Phosphorylation Of p27Kip1 Research Articles

Jun activation domain-binding protein 1 negatively regulate p27kip1 in non-Hodgkin’s lymphomas

Objective: Recent evidences suggest that Jun activation domain-binding protein 1(Jab1) specifically interacts with the Cdk inhibitor p27kip1 and induces nuclear export and subsequent degradation of p27kip1. The purpose of this study is to investigate whether Jab1 expression is correlated with p27kip1 level in Non-Hodgkin's Lymphomas(NHLs) and how it influenced the stability of p27kip1, as well as whether Jab1 expression is associated with clinicopathologic variables and prognosis of NHLs.Methods: Immunohistochemical and/or Western blot analysis was done in 116 cases of NHLs and Jurkat cells.Results: Immunohistochemical analysis showed that Jab1 expression was negatively associated with p27kip1 level and significantly associated with unfavorable clinicopathologic variables. Overexpression Jab1 in lymphoma cell lines Jurkat resulted in decreased p27kip1 level and advanced progression from G1 to S phase of the cell cycle. Subcellular fractionation confirmed Jab1 could lead to nuclear export of p27kip1. Phosphorylation of p27kip1 at Ser10 and Thr157 was significantly increased after Jab1 transient transfected, while Thr187 phosphorylation was decreased. Immunoprecipitation demonstrated that Jab1 overexpression facilitated p27kip1 to dissociate from Cdk2 and associated with Cdk4. Finally, Survival analysis revealed that Jab1 overexpression is significantly associated with overall survival (P=0.000). When Jab1 and p27kip1 are combined, patients with Jab1(+)/p27kip1(-) revealed poorer overall survival (P=0.001). Multivariate analysis revealed that Jab1 protein is an independent prognostic indicator for overall survival.Conclusions: These findings suggest that Jab1 protein may contribute to the tumor progression through Jab1-mediated p27kip1 degradation and that control of Jab1 expression is a novel therapeutic target in patients with NHLs.

(1′ S)-Acetoxychavicol acetate and its enantiomer inhibit tumor cells proliferation via different mechanisms

Elucidation of the mechanisms underlying potential anticancer drugs continues and unraveling these mechanisms would not only provide a conceptual framework for drug design but also promote use of natural products for chemotherapy. The biological effects of (1′ S)-acetoxychavicol acetate (( S)-ACA) have been widely investigated. However, in most cases, a natural product or synthetic racemic compound was used in the study. In this study, we prepared ( S)-ACA and its enantiomer ( R)-ACA by a lipase-catalyzed esterification method and sought to determine the mechanisms of action of ( S)-ACA and ( R)-ACA in the growth inhibitory effect in Ehrlich ascites tumor cells (EATC). ( S)-ACA caused an accumulation of tumor cells in the G1 phase of the cell cycle, which was accompanied by a decrease in phosphorylated retinoblastoma protein (Rb), an increase in Rb and a decrease in the phosphorylation of p27 kip1. However, ( R)-ACA caused an accumulation of tumor cells in the G2 phase of the cell cycle, an increase in hyperphosphorylated Rb and an increase in the phosphorylation of p27 kip1. The results obtained in the present study demonstrate for the first time, to the best of our knowledge, that both ( S)-ACA and ( R)-ACA caused the inhibition of tumor cells growth but the inhibition was caused via different mechanisms.

Mechanism of the Anti-Cancer Activity of Zizyphus jujuba in HepG2 Cells

The Zizyphus jujuba fruit has been used as a traditional Chinese medicinal herb and considered to affect various physiological functions in the body for thousands of years. However, its anti-cancer activity and mechanism of action remain to be elucidated. We investigated the anti-cancer activity of Zizyphus jujuba Mill and its underlining mechanisms of action in human hepatoma cells (HepG2) and found that the extract of Z. jujuba decreased the viability of the cells. Further extraction of the initial Z. jujuba extract with organic solvents revealed that the chloroform fraction (CHCl(3)-F) was the most effective. Interestingly, the CHCl(3)-F induced not only apoptosis but also G1 arrest at a low concentration (100 mug/ml) and G2/M arrest at a higher concentration (200 mug/ml) by cell cycle assay. Apoptosis, an increase in intracellular ROS (reactive oxygen species) level, a decline of mitochondrial membrane potential at low Z. jujuba concentrations, and a ROS-independent mitochondrial dysfunction pathway at high concentrations were all observed. CHCl(3)-F-induced G1 arrest in HepG2 cells was associated with an increase in hypohosphorylation of Rb and p27(Kip1), and a decrease of phosphorylated Rb. However, CHCl(3)-F-induced G2/M arrest in HepG2 cells correlated with a decrease of the p27(Kip1) levels and generation of the phosphorylation of p27(Kip1), however the hypohosphorylation of Rb protein remained. Collectively, our findings suggest that the CHCl(3)-F extract of Z. jujuba extract induced a concentration dependent effect on apoptosis and a differential cell cycle arrest in HepG2 cells.

Cyclin D1 Induction of Cellular Migration Requires p27KIP1

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 NH(2)-terminal residues 46 to 90. These mutations of cyclin D1 abrogated physical interaction with p27(KIP1). Cyclin D1(-/-) cells were p27(KIP1) deficient and the defect in migration was rescued by p27(KIP1) reintroduction. Conversely, the cyclin D1 rescue of cyclin D1(-/-) cellular migration was reversed by p27(KIP1) small interfering RNA. Cyclin D1 regulated p27(KIP1) abundance at the posttranslational level, inhibiting the Skp2 promoter, Skp2 abundance, and induced p27(KIP1) phosphorylation at Ser(10). Together, these studies show cyclin D1 promotes mammary epithelial cell migration. p27(KIP1) is required for cyclin D1-mediated cellular migration.

Constitutive activation of Akt contributes to the pathogenesis and survival of mantle cell lymphoma

To determine whether the PI3K/Akt signaling pathway is involved in the pathogenesis of mantle cell lymphoma (MCL), we investigated the phosphorylation status of Akt and multiple downstream targets in primary MCL cases and cell lines. Akt was phosphorylated in 12 of 12 aggressive blastoid MCL variants and in 4 of 4 MCL cell lines. In contrast, phosphorylated Akt was present in only 5 of 16 typical MCL, 3 at comparable levels to the blastoid cases, and 2 at low levels. The presence of p-Akt was accompanied by the phosphorylation of p27(kip1), FRKHL-1, MDM2, Bad, mTOR, and p70S6K. Inhibition of the PI3K/Akt pathway in the MCL cell lines abrogated or reduced the phosphorylation of Akt, p27(kip1), FRKHL-1, MDM2, Bad, mTOR, GSK-3beta, IkappaB, and led to cell-cycle arrest and apoptosis. Six MCL cases (5 with activated Akt and 1 with inactive Akt) and 3 of 4 cell lines showed loss of PTEN expression. PIK3CA mutations were not detected. We conclude that constitutive activation of the PI3K/Akt pathway contributes to the pathogenesis of MCL and preferentially occurs in blastoid variants. One possible mechanism of activation is loss of PTEN expression. These data suggest that PI3K/Akt inhibitors may be effective in the treatment of Akt-activated MCL.

IKKα controls p52/RelB at the skp2 gene promoter to regulate G1- to S-phase progression

The IkappaB-inducing kinase (IKK) is composed of two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma. IKK-regulated signaling pathways are believed to promote the proliferation of normal cells as well as the aberrant proliferation of cancer cells. The molecular mechanisms linking the IKK signaling pathway components to the cell cycle machinery are not entirely understood. To study the function(s) of the catalytic subunits of the IKK complex, we used pancreatic cancer cells, with constitutive IKK activity. We show that the G1 phase of the cell cycle is specifically regulated by the IKKalpha subunit, which regulates the stability of the cyclin-dependent kinase inhibitor p27(Kip1). Increased p27(Kip1) protein levels following the transfection of IKKalpha-specific siRNAs are a result of the downregulation of the F-box protein S-phase kinase-associated protein 2 (skp2). Additionally, we demonstrate that IKKalpha signaling regulates the transcription of the skp2 gene by controlling the composition of a RelB-containing NF-kappaB complex. Together, this work defines a novel IKKalpha-regulated growth pathway involving the p52/RelB-dependent transcriptional regulation of the skp2 gene.

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.

Farnesyltransferase Inhibitor, ABT-100, Is a Potent Liver Cancer Chemopreventive Agent

Treatment of hepatocellular carcinoma raised on cirrhotic liver represents a major endeavor because surgery and chemotherapeutic management fail to improve the clinical course of the disease. Chemoprevention could represent an important means to inhibit the process of hepatocarcinogenesis. Farnesyltransferase inhibitors are a class of drugs blocking the growth of tumor cells with minimal toxicity towards normal cells. In the present study, we investigated the effects of a novel farnesyltransferase inhibitor, ABT-100, on human liver cancer cell lines, HepG2 and Huh7, and on an animal model of hepatocarcinogenesis. ABT-100 inhibited HepG2 and Huh7 cell growth as well as the invading ability of Huh7 on Matrigel. In HepG2 and Huh7 cells, ABT-100 inhibited growth factor-stimulated phosphoinositide 3-kinase and Akt/protein kinase B activity. Furthermore, ABT-100 inhibited Akt-dependent p27(Kip1) phosphorylation and this event was associated with increased levels of p27(Kip1) in the nucleus and reduced activity of the cyclin-dependent kinase 2. Moreover, ABT-100 treatment resulted in a significant reduction in tumor incidence and multiplicity. Taken together, these findings identify a mechanism of ABT-100 function and show the efficacy of ABT-100 as a chemopreventive agent of hepatocellular carcinoma.

Posttranslational modifications of p27kip1 determine its binding specificity to different cyclins and cyclin-dependent kinases in vivo

AbstractUsing 2-dimensional gel electrophoresis (2D-gel) analysis, we show here that cell-cycle entry is associated with a significant increase in p27kip1 phosphorylation in human primary B cells. A similar pattern of increase in p27kip1 phosphorylation was also seen in 2 fast-growing tumor cell lines, Burkitt lymphoma cell line BL40 and breast carcinoma cell line Cal51, where inactive p27kip1 is expressed at high levels. Detailed analysis revealed for the first time that different cyclins and cyclin-dependent kinases (cdk's) interact with distinct posttranslationally modified isoforms of p27kip1 in vivo. Cyclin E but not cyclin A selectively interacts with phosphorylated p27kip1 isoforms, while cyclin D1 and D2 favor unphosphorylated p27kip1 isoforms in vivo. Interestingly, cyclin D3 and cdk4 selectively interact with phosphorylated p27kip1 in BL40 cells. Among all D-type cyclin/cdk4 and cdk6 complexes, cyclin D3/cdk4 is most active in sequestering the inhibitory activity of p27kip1 in vitro in a cyclinE/cdk2 kinase assay. This novel feature of the binding specificity of p27kip1 to cyclins and cdk's in vivo is interpreted in the context of overexpression of cyclin D3 in the presence of high levels of p27kip1 in human B-cell lymphomas with adverse clinical outcome.

Phosphorylation of p27Kip1 at Thr-157 Interferes with Its Association with Importin α during G1 and Prevents Nuclear Re-entry

We have studied mechanisms of Akt-mediated phosphorylation and regulation of cellular localization of p27. Akt phosphorylates Thr-157 in p27 and retains it in the cytosol. In cells arrested in G(1) and then synchronized to enter into S phase, Akt-mediated phosphorylation of Thr-157 p27 occurred in the cytosol during G(1) phase of the cell cycle. Both T157A and S10A p27 mutants localized in the nucleus in all phases of the cell cycle regardless of the expression of active Akt. Thr-157 phosphorylation was undetectable in S10A-p27, suggesting that Ser-10 phosphorylation is required for p27 localization in the cytosol and subsequent phosphorylation at Thr-157. Phosphorylation at Thr-157 interrupted the association of p27 with importin alpha. A T157A-p27 mutant protein exhibited higher association with importin alpha than wild-type-p27. Treatment of transfected and endogenous p27 with alkaline phosphatase rescued its association with importin alpha. Leptomycin B inhibited cytosolic Thr-157 P-p27 staining, implying that CRM1-dependent nuclear export is required for Akt-mediated Thr-157 phosphorylation. Heterokaryon shuttling assays with NIH3T3 (mouse) cells transfected with FLAG-p27 and HeLa (human) cells revealed that both wild type and T157A-p27 shuttled from NIH3T3 to HeLa cell nuclei with similar frequencies. However, S10A-p27 was found only in the NIH3T3 nuclei of NIH3T3-HeLa cell fusions. These results suggest that 1) Ser-10 phosphorylation is required for nuclear export of p27, 2) subsequent Akt-mediated phosphorylation at Thr-157 during G(1) phase corrals p27 in the cytosol, and 3) Thr-157 phosphorylation inhibits the association of p27 with importin alpha thus preventing its re-entry into the nucleus.

Akt/PKB Activation-Induced Cytoplasmic Mislocalization of p27Kip1 Protein Is Mediated by Jab1 Shuttling in Human Myeloid Leukemia Cells.

We previously demonstrated the cytoplasmic mislocalization of p27Kip1 protein was highly correlated with constitutive Akt/PKB phosphorylation and poor prognosis in primary acute myeloid leukemia. However, mechanism involved in the Akt/PKB-mediated nuclear export of p27Kip1 in leukemia cells is not fully understood. Jun activation domain-binding protein 1 (Jab1)/CSN5 is the fifth component of the COP9 signalosome complex, which is involved in modulating signal transduction, ubiquitin-proteasome pathway, and nuclear transport. We examined the effects of Akt/PKB activation on the phosphorylation and localization of p27Kip1 protein in relation to Jab1 shuttling in K562 leukemia cells. Akt/PKB activation did not affect the phosphorylation at threonine 187 residue (T187) of p27Kip1 protein in K562 cells. However, the phosphorylation of p27Kip1 at the serine 10 (S10) and threonine T157 (T157) residue was markedly increased in the constitutively active Akt/PKB cDNA-transfected K562 (Myr-Akt/PKB-K562) cells. In the Myr-Akt/PKB-K562 cells, the S10- and T157-phosphorylated form of p27Kip1 localized almost exclusively to the cytoplasm. Leptomycin-B (LMB) pretreatment of Myr-Akt/PKB-K562 cells led to restoration of S10- and T157-phosphorylated p27Kip1 to the nucleus, indicating that Akt/PKB activation-induced cytoplasmic translocation of S10- and T157-phosphorylated p27Kip1 was mediated by CRM1-dependent pathway. We found that Akt/PKB activation did not alter the total cellular levels of Jab1 protein. However, Jab1 protein was exclusively localized to the cytoplasm in the Myr-Akt/PKB-K562 cells, whereas it was preferentially localized to the nucleus in the dominant negative Akt/PKB cDNA-transfected K562 cells. In Myr-Akt/PKB-K562 cells pretreated with LMB, the localization of the Jab1 was essentially nuclear. Jab1 silencing led to an accumulation of S10- and T157-phosphorylated p27Kip1 to the nucleus in the Myr-Akt/PKB-K562 cells, which was accompanied by a prominent decrease in the cytoplasmic level of p27Kip1. Taken together, we demonstrate that Akt/PKB activation-induced cytoplasmic mislocalization of S10- and T157-phosphorylated p27Kip1 protein is mediated by Jab1 shuttling in human myeloid leukemia cells. Apart from our understanding of the mechanism implicated in the regulation of phosphorylation and localization of p27Kip1 protein in leukemia cells, all these data might suggest possibilities for designing leukemia therapy since it should be possible to restore the function of a misplaced p27Kip1 protein.

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.

Tumor suppressor ARF inhibits HER-2/neu-mediated oncogenic growth.

HER2/neu, a receptor tyrosine kinase oncogene, promotes mitogenic growth and antiapoptotic activity in cancer cells. Strong expression of HER2/neu in cancers has been associated with poor prognosis. Alternative reading frame protein (ARF), a tumor suppressor protein encoded by a gene located in the Ink4a/ARF gene locus, is frequently inactivated in human cancers. Little is known about the tumor suppressor role of ARF in HER2/neu-overexpressing cancers. Here, we applied the ARF gene as a tumor-suppressive agent for HER2/neu-overexpressing cells under the control of a tetracycline (tet)-regulated gene expression system. We found that ARF antagonized protein kinase B (PKB)/Akt-mediated p27Kip1 phosphorylation and increased p27 stability in HER2/neu-overexpressing cells. ARF expression also led to decreased levels of Cul1 and Skp2, two proteins involved in p27 degradation. We also found that ARF caused apoptosis in HER2/neu-overexpressing cells, and sensitized cells to apoptosis induced by the chemotherapeutic agents taxol and 2-methoxyestradiol. Most significantly for clinical application, we found that ARF inhibited HER2/neu-mediated cell growth, transformation, and tumorigenesis. These findings indicate that modulation of ARF activity may be a useful therapeutic intervention in HER2-overexpressing cancers.

Phosphorylation of p27Kip1 at Threonine 198 by p90 Ribosomal Protein S6 Kinases Promotes Its Binding to 14-3-3 and Cytoplasmic Localization

The cyclin-dependent kinase inhibitor p27Kip1 plays an important role in cell cycle regulation. The cyclin-dependent kinase-inhibitory activity of p27Kip1 is regulated by changes in its concentration and its subcellular localization. Several reports suggest that phosphorylation of p27Kip1 at serine 10, threonine 157, and threonine 187 regulate its localization. We have previously identified that carboxyl-terminal threonine 198 (Thr198) in p27Kip1 is a novel phosphorylation site and that Akt is associated with the phosphorylation at the site (Fujita, N., Sato, S., Katayama, K., and Tsuruo, T. (2002) J. Biol. Chem. 277, 28706-28713). We show herein that activation of the Ras/Raf/mitogen-activated protein kinase kinase (MAPK kinase/MEK) pathway also regulates phosphorylation of p27Kip1 at Thr198. MAPKs were not directly associated with p27Kip1 phosphorylation at Thr198, but the p90 ribosomal protein S6 kinases (RSKs) could bind to and directly phosphorylate p27Kip1 at Thr198 in a Ras/Raf/MEK-dependent manner. RSK-dependent phosphorylation promoted the p27Kip1 binding to 14-3-3 and its cytoplasmic localization. To prove the direct relationship between 14-3-3 binding and cytoplasmic localization, we constructed a p27Kip1-R18 fusion protein in which the R18 peptide was fused to the carboxyl-terminal region of p27Kip1. The R18 peptide is known to interact with 14-3-3 independent of phosphorylation. The p27Kip1-R18 distributed mainly in the cytosol, whereas mutant p27Kip1-R18 (p27Kip1-R18-K2) that had no 14-3-3 binding capability existed mainly in the nucleus. These results indicate that RSKs play a crucial role in cell cycle progression through translocation of p27Kip1, in addition to Akt, to the cytoplasm in a phosphorylation and 14-3-3 binding-dependent manner.

P27KIP1 phosphorylation by PKB/Akt leads to poor breast cancer prognosis.

Three papers published concurrently in the October issue of Nature Medicine have demonstrated new links between mitogenic cytokine signalling and the cell cycle inhibitor p27KIP1, and have elucidated how this link may contribute to breast cancer patient prognosis [1-3]. Cell cycle control occurs through cyclin binding to the cyclin-dependent kinases (CDKs) that stimulate G1 progression [4]. p27KIP1 is a member of the kinase inhibitor protein (KIP) family that inhibit cell cycle progression by binding to cyclin-CDK complexes [5]. In cancers where protein kinase B (PKB)/Akt is constitutively activated by upstream oncogenes, Akt decreases p27KIP1 by increasing proteolysis and reducing transcription [6]. These three articles describe a third way in which Akt resists G1 arrest, namely by a p27KIP1 phosphorylation that impairs nuclear import and prevents its inhibition of cyclin-CDK complexes. The authors of these studies conclude that exclusion of p27KIP1 from the nucleus leads to a poor patient outcome in breast cancer [1-3].

Phosphorylation of p27Kip1 on Serine 10 Is Required for Its Binding to CRM1 and Nuclear Export

Phosphorylation of the cyclin-dependent kinase inhibitor p27(Kip1) has been thought to regulate its stability. Ser(10) is the major phosphorylation site of p27(Kip1), and phosphorylation of this residue affects protein stability. Phosphorylation of p27(Kip1) on Ser(10) has now been shown to be required for the binding of CRM1, a carrier protein for nuclear export. The p27(Kip1) protein was translocated from the nucleus to the cytoplasm at the G(0)-G(1) transition of the cell cycle, and this export was inhibited by leptomycin B, a specific inhibitor of CRM1-dependent nuclear export. The nuclear export and subsequent degradation of p27(Kip1) at the G(0)-G(1) transition were observed in cells lacking Skp2, the F-box protein component of an SCF ubiquitin ligase complex, indicating that these early events are independent of Skp2-mediated proteolysis. Substitution of Ser(10) with Ala (S10A) markedly reduced the extent of p27(Kip1) export, whereas substitution of Ser(10) with Asp (S10D) or Glu (S10E) promoted export. Co-immunoprecipitation analysis showed that CRM1 preferentially interacted with S10D and S10E but not with S10A, suggesting that the phosphorylation of p27(Kip1) on Ser(10) is required for its binding to CRM1 and for its subsequent nuclear export.

Cell cycle-independent upregulation of p27Kip1 by p21Waf1 in K562 cells.

Cellular differentiation frequently involves sequential peaks in the expression of cyclin-dependent kinase inhibitors (cdki's). For example, an increase in levels of the cdki p27Kip1 follows upregulation of p21Waf1 in several cell types induced to differentiate by diverse stimuli. In this study, we have investigated whether p21Waf1 expression itself, rather than the differentiating agent, could be increasing p27Kip1 protein levels. We used an inducible p21Waf1 expression vector in a K562 leukemic cell model which we had previously shown to initiate differentiation following p21Waf1 upregulation. The current study reports that p21Waf1 upregulated p27Kip1 protein without altering p27Kip1 mRNA levels. This effect did not depend on G1-phase arrest-the increase in p27Kip1 occurred at all phases of the cell cycle. p21Waf1-expressing extracts inhibited phosphorylation of p27Kip1 on threonine-187, leading to decreased ubiquitination and decreased proteasomal destruction of p27Kip1. In K562 cells, upregulation of p27Kip1 by p21Waf1 during differentiation facilitated an ordered transition between these two cdki's, each of which may distinctly influence the differentiation process.

Gamma-Linolenic acid blocks cell cycle progression by regulating phosphorylation of p27kip1 and p57kip2 and their interactions with other cycle regulators in cancer cells.

gamma-Linolenic acid (gamma-LA), a n-6 essential fatty acid, has been previously shown to affect cell cycle and growth of cancer cells. This study examined the effects of gamma-LA on the cell cycle and cycle regulators in human colon cancer HT115 and breast cancer MCF7 cells. Brief treatment of cancer cells (<2 h) with gamma-LA resulted in a decrease in the phosphorylation of both cell cycle inhibitors, p27kip1 and p57kip2 as shown by immunoprecipitation and Western blotting. Protein levels of both inhibitors were increased following a prolonged culture of cells with the fatty acid. A co-precipitation study showed that in cells treated with gamma-LA there was an increase in the binding of these inhibitors with CDK4, CDC2, and cyclin E. Flow cytometry study indicated an inhibition of cell cycle progression by gamma-LA (G0/G1 -45.4%, S - 34.6%, G2+M - 20.0% in control, and 70.5%, 21.0%, and 8.5%, respectively, in gamma-LA treated cells). It is concluded that gamma-linolenic acid inhibits cell cycle progression in the cancer cell lines investigated, via its regulation of the phosphorylation and subsequent degradation of p27kip1 and p57kip2 and their interactions with other cycle regulators.