Calcineurin regulates cyclin D1 accumulation in growth-stimulated fibroblasts

Calcium (Ca(2+)) and calmodulin (CaM) are required for progression of mammalian cells from quiescence into S phase. In multiple cell types, cyclosporin A causes a G(1) cell cycle arrest, implicating the serine/threonine phosphatase calcineurin as one Ca(2+)/CaM-dependent enzyme required for G(1) transit. Here, we show, in diploid human fibroblasts, that cyclosporin A arrested cells in G(1) before cyclin D/cdk4 complex activation and retinoblastoma hyperphosphorylation. This arrest occurred in early G(1) with low levels of cyclin D1 protein. Because cyclin D1 mRNA was induced normally in the cyclosporin A-treated cells, we analyzed the half-life of cyclin D1 in the presence of cyclosporin A and found no difference from control cells. However, cyclosporin A treatment dramatically reduced cyclin D1 protein synthesis. Although these pharmacological experiments suggested that calcineurin regulates cyclin D1 synthesis, we evaluated the effects of overexpression of activated calcineurin on cyclin D1 synthesis. In contrast to the reduction of cyclin D1 with cyclosporin A, ectopic expression of calcium/calmodulin-independent calcineurin promoted synthesis of cyclin D1 during G(1) progression. Therefore, calcineurin is a Ca(2+)/CaM-dependent target that regulates cyclin D1 accumulation in G(1).     

Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport

We have used immunofluorescence staining to study the subcellular distribution of cyclin A and B1 during the somatic cell cycle. In both primary human fibroblasts and in epithelial tumor cells, we find that cyclin A is predominantly nuclear from S phase onwards. Cyclin A may associated with condensing chromosomes in prophase, but is not associated with condensed chromosomes in metaphase. By contrast, cyclin B1 accumulates in the cytoplasm of interphase cells and only enters the nucleus at the beginning of mitosis, before nuclear lamina breakdown. In mitotic cells, cyclin B1 associates with condensed chromosomes in prophase and metaphase, and with the mitotic apparatus. Cyclin A is degraded during metaphase and cyclin B1 is precipitously destroyed at the metaphase----anaphase transition. Cell fractionation and immunoprecipitation studies showed that both cyclin A and cyclin B1 are associated with PSTAIRE-containing proteins. The nuclear, but not the cytoplasmic form, of cyclin A is associated with a 33-kD PSTAIRE-containing protein. Cyclin B1 is associated with p34cdc2 in the cytoplasm. Thus we propose that the different localization of cyclin A and cyclin B1 in the cell cycle could be the means by which the two types of mitotic cyclin confer substrate specificity upon their associated PSTAIRE-containing protein kinase subunit.     

A novel inhibitor of cyclin-Cdk activity detected in transforming growth factor beta-arrested epithelial cells.

Transforming growth factor beta (TGF-beta) is a potent inhibitor of epithelial cell growth. Cyclins E and A in association with Cdk2 have been shown to play a role in the G1-to-S phase transition in mammalian cells. We have studied the effects of TGF-beta-mediated growth arrest on G1/S cyclins E and A. Inhibition of cyclin A-associated kinase by TGF-beta is primarily due to a decrease in cyclin A mRNA and protein. By contrast, while TGF-beta inhibits accumulation of cyclin E mRNA, the reduction in cyclin E protein is minimal. Instead, we find that the activation of cyclin E-associated kinase that normally accompanies the G1-to-S phase transition is inhibited. A novel inhibitor of cyclin-Cdk complexes was detected in TGF-beta-treated cell lysates. Inhibition is mediated by a heat-stable protein that targets both Cdk2 and Cdc2 kinases. In G0-arrested cells, a similar inhibitor of Cdk2 kinase was detected. These data suggest the existence of an inhibitor of cyclin-dependent kinases induced under different conditions to mediate antiproliferative responses.     

Characterization of cyclin L2, a novel cyclin with an arginine/serine-rich domain: phosphorylation by DYRK1A and colocalization with splicing factors

A novel method employing filter arrays of a cDNA expression library for the identification of substrates for protein kinases was developed. With this technique, we identified a new member of the cyclin family, cyclin L2, as a substrate of the nuclear protein kinase DYRK1A. Cyclin L2 contains an N-terminal cyclin domain and a C-terminal arginine/serine-rich domain (RS domain), which is a hallmark of many proteins involved in pre-mRNA processing. The gene for cyclin L2 encodes the full-length cyclin L2, which is predominantly expressed in testis, as well as a truncated splicing variant (cyclin L2S) that lacks the RS domain and is ubiquitously expressed in human tissues. Full-length cyclin L2, but not cyclin L2S, was associated with the cyclin-dependent kinase PITSLRE. Cyclin L2 interacted with splicing factor 2 in vitro and was co-localized with the splicing factor SC35 in the nuclear speckle compartment. Photobleaching experiments showed that a fusion protein of green fluorescent protein and cyclin L2 in nuclear speckles rapidly exchanged with unbleached molecules in the nucleus, similar to other RS domain-containing proteins. In striking contrast, the closely related green fluorescent protein-cyclin L1 was immobile in the speckle compartment. DYRK1A interacted with cyclin L2 in pull-down assays, and overexpression of DYRK1A stimulated phosphorylation of cyclin L2 in COS-7 cells. These data characterize cyclin L2 as a highly mobile component of nuclear speckles and suggest that DYRK1A may regulate splicing by phosphorylation of cyclin L2.     

Fbxw8 is involved in the proliferation of human choriocarcinoma JEG-3 cells

Fbxw8 is the F-box component of a SCF-like E3 ubiquitin ligase complex. Mice lacking Fbxw8 exhibit pathological defects in placenta and embryo similar to fetal growth retardation, suggesting a role of Fbxw8 in placentation. Proliferative capacity of trophoblast cells is very important in placental development. In this context, we revealed that Fbxw8 was expressed in four different human trophoblast cell lines. Silencing of Fbxw8 expression by siRNA inhibited the growth of choriocarcinoma JEG-3 cells. By Western blotting, cell cycle analysis, we showed that down-regulation of Fbxw8 by RNAi induced cell-growth arrest at G2/M phase through decreasing the levels of CDK1, CDK2, cyclin A and cyclin B1 and up-regulation of p27 at protein level. Conversely, over-expression of Fbxw8 led to the opposite effect. These results suggest that Fbxw8 plays an essential role in the proliferation of human trophoblast cells, especially JEG-3 cells, via G2/M phase transition in association with regulation of CDK1, CDK2, cyclin A, cyclin B1 and p27 expression.     

Production of the kringle fragments of human apolipoprotein(a) by continuous lactose induction strategy

A novel lactose induction strategy for the production of rhLK68, the kringle fragments of human apolipoprotein(a) (apo(a)) as a novel anti-angiogenic protein, was investigated. A scale-up of the production was accompanied by a decrease in expression level, and severe aggregation occurred during the solubilization of rhLK68 from the inclusion body during a conventional single introduction of lactose. To overcome this problem, a continuous induction strategy was applied where lactose was mixed with glycerol and fed continuously in a dissolved oxygen (DO)-stat manner. With the sub-optimal feed medium consisted of 1:50 of lactose/glycerol (w/w), the expression level reached 16% of the total cellular protein, which was 1.6-fold higher than that obtained from the conventional lactose induction. Moreover, the solubilization yield of rhLK68 from the inclusion body increased from 30 +/- 5 to 85 +/- 3% compared to the conventional single introduction of lactose. This result suggests that the continuous lactose induction strategy beneficially influenced the expression level of rhLK68 and the quality of its inclusion body.     

Improved Recovery of a Fusion Protein Containing the Antigenic Domain 1 of the Human Cytomegalovirus Glycoprotein B

Heterologous expression in Escherichia coli often leads to production of the expressed proteins as insoluble and inactive inclusion bodies. The general strategy for protein recovery includes isolation and washing of inclusion bodies, solubilization of aggregated protein and refolding of solubilized protein. The process of refolding, as well as the other steps involved in inclusion body recovery, must be optimized according to the characteristics of each protein. For the development of reliable and inexpensive serodiagnostic tests, the antigenic domain 1 (AD-1) of human cytomegalovirus glycoprotein B was expressed in E. coli and a process was developed to increase recovery of the fusion protein containing AD-1. A comparison of disruption methods and different conditions involved in recovery of this fusion protein from inclusion bodies is presented. The developed method gives a high yield of the fusion protein with a purity sufficient for use in diagnostic tests.     

On the concentrations of cyclins and cyclin-dependent kinases in extracts of cultured human cells

Cyclins and cyclin-dependent kinases (CDKs) are key regulators of the human cell cycle. Here we have directly measured the concentrations of the G(1) and G(2) cyclins and their CDK partners in highly synchronized human cervical carcinoma cells (HeLa). To determine the exact concentrations of cyclins and CDKs in the cell extracts, we developed a relatively simple method that combined the use of (35)S-labeled standards produced in rabbit reticulocyte lysates and immunoblotting with specific antibodies. Using this approach, we formally demonstrated that CDC2 and CDK2 are in excess of their cyclin partners. We found that the concentrations of cyclin A2 and cyclin B1 (at their peak levels in the G(2) phase) were about 30-fold less than that of their partner CDC2. The peak levels of cyclin A2 and cyclin E1, at the G(2) phase and G(1) phase, respectively, were only about 8-fold less than that of their partner CDK2. These ratios are in good agreement with size fractionation analysis of the relative amount of monomeric and complexed forms of CDC2 and CDK2 in the cell. All the cyclin A2 and cyclin E1 are in complexes with CDC2 and CDK2, but there are some indications that a significant portion of cyclin B1 may not be in complex with CDC2. Furthermore, we also demonstrated that the concentration of the CDK inhibitor p21(CIP1/WAF1) induced after DNA damage is sufficient to overcome the cyclin-CDK2 complexes in MCF-7 cells. These direct quantitations formally confirmed the long-held presumption that CDKs are in excess of the cyclins in the cell. Moreover, similar approaches can be used to measure the concentration of any protein in cell-free extracts.     

Human p55(CDC)/Cdc20 associates with cyclin A and is phosphorylated by the cyclin A-Cdk2 complex

The initiation of anaphase and exit from mitosis depend on the activation of the anaphase-promoting complex/cyclosome (APC/C), a multicomponent, ubiquitin-protein ligase. The WD-repeat protein called p55(CDC)(Cdc20) directly binds to and activates APC/C. By using yeast two-hybrid screening, we found that cyclin A, a critical cell cycle regulator in the S and G2/M phases, specifically interacts with p55(CDC). Ectopically expressed p55(CDC) and cyclin A form a stable protein complex in mammalian cells. The p55(CDC)-cyclin A interaction occurs through the region containing the WD repeats of p55(CDC) and the region between the destruction box and the cyclin box of cyclin A. In addition to the physical interaction, p55(CDC) is phosphorylated by cyclin A-associated kinase. These findings suggest that the function of p55(CDC) is mediated or regulated by its complex formation with cyclin A.     

The differential localization of human cyclins A and B is due to a cytoplasmic retention signal in cyclin B.

We have shown previously that human cyclins A and B1 are localized differentially in the cell during interphase; cyclin A is nuclear and cyclin B1 is a cytoplasmic protein. To understand the basis of this difference we created deletion mutants and various chimeras between the two types of cyclin and expressed them in tissue culture cells by transient transfection. We find that the N-terminus of cyclin B1 contains a 42 amino acid region that is sufficient to retain the normally nuclear cyclin A in the cytoplasm. Conversely, deleting the cytoplasmic retention signal region from cyclin B1 causes the protein to become nuclear. Although the cytoplasmic retention signal region is outside the cyclin box, its sequence is well conserved in human cyclin B2, and is both necessary and sufficient to keep cyclin B2 in the cytoplasm. Thus we propose that the subcellular distribution of the B-type cyclins is determined primarily by a small region of the N-terminus which targets the cyclin--CDK complexes to particular structures in the cytoplasm.     

Role of the human and murine cyclin T proteins in regulating HIV-1 tat-activation.

Human cyclin T1 markedly stimulates tat-activation in rodent cells which are normally poorly responsive to the effects of Tat. This result suggests that there are likely to be critical differences in the murine and human cyclin T1 proteins. Here, we analyzed the role of the murine and human cyclin T1 proteins in addition to the human cyclin T2a and T2b proteins on regulating tat-activation. Only the human cyclin T1 protein efficiently formed a complex with Tat bound to TAR RNA. This difference in function was due to the presence of a cysteine residue in human cyclin T1 at position 261 rather than a tyrosine or asparagine residue which are found in the murine cyclin T1 protein and the human cyclin T2a and T2b proteins, respectively. A mouse cyclin T1 protein containing a substitution of tyrosine residue 261 with a cysteine residue, was able to interact with Tat and stimulate tat-transactivation in rodent cells. Likewise, substitution of a cysteine residue for an asparagine residue at position 260 of the cyclin T2a and T2b proteins also resulted in their ability to interact with Tat and stimulate tat-activation in rodent cells. The data indicate that a specific residue in the cyclin T proteins is required for their in vitro interaction with Tat and their ability to stimulate in vivo tat-activation.     

融合干扰素-BLA（IFN-BLA）的构建、表达、纯化及抗病毒活性测定

干扰素-BLA(IFN-BLA)由干扰素-beta-1b(IFN-beta-1b)和干扰素-alpha-2b（IFN-alpha-2b）通过连接肽-GGGS-融合而成。优化了其在大肠感菌BL21 CodonPlus (DE3)-RIL中的实验室表达条件,表达的目的蛋白占菌体总蛋白的35％以上并且主要以包涵体的形式存在。对包涵体的复性条件进行了摸索,建立了IFN-BLA的复性及纯化方法,纯化后的蛋白产量约为45 mg/L, 纯度在90％以上。抗病毒活性分析表明这一新的融合蛋白可能具有协同或加成活性。     

The C-terminal regulatory domain of p53 contains a functional docking site for cyclin A

Radiation injury to cells enhances C-terminal phosphorylation of p53 at both Ser315 and Ser392 in vivo, suggesting the existence of two cooperating DNA damage-responsive pathways that play a role in stimulating p53-dependent gene expression. Our previous data has shown that cyclin A-cdk2 is the major enzyme responsible for modifying p53 at Ser315 in vivo after irradiation damage and in this report we dissect the mechanism of cyclinA-cdk2 binding to and phosphorylation of p53. Although cyclin B(1)-dependent protein kinases can phosphorylate small peptides containing the Ser315 site, cyclin A-cdk2 does not phosphorylate such small peptides suggesting that additional determinants are required for cyclin A-cdk2 interaction with p53. Peptide competition studies have localized a cyclin A interaction site to a Lys381Lys382Leu383Met384Phe385 sequence within C-terminal negative regulatory domain of human p53. An alanine mutation at any one of four key positions abrogates the efficacy of a synthetic peptide containing this motif as an inhibitor of cyclin A-cdk2 phosphorylation of p53 protein. Single amino acid mutations of full-length p53 protein at Lys382, Leu383, or Phe385 decreases cyclin A-cdk2 dependent phosphorylation at Ser315. Cyclin B(1)-cdk2 complexes are not inhibited by KKLMF motif-containing peptides nor is p53 phosphorylation by cyclin B-cdk2 reduced by mutation of the cyclin A interaction site. These data identifying a KKLMF cyclin A docking site on p53 protein highlight a common cyclin A interaction motif that is shared between the tumour suppressor proteins pRb and p53.     

提高成纤维细胞生长因子-21产率和纯度

成纤维细胞生长因子-21(FGF-21)作为近期发现的新型代谢调节因子,因其具有独立于胰岛素调节糖脂代谢、增加胰岛素敏感性等作用,有望成为治疗糖尿病的新型药物。包涵体形式表达外源蛋白表达量及纯度高,但是以pET载体表达时,FGF-21以包涵体形式表达,且复性率及产率低,蛋白活性降低[1]。针对这一瓶颈问题,用SUMO载体首次以包涵体形式表达带有SUMO标签的hFGF-21,通过优化培养条件,并应用中空纤维柱膜过滤技术对菌体进行富集,对包涵体进行洗涤、变性及复性,经过亲和层析、凝胶过滤层析的纯化方法,得到了成熟的hFGF-21,在保证蛋白活性的同时增加了蛋白的产量及纯度。通过检测HepG2细胞葡萄糖吸收及2型糖尿病db/db小鼠短期及长期血糖变化鉴定其降糖生物学活性。结果表明,以包涵体形式表达hFGF-21(ihFGF-21)的表达量是可溶形式表达的hFGF-21(shFGF-21)的3倍,最终ihFGF-21的收率为20 mg/L,而shFGF-21的收率仅为6 mg/L。ihFGF-21的纯度可达到95%以上,而shFGF-21仅能达到90%左右;在细胞水平和动物水平上两者的降糖生物学活性一致。在保证hFGF-21生物学活性的前提下,与传统包涵体途径提取目的蛋白的方法相比,应用中空纤维柱膜过滤技术使hFGF-21的生产周期缩短了约1/3左右。综上所述,此法为FGF-21中试及工业化生产提供了高效、经济的策略。     

The mitogenic signaling pathway for fibroblast growth factor-2 involves the tyrosine phosphorylation of cyclin D2 in MCF-7 human breast cancer cells

Fibroblast growth factor-2 (FGF-2) is mitogenic for the human breast cancer cell line MCF-7; here we investigate some of the signaling pathways subserving this activity. FGF-2 stimulation of MCF-7 cells resulted in a global increase of intracellular tyrosine phosphorylation of proteins, particularly FGF receptor substrate-2, the protooncogene product Src and the mitogen-activated protein kinase (MAP kinase) cascade. A major increase in the tyrosine phosphorylation of a 30-kDa protein species was also found. This protein was identified as cyclin D2 by mass spectrometry after trypsin digestion. Immunoprecipitation of cyclin D2 and immunoblotting with anti-phosphotyrosine antibodies confirmed that the tyrosine phosphorylation of cyclin D2 was indeed induced by FGF-2 stimulation. In addition, pharmacological inhibition of Src (with herbimycin A and PP2), and of the MAP kinase cascade (with PD98059), confirmed that Src activity is required for the FGF-2-induced phosphorylation of cyclin D2 whereas MAP kinase activity is not. Thus, tyrosine phosphorylation of cyclin D2 may be a key regulatory target for FGF-2 signaling.     

Effect of elevated levels of ornithine decarboxylase on cell cycle progression in skin.

By crossing TG.AC v-Ha-ras and K6/ODC transgenic mice, we found previously that an activated ras and follicular ornithine decarboxylase (ODC) overexpression cooperate to generate spontaneous tumors in the skin. Cellular proliferation was dramatically increased in the K6/ODC transgenic skin, as evidenced by elevated proliferating cell nuclear antigen and Ki67 expression compared with nontransgenic littermates. Keratinocytes isolated from transgenic skin also displayed increased clonal growth. Paradoxically, expression of the growth inhibition-associated proteins p53, p21Waf1, p27Klp1, and Bax was increased with ODC overexpression in the skin. ODC overexpression did not affect cyclin D/cyclin-dependent kinase 4 (Cdk4)-dependent phosphorylation of retinoblastoma protein but stimulated cyclin E/Cdk2 and cyclin A/Cdk2-associated kinase activity, with minimal effect on the levels of these proteins. Thus, ODC/polyamine-induced activation of cyclin E/Cdk2 and cyclin A/Cdk2-associated kinase activity may cooperate with the ras induction of cyclin D/Cdk4/6-associated retinoblastoma protein phosphorylation to not only stimulate proliferation but ultimately contribute to tumor development.