ERK phosphorylates p66shcA on Ser36 and subsequently regulates p27kip1 expression via the Akt-FOXO3a pathway: implication of p27kip1 in cell response to oxidative stress

Mice deficient for p66shcA represent an animal model to link oxidative stress and aging. p66shcA is implicated in oxidative stress response and mitogenic signaling. Phosphorylation of p66shcA on Ser36 is critical for its function in oxidative stress response. Here we report the identification of ERK as the kinase phosphorylating p66shcA on Ser36. Activation of ERKs was necessary and sufficient for Ser36 phosphorylation. p66shcA interacted with ERK and was demonstrated to be a substrate for ERK, with Ser36 being the major phosphorylation site. Furthermore, in response to H2O2, inhibition of ERK activation repressed p66shcA-dependent phosphorylation of FOXO3a and the down-regulation of its target gene p27kip1. Down-regulation of p27 might promote cell survival, as p27 played a proapoptotic role in oxidative stress response. As a feedback regulation, Ser36 phosphorylated p66shcA attenuated H2O2-induced ERK activation, whereas p52/46shcA facilitated ERK activation, which required tyrosine phosphorylation of CH1 domain. p66shcA formed a complex with p52/46ShcA, which may provide a platform for efficient signal propagation. Taken together, the data suggest there exists an interplay between ERK and ShcA proteins, which modulates the expression of p27 and cell response to oxidative stress.     

p27~(kip1)及相关分子skp2在肺癌组织中的表达及其临床意义

目的:研究细胞周期素依赖性激酶抑制蛋白27(p27kip1)和细胞S相激酶相关蛋白2(skp2)在肺癌癌组织中的表达及意义。方法:选取于我院就诊的72例肺癌患者的肺癌组织和20例癌旁正常肺组织,采用免疫组化技术检测标本中p27kip1和skp2的表达,并分析其与患者的临床病理之间的关系。结果:skp2在肺癌组织中的表达高于正常肺组织,而p27kip1在肺癌组织中的表达低于正常肺组织,差异均有统计学意义(P0.05),且两者的表达呈负相关关系,相关系数r=-0.855(P0.05),skp2的表达与肺癌组织学类型、分化程度、TNM分期、淋巴结有无转移、吸烟与否及p27kip1蛋白表达有关(P0.05)。结论:p27kip1低表达和skp2高表达可能是肺癌发生发展的重要原因,可应用于临床诊治肺癌患者和判断预后。     

C-terminal phosphorylation controls the stability and function of p27kip1

Entry of cells into the cell division cycle requires the coordinated activation of cyclin-dependent kinases (cdks) and the deactivation of cyclin kinase inhibitors. Degradation of p27kip1 is known to be a central component of this process as it allows controlled activation of cdk2-associated kinase activity. Turnover of p27 at the G1/S transition is regulated through phosphorylation at T187 and subsequent SCF(skp2)-dependent ubiquitylation. However, detailed analysis of this process revealed the existence of additional pathways that regulate the abundance of the protein in early G1 and as cells exit quiescence. Here, we report on a molecular mechanism that regulates p27 stability by phosphorylation at T198. Phosphorylation of p27 at T198 prevents ubiquitin-dependent degradation of free p27. T198 phosphorylation also controls progression through the G1 phase of the cell cycle by regulating the association of p27 with cyclin-cdk complexes. Our results unveil the molecular composition of a pathway, which regulates the abundance and activity of p27kip1 during early G1. They also explain how the T187- and the T198-dependent turnover systems synergize to allow cell cycle progression in G1.     

A p27(kip1)-binding protein, p27RF-Rho, promotes cancer metastasis via activation of RhoA and RhoC

Rho family proteins regulate multiple cellular functions including motility and invasion through regulation of the actin cytoskeleton and gene expression. Activation of Rho proteins is controlled precisely by multiple regulators in a spatiotemporal manner. RhoA and/or RhoC are key players that regulate the metastatic activity of malignant tumor cells, and it is therefore of particular interest to understand how activation of these Rho proteins is controlled. We recently identified an upstream regulator of RhoA activation, p27RF-Rho (p27(kip1) releasing factor from RhoA) that acts by freeing RhoA from inhibition by p27(kip1). p27(kip1) is a cell cycle regulator when it is localized to the nucleus, but it binds RhoA and inhibits activation of the latter when it is localized to the cytoplasm. Here, we show that a metastatic variant of mouse melanoma B16 cells (F10) exhibits greater expression of p27RF-Rho, RhoA, and RhoC than the nonmetastatic parental cells (F0). Injection of F10 cells into mouse tail vein resulted in the formation of metastatic lung colonies, whereas prior knockdown of expression of either one of the three proteins using specific shRNA sequences decreased metastasis markedly. p27RF-Rho regulated the activation of RhoA and RhoC and thereby modulated cellular adhesion and motility, in addition to pericellular proteolysis. The Rho activities enhanced by p27RF-Rho had a marked effect upon efficiency of lodging of F10 cells in the lung, which represents an early step of metastasis. p27RF-Rho also regulated metastasis of human melanoma and fibrosarcoma cells. Thus, p27RF-Rho is a key upstream regulator of RhoA and RhoC that controls spreading of tumor cells.     

PP2A通过Akt/Skp2通路调控p27~(Kip1)的表达并抑制肺癌细胞的致瘤能力

蛋白磷酸酶2A(PP2A)是由36 k Da的催化亚基C(PP2Ac)和65 k Da的结构亚基A(PP2Aα/β)一起组成PP2A的核心酶,并且和各种不同的调节亚基B形成具有不同功能的PP2A全酶复合体。在细胞中PP2A发挥着重要作用,特别是在抑制肿瘤的形成当中,编码PP2Aα/β基因的突变将导致肿瘤的形成和其他疾病。当非小细胞肺癌细胞H1299中过表达PP2A-Aα时,细胞生长被抑制,细胞周期停留在G0/G1期,致瘤能力也同时被抑制。进一步研究证明当PP2A-Aα过表达时,Akt被去磷酸化失活使Skp2的表达下调,从而导致细胞周期抑制因子p27kip1的表达上调。肿瘤细胞软琼脂克隆形成实验的结果表明过表达PP2A-Aα之后H1299细胞的锚定非依赖性生长能力明显的降低,形成的克隆细胞团也较小,这些结果和裸鼠成瘤实验的结果是一致的。     

Roles for p21waf1/cip1 and p27kip1 during the adaptation response to massive intestinal resection

The magnitude of gut adaptation is a decisive factor in determining whether patients are able to live independent of parenteral nutrition after massive small bowel loss. We previously established that the cyclin-dependent kinase inhibitor (CDKI) p21(waf1/cip1) is necessary for enterocyte proliferation and a normal adaptation response. In the present study, we have further elucidated the role of this CDKI in the context of p27(kip1), another member of the Cip/Kip CDKI family. Small bowel resections (SBRs) or sham operations were performed in control (C57/BL6), p21(waf1/cip1)-null, p27(kip1)-null, and p21(waf1/cip1)/p27(kip1) double-null mice. Morphological (villus height/crypt depth) alterations in the mucosa, the kinetics of enterocyte turnover (rates of enterocyte proliferation and apoptosis), and the protein expression of various cell cycle-regulatory proteins were recorded at various postoperative times. Enterocyte compartment-specific mRNA expression was investigated using laser capture microdissection. Resection-induced adaptation in control mice coincided with increased protein expression of p21(waf1/cip1) and decreased p27(kip1) within 3 days postoperatively. Identical changes in mRNA expression were detected in crypt but not in villus enterocytes. Adaptation occurred normally in control and p27(kip1)-null mice; however, mice deficient in both p21(waf1/cip1) and p27(kip1) failed to increase baseline rates of enterocyte proliferation and adaptation. The expression of p21(waf1/cip1) protein and mRNA in the proliferative crypt compartment is necessary for resection-induced enterocyte proliferation and adaptation. The finding that deficient expression of p27(kip1) does not affect adaptation suggests that these similar CDKI family members display distinctive cellular functions during the complex process of intestinal adaptation.     

Eight Functional Polymorphisms in the Estrogen Receptor 1 Gene and Endometrial Cancer Risk: A Meta-Analysis

Background and Objective

Emerging evidence indicates that common functional polymorphisms in the estrogen receptor 1 (ESR1) gene may have an impact on an individual’s susceptibility to endometrial cancer, but individually published results are inconclusive. The aim of this meta-analysis is to derive a more precise estimation of the associations between eight polymorphisms in the ESR1 gene and endometrial cancer risk.

Methods

A literature search of PubMed, Embase, Web of Science and China Biology Medicine (CBM) databases was conducted on publications published before November 1^st, 2012. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Statistical analyses were performed using the STATA 12.0 software.

Results

Thirteen case-control studies were included with a total of 7,649 endometrial cancer cases and 16,855 healthy controls. When all the eligible studies were pooled into the meta-analysis, the results indicated that PvuII (C>T) polymorphism was associated with an increased risk of endometrial cancer, especially among Caucasian populations. There were also significant associations between rs3020314 (C>T) polymorphism and an increased risk of endometrial cancer. Furthermore, rs2234670 (S/L) polymorphism may decrease the risk of endometrial cancer. However, no statistically significant associations were found in XbaI (A>G), Codon 325 (C>G), Codon 243 (C>T), VNTR (S/L) and rs2046210 (G>A) polymorphisms.

Conclusion

The current meta-analysis suggests that PvuII (C>T) and rs3020314 (C>T) polymorphisms may be risk factors for endometrial cancer, especially among Caucasian populations.     

Regulated expression of cofilin and the consequent regulation of p27kip1 are essential for G1 phase progression

Cofilin, a ubiquitously expressed actin binding protein, is responsible for the formation of the actin cytoskeleton and is indispensable for cell cycle control. However, the association between cofilin expression and the cell cycle remains to be elucidated. In this study, we found that the expression level of cofilin up-regulated in G1 phase-arrested confluent cells, while knockdown of cofilin expression by small interference RNA (siRNA) in these cells led to a reduction in the population of G1 cells. To investigate the role of cofilin in the control of G1 phase progression, a tet-on gene expression system was introduced to over-express different concentrations of cofilin in cells. The results showed that G1 phase progression was blocked following induction of exogenous cofilin. A survey of the cell cycle proteins controlling the G1 phase progression revealed that the cyclin-dependent kinase inhibitor (CKI) p27kip1 was the primary molecule induced by over-expressed cofilin in a time and dose dependent manner. Up-regulated p27kip1 repressed phosphorylation of the retinoblastoma protein (Rb) mediated by cyclin D1/CDK4 activity. Conversely, siRNA against p27kip1 expression in the cofilin over-expressing cells released the G1 phase arrest. Furthermore, we found that over-expression of cofilin led to induction of p27kip1 gene promoter transactivation using luciferase reporter gene assay. This effect was associated with increase of p27kip1 mRNA transiently. In addition, inhibition of threonine-187 phosphorylation of p27kip1 protein for ubiquitinyl-proteasomal mediated degradation was also involved in up-regulation of p27 kip1. These data suggest that cofilin expression and its regulation of p27kip1 expression is important for the control of G1 phase progression.     

Glycogen Synthase Kinase - 3 Phosphorylates and Regulates the Stability of p27kip1 Protein

p27 Kip1 is a critical regulator of the eukaryotic cell cycle. It acts as a check point proteinand regulates cell cycle progression at the G1 and G1/S phase as well as predominantlyblocks cell cycle progression in the absence of growth factors. Intracellular turnover of p27is tightly regulated at the level of translation as well as by posttranslational modification.The mechanism by which p27 protein is rapidly degraded during the G1 and G1/S phasetransition is well characterized. However, the process by which p27 remains extremelystable in the absence of growth factors remains unknown. Here, we report that GSK-3dependent phosphorylation of p27 protein is essential for its enhanced stability. p27 proteinharbours 2 functional GSK-3 phosphorylation sites at the C- terminus, which was found tobe effectively phosphorylated by the cognate enzyme both in vitro and in vivo. Combinedwith earlier observation which shows that it phosphorylates and triggers cyclin Ddegradation; GSK-3 now appears to be a central mediator of the cell-cycle regulatorynetwork, where it acts as a two-way switch, phosphorylating and targeting pro-proliferativefactors for degradation on one hand and simultaneously phosphorylating and stabilizing ananti-proliferative factor on the other hand. This dual mode of activity may doubly ensurethat cell cycle progression is aptly prohibited under conditions of limited growth factoravailability.     

Temporal-spatial expressions of p27kip1 and its phosphorylation on Serine-10 after acute spinal cord injury in adult rat: Implications for post-traumatic glial proliferation

P27kip1, as a member of Cip/Kip family of cyclin-dependent kinase inhibitors, plays important roles in cell cycle regulation and neurogenesis in the developing central nervous system. Serine-10 is the major phosphorylation site of p27kip1, and post-translational regulation of p27kip1 by different phosphorylation events is critical for its function. To elucidate the expressions and possible functions of p27kip1 and its phosphorylation in central nervous system lesion and repair, we performed an acute spinal cord contusion injury model in adult rats. Our work studied the temporal-spatial expression patterns of p27kip1 and Serine-10 phosphorylated p27kip1 (p-p27s10). Western blot analysis showed p27kip1 level significantly decreased at day 3 after damage, while p-p27s10 was detected at a high-level at the same time reaching the uninjured level. Moreover, immunofluorescence double labeling suggested these changes were striking in microglia and astrocytes, which were largely proliferated. Immunohistochemical analysis revealed subcellular localization changes of p27kip1 and p-p27s10 staining between nucleus and cytoplasm after injury in about 20% of total positive cells including neurons and glial cells. We also investigated the increased interactions of p27kip1 and p-p27s10 with CRM1 3 days after injury by co-immunoprecipitation studies. Taken together, we hypothesized spinal cord injury stimulated mitogenic signals to induce a serine-threonine kinase KIS (kinase interacting stathmin) to phosphorylate p27kip1 on Serine-10, so that p27kip1 could bind to CRM1 and be exported from nuclei for degradation. Such an event facilitated cell cycle progression of glial cells, especially microglia and astrocytes which had a prevalent proliferation.     

p27Kip1 and Cyclin Dependent Kinase 2 Regulate Passage Through the Restriction Point

When quiescent cells are stimulated to re-enter the cell cycle, growth factors are required only until the restriction point in G1 phase. After this point the cell no longer requires growth factors, proliferative signaling molecules, or even protein synthesis in order to initiate DNA synthesis, which starts several hours later. Consequently, understanding the molecular nature of the restriction point constitutes one of the major goals in studies of growth regulation. We recently demonstrated that p27Kip1 (p27) regulates passage through G1 phase in actively proliferating cultures, and initiated these studies to determine if it is also involved in passage through the restriction point following stimulation of quiescent cells. In support of this suggestion, we found that passage through the restriction point requires mitogen-dependent suppression of the high p27 levels normally present in quiescent cells. Moreover, as the culture progresses to mid-G1 phase, the proportion of cells that pass the restriction point is increased by artificial suppression of p27 levels, while this proportion is reduced by elevation of p27 levels. p27 performs this critical function by regulating the subsequent activating phosphorylation of cyclin dependent kinase (CDK)2, which we also show is necessary for and closely associated with the initiation of DNA synthesis. We conclude that the p27 expression level at mid-G1 phase determines when a cell passes through the restriction point, and does so by regulating subsequent CDK2 activation.     

The COP9 signalosome inhibits p27(kip1) degradation and impedes G1-S phase progression via deneddylation of SCF Cul1

The COP9 signalosome (CSN) is a conserved protein complex with homologies to the lid subcomplex of the 26S proteasome. It promotes cleavage of the Nedd8 conjugate (deneddylation) from the cullin component of SCF ubiquitin ligases. We provide evidence that cullin neddylation and deneddylation is highly dynamic, that its equilibrium can be effectively modulated by CSN, and that neddylation allows Cul1 to form larger protein complexes. CSN2 integrates into the CSN complex via its C-terminal region and its N-terminal half region is necessary for direct interaction with Cul1. The polyclonal antibodies against CSN2 but not other CSN subunits cause accumulation of neddylated Cul1/Cul2 in HeLa cell extract, indicating that CSN2 is essential in cullin deneddylation. Further, CSN inhibits ubiquitination and degradation of the cyclin-dependent kinase inhibitor p27(kip1) in vitro. Microinjection of the CSN complex impeded the G1 cells from entering the S phase. Moreover, anti-CSN2 antibodies negate the CSN-dependent p27 stabilization and the G1/S blockage, suggesting that these functions require the deneddylation activity. We conclude that CSN inhibits SCF ubiquitin ligase activity in targeting p27 proteolysis and negatively regulates cell cycle at the G1 phase by promoting deneddylation of Cul1.     

Opposing roles for the cyclin-dependent kinase inhibitor p27kip1 in the control of CD4+ T cell proliferation and effector function

Cell division drives T cell clonal expansion and differentiation, and is the result of concerted signaling from Ag, costimulatory, and growth factor receptors. How these mitogenic signals are coupled to the cell cycle machinery in primary T cells is not clear. We have focused on the role of p27kip1, a major cyclin-dependent kinase binding protein expressed by CD4+ T cells. Our studies using p27kip1 gene dosage demonstrate that early after activation, p27kip1 acts to promote, rather than inhibit, G1 to S phase progression within the first division cycle. However, throughout subsequent cell divisions p27kip1 behaves as a negative regulator, directly establishing the threshold amount of growth factor signaling required to support continued cell division. During this phase, signals from CD28 and IL-2R cooperate with the TCR to "tune" this threshold by inducing the degradation of p27kip1 protein, and we show that agents that block these pathways require elevated p27kip1 levels for their full antiproliferative activity. Finally, we show that p27kip1 opposes the development of CD4+ T cell effector function, and is required for the full development of anergy in response to a tolerizing stimulus. Our results suggest that p27kip1 plays a complex and important role in the regulation of cell division and effector function in primary CD4+ T cells.     

Cell-Free Degradation of p27kip1, a G1 Cyclin-Dependent Kinase Inhibitor, Is Dependent on CDK2 Activity and the Proteasome

Entry into S phase is dependent on the coordinated activation of CDK4,6 and CDK2 kinases. Once a cell commits to S phase, there must be a mechanism to ensure the irreversibility of this decision. The activity of these kinases is inhibited by their association with p27. In many cells, p27 plays a major role in the withdrawal from the cell cycle in response to environmental cues. Thus, it is likely that p27 is a target of the machinery required to ensure the irreversibility of S-phase entry. We have been interested in understanding the mechanisms regulating p27 at the G₁/S transition. In this report, we define a cell-free degradation system which faithfully recapitulates the cell cycle phase-specific degradation of p27. We show that this reaction is dependent on active CDK2 activity, suggesting that CDK2 activity is directly required for p27 degradation. In addition to CDK2, other S-phase-specific factors are required for p27 degradation. At least some of these factors are ubiquitin and proteasome dependent. We discuss the relationships between CDK2 activity, ubiquitin-dependent, and possibly ubiquitin-independent proteasomal activities in S-phase extracts as related to p27.