Localization Of p27Kip1 Research Articles

Tyrosine phosphorylation modulates binding preference to cyclin-dependent kinases and subcellular localization of p27Kip1 in the acute promyelocytic leukemia cell line NB4

AbstractWe have investigated the role of tyrosine phosphorylation of the cyclin-dependent kinase (cdk) inhibitor p27Kip1 using the acute promyelocytic leukemia cell line NB4 together with granulocyte colony-stimulating factor (G-CSF). Short-term G-CSF stimulation resulted in a rapid tyrosine dephosphorylation of p27Kip1 accompanied by a change in its binding preferences to cdks. On G-CSF stimulation, p27Kip1 dissociated from cdk4 and associated with cdk2. Binding assays with recombinant p27Kip1 confirmed that tyrosine-phosphorylated p27Kip1 preferentially bound to cdk4, whereas unphosphorylated protein preferentially associated with cdk2. In addition, studies with p27Kip1 point mutations revealed a decisive role of Tyr88 and Tyr89 in binding to cdk4. Furthermore, phosphorylation of Tyr88 and Tyr89 was accompanied by strong nuclear translocation of p27Kip1. Taken together, this report provides the first evidence that tyrosine phosphorylation of p27Kip1 plays a crucial role in binding to cdks and its subcellular localization. Moreover, both effects are mediated by application of G-CSF.

Increased expression of MDM2, cyclin D1, and p27Kip1 in carcinogen‐induced rat mammary tumors

It is thought that environmental pollutants, such as polycyclic aromatic hydrocarbons (PAH), contribute to human breast tumorigenesis, yet their roles remain incompletely elucidated. The prototypical PAH 7,12-dimethylbenz(alpha)anthracene (DMBA) specifically and effectively induces mammary tumor formation in rodent models. In an attempt to explore the molecular mechanisms by which PAH initiates and promotes mammary tumorigenesis, we examined the expression of several cell cycle regulators in rat mammary tumors induced by DMBA. Expression of cyclin D1, murine double minute-2 (MDM2), and Akt was up-regulated in tumors in comparison to normal mammary glands, as indicated by RT-PCR, Western blot analysis, and immunohistochemical staining. Expression of p27Kip1 protein was also elevated in the tumors with increased cytoplasmic localization. However, RB protein remained hyperphosphorylated. To directly test the effects of DMBA, the MCF-7 human breast cancer cells were treated. DMBA induced MDM2 expression in a dose- and time-dependent fashion in the MCF-7 cells, and this activation appeared to be p53 dependent. These data suggest that activation of cyclin D1, MDM2, and AKT as well as increased expression and cytoplasmic localization of p27Kip1 may play a role in this model of environmental pollutant-induced mammary tumorigenesis.

Subcellular Localization of p27kip1 Expression Predicts Poor Prognosis in Human Ovarian Cancer

The cyclin-dependent kinase inhibitor p27(kip1) regulates cellular progression from G(1) to S phase. Several studies have shown that loss of p27(kip1) protein expression is associated with disease progression in various malignancies. The purpose of this study was to evaluate the subcellular localization of this cyclin-dependent kinase inhibitor in a large cohort of primary ovarian carcinomas and compare the results with clinicopathologic variables and overall survival. Subcellular localization of p27(kip1) was first assessed by Western blotting in nuclear and cytoplasmic extract from 13 cases of ovarian carcinoma. Subcellular localization of the p27(kip1) protein was evaluated using tissue microarrays containing 421 cases of ovarian carcinoma. The presence of p27(kip1) in the cytoplasm regardless of the nuclear stain correlated strongly with late-stage disease (P < 0.03), extent of cytoreduction (P = 0.03), and shorter disease-specific survival (P < 0.0001). Cytoplasmic localization of p27(kip1) predicts poorer prognosis in ovarian carcinoma, particularly in late-stage disease.

NPM/ALK downregulates p27 Kip1 in a PI-3K-dependent manner

Anaplastic large-cell lymphomas (ALCL) are frequently associated with the chromosomal translocation t(2;5) (p23;q35) resulting in the NPM/ALK fusion gene that encodes a constitutively activated tyrosine kinase. We showed that NPM/ALK stimulated cell proliferation and that PI-3K/AKT pathway played an important role in this effect. p27Kip1 is a member of the CDK family inhibitory proteins regulating the entry into S phase. It was reported that p27Kip1 function is impaired in many tumors. In this study we investigated the role of PI-3K/AKT in NPM/ALK-dependent downregulation of p27Kip1 protein. To investigate this phenomenon the pro-B cell line BaF3, BaF3 cell line stably expressing NPM/ALK, and ALCL SUP-M2 cell line were used. The p27Kip1 protein expression before and after LY294002, wortmannin, or epoxomicin treatment and phosphorylation status of AKT were measured in parental and NPM/ALK+ cells by Western analysis. Also, the localization of p27Kip1 protein was analyzed by fractionation and immunoblotting. p27Kip1 was found to be downregulated in NPM/ALK-transformed hematopoietic cells, but inhibition of proteasome-dependent degradation pathway by epoxomicin reversed this effect. In addition, treatment of NPM/ALK+ cells with LY294002, the PI-3K inhibitor, caused elevation of p27Kip1 protein expression and its nuclear localization. Taken together, we postulate that NPM/ALK-PI-3K pathway stimulates cell proliferation by regulation of the expression and nuclear localization of p27Kip1.

Cytoplasmic mislocalization of p27Kip1 protein is associated with constitutive phosphorylation of Akt or protein kinase B and poor prognosis in acute myelogenous leukemia.

Cyclin-dependent kinase inhibitor p27Kip1 functions at the nuclear level by binding to cyclin E/cyclin-dependent kinase-2. It was shown that Akt or protein kinase B (Akt/PKB)-dependent phosphorylation of p27Kip1 led to the cytoplasmic mislocalization of p27Kip1, suggesting the potential abrogation of its activity. Here, we evaluated the localization of p27Kip1 protein in leukemic blasts in relation to Akt/PKB phosphorylation and clinical outcomes in acute myelogenous leukemia (AML). Western blot analysis of the nuclear and cytoplasmic fractions revealed a heterogenous localization pattern of p27Kip1 in AML. Cytoplasmic mislocalization of p27Kip1 was significantly associated with the constitutive serine(473) Akt/PKB phosphorylation in AML cells (P < 0.05). Transfection of U937 cells with an expression construct encoding the constitutively active form of Akt/PKB resulted in a remarkable increase in the levels of cytoplasmic p27Kip1. Whereas the transfection of U937 cells with a construct encoding dominant-negative Akt/PKB resulted in a recovery of nuclear localization of p27Kip1. Both the disease-free survival and overall survival are significantly shorter in AML cases with high cytoplasmic to nuclear ratio of p27Kip1 localization compared with the cases with low cytoplasmic to nuclear ratio (P = 0.0353, P = 0.0023, respectively). Multivariate analysis indicated that the cytoplasmic to nuclear ratio of p27Kip1 localization was an independent prognostic variable for both disease-free survival and overall survival (P = 0.043, P = 0.008, respectively). These findings additionally extend our understanding of the role of p27Kip1 in AML, and buttress the case of p27Kip1 mislocalization as a prognostic indicator and Akt/PKB/p27Kip1 pathway as a ready target for antileukemia therapy.

Estrogens Down-regulate p27 Kip1 in Breast Cancer Cells through Skp2 and through Nuclear Export Mediated by the ERK Pathway

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. 17beta-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.

Expression and Subcellular Localization of the Cyclin-Dependent Kinase Inhibitor p27Kip1 in Epithelial Ovarian Cancer

Objective. To determine if p27Kip1 expression was altered in epithelial ovarian cancers as compared to normal ovarian surface epithelial (NOSE) cells and to determine if subcellular localization of p27Kip1 was an important feature.Methods. Thirteen tumor samples (1 Stage IC [early] and 12 Stage III/IV [advanced]) from patients with epithelial ovarian cancer and five NOSE samples were evaluated. Samples were surgically dissected to obtain an enriched population (90%) of cancer cells. The level of p27Kip1 protein expression was determined by Western blot analysis. Actin was used as a loading control, and results were quantified by scanning densitometry using the ratio of the p27Kip1 signal to the actin signal for comparison. To evaluate the subcellular localization of p27Kip1, immunocytochemical staining was performed. Clinical pathological parameters were correlated to nuclear p27Kip1 staining to establish if any association existed.Results. When comparing the expression of p27Kip1 between NOSE and ovarian cancer samples, only 2 of 13 ovarian cancer samples had altered p27Kip1 expression. No correlation was found between the expression level of p27Kip1 on Western blot and clinical pathological correlates. While no correlation between expression level of p27Kip1 and subcellular localization was found, decreased nuclear staining (1+) was associated with shorter survivals using the log-rank test (P < 0.001). More importantly, in all tumor samples examined under the microscope, no nuclear p27Kip1 staining was noted in cells that were undergoing mitosis.Conclusions. p27Kip1 protein degradation may not be modified in ovarian cancer cells undergoing mitosis. Altered expression of p27Kip1 is not an overwhelming feature in certain epithelial ovarian cancers. Decreased nuclear staining of p27Kip1 is associated with poor survival in some epithelial ovarian cancers.

Differentiation-associated expression and intracellular localization of cyclin-dependent kinase inhibitor p27KIP1 and c-Jun co-activator JAB1 in neuroblastoma.

Neuroblastoma is a unique pediatric cancer, which spontaneously regress in some infants and undergo maturation in older children. The cyclin-dependent kinase inhibitor p27KIP1 negatively control cell cycle progression and its expression is reported to be associated with differentiation and prognosis of some human cancers. To examine whether p27KIP1 is involved in differentiation of neuroblastomas, expression and localization of p27KIP1 in 30 cases of neuroblastic tumors were determined with immunohistochemistry. p27KIP1 was expressed in all cases, but staining intensity and intracellular localization varied in association with tumor differentiation. Primitive small round neuroblasts showed negative or only weak nuclear staining, while differentiating tumor cells displayed a novel, intense cytoplasmic positivity besides the nuclear staining, and mature ganglion cells showed intense positive reaction confined to the nucleus. A neuroblastoma cell line TGW was also immunostained positively for p27KIP1 in the cytoplasm after differentiation induction, and western blot analysis revealed an increase of p27KIP1 in these cells, corroborating the in vivo observations. JAB1, which is thought to bind p27KIP1 and transport it from the nucleus to the cytoplasm for proteasome/ubiquitin-mediated degradation, was found to be localized both in the cytoplasm and the nucleus in undifferentiated and differentiating tumors whereas located predominantly in the nucleus of differentiated tumor cells. These data indicate that the cytoplasmic localization of p27KIP1 in the process of differentiation is due to upregulation of p27KIP1 synthesis and subsequent degradation and suggest a role of p27KIP1 in differentiation of neuroblastoma.

Minimal Requirements for the Nuclear Localization of p27Kip1, a Cyclin-Dependent Kinase Inhibitor

p27Kip1 is a cyclin-dependent kinase inhibitor, and its nuclear localization is a prerequisite for it to function as a cell cycle regulator. In the present study, the minimal requirement for the nuclear localization signal (NLS) of p27Kip1 was determined by analyzing the localization of various mutants of p27Kip1 tagged with green fluorescent protein (GFP) in HeLa cells and porcine aortic endothelial cells. Wild-type p27Kip1 exclusively localized into nucleus, while GFP alone localized in both cytosol and nucleus. A comparison of various truncation mutants revealed residues 153–166 to be the minimal region necessary for nuclear localization. However, a fusion of this region to GFP showed cytoplasmic retention in addition to nuclear localization, thus suggesting that some extension flanking this region is required to achieve a full function of NLS. The site-directed mutation of the full-length p27Kip1 therefore showed that four basic residues (K153, R154, K165, R166), especially R166, play a critical role in the nuclear localization of p27Kip1.

The cyclin-dependent kinase inhibitor p27Kip1 is localized to the cytosol in Swiss/3T3 cells.

p27Kip1 plays an important role in cell cycle progression by negatively regulating the activity of cyclin-Cdk complexes. To understand how p27Kip1 functions, the level and subcellular location of p27Kip1 in Swiss/3T3 cells following serum stimulation of quiescent cells was examined. Surprisingly, p27Kip1 was observed exclusively in the cytosol throughout G1 and into early S phase. However, as expected, p27Kip1 in the cytosolic fraction was greatly reduced following serum stimulation and reached very low levels by late G1. The decline in the level of p27Kip1 corresponded in time to an increase in the nuclear level of both Cdk2 and cyclin E. In quiescent 3T3 cells Cdk2 was inactive and co-precipitated with p27Kip1. After serum stimulation, both nuclear and cytosolic Cdk2 was activated and this corresponded to the decline in p27Kip1. Overexpression of p27Kip1 allowed accumulation of the inhibitor in the nucleus but inhibited entry of Cdk2 into the nucleus following serum stimulation. The subcellular localization of p27Kip1 was also examined in a variety of other mammalian cells. In all the cell lines examined the preponderance of p27Kip1 was found in the cytosolic fraction. However, a substantial level of nuclear p27Kip1 was observed for several cell lines. In a primary mixed glial cell culture p27Kip1 was localized to the nucleus. The results suggest that cytosolic p27Kip1 has a functional role in regulating cell cycle progression, possibly through inhibiting transport of cyclin E-Cdk 2 complexes into the nucleus.

P27Kip1 is inactivated in human colorectal cancer by cytoplasmic localization associated with activation of Akt/PKB

p27Kip1 is a nuclear member of the Kip/Cip family of cyclin-dependent kinase inhibitors and is a negative cell cycle regulator that is thought to play a role in tumour suppression. Reduced levels of p27Kip1 are frequent in human cancers and these have been associated with poor prognosis. We have analysed p27Kip1 expression and intracellular localization in 70 human colorectal cancers by western blotting and immunohistochemistry and the results related to Akt expression and clinical pathological parameters. p27Kip1 protein expression, as evaluated by western blotting, was absent or reduced in about 63% of colorectal cancers compared with both peritumoral and normal tissue. Cytoplasmic p27Kip1 was detected, by immunohistochemical analysis, in 30% (21 of 70) of cases indicating that translocation of p27Kip1 protein into the cytoplasm may be responsible for p27Kip1 inactivation. The analysis of phosphorylated Akt by western blotting indicated that it was expressed in 38% (8 of 21) of tumours showing cytoplasmic p27Kip1. Patients whose cancer presented accumulation of cytoplasmic p27Kip1 showed poorer outcomes for cancer-related relapse and survival. These results suggest that cytoplasmic p27Kip1 localization, associated or not with Akt activation, may contribute to colorectal tumorigenesis and metastasis and it may be useful as a negative prognostic factor for the outcome of patients with colorectal cancer.