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.

Ca2+/calmodulin-dependent kinase II mediates simultaneous enhancement of gap-junctional conductance and glutamatergic transmission

While chemical synapses are very plastic and modifiable by defined activity patterns, gap junctions, which mediate electrical transmission, have been classically perceived as passive intercellular channels. Excitatory transmission between auditory afferents and the goldfish Mauthner cell is mediated by coexisting gap junctions and glutamatergic synapses. Although an increased intracellular Ca2+ concentration is expected to reduce gap junctional conductance, both components of the synaptic response were instead enhanced by postsynaptic increases in Ca2+ concentration, produced by patterned synaptic activity or intradendritic Ca2+ injections. The synaptically induced potentiations were blocked by intradendritic injection of KN-93, a Ca2+/calmodulin-dependent kinase (CaM-K) inhibitor, or CaM-KIINtide, a potent and specific peptide inhibitor of CaM-KII, whereas the responses were potentiated by injection of an activated form of CaM-KII. The striking similarities of the mechanisms reported here with those proposed for long-term potentiation of mammalian glutamatergic synapses suggest that gap junctions are also similarly regulated and indicate a primary role for CaM-KII in shaping and regulating interneuronal communication, regardless of its modality.

Ca2+-induced rebound potentiation of γ-aminobutyric acid-mediated currents requires activation of Ca2+/calmodulin-dependent kinase II

In cerebellar Purkinje neurons, γ-aminobutyric acid (GABA)-mediated inhibitory synaptic transmission undergoes a long-lasting “rebound potentiation” after the activation of excitatory climbing fiber inputs. Rebound potentiation is triggered by the climbing-fiber-induced transient elevation of intracellular Ca2+ concentration and is expressed as a long-lasting increase of postsynaptic GABAA receptor sensitivity. Herein we show that inhibitors of the Ca2+/calmodulin-dependent protein kinase II (CaM-KII) signal transduction pathway effectively block the induction of rebound potentiation. These inhibitors have no effect on the once established rebound potentiation, on voltage-gated Ca2+ channel currents, or on the basal inhibitory transmission itself. Futhermore, a protein phosphatase inhibitor and the intracellularly applied CaM-KII markedly enhanced GABA-mediated currents in Purkinje neurons. Our results demonstrate that CaM-KII activation and the following phosphorylation are key steps for rebound potentiation.

An artificial cell-cycle inhibitor isolated from a combinatorial library

Understanding the genetic networks that operate inside cells will require the dissection of interactions among network members. Here we describe a peptide aptamer isolated from a combinatorial library that distinguishes among such interactions. This aptamer binds to cyclin-dependent kinase 2 (Cdk2) and inhibits its kinase activity. In contrast to naturally occurring inhibitors, such as p21Cip1, which inhibit the activity of Cdk2 on all its substrates, inhibition by pep8 has distinct substrate specificity. We show that the aptamer binds to Cdk2 at or near its active site and that its mode of inhibition is competitive. Expression of pep8 in human cells retards their progression through the G1 phase of the cell cycle. Our results suggest that the aptamer inhibits cell-cycle progression by blocking the activity of Cdk2 on substrates needed for the G1-to-S transition. This work demonstrates the feasibility of selection of artificial proteins to perform functions not developed during evolution. The ability to select proteins that block interactions between a gene product and some partners but not others should make sophisticated genetic manipulations possible in human cells and other currently intractable systems.

A cytosolic activity distinct from Crm1 mediates nuclear export of protein kinase inhibitor in permeabilized cells

The leucine-rich nuclear export signal (NES) is used by a variety of proteins to facilitate their delivery from the nucleus to the cytoplasm. One of the best-studied examples, protein kinase inhibitor (PKI), binds to the catalytic subunit of protein kinase A in the nucleus and mediates its rapid export to the cytoplasm. We developed a permeabilized cell assay that reconstitutes nuclear export mediated by PKI, and we used it to characterize the cytosolic factors required for this process. The two-step assay involves an import phase and an export phase, and quantitation is achieved by digital fluorescence microscopy. During the import phase, a fluorescent derivative of streptavidin is imported into the nuclei of digitonin-permeabilized HeLa cells. During the export phase, biotinylated PKI diffuses into the nucleus, binds to fluorescent streptavidin, and mediates export of the complex to the cytoplasm. Nuclear export of the PKI complex is cytosol dependent and can be stimulated by addition of the purified NES receptor, Crm1. HeLa cell cytosol treated with N-ethylmaleimide (NEM) or phenyl-Sepharose to inactivate or deplete Crm1, respectively, is still fully active in the PKI export assay. Significantly, the export activity can be depleted from cytosol by preadsorption with a protein conjugate that contains a functional NES. These data indicate that cytosol contains an export activity that is distinct from Crm1 and is likely to correspond to an NES receptor.

Cyclin E, a redundant cyclin in breast cancer

Cyclin E is an important regulator of cell cycle progression that together with cyclin-dependent kinase (cdk) 2 is crucial for the G1/S transition during the mammalian cell cycle. Previously, we showed that severe overexpression of cyclin E protein in tumor cells and tissues results in the appearance of lower molecular weight isoforms of cyclin E, which together with cdk2 can form a kinase complex active throughout the cell cycle. In this study, we report that one of the substrates of this constitutively active cyclin E/cdk2 complex is retinoblastoma susceptibility gene product (pRb) in populations of breast cancer cells and tissues that also overexpress p16. In these tumor cells and tissues, we show that the expression of p16 and pRb is not mutually exclusive. Overexpression of p16 in these cells results in sequestering of cdk4 and cdk6, rendering cyclin D1/cdk complexes inactive. However, pRb appears to be phosphorylated throughout the cell cycle following an initial lag, revealing a time course similar to phosphorylation of glutathione S-transferase retinoblastoma by cyclin E immunoprecipitates prepared from these synchronized cells. Hence, cyclin E kinase complexes can function redundantly and replace the loss of cyclin D-dependent kinase complexes that functionally inactivate pRb. In addition, the constitutively overexpressed cyclin E is also the predominant cyclin found in p107/E2F complexes throughout the tumor, but not the normal, cell cycle. These observations suggest that overexpression of cyclin E in tumor cells, which also overexpress p16, can bypass the cyclin D/cdk4-cdk6/p16/pRb feedback loop, providing yet another mechanism by which tumors can gain a growth advantage.

Cyclin-dependent kinases as a therapeutic target for stroke

Cyclin-dependent kinases (CDKs) are commonly known to regulate cell proliferation. However, previous reports suggest that in cultured postmitotic neurons, activation of CDKs is a signal for death rather than cell division. We determined whether CDK activation occurs in mature adult neurons during focal stroke in vivo and whether this signal was required for neuronal death after reperfusion injury. Cdk4/cyclin D1 levels and phosphorylation of its substrate retinoblastoma protein (pRb) increase after stroke. Deregulated levels of E2F1, a transcription factor regulated by pRb, are also observed. Administration of a CDK inhibitor blocks pRb phosphorylation and the increase in E2F1 levels and dramatically reduces neuronal death by 80%. These results indicate that CDKs are an important therapeutic target for the treatment of reperfusion injury after ischemia.

Differential Expression of Genes for Cyclin-Dependent Protein Kinases in Rice Plants1

Cyclin-dependent protein kinases (CDKs) play key roles in regulating the eukaryotic cell cycle. We have analyzed the expression of four rice (Oryza sativa) CDK genes, cdc2Os1, cdc2Os2, cdc2Os3, and R2, by in situ hybridization of sections of root apices. Transcripts of cdc2Os1, cdc2Os2, and R2 were detected uniformly in the dividing region of the root apex. cdc2Os1 and cdc2Os2 were also expressed in differentiated cells such as those in the sclerenchyma, pericycle, and parenchyma of the central cylinder. By contrast, signals corresponding to transcripts of cdc2Os3 were distributed only in patches in the dividing region. Counterstaining of sections with 4′,6-diamidino-2-phenylindole and double-target in situ hybridization with a probe for histone H4 transcripts revealed that cdc2Os3 transcripts were abundant from the G2 to the M phase, but were less abundant or absent during the S phase. The levels of the Cdc2Os3 protein and its associated histone H1-kinase activity were reduced by treatment of cultured cells with hydroxyurea, which blocks cycling cells at the onset of the S phase. Our results suggest that domains other than the conserved amino acid sequence (the PSTAIRE motif) have important roles in the function of non-PSTAIRE CDKs in distinct cell-cycle phases.

p34cdc2 kinase activity is maintained upon activation of the replication checkpoint in Schizosaccharomyces pombe.

All eukaryotes use feedback controls to order and coordinate cell cycle events. In Schizosaccharomyces pombe, several classes of checkpoint genes serve to ensure that DNA replication is complete and free of error before the onset of mitosis. Wild-type cells normally arrest upon inhibition of DNA synthesis or in response to DNA damage, although the exact mechanisms controlling this arrest are unclear. Genetic evidence in fission yeast suggests that the dependence of mitosis upon completion of DNA replication is linked to the regulation of the p34cdc2 cyclin-dependent kinase. It has been hypothesized that inhibition of DNA synthesis triggers down-regulation of p34cdc2 kinase activity, although this has never been shown biochemically. We analyzed the activity of p34cdc2 in wild-type and checkpoint-defective cells treated with a DNA synthesis inhibitor. Using standard in vitro assays we demonstrate that p34cdc2 kinase activity is maintained in wild-type cells arrested at the replication checkpoint. We also used a novel in vivo assay for p34cdc2 kinase activity, in which we expressed a fragment of the human retinoblastoma tumor suppressor protein in fission yeast. Phosphorylation of this fragment of the human retinoblastoma tumor suppressor protein is dependent on p34cdc2 kinase activity, and this activity is also maintained in cells arrested at the replication checkpoint. These data suggest that the mechanism for cell-cycle arrest in response to incomplete DNA synthesis is not dependent on the attenuation of p34cdc2 activity.

Involvement of the cell-cycle inhibitor Cip1/WAF1 and the E1A-associated p300 protein in terminal differentiation.

The mechanism of cell cycle withdrawal during terminal differentiation is poorly understood. We report here that the cyclin-dependent kinase (CDK) inhibitor p21Cip1/WAF1 is induced at early times of both keratinocyte and myoblast differentiation. p21Cip1/WAF1 induction is accompanied by a drastic inhibition of total Cdk2, as well as p21Cip1/WAF1-associated CDK kinase activities. p21Cip1/WAF1 has been implicated in p53-mediated G1 arrest and apoptosis. In keratinocyte differentiation, Cip1/WAF1 induction is observed even in cells derived from p53-null mice. Similarly, keratinocyte differentiation is associated with induction of Cip1/WAF1 promoter activity in both wild-type and p53-negative keratinocytes. Induction of the Cip1/WAF1 promoter upon differentiation is abolished by expression of an adenovirus E1A oncoprotein (d1922/947), which is unable to bind p105-Rb, p107, or cyclin A but which still binds the nuclear phosphoprotein p300. Overexpression of p300 can suppress the E1A effect, independent of its direct binding to E1A. Thus, terminal differentiation-induced growth arrest in both keratinocyte and myoblast systems is associated with induction of Cip1/WAF1 expression. During keratinocyte differentiation, Cip1/WAF1 induction does not require p53 but depends on the transcriptional modulator p300.

Selective inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine kinase inhibitor of the 2-phenylaminopyrimidine class.

The platelet-derived growth factor (PDGF) receptor is a member of the transmembrane growth factor receptor protein family with intrinsic protein-tyrosine kinase activity. We describe a potent protein-tyrosine kinase inhibitor (CGP 53716) that shows selectivity for the PDGF receptor in vitro and in the cell. The compound shows selectivity for inhibition of PDGF-mediated events such as PDGF receptor autophosphorylation, cellular tyrosine phosphorylation, and c-fos mRNA induction in response to PDGF stimulation of intact cells. In contrast, ligand-induced autophosphorylation of the epidermal growth factor (EGF) receptor, insulin receptor, and the insulin-like growth factor I receptor, as well as c-fos mRNA expression induced by EGF, fibroblast growth factor, and phorbol ester, was insensitive to inhibition by CGP 53716. In antiproliferative assays, the compound was approximately 30-fold more potent in inhibiting PDGF-mediated growth of v-sis-transformed BALB/c 3T3 cells relative to inhibition of EGF-dependent BALB/Mk cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line. When tested in vivo using highly tumorigenic v-sis- and human c-sis-transformed BALB/c 3T3 cells, CGP 53716 showed antitumor activity at well-tolerated doses. In contrast, CGP 53716 did not show antitumor activity against xenografts of the A431 tumor, which overexpresses the EGF receptor. These findings suggest that CGP 53716 may have therapeutic potential for the treatment of diseases involving abnormal cellular proliferation induced by PDGF receptor activation.

Photodynamic therapy results in induction of WAF1/CIP1/P21 leading to cell cycle arrest and apoptosis

Photodynamic therapy (PDT) is a promising new modality that utilizes a combination of a photosensitizing chemical and visible light for the management of a variety of solid malignancies. The mechanism of PDT-mediated cell killing is not well defined. We investigated the involvement of cell cycle regulatory events during silicon phthalocyanine (Pc4)-PDT-mediated apoptosis in human epidermoid carcinoma cells A431. PDT resulted in apoptosis, inhibition of cell growth, and G0-G1 phase arrest of the cell cycle, in a time-dependent fashion. Western blot analysis revealed that PDT results in an induction of the cyclin kinase inhibitor WAF1/CIP1/p21, and a down-regulation of cyclin D1 and cyclin E, and their catalytic subunits cyclin-dependent kinase (cdk) 2 and cdk6. The treatment also resulted in a decrease in kinase activities associated with all the cdks and cyclins examined. PDT also resulted in (i) an increase in the binding of cyclin D1 and cdk6 toward WAF1/CIP1/p21, and (ii) a decrease in the binding of cyclin D1 toward cdk2 and cdk6. The binding of cyclin E and cdk2 toward WAF1/CIP1/p21, and of cyclin E toward cdk2 did not change by the treatment. These data suggest that PDT-mediated induction of WAF1/CIP1/p21 results in an imposition of artificial checkpoint at G1 → S transition thereby resulting in an arrest of cells in G0-G1 phase of the cell cycle through inhibition in the cdk2, cdk6, cyclin D1, and cyclin E. We suggest that this arrest is an irreversible process and the cells, unable to repair the damages, ultimately undergo apoptosis.

Effectors of a developmental mitogen-activated protein kinase cascade revealed by expression signatures of signaling mutants

Despite the importance of mitogen-activated protein kinase (MAPK) signaling in eukaryotic biology, the mechanisms by which signaling yields phenotypic changes are poorly understood. We have combined transcriptional profiling with genetics to determine how the Kss1 MAPK signaling pathway controls dimorphic development in Saccharomyces cerevisiae. This analysis identified dozens of transcripts that are regulated by the pathway, whereas previous work had identified only a single downstream target, FLO11. One of the MAPK-regulated genes is PGU1, which encodes a secreted enzyme that hydrolyzes polygalacturonic acid, a structural barrier to microbial invasion present in the natural plant substrate of S. cerevisiae. A third key transcriptional target is the G1 cyclin gene CLN1, a morphogenetic regulator that we show to be essential for pseudohyphal growth. In contrast, the homologous CLN2 cyclin gene is dispensable for development. Thus, the Kss1 MAPK cascade programs development by coordinately modulating a cell adhesion factor, a secreted host-destroying activity, and a specialized subunit of the Cdc28 cyclin-dependent kinase.

Postnatal neuronal proliferation in mice lacking Ink4d and Kip1 inhibitors of cyclin-dependent kinases

Development of the central nervous system requires proliferation of neuronal and glial cell precursors followed by their subsequent differentiation in a highly coordinated manner. The timing of neuronal cell cycle exit and differentiation is likely to be regulated in part by inhibitors of cyclin-dependent kinases. Overlapping and sustained patterns of expression of two cyclin-dependent kinases, p19Ink4d and p27Kip1, in postmitotic brain cells suggested that these proteins may be important in actively repressing neuronal proliferation. Animals derived from crosses of Ink4d- null with Kip1-null mice exhibited bradykinesia, proprioceptive abnormalities, and seizures, and died at about 18 days after birth. Metabolic labeling of live animals with bromodeoxyuridine at postnatal days 14 and 18, combined with immunolabeling of neuronal markers, showed that subpopulations of central nervous system neurons were proliferating in all parts of the brain, including normally dormant cells of the hippocampus, cortex, hypothalamus, pons, and brainstem. These cells also expressed phosphorylated histone H3, a marker for late G2 and M-phase progression, indicating that neurons were dividing after they had migrated to their final positions in the brain. Increased proliferation was balanced by cell death, resulting in no gross changes in the cytoarchitecture of the brains of these mice. Therefore, p19Ink4d and p27Kip1 cooperate to maintain differentiated neurons in a quiescent state that is potentially reversible.

Targeting cyclin-dependent kinases in Drosophila with peptide aptamers

Two-hybrid technology provides a simple way to isolate small peptide aptamers that specifically recognize and strongly bind to a protein of interest. These aptamers have the potential to dominantly interfere with specific activities of their target proteins and, therefore, could be used as in vivo inhibitors. Here we explore the ability to use peptide aptamers as in vivo inhibitors by expressing aptamers directed against cell cycle regulators in Drosophila. We expressed two peptide aptamers, each of which specifically recognizes one of the two essential cyclin-dependent kinases (Cdks), DmCdk1 and DmCdk2, in Drosophila. Expression of each Cdk aptamer during organogenesis caused adult eye defects typical of those caused by cell cycle inhibition. Co-overexpression of DmCdk1 or DmCdk2 resulted in suppression of the eye phenotypes, indicating that each aptamer interacts with a Cdk target in vivo and suggesting that these peptides disrupt normal eye development by inhibiting Cdk function. Moreover, the specificity of each aptamer for one of the two Cdks as determined in two-hybrid assays was retained in Drosophila. Combined, our results demonstrate that peptide aptamers generated by yeast two-hybrid methods can serve as inhibitory reagents to target specific proteins in vivo.