蛙皮素对人胃黏膜上皮永生细胞系cyclin D1/CDK4的影响

目的 研究蛙皮素对正常胃黏膜上皮细胞的促生长作用及其与细胞调控因子之间的关系。方法 应用人胃黏膜上皮永生细胞株GES－1,经蛙皮素及受体拮抗剂不同处理后,以MTT为指标研究对细胞增殖的影响;流式细胞仪观察对细胞cyclin D1表达的影响;利用Western印迹和增强化学发光法观察CDK4蛋白表达的变化;利用Sepharose微珠免疫沉淀方法检测对CDK4活性的影响。结果 蛙皮素在浓度为10^-7mol/L时对GES－1细胞有明显的促生长作用,对cyclin D1表达有促进作用;蛙皮素受体拮抗剂均抑制这些作用;同时还发现蛙皮素可使人胃黏膜上皮永生细胞系细胞的CDK4蛋白表达及其激酶活性增加。结论 蛙皮素促进人胃黏膜上皮细胞生长是通过使细胞周期调控因子cyclin D1和CDK4蛋白表达升高,CDK4激酶活性增加,促进细胞周期的进展而实现的。     

Critical role of cyclin D1 nuclear import in cardiomyocyte proliferation

Mammalian cardiomyocytes irreversibly lose their capacity to proliferate soon after birth, yet the underlying mechanisms have been unclear. Cyclin D1 and its partner, cyclin-dependent kinase 4 (CDK4), are important for promoting the G1-to-S phase progression via phosphorylation of the retinoblastoma (Rb) protein. Mitogenic stimulation induces hypertrophic cell growth and upregulates expression of cyclin D1 in postmitotic cardiomyocytes. In the present study, we show that, in neonatal rat cardiomyocytes, D-type cyclins and CDK4 were predominantly cytoplasmic, whereas Rb remained in an underphosphorylated state. Ectopically expressed cyclin D1 localized in the nucleus of fetal but not neonatal cardiomyocytes. To target cyclin D1 to the nucleus efficiently, we constructed a variant of cyclin D1 (D1NLS), which directly linked to nuclear localization signals (NLSs). Coinfection of recombinant adenoviruses expressing D1NLS and CDK4 induced Rb phosphorylation and CDK2 kinase activity. Furthermore, D1NLS/CDK4 was sufficient to promote the reentry into the cell cycle, leading to cell division. The number of cardiomyocytes coinfected with these viruses increased 3-fold 5 days after infection. Finally, D1NLS/CDK4 promoted cell cycle reentry of cardiomyocytes in adult hearts injected with these viruses, evaluated by the expression of Ki-67, which is expressed in proliferating cells in all phases of the cell cycle, and BrdU incorporation. Thus, postmitotic cardiomyocytes have the potential to proliferate provided that cyclin D1/CDK4 accumulate in the nucleus, and the prevention of their nuclear import plays a critical role as a physical barrier to prevent cardiomyocyte proliferation. Our results provide new insights into the development of therapeutics strategies to induce regeneration of cardiomyocytes. The full text of this article is available at http://www.circresaha.org.     

Nitric oxide-induced cytostasis and cell cycle arrest of a human breast cancer cell line (MDA-MB-231): potential role of cyclin D1

DETA-NONOate, a nitric oxide (NO) donor, induced cytostasis in the human breast cancer cells MDA-MB-231, and the cells were arrested in the G(1) phase of the cell cycle. This cytostatic effect of the NO donor was associated with the down-regulation of cyclin D1 and hypophosphorylation of the retinoblastoma protein. No changes in the levels of cyclin E or the catalytic partners of these cyclins, CDK2, CDK4, or CDK6, were observed. This NO-induced cytostasis and decrease in cyclin D1 was reversible for up to 48 h of DETA-NONOate (1 mM) treatment. DETA-NONOate (1 mM) produced a steady-state concentration of 0.5 microM of NO over a 24-h period. Synchronized population of the cells exposed to DETA-NONOate remained arrested at the G(1) phase of the cell cycle whereas untreated control cells progressed through the cell cycle after serum stimulation. The cells arrested at the G(1) phase after exposure to the NO donor had low cyclin D1 levels compared with the control cells. The levels of cyclin E and CDK4, however, were similar to the control cells. The decline in cyclin D1 protein preceded the decrease of its mRNA. This decline of cyclin D1 was due to a decrease in its synthesis induced by the NO donor and not due to an increase in its degradation. We conclude that down-regulation of cyclin D1 protein by DETA-NONOate played an important role in the cytostasis and arrest of these tumor cells in the G(1) phase of the cell cycle.     

Functional regulation of D-type cyclins by insulin-like growth factor-I and serum in multiple myeloma cells

D-type cyclin genes are universally dysregulated in multiple myeloma (MM), but the functional consequences are unclear as D-type cyclin gene expression does not correlate with proliferation or disease progression. We examined the protein expression and regulation of D-type cyclins and other cell cycle regulators in human myeloma cell lines and primary CD138(+) plasma cells (PCs). Cyclin D1, cyclin D2, cyclin dependent kinase (CDK) 4, CDK6, p27(Kip1) p18(INK4C) and retinoblastoma protein (pRb) were absent in normal PCs, heterogeneously expressed in primary MM cells and positively correlated with disease activity/progression. Cyclins D1 and D2 complexed with both CDK4 and CDK6, suggesting that both phosphorylate pRb in MM. Furthermore, cyclin D2 expressed via either t(14;16) or t(4;14) IgH translocations was functionally upregulated by fetal calf serum or insulin-like growth factor-I, leading to pRb phosphorylation and cell cycle entry/progression, and in some cases inversely correlated with p27(Kip1). However, pRb phosphorylation and cell cycle progression mediated by cyclin D1 expressed via t(11;14) was less dependent on exogenous stimuli. These data suggest that the presence or absence of specific IgH translocations underlying aberrant D-type cyclin expression may influence their response to mitogens in the bone marrow microenvironment. We showed for the first time that D-type cyclins are functionally regulated in MM, differentially responsive to exogenous growth factors and upregulated with disease progression.     

G1 arrest and down-regulation of cyclin E/cyclin-dependent kinase 2 by the protein kinase inhibitor staurosporine are dependent on the retinoblastoma protein in the bladder carcinoma cell line 5637.

The protein kinase inhibitor staurosporine has been shown to induce G1 phase arrest in normal cells but not in most transformed cells. Staurosporine did not induce G1 phase arrest in the bladder carcinoma cell line 5637 that lacks a functional retinoblastoma protein (pRB-). However, when infected with a pRB-expressing retrovirus [Goodrich, D. W., Chen, Y., Scully, P. & Lee, W.-H. (1992) Cancer Res. 52, 1968-1973], these cells, now pRB+, were arrested by staurosporine in G1 phase. This arrest was accompanied by the accumulation of hypophosphorylated pRB. In both the pRB+ and pRB- cells, cyclin D1-associated kinase activities were reduced on staurosporine treatment. In contrast, cyclin-dependent kinase (CDK) 2 and cyclin E/CDK2 activities were inhibited only in pRB+ cells. Staurosporine treatment did not cause reductions in the protein levels of CDK4, cyclin D1, CDK2, or cyclin E. The CDK inhibitor proteins p21(Waf1/Cip1) and p27 (Kip1) levels increased in staurosporine-treated cells. Immunoprecipitation of CDK2, cyclin E, and p2l from staurosporine-treated pRB+ cells revealed a 2.5- to 3-fold higher ratio of p2l bound to CDK2 compared with staurosporine-treated pRB- cells. In pRB+ cells, p2l was preferentially associated with Thrl6O phosphorylated active CDK2. In pRB- cells, however, p2l was bound preferentially to the unphosphorylated, inactive form of CDK2 even though the phosphorylated form was abundant. This is the first evidence suggesting that G1 arrest by 4 nM staurosporine is dependent on a functional pRB protein. Cell cycle arrest at the pRB- dependent checkpoint may prevent activation of cyclin E/CDK2 by stabilizing its interaction with inhibitor proteins p2l and p27.     

Cyclin D type does not influence cell cycle response to DNA damage caused by ionizing radiation in multiple myeloma tumours

Multiple myeloma (MM) is characterized by over‐expression of cyclin D1 (CCND1) or D2 (CCND2), which control G1 phase cell‐cycle progression. Proteolytic degradation of CCND1 (but not CCND2), resulting in G1 arrest, is reported in non‐MM cells post‐DNA damage, affecting DNA repair and survival. We examined the effect of ionizing radiation (IR) on D‐cyclin levels and cell‐cycle kinetics of MM cells, exploring differences based on D‐cyclin expression. We showed that CCND1 is downregulated, whereas CCND2 is not, following IR. This did not lead to hypo‐phosphorylation of retinoblastoma protein or G1 arrest. Both CCND1‐ and CCND2‐expressing MM cells arrested in S/G2/M, and did not differ in other cell‐cycle proteins or sensitivity to IR. When treated with a CDK4/6 inhibitor, both CCND1 and CCND2 MM cells arrested in G1 and therefore are subject to physiological regulation at this checkpoint. Immunoprecipitation showed that, despite CCND1 degradation following IR, sufficient protein remains bound to CDK4/6 to prevent G1 arrest. Aberrant expression of CCND1 driven from the IGH promoter in t(11;14) MM cells maintains progression through G1 to arrest in S/G2/M. Differential expression of D‐cyclin does not appear to affect cell‐cycle response to IR, and is unlikely to underlie differential sensitivity to DNA damage.     

Effects of tachyplesin on the regulation of cell cycle in human hepatocarcinoma SMMC-7721 cells

AIM: To investigate the effects of tachyplesin on the cell cycle regulation in human hepatcarcinoma cells. METHODS: Effects of tachyplesin on the cell cycle in human hepatocarcinoma SMMC-7721 cells were assayed with flow cytometry. The protein levels of p53, p16, cyclin D1 and CDK4 were assayed by immunocytochemistry. The mRNA levels of p21(WAF1/CIP1) and c-myc genes were examined with in situ hybridization assay. RESULTS: After tachyplesin treatment, the cell cycle arrested at G0/G1 phase, the protein levels of mutant p53, cyclin D1 and CDK4 and the mRNA level of c-myc gene were decreased, whereas the levels of p16 protein and p21(WAF1/CIP1) mRNA increased. CONCLUSION: Tachyplesin might arrest the cell at G0/G1 phase by upregulating the levels of p16 protein and p21(WAF1/CIP1) mRNA and downregulating the levels of mutant p53, cyclin D1 and CDK4 proteins and c-myc mRNA, and induce the differentiation of human hepatocacinoma cells.     

Regulation of cell cycle molecules by the Ras effector system

Eukaryotic cell cycle progression is driven by an ordered array of phosphorylation events that are specifically catalyzed by members of CDK (cyclin-dependent kinase) family serine/threonine protein kinases, each consisting of a catalytic subunit CDK and a positive regulatory subunit cyclin. In mammalian somatic cells extracellular cues act mainly during the G1 phase to regulate the activity of D type cyclin-dependent CDKs, which, in turn, serve as key regulators of G1--S phase progression by phosphorylating and functionally inactivating the tumor suppressor retinoblastoma (Rb) protein. The small molecular weight G protein Ras has been implicated as a crucial molecule that transduces extracellular growth stimuli into intracellular signals. Recent studies, including our own, have demonstrated that maintained cellular Ras activity is required until late in the G1 phase for inactivation of the Rb protein and the G1/S transition and mediates both upregulation of cyclin D1 and downregulation of p27kip1 CDK inhibitor.     

氧化苦参碱对人结肠癌细胞株SW1116细胞周期通路相关调控因子的影响

背景：氧化苦参碱为中药苦参的主要有效成分，研究显示其对结肠癌细胞具有杀伤作用。目的：观察氧化苦参碱对人结肠癌细胞株SW1116细胞周期通路相关调控因子的影响，阐明其抗结肠癌作用的分子机制。方法：分别以2、3、4mg／m1氧化苦参碱干预SW1116细胞24h或48h，以流式细胞仪检测细胞周期，以逆转录聚合酶链反应（RTPCR）和蛋白质印迹法检测细胞周期蛋白（cyclin）D1、细胞周期蛋白依赖性激酶4（CDK4）、CDK抑制剂p53、核转录岗子E2F1和S期激酶相关蛋白2（Skp2）mRNA和蛋白水平的表达。结果：氧化苦参碱干预可使SW1116细胞周期阻滞于G0／G1期，cyc1inD1、CDK4、E2F1和Skp2mRNA和蛋白表达显著下降，p53mRNA和蛋白表达显著上升，作用呈剂量和时间依赖性（P〈0．05）。结论：氧化苦参碱可通过影响细胞周期相关通路以抑制人结肠癌细胞增殖。     

Melatonin delays cell proliferation by inducing G1 and G2 /M phase arrest in a human osteoblastic cell line hFOB 1.19

Abstract: A recent prospective study indicated that melatonin supplements may reduce the progression of idiopathic scoliosis, the most common deformity of the spine. This form of scoliosis occurs during rapid skeletal growth. To date, however, there is no direct evidence regarding an antiproliferative effect of melatonin at the level of osteoblasts. Herein, we investigated whether melatonin inhibits cell proliferation in a normal human fetal osteoblastic cell line hFOB 1.19. MTT staining showed that at 1 mm concentrations, melatonin significantly inhibited osteoblast proliferation in time‐dependent manner. Flow cytometry demonstrated that melatonin significantly increased the fraction of cells in G₀/G₁ phase of the cell cycle, while simultaneously reducing the proportion in the G₂/M phase rather than the S phase. Western blot and real‐time PCR analyses further confirmed that melatonin’s inhibitory effect was possibly because of downregulation of cyclin D1 and CDK4, related to the G₁ phase, and of cyclin B1 and CDK1, related to the G₂/M phase. There was no downregulation of cyclin E, CDK2, and cyclin A, which are related to G₁/S transition and S phase. In addition, the trypan blue dye exclusion assay showed that cell viability was not changed by melatonin relative to control cells. These findings provide evidence that melatonin may significantly delay osteoblast proliferation in a time‐dependent manner and this inhibition involves the downregulation of cyclin D1 and CDK4, related to the G₁ phase, and of cyclin B1 and CDK1, related to the G₂/M phase.     

High expression of cyclin E and G1 CDK and loss of function of p57KIP2 are involved in proliferation of malignant sporadic adrenocortical tumors

Maternal loss of heterozygosity (LOH) of the 11p15 region and overexpression of the insulin-like growth factor (IGF)-II gene are associated with the malignant phenotype in sporadic adrenocortical tumors. In the imprinted 11p15 region, the p57KIP2 gene is maternally expressed and encodes a cyclin-dependent kinase (CDK) inhibitor involved in G1/S phase of the cell cycle. We hypothesized that maternal LOH in malignant adrenocortical tumors could be responsible for loss of p57KIP2 gene expression and, thus, could favor progression through the cell cycle. We investigated 3 normal adrenals, 31 adrenocortical tumors [11 tumors with normal expression of the IGF-II gene (mainly benign) and 20 with IGF-II gene overexpression (mainly malignant)], and the human adrenocortical tumor cell line NCI H295R for expression of the p57KIP2 gene, G1 cyclins (cyclin D2 and E) and G1 CDK (CDK2, CDK3 and CDK4) protein contents and for kinase activity of G1 cyclin-CDK complexes. The expression of p57KIP2, G1 cyclins, and G1 CDKs in benign tumors was similar to that in normal adrenal tissues, as were kinase activities of G1 cyclin-CDK complexes. By contrast, abrogation of the p57KIP2 gene expression and increased expression of G1 cyclins (cyclin E) and G1 CDKs (CDK2 and CDK4) were associated with high activity of G1 cyclin-CDK complexes in malignant tumors and in the H295R cell line. These data suggest that the p57KIP2 gene might act as a tumor suppressor gene in adrenocortical tumors. Maternal LOH with duplication of the paternal allele or pathological functional imprinting of the 11p15 region are responsible for loss of expression of the p57KIP2 gene and increased expression of the IGF-II gene. Consequently, both events favor cell proliferation in malignant adrenocortical tumors.     

Adhesion to fibronectin stimulates proliferation of wild-type and bcr/abl-transfected murine hematopoietic cells

Cells of most tissues require adhesion to a surface to grow. However, for hematopoietic cells, both stimulation and inhibition of proliferation by adhesion to extracellular matrix components have been described. Furthermore, it has been suggested that progenitor cells from chronic myelogenous leukemia show decreased beta1 integrin-mediated adhesion to fibronectin, resulting in increased proliferation and abnormal trafficking. However, we show here that the chronic myelogenous leukemia-specific fusion protein p210bcr/abl stimulates the expression of alpha5beta1 integrins and induces adhesion to fibronectin when expressed in the myeloid cell line 32D. Moreover, proliferation of both p210bcr/abl-transfected 32D (32Dp210) cells and untransfected 32D cells is stimulated by immobilized fibronectin. Cell cycle analysis revealed that nonadherent 32D and 32Dp210 cells are arrested in late G1 or early S phase, whereas the adherent fractions continue cycling. Although both adherent and nonadherent p210bcr/abl-transfected and parental 32D cells express equal amounts of cyclin A, a protein necessary for cell cycle progression at the G1/S boundary, cyclin A complexes immunoprecipitated from 32D cells cultured on immobilized fibronectin were found to be catalytically inactive in nonadherent but not in adherent cells. In addition, as compared with untransfected 32D cells, cyclin A immunoprecipitates from 32Dp210 cells exhibited a greatly elevated kinase activity and remained partially active irrespective of the adhesion status. The lack of cyclin A/cyclin-dependent kinase (CDK) 2 activity in nonadherent 32D cells appeared to result from increased expression and cyclin A complex formation of the CDK inhibitor p27(Kip1). Taken together, our results indicate that adhesion stimulates cell cycle progression of hematopoietic cells by down-regulation of p27(Kip1), resulting in activation of cyclin A/CDK2 complexes and subsequent transition through the G1/S adhesion checkpoint.     

XIAP通过调节CDK4／CDK6／cyclinD1复合物表达促进肝癌细胞增殖

目的研究肝癌细胞中XIAP与cDK4／cDK6／cyclinD1复合物之间的调控关系及在肝癌细胞增殖中的作用。方法前期研究用PathwayArray进行肝癌组织异常表达蛋白筛选,后期XIAP特异性抑制剂embelin处理Huh7细胞进行增殖、周期分析。Westernblot验证XIAP与cDK4／cDK6／cyclinD1复合物之间的调控关系。结果PathwayArray提示XIAP与CDK4、CDK6、cyclinD1之间存在联合表达。embelin引起Huh7细胞G1期阻滞、S期分布减少,抑制细胞增殖。XIAP可以调节Huh7细胞G1期蛋白CDK4、CDK6、cyclinD1表达。结论XIAP通过调节肝癌细胞G1期蛋白CDK4／CDK6／eyelinD1复合物的表达,促使细胞进入G1周期,从而促进其增殖。     

Oil A induces apoptosis of pancreatic cancer cells via caspase activation, redistribution of cell cycle and GADD expression

AIM: To explore the mechanisms of effects of oil A on apoptosis of human pancreatic cancer cells. METHODS: Cellular DNA content was analyzed by flow cytometry. Western blotting was used for caspase-3 and PARP, caspase-7, caspase-9, cytochrome c, Bcl-2, Bax, Mcl-1, cyclinA, cyclin B1, cyclin D1, cyclin E, CDK2, CDK4, CDK6, P21, P27, GADD45, GADD153. RESULTS: The caspase-3, caspase-7, and caspase-9 activities were significantly increased as well as the cleavage of caspase-3, downstream substrate poly-ADP ribose polymerase (PARP) was induced. The amount of cytochrome c in the cytosolic fraction was increased, while the amount of cytochrome c in the mitochondrial fraction was decreased after oil A treatment. The anti-apoptosis proteins Bcl-2 and Mcl-1 were decreased in parallel and Bax increased, indicating that Bcl-2 family proteins-mitochondria-caspase cascade was responsible for oil-induced apoptosis. The proportion of cells in the G0/G1 decreased in MiaPaCa-2 and AsPC-1 cells after the treatment of oil A for 24 hours. The number of cells in S phase was increased in two cancer cell lines at 24 hours. Therefore, cells were significantly accumulated in G2/M phase. The cells with a sub-G0/G1 DNA content, a hallmark of apoptosis, were seen at 24 hours both in MiaPaCa-2 and AsPC-1 cells following exposure to oil A. The expression of cyclin A and cyclin B1 was slightly decreased and cyclin D1 levels were markedly lowered in MiaPaCa-2 cells. The expression of cyclin A and cyclin B1 was markedly decreased and cyclin D1 levels were slightly lowered in AsPC-1 cells, while cyclin E was not affected and the levels of CDK2, CDK4, and CDK6 were unchanged in MiaPaCa-2 and AsPC-1 cells. In response to oil A, P21 expression was increased, but P27 expression was not affected. The expression of both GADD45 and GADD153 was increased in two cell lines following oil A treatment. CONCLUSION: Oil A induces apoptosis of pancreatic cancer cells via activating caspase cascade, modifying cell cycle progress and changing cell cycle-regulating proteins and GADD expression.     

Molecular mechanism of progesterone-induced antiproliferation in rat aortic smooth muscle cells

Previously we demonstrated that progesterone at physiologic levels dose dependently inhibited cell proliferation in cultured rat aortic smooth muscle cells (RASMCs). However, the molecular mechanism underlying of progesterone-induced antiproliferation was not clear. Here we demonstrated that progesterone induced a reduction of the [(3)H]thymidine incorporation into RASMCs during the S-phase of the cell cycle. Western blotting analysis revealed that the protein levels of cyclin A, cyclin E, and cyclin-dependent-kinase (CDK) 2 but not cyclin D1 and CDK4 decreased after progesterone treatment, but those of CDK-inhibitory proteins, p21 and p27, increased. Immunoprecipitation showed that the formations of the CDK2-p21 and CDK2-p27 complex were increased and the assayable CDK2 kinase activity was decreased in the progesterone-treated RASMCs. In contrast, the formations of the CDK4-p21 and CDK4-p27 complex and the assayable CDK4 kinase activity were not changed significantly by progesterone treatment. Pretreatment of RASMCs with a p21 or p27 antisense oligonucleotide reduced the progesterone-induced inhibition of [(3)H]thymidine incorporation into RASMCs. In conclusion, these data suggest that progesterone inhibits RASMCs proliferation by increasing the levels of p21 and p27 protein, which in turn inhibit CDK2 kinase activity, and finally interrupt the cell cycle.     

Recombinant human prothrombin kringle-2 induces bovine capillary endothelial cell cycle arrest at G0-G1 phase through inhibition of cyclin D1/CDK4 complex: modulation of reactive oxygen species generation and up-regulation of cyclin-dependent kinase inhibitors

Prothrombin is a plasma glycoprotein involved in blood coagulation and, as we have previously reported, prothrombin kringles inhibit BCE (bovine capillary endothelial) cell proliferation. To reveal the mechanism, we investigated the influence of rk-2 (recombinant human prothrombin kringle-2) on the BCE cell cycle progression and ROS (reactive oxygen species) generation using FACS (fluorescence-activated cell sorter) analysis. Cell cycle analysis showed a decrease of G(1) phase cells in cells treated with bFGF (basic fibroblast growth factor) and an increase in cells treated with rk-2, as compared with the control cells. But, the portion of the S phase was reversed. In Western blot analysis, bFGF induced cytoplasmic translocation of p21(Waf1/Cip1) and p27(Kip1) and phosphorylation of p27(Kip1) but rk-2 treatment inhibited translocation of p21(Waf1/Cip1) and p27(Kip1) from nucleus to cytoplasm and phosphorylation of p27(Kip1). Also, rk-2 induced up-regulation of p53 and nuclear p21(Waf1/Cip1) and inhibited the cyclin D1/CDK4 (cyclin-dependent kinase 4) complex. The ROS level of rk-2-treated BCE cells was increased 2-fold when compared with the control, but treatment with NAC (N-Acetyl-L: -cysteine), an anti-oxidant, decreased ROS generation about 55% as compared with the rk-2 treatment. NAC treatment also restored cell cycle progression inhibited by rk-2 and down-regulated p53 and nuclear p21(Waf1/Cip1) expression induced by rk-2.These data suggest that rk-2 induces the BCE cell cycle arrest at G(0)-G(1) phase through inhibition of the cyclin D1/CDK4 complex caused by increase of ROS generation and nuclear cyclin-dependent kinase inhibitors.     

A calmodulin-binding site on cyclin E mediates Ca2+-sensitive G1/s transitions in vascular smooth muscle cells

Calcium transients are known to control several transition points in the eukaryotic cell cycle. For example, we have previously shown that a coordinate elevation in the intracellular free calcium ion concentration is required for G1- to S-phase cell cycle progression in vascular smooth muscle cells (VSMC). However, the molecular basis for this Ca2+ sensitivity was not known. Using buffers with differing [Ca2+], we found that the kinase activity of mouse and human cyclin E/CDK2, but not other G1/S-associated cell cycle complexes, was responsive to physiological changes in [Ca2+]. We next determined that this Ca2+-responsive kinase activity was dependent on a direct interaction between calmodulin (CaM), one of the major Ca2+-signal transducers of eukaryotic cells, and cyclin E. Pharmacological inhibition of CaM abrogated the Ca2+ sensitivity of cyclin E/CDK2 and retarded mouse VSMC proliferation by causing G1 arrest. We next defined the presence of a highly conserved 22 amino acid N-terminal CaM-binding motif in mammalian cyclin E genes (dissociation constant, 1.5+/-0.1 micromol/L) and showed its essential role in mediating Ca2+-sensitive kinase activity of cyclin E/CDK2. Mutant human cyclin E protein, lacking this CaM-binding motif, was incapable of binding CaM or responding to [Ca2+]. Taken together, these findings reveal CaM-dependent cyclin E/CDK2 activity as a mediator of the known Ca2+ sensitivity of the G1/S transition of VSMC.