Regulation of estrogen receptor transcriptional enhancement by the cyclin A/Cdk2 complex

We have found that ectopic expression of cyclin A increases hormone-dependent and hormone-independent transcriptional activation by the estrogen receptor in vivo in a number of cell lines, including HeLa cells, U-2 OS osteosarcoma cells and Hs 578Bst breast epithelial cells. This effect can be further enhanced in HeLa cells by the concurrent expression of the cyclin-dependent kinase activator, cyclin H, and cdk7, and abolished by expression of the cdk inhibitor, p27KIP1, or by the expression of a dominant negative catalytically inactive cdk2 mutant. ER is phosphorylated between amino acids 82 and 121 in vitro by the cyclin A/cdk2 complex and incorporation of phosphate into ER is stimulated by ectopic expression of cyclin A in vivo. Together, these results strongly suggest a direct role for the cyclin A/cdk2 complex in phosphorylating ER and regulating its transcriptional activity.

Posttranslational regulation of cyclin D1 by retinoic acid: A chemoprevention mechanism

The retinoids are reported to reduce incidence of second primary aerodigestive cancers. Mechanisms for this chemoprevention are previously linked to all-trans retinoic acid (RA) signaling growth inhibition at G1 in carcinogen-exposed immortalized human bronchial epithelial cells. This study investigated how RA suppresses human bronchial epithelial cell growth at the G1-S cell cycle transition. RA signaled growth suppression of human bronchial epithelial cells and a decline in cyclin D1 protein but not mRNA expression. Exogenous cyclin D1 protein also declined after RA treatment of transfected, immortalized human bronchial epithelial cells, suggesting that posttranslational mechanisms were active in this regulation of cyclin D1 expression. Findings were extended by showing treatment with ubiquitin-dependent proteasome inhibitors: calpain inhibitor I and lactacystin each prevented this decreased cyclin D1 protein expression, despite RA treatment. Treatment with the cysteine proteinase inhibitor, E-64, did not prevent this cyclin D1 decline. High molecular weight cyclin D1 protein species appeared after proteasome inhibitor treatments, suggesting that ubiquitinated species were present. To learn whether RA directly promoted degradation of cyclin D1 protein, studies using human bronchial epithelial cell protein extracts and in vitro-translated cyclin D1 were performed. In vitro-translated cyclin D1 degraded more rapidly when incubated with extracts from RA treated vs. untreated cells. Notably, this RA-signaled cyclin D1 proteolysis depended on the C-terminal PEST sequence, a region rich in proline (P), glutamate (E), serine (S), and threonine (T). Taken together, these data highlight RA-induced cyclin D1 proteolysis as a mechanism signaling growth inhibition at G1 active in the prevention of human bronchial epithelial cell transformation.

Regulation of the Cyclin B Degradation System by an Inhibitor of Mitotic Proteolysis

The initiation of anaphase and exit from mitosis depend on the anaphase-promoting complex (APC), which mediates the ubiquitin-dependent proteolysis of anaphase-inhibiting proteins and mitotic cyclins. We have analyzed whether protein phosphatases are required for mitotic APC activation. In Xenopus egg extracts APC activation occurs normally in the presence of protein phosphatase 1 inhibitors, suggesting that the anaphase defects caused by protein phosphatase 1 mutation in several organisms are not due to a failure to activate the APC. Contrary to this, the initiation of mitotic cyclin B proteolysis is prevented by inhibitors of protein phosphatase 2A such as okadaic acid. Okadaic acid induces an activity that inhibits cyclin B ubiquitination. We refer to this activity as inhibitor of mitotic proteolysis because it also prevents the degradation of other APC substrates. A similar activity exists in extracts of Xenopus eggs that are arrested at the second meiotic metaphase by the cytostatic factor activity of the protein kinase mos. In Xenopus eggs, the initiation of anaphase II may therefore be prevented by an inhibitor of APC-dependent ubiquitination.

Abnormal integrin-mediated regulation of chronic myelogenous leukemia CD34+ cell proliferation: BCR/ABL up-regulates the cyclin-dependent kinase inhibitor, p27Kip, which is relocated to the cell cytoplasm and incapable of regulating cdk2 activity

β1-integrin engagement on normal (NL) CD34+ cells increases levels of the cyclin-dependent kinase inhibitor (cdki), p27Kip, decreases cdk2 activity, and inhibits G1/S-phase progression. In contrast, β1-integrin engagement on chronic myelogenous leukemia (CML) CD34+ cells does not inhibit G1/S progression. We now show that, in CML, baseline p27Kip levels are significantly higher than in NL CD34+ cells, but adhesion to fibronectin (FN) does not increase p27Kip levels. p27Kip mRNA levels are similar in CML and NL CD34+ cells and remain unchanged after adhesion, suggesting posttranscriptional regulation. Despite the elevated p27Kip levels, cdk2 kinase activity is similar in CML and NL CD34+ cells. In NL CD34+ cells, >90% of p27Kip is located in the nucleus, where it binds to cdk2 after integrin engagement. In CML CD34+ cells, however, >80% of p27Kip is located in the cytoplasm even in FN-adherent cells, and significantly less p27Kip is bound to cdk2. Thus, presence of BCR/ABL induces elevated levels of p27Kip and relocation of p27Kip to the cytoplasm, which contributes to the loss of integrin-mediated proliferation inhibition, characteristic of CML.

Specific down-regulation of an engineered human cyclin D1 promoter by a novel DNA-binding ligand in intact cells

Cyclin D1 is expressed at abnormally high levels in many cancers and has been specifically implicated in the development of breast cancer. In this report we have extensively analyzed the cyclin D1 promoter in a variety of cancer cell lines that overexpress the protein and identified two critical regulatory elements (CREs), a previously identified CRE at –52 and a novel site at –30. In vivo footprinting experiments demonstrated factors binding at both sites. We have used a novel DNA-binding ligand, GL020924, to target the site at –30 (–30–21) of the cyclin D1 promoter in MCF7 breast cancer cells. A binding site for this novel molecule was constructed by mutating 2 bp of the wild-type cyclin D1 promoter at the –30–21 site. Treatment with GL020924 specifically inhibited expression of the targeted cyclin D1 promoter construct in MCF7 cells in a concentration-dependent manner, thus validating the –30–21 site as a target for minor groove-binding ligands. In addition, this result validates our approach to regulating the expression of genes implicated in disease by targeting small DNA-binding ligands to key regulatory elements in the promoters of those genes.

Evidence for regulation of protein synthesis at the elongation step by CDK1/cyclin B phosphorylation

Eukaryotic elongation factor 1 (eEF-1) contains the guanine nucleotide exchange factor eEF-1B that loads the G protein eEF-1A with GTP after each cycle of elongation during protein synthesis. Two features of eEF-1B have not yet been elucidated: (i) the presence of the unique valyl-tRNA synthetase; (ii) the significance of target sites for the cell cycle protein kinase CDK1/cyclin B. The roles of these two features were addressed by elongation measurements in vitro using cell-free extracts. A poly(GUA) template RNA was generated to support both poly(valine) and poly(serine) synthesis and poly(phenylalanine) synthesis was driven by a poly(uridylic acid) template. Elongation rates were in the order phenylalanine > valine > serine. Addition of CDK1/cyclin B decreased the elongation rate for valine whereas the rate for serine and phenylalanine elongation was increased. This effect was correlated with phosphorylation of the eEF-1δ and eEF-1γ subunits of eEF-1B. Our results demonstrate specific regulation of elongation by CDK1/cyclin B phosphorylation.

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.

Regulation of the retinoblastoma protein-related protein p107 by G1 cyclin-associated kinases.

p107 is a retinoblastoma protein-related phosphoprotein that, when overproduced, displays a growth inhibitory function. It interacts with and modulates the activity of the transcription factor, E2F-4. In addition, p107 physically associates with cyclin E-CDK2 and cyclin A-CDK2 complexes in late G1 and at G1/S, respectively, an indication that cyclin-dependent kinase complexes may regulate, contribute to, and/or benefit from p107 function during the cell cycle. Our results show that p107 phosphorylation begins in mid G1 and proceeds through late G1 and S and that cyclin D-associated kinase(s) contributes to this process. In addition, E2F-4 binds selectively to hypophosphorylated p107, and G1 cyclin-dependent p107 phosphorylation leads to the dissociation of p107-E2F-4 complexes as well as inactivation of p107 G1 blocking function.

Regulation of the cyclin E gene by transcription factor E2F1.

A variety of results point to the transcription factor E2F as a critical determinant of the G1/S-phase transition during the cell cycle in mammalian cells, serving to activate the transcription of a group of genes that encode proteins necessary for DNA replication. In addition, E2F activity appears to be directly regulated by the action of retinoblastoma protein (RB) and RB-related proteins and indirectly regulated through the action of G1 cyclins and associated kinases. We now show that the accumulation of G1 cyclins is regulated by E2F1. E2F binding sites are found in both the cyclin E and cyclin D1 promoters, both promoters are activated by E2F gene products, and at least for cyclin E, the E2F sites contribute to cell cycle-dependent control. Most important, the endogenous cyclin E gene is activated following expression of the E2F1 product encoded by a recombinant adenovirus vector. These results suggest the involvement of E2F1 and cyclin E in an autoregulatory loop that governs the accumulation of critical activities affecting the progression of cells through G1.

Cell cycle regulation of the cyclin A gene promoter is mediated by a variant E2F site.

Cyclin A is involved in the control of S phase and mitosis in mammalian cells. Expression of the cyclin A gene in nontransformed cells is characterized by repression of its promoter during the G1 phase of the cell cycle and its induction at S-phase entry. We show that this mode of regulation is mediated by the transcription factor E2F, which binds to a specific site in the cyclin A promoter. It differs from the prototype E2F site in nucleotide sequence and protein binding; it is bound by E2F complexes containing cyclin E and p107 but not pRB. Ectopic expression of cyclin D1 triggers premature activation of the cyclin A promoter by E2F, and this effect is blocked by the tumor suppressor protein p16INK4.

Oncogenic stimulation recruits cyclin-dependent kinase in the cell cycle start in rat fibroblast

The rat fibroblast NRK cells are transformed reversibly by a combination of growth factors. When stimulated with serum, NRK cells rely on cyclin-dependent kinase 4 (Cdk4) for their S phase entry. However, when stimulated with serum containing oncogenic growth factors, they come to rely on either Cdk4 or Cdk6, and their S phase entry cannot be blocked unless both Cdk4 and Cdk6 are immunodepleted. Such change of dependence does not occur in the NRK cell mutants defective in an oncogenic signal pathway and, therefore, deficient in anchorage-independent cell cycle start ability, correlating Cdk6 dependence with this remarkable, cancer-associated phenotype. However, both Cdk4 and Cdk6 are activated upon serum stimulation, and neither the amounts of Cdk6, Cdk4, cyclin D1, and cyclin-dependent kinase inhibitors nor the activities or subcellular localization of Cdk6 and Cdk4 are significantly influenced by oncogenic stimulation. Thus, oncogenic stimulation invokes Cdk6 to participate in a critical step of the cell cycle start in a rat fibroblast, but by a mechanism seemingly unrelated to the regulation of the kinase. Given that many hematopoietic cells employ predominantly Cdk6 for the cell cycle start and perform anchorage-independent growth by nature, our results raise the possibility that the oncogenic stimulation-induced anchorage-independent cell cycle start of NRK is elicited by a mechanism similar to the one used for hematopoietic cell proliferation.

Mammalian Cdk5 is a functional homologue of the budding yeast Pho85 cyclin-dependent protein kinase

Mammalian Cdk5 is a member of the cyclin-dependent kinase family that is activated by a neuron-specific regulator, p35, to regulate neuronal migration and neurite outgrowth. p35/Cdk5 kinase colocalizes with and regulates the activity of the Pak1 kinase in neuronal growth cones and likely impacts on actin cytoskeletal dynamics through Pak1. Here, we describe a functional homologue of Cdk5 in budding yeast, Pho85. Like Cdk5, Pho85 has been implicated in actin cytoskeleton regulation through phosphorylation of an actin-regulatory protein. Overexpression of CDK5 in yeast cells complemented most phenotypes associated with pho85Δ, including defects in the repression of acid phosphatase expression, sensitivity to salt, and a G1 progression defect. Consistent with the functional complementation, Cdk5 associated with and was activated by the Pho85 cyclins Pho80 and Pcl2 in yeast cells. In a reciprocal series of experiments, we found that Pho85 associated with the Cdk5 activators p35 and p25 to form an active kinase complex in mammalian and insect cells, supporting our hypothesis that Pho85 and Cdk5 are functionally related. Our results suggest the existence of a functionally conserved pathway involving Cdks and actin-regulatory proteins that promotes reorganization of the actin cytoskeleton in response to regulatory signals.

Cyclin B Proteolysis and the Cyclin-dependent Kinase Inhibitor rum1p Are Required for Pheromone-induced G1 Arrest in Fission Yeast

The blocking of G1 progression by fission yeast pheromones requires inhibition of the cyclin-dependent kinase cdc2p associated with the B-cyclins cdc13p and cig2p. We show that cyclosome-mediated degradation of cdc13p and cig2p is necessary for down-regulation of B-cyclin–associated cdc2p kinase activity and for phermone-induced G1 arrest. The cyclin-dependent kinase inhibitor rum1p is also required to maintain this G1 arrest; it binds both cdc13p and cig2p and is specifically required for cdc13p proteolysis. We propose that rum1p acts as an adaptor targeting cdc13p for degradation by the cyclosome. In contrast, the cig2p–cdc2p kinase can be down-regulated, and the cyclin cig2p can be proteolyzed independently of rum1p. We suggest that pheromone signaling inhibits the cig2p–cdc2p kinase, bringing about a transient G1 arrest. As a consequence, rum1p levels increase, thus inhibiting and inducing proteolysis of the cdc13p–cdc2p kinase; this is necessary to maintain G1 arrest. We have also shown that pheromone-induced transcription occurs only in G1 and is independent of rum1p.

Regulation of the Anaphase-promoting Complex/Cyclosome by bimAAPC3 and Proteolysis of NIMA

Surprisingly, although highly temperature-sensitive, the bimA1APC3 anaphase-promoting complex/cyclosome (APC/C) mutation does not cause arrest of mitotic exit. Instead, rapid inactivation of bimA1APC3 is shown to promote repeating oscillations of chromosome condensation and decondensation, activation and inactivation of NIMA and p34cdc2 kinases, and accumulation and degradation of NIMA, which all coordinately cycle multiple times without causing nuclear division. These bimA1APC3-induced cell cycle oscillations require active NIMA, because a nimA5 + bimA1APC3 double mutant arrests in a mitotic state with very high p34cdc2 H1 kinase activity. NIMA protein instability during S phase and G2 was also found to be controlled by the APC/C. The bimA1APC3 mutation therefore first inactivates the APC/C but then allows its activation in a cyclic manner; these cycles depend on NIMA. We hypothesize that bimAAPC3 could be part of a cell cycle clock mechanism that is reset after inactivation of bimA1APC3. The bimA1APC3 mutation may also make the APC/C resistant to activation by mitotic substrates of the APC/C, such as cyclin B, Polo, and NIMA, causing mitotic delay. Once these regulators accumulate, they activate the APC/C, and cells exit from mitosis, which then allows this cycle to repeat. The data indicate that bimAAPC3 regulates the APC/C in a NIMA-dependent manner.

Two Distinct Mechanisms Control the Accumulation of Cyclin B1 and Mos in Xenopus Oocytes in Response to Progesterone

Progesterone-induced meiotic maturation of Xenopus oocytes requires the synthesis of new proteins, such as Mos and cyclin B. Synthesis of Mos is thought to be necessary and sufficient for meiotic maturation; however, it has recently been proposed that newly synthesized proteins binding to p34cdc2 could be involved in a signaling pathway that triggers the activation of maturation-promoting factor. We focused our attention on cyclin B proteins because they are synthesized in response to progesterone, they bind to p34cdc2, and their microinjection into resting oocytes induces meiotic maturation. We investigated cyclin B accumulation in response to progesterone in the absence of maturation-promoting factor–induced feedback. We report here that the cdk inhibitor p21cip1, when microinjected into immature Xenopus oocytes, blocks germinal vesicle breakdown induced by progesterone, by maturation-promoting factor transfer, or by injection of okadaic acid. After microinjection of p21cip1, progesterone fails to induce the activation of MAPK or p34cdc2, and Mos does not accumulate. In contrast, the level of cyclin B1 increases normally in a manner dependent on down-regulation of cAMP-dependent protein kinase but independent of cap-ribose methylation of mRNA.