyclin A-Cdk2 and cyclin D2–4. J Biol Chem 1997;272:25863–72.[Abstract/Free Full Text]
  12. LaBaer J, Garrett MD, Stevenson LF, et al. New functional activities for the p21 family of CDK inhibitors. Genes Dev 1997;11:847–62.[Abstract/Free Full Text]
  13. Martin A, Odajima J, Hunt SL, et al. Cdk2 is dispensable for cell cycle inhibition and tumor suppression mediated by p27(Kip1) and p21(Cip1). Cancer Cell 2005;7:591–8.[CrossRef][Medline]
  14. Loda M, Cukor B, Tam SW, et al. Increased proteasome-dependent degradation of the cyclin-dependent kinase inhibitor p27 in aggressive colorectal carcinomas. Nat Med 1997;3:231–4.[CrossRef][Medline]
  15. Porter PL, Malone KE, Heagerty PJ, et al. Expression of cell-cycle regulators p27Kip1 and cyclin E, alone and in combination, correlate with survival in young breast cancer patients. Nat Med 1997;3:222–5.[CrossRef][Medline]
  16. Catzavelos C, Bhattacharya N, Ung YC, et al. Decreased levels of the cell-cycle inhibitor p27Kip1 protein: prognostic implications in primary breast cancer. Nat Med 1997;3:227–30.[CrossRef][Medline]
  17. Besson A, Gurian-West M, Schmidt A, Hall A, Roberts JM. p27Kip1 modulates cell migration through the regulation of RhoA activation. Genes Dev 2004;18:862–76.[Abstract/Free Full Text]
  18. Denicourt C, Dowdy SF. Cip/Kip proteins: more than just CDKs inhibitors. Genes Dev 2004;18:851–5.[Free Full Text]
  19. Goukassian D, Diez-Juan A, Asahara T, et al. Overexpression of p27(Kip1) by doxycycline-regulated adenoviral vectors inhibits endothelial cell proliferation and migration and impairs angiogenesis. FASEB J 2001;15:1877–85.[Abstract/Free Full Text]
  20. Sun J, Marx SO, Chen HJ, Poon M, Marks AR, Rabbani LE. Role for p27(Kip1) in vascular smooth muscle cell migration. Circulation 2001;103:2967–72.[Abstract/Free Full Text]
  21. McAllister SS, Becker-Hapak M, Pintucci G, Pagano M, Dowdy SF. Novel p27(kip1) C-terminal scatter domain mediates Rac-dependent cell migration independent of cell cycle arrest functions. Mol Cell Biol 2003;23:216–28.[Abstract/Free Full Text]
  22. Baldassarre G, Belletti B, Nicoloso MS, et al. p27(Kip1)-stathmin interaction influences sarcoma cell migration and invasion. Cancer Cell 2005;7:51–63.[CrossRef][Medline]
  23. Li Z, Wang C, Jiao X, et al. Cyclin D1 regulates cellular migration through the inhibition of thrombospondin 1 and ROCK signaling. Mol Cell Biol 2006;26:4240–56.[Abstract/Free Full Text]
  24. Wang C, Pattabiraman N, Fu M, et al. Cyclin D1 repression of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) expression and transactivation. Mol Cell Biol 2003;23:6159–73.[Abstract/Free Full Text]
  25. Neumeister P, Pixley FJ, Xiong Y, et al. Cyclin D1 governs adhesion and motility of macrophages. Mol Biol Cell 2003;14:2005–15.[Abstract/Free Full Text]
  26. Bromberg JF, Wrzeszczynska MH, Devgan G, et al. Stat3 as an oncogene. Cell 1999;98:295–303.[CrossRef][Medline]
  27. Manes T, Zheng DQ, Tognin S, Woodard AS, Marchisio PC, Languino LR. {alpha}(v)ß3 integrin expression up-regulates cdc2, which modulates cell migration. J Cell Biol 2003;161:817–26.[Abstract/Free Full Text]
  28. Geng Y, Yu Q, Sicinska E, Das M, Bronson RT, Sicinski P. Deletion of the p27 Kip1 gene restores normal development in cyclin D1-deficient mice. Proc Natl Acad Sci U S A 2001;98:194–9.[Abstract/Free Full Text]
  29. Iancu-Rubin C, Atweh GF. p27(Kip1) and stathmin share the stage for the first time. Trends Cell Biol 2005;15:346–8.[CrossRef][Medline]
  30. Vlach J, Hennecke S, Alevizopoulis K, Conti D, Amati B. Growth arrest by the cyclin-dependent kinase inhibitor p27Kip1 is abrogated by c-Myc. EMBO J 1996;15:6595–604.[Medline]
  31. Tsvetkov LM, Yeh KH, Lee SJ, Sun H, Zhang H. p27(Kip1) ubiquitination and degradation is regulated by the SCF(Skp2) complex through phosphorylated Thr187 in p27. Curr Biol 1999;9:661–4.[CrossRef][Medline]
  32. Imaki H, Nakayama K, Delehouzee S, et al. Cell cycle-dependent regulation of the Skp2 promoter by GA-binding protein. Cancer Res 2003;63:4607–13.[Abstract/Free Full Text]
  33. Kotake Y, Nakayama K, Ishida N, Nakayama KI. Role of serine 10 phosphorylation in p27 stabilization revealed by analysis of p27 knock-in mice harboring a serine 10 mutation. J Biol Chem 2005;280:1095–102.[Abstract/Free Full Text]
  34. Ishida N, Hara T, Kamura T, Yoshida M, Nakayama K, Nakayama KI. Phosphorylation of p27Kip1 on serine 10 is required for its binding to CRM1 and nuclear export. J Biol Chem 2002;277:14355–8.[Abstract/Free Full Text]
  35. Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 1999;13:1501–12.[Free Full Text]
  36. Wu FY, Wang SE, Sanders ME, et al. Reduction of cytosolic p27(Kip1) inhibits cancer cell motility, survival, and tumorigenicity. Cancer Res 2006;66:2162–72.[Abstract/Free Full Text]
  37. Watanabe G, Albanese C, Lee RJ, et al. Inhibition of cyclin D1 kinase activity is associated with E2F-mediated inhibition of cyclin D1 promoter activity through E2F and Sp1. Mol Cell Biol 1998;18:3212–22.[Abstract/Free Full Text]
  38. Polzin RG, Benlhabib H, Trepel J, Herrera JE. E2F sites in the Op18 promoter are required for high level of expression in the human prostate carcinoma cell line PC-3-M. Gene 2004;341:209–18.[CrossRef][Medline]
  39. Carrano A, Eytan E, Hershko A, Pagano M. SKP2 is required for ubiquitin-mediated degradation of the Cdk inhibitor p27. Nat Cell Biol 1999;1:193–9.[CrossRef][Medline]
  40. Sutterluty H, Chatelain E, Marti A, et al. p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells. Nat Cell Biol 1999;1:207–14.[CrossRef][Medline]
  41. Nakayama K, Nagahama H, Minamishima YA, et al. Targeted disruption of Skp2 results in accumulation of cyclin E and p27(Kip1), polyploidy and centrosome overduplication. EMBO J 2000;19:2069–81.[CrossRef][Medline]
  42. Deng X, Mercer SE, Shah S, Ewton DZ, Friedman E. The cyclin-dependent kinase inhibitor p27Kip1 is stabilized in G(0) by Mirk/dyrk1B kinase. J Biol Chem 2004;279:22498–504.[Abstract/Free Full Text]
  43. Boehm M, Yoshimoto T, Crook MF, et al. A growth factor-dependent nuclear kinase phosphorylates p27(Kip1) and regulates cell cycle progression. EMBO J 2002;21:3390–401.[CrossRef][Medline]
  44. Fujita N, Sato S, Katayama K, Tsuruo T. Akt-dependent phosphorylation of p27Kip1 promotes binding to 14–3-3 and cytoplasmic localization. J Biol Chem 2002;277:28706–13.[Abstract/Free Full Text]
  45. Kawauchi T, Chihama K, Nabeshima Y, Hoshino M. Cdk5 phosphorylates and stabilizes p27kip1 contributing to actin organization and cortical neuronal migration. Nat Cell Biol 2006;8:17–26.[CrossRef][Medline]
  46. Tomoda K, Kubota Y, Kato J. Degradation of the cyclin-dependent-kinase inhibitor p27Kip1 is instigated by Jab1. Nature 1999;398:160–5.[CrossRef][Medline]
  47. Rodier G, Montagnoli A, Di Marcotullio L, et al. p27 cytoplasmic localization is regulated by phosphorylation on Ser10 and is not a prerequisite for its proteolysis. EMBO J 2001;20:6672–82.[CrossRef][Medline]



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