Activation of cyclin-dependent kinase 2 (Cdk2) in growth-stimulated rat astrocytes. Geranylgeranylated Rho small GTPase(s) are essential for the induction of cyclin E gene expression

The role of the mevalonate cascade in the control of cell cycle progression in astrocytes has been investigated. Serum stimulation of rat astrocytes in primary culture induces the expression of cyclin E followed by the activation of cyclin-dependent kinase 2 (Cdk2) during G1/S transition. The expression of p27, cyclin D1, and the activities of Cdk4 and Cdk-activating kinase (CAK), composed of Cdk7 and cyclin H, were not affected. Serum did, however, stimulate the expression of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA at mid-G1 phase. Moreover, an inhibitor of HMG-CoA reductase, pravastatin, reduced cyclin E expression and Cdk2 activation and caused G1 arrest in the astrocytes. In contrast, mevalonate and its metabolite, geranylgeranylpyrophosphate (GGPP) but not farnesylpyrophosphate (FPP), reversed the inhibitory effects of pravastatin on cyclin E expression and Cdk2 activation and allowed G1/S transition. Rho small GTPase(s) were geranylgeranylated and translocated to membranes in the presence of GGPP during G1/S transition. The effect of GGPP on cyclin E expression was abolished by botulinum C3 exoenzyme, which specifically inactivates Rho. These data indicate that geranylgeranylated Rho small GTPase(s) are essential for the induction of cyclin E expression, Cdk2 activation, and G1/S transition in rat astrocytes.     

The muscle regulatory factors MyoD and myf-5 undergo distinct cell cycle-specific expression in muscle cells

The muscle regulators MyoD and Myf-5 control cell cycle withdrawal and induction of differentiation in skeletal muscle cells. By immunofluorescence analysis, we show that MyoD and Myf-5 expression patterns become mutually exclusive when C2 cells are induced to differentiate with Myf-5 staining present in cells which fail to differentiate. Isolation of these undifferentiated cells reveals that upon serum stimulation they reenter the cell cycle, express MyoD and downregulate Myf-5. Similar regulations of MyoD and Myf-5 were observed using cultured primary myoblasts derived from satellite cells. To further analyze these regulations of MyoD and Myf-5 expression, we synchronized proliferating myoblasts. Analysis of MyoD and Myf-5 expression during cell cycle progression revealed distinct and contrasting profiles of expression. MyoD is absent in G0, peaks in mid-G1, falls to its minimum level at G1/S and reaugments from S to M. In contrast, Myf-5 protein is high in G0, decreases during G1 and reappears at the end of G1 to remain stable until mitosis. These data demonstrate that the two myogenic factors MyoD and Myf-5 undergo specific and distinct cell cycle-dependent regulation, thus establishing a correlation between the cell cycle-specific ratios of MyoD and Myf-5 and the capacity of cells to differentiate: (a) in G1, when cells express high levels of MyoD and enter differentiation; (b) in G0, when cells express high levels of Myf-5 and fail to differentiate.     

Modulation of Sp1 phosphorylation by human immunodeficiency virus type 1 Tat

We previously reported (K. T. Jeang, R. Chun, N. H. Lin, A. Gatignol, C. G. Glabe, and H. Fan, J. Virol. 67: 6224-6233, 1993) that human immunodeficiency virus type 1 (HIV-1) Tat and Sp1 form a protein-protein complex. Here, we have characterized the physical interaction and a functional consequence of Tat-Sp1 contact. Using in vitro protein chromatography, we mapped the region in Tat that contacts Sp1 to amino acids 30 to 55. We found that in cell-free reactions, Tat augmented double-stranded DNA-dependent protein kinase (DNA-PK)-mediated Sp1 phosphorylation in a contact-dependent manner. Tat mutants that do not bind Sp1 failed to influence phosphorylation of the latter. In complementary experiments, we also found that Tat forms protein-protein contacts with DNA-PK. We confirmed that in HeLa and Jurkat cells, Tat expression indeed increased the intracellular amount of phosphorylated Sp1 in a manner consistent with the results of cell-free assays. Furthermore, using two phosphatase inhibitors and a kinase inhibitor, we demonstrated a modulation of reporter gene expression as a consequence of changes in Sp1 phosphorylation. Taken together, these findings suggest that activity at the HIV-1 promoter is influenced by phosphorylation of Sp1 which is affected by Tat and DNA-PK.     

p53 regulates insulin-like growth factor-I (IGF-I) receptor expression and IGF-I-induced tyrosine phosphorylation in an osteosarcoma cell line: interaction between p53 and Sp1

The insulin-like growth factor-I receptor (IGF-IR) is involved in tumorigenesis. The aim of the present study was to investigate whether the IGF-IR is a physiological target for p53 in osteosarcoma cells. The p53-induced regulation of IGF-IR levels was studied in a tetracycline-regulated expression system. When expressed in Saos-2, osteosarcoma cells that lack p53, wild-type p53 decreased, whereas mutated p53 increased IGF-IR expression, and IGF-I-induced tyrosine phosphorylation of the IGF-IR. Similarly, wild-type p53 decreased IGF-I-induced tyrosine phosphorylation of IRS-1. A functional and physical interaction between p53 and Sp1, in the regulation of the IGF-R, was studied in osteosarcoma cells. Expression of p53 decreased IGF-IR promoter activity, whereas no effect on promoter activity was seen by Sp1 expressed alone. However, Sp1 counteracted the inhibitory effect of p53 on IGF-IR promoter activity in a dose-dependent manner. Furthermore, wild-type and mutated p53 were coimmunoprecipitated with Sp1, indicating a physical interaction between p53 and Sp1. In conclusion, p53 regulates IGF-IR expression, as reflected by a reduction in IGF-IR protein and a parallel reduction in IGF-I-induced tyrosine phosphorylation of the IGF-IR and IRS-1 in an osteosarcoma cell line. These data indicate that the IGF-I receptor is a physiological target for p53 in osteosarcoma cells. Furthermore, data supporting an interaction between p53 and Sp1 in the regulation of the promoter activity of IGF-IR are presented.     

Control elements of Dnmt1 gene are regulated in cell-cycle dependent manner

The Dnmt1 gene is constitutively expressed and is required for the maintenance of global methylation after DNA replication. We investigated here the effects of histone deacetylase (HDAC) inhibitor and DNA demetylation agent on promoter activity of mouse Dnmt1 gene in somatic cells. The promoter activity of Dnmt1 gene was increased approximately 2-fold in the treatment of cells by Tricostatin A (TSA) at 1 x 10(-8) M, as compared with that without treatment of TSA. By contrast, treatment with 5-azacytidne (5aza-C) did not affect the promoter activity of the Dnmt1 gene. This result indicates the Dnmt1 gene is possibly regulated by histone acetylation. We also examined the expression levels of Dnmt1 gene and of its control elements like Sp1, Sp3 and p300 by the chromatin immunoprecipitation and Western blot analysis. The expression of Dnmt1 gene is observed at early S phase. Sp1 is recruited mainly at the G1 phase and Sp3 is recruited at the early S phase. p300 is also obviously recruited at the second S phase. These data indicated that the regulators of Dnmt1 gene were controlled in cell-cycle dependent manner.     

Effects of guanine nucleotide depletion on cell cycle progression in human T lymphocytes

Depletion of guanine nucleotide pools after inhibition of inosine monophosphate dehydrogenase (IMPDH) potently inhibits DNA synthesis by arresting cells in G1 and has been shown to induce the differentiation of cultured myeloid and erythroid cell lines, as well as chronic granulocytic leukemic cells after blast transformation. Inhibitors of IMPDH are also highly effective as immunosuppressive agents. The mechanism underlying these pleiotropic effects of depletion of guanine nucleotides is unknown. We have examined the effects of mycophenolic acid (MPA), a potent IMPDH inhibitor, on the cell cycle progression of activated normal human T lymphocytes. MPA treatment resulted in the inhibition of pRb phosphorylation and cell entry into S phase. The expression of cyclin D3, a major component of the cyclin-dependent kinase (CDK) activity required for pRb phosphorylation, was completely abrogated by MPA treatment of T cells activated by interleukin-2 (IL-2) and leucoagglutinin (PHA-L), whereas the expression of cyclin D2, CDK6, and CDK4 was more mildly attenuated. The direct kinase activity of a complex immunoprecipitated with anti-CDK6 antibody was also inhibited. In addition, MPA prevented the IL-2-induced elimination of p27(Kip1), a CDK inhibitor, and resulted in the retention of high levels of p27(Kip1) in IL-2/PHA-L-treated T cells bound to CDK2. These results indicate that inhibition of the de novo synthesis of guanine nucleotides blocks the transition of normal peripheral blood T lymphocytes from G0 to S phase in early- to mid-G1 and that this cell cycle arrest results from inhibition of the induction of cyclin D/CDK6 kinase and the elimination of p27(Kip1) inhibitory activity.     

Phosphorylation of elongation factor 1 and ribosomal protein S6 by multipotential S6 kinase and insulin stimulation of translational elongation

Stimulation of protein synthesis in response to insulin is concomitant with increased phosphorylation of initiation factors 4B and 4G and ribosomal protein S6 (Morley, S. J., and Traugh, J. A. (1993) Biochimie 75, 985-989) and is due at least in part to multipotential S6 kinase. When elongation factor 1 (EF-1) from rabbit reticulocytes was examined as substrate for multipotential S6 kinase, up to 1 mol/mol of phosphate was incorporated into the alpha, beta, and delta subunits. Phosphorylation of EF-1 resulted in a 2-2. 6-fold stimulation of EF-1 activity, as measured by poly(U)-directed polyphenylalanine synthesis. The rate of elongation was also stimulated by approximately 2-fold with 80 S ribosomes phosphorylated on S6 by multipotential S6 kinase. When the rates of elongation in extracts from serum-fed 3T3-L1 cells and cells serum-deprived for 1.5 h were compared, a 40% decrease was observed upon serum deprivation. The addition of insulin to serum-deprived cells for 15 min stimulated elongation to a rate equivalent to that of serum-fed cells. Similar results were obtained with partially purified EF-1, with both EF-1 and ribosomes contributing to stimulation of elongation. These data are consistent with a ribosomal transit time of 3.2 min for serum-deprived cells and 1.6 min following the addition of insulin for 15 min. Taken together, the data suggest that insulin stimulation involves coordinate regulation of EF-1 and ribosomes through phosphorylation by multipotential S6 kinase.     

Phosphorylation of the G protein gamma12 subunit regulates effector specificity

Although the G protein betagamma dimer is an important mediator in cell signaling, the mechanisms regulating its activity have not been widely investigated. The gamma12 subunit is a known substrate for protein kinase C, suggesting phosphorylation as a potential regulatory mechanism. Therefore, recombinant beta1 gamma12 dimers were overexpressed using the baculovirus/Sf9 insect cell system, purified, and phosphorylated stoichiometrically with protein kinase C alpha. Their ability to support coupling of the Gi1 alpha subunit to the A1 adenosine receptor and to activate type II adenylyl cyclase or phospholipase C-beta was examined. Phosphorylation of the beta1 gamma12 dimer increased its potency in the receptor coupling assay from 6.4 to 1 nM, changed the Kact for stimulation of type II adenylyl cyclase from 14 to 37 nM, and decreased its maximal efficacy by 50%. In contrast, phosphorylation of the dimer had no effect on its ability to activate phospholipase C-beta. The native beta1gamma10 dimer, which has 4 similar amino acids in the phosphorylation site at the N terminus, was not phosphorylated by protein kinase C alpha. Creation of a phosphorylation site in the N terminus of the protein (Gly4 --> Lys) resulted in a beta1 gamma10G4K dimer which could be phosphorylated. The activities of this beta gamma dimer were similar to those of the phosphorylated beta1 gamma12 dimer. Thus, phosphorylation of the beta1 gamma12 dimer on the gamma subunit with protein kinase C alpha regulates its activity in an effector-specific fashion. Because the gamma12 subunit is widely expressed, phosphorylation may be an important mechanism for integration of the multiple signals generated by receptor activation.     

Repression of the CDK activator Cdc25A and cell-cycle arrest by cytokine TGF-beta in cells lacking the CDK inhibitor p15

The activity of the cyclin-dependent kinases (CDKs) that control cell growth and division can be negatively regulated by tyrosine phosphorylation or by the binding of various CDK inhibitors. Whereas regulation by tyrosine phosphorylation is well documented in CDKs that function during mitosis, little is known about its role in the regulation of CDKs that act in the G1 phase of the cell cycle. In contrast, much evidence has accumulated on the regulation of G1 CDKs by CDK inhibitors. The cytokine TGF-beta inhibits growth by causing cell-cycle arrest as a result of increasing the concentration of the Cdk4/6 inhibitor p15(INK4B/MTS2) (refs 3, 4). Here we report that TGF-beta can also cause the inhibition of Cdk4 and Cdk6 by increasing their level of tyrosine phosphorylation. Tyrosine phosphorylation and inactivation of Cdk4/6 in a human mammary epithelial cell line are shown to result from the ability of TGF-beta to repress expression of the CDK tyrosine phosphatase Cdc25A. Repression of Cdc25A and induction of p15 are independent effects mediating the inhibition of Cdk4/6 by TFG-beta.     

Characterization of the human aldose reductase gene promoter

The promoter region of the human aldose reductase gene has been identified upstream of the translation start ATG codon. The promoter contains a TATA box, a CCAAT promoter element, and three Sp1 protein binding consensus sequences upstream of the capsite. A 640-base pair insert spanning +31 to -609 directs expression of the reporter gene chloramphenicol acetyltransferase in an orientation-specific manner in transfected Hep G2 cells. The promoter activity remained constant with deletions from base pairs -609 to -186. The TATA and the CCAAT consensus sequences show significant promoter activity, whereas the three Sp1 binding consensus sequences, individually, have no significant promoter activity. A GA-rich region (-186 to -146) contains two CGGAAA/G motifs, which show promoter activity and interaction with Hep G2 nuclear extract and GA-binding proteins (GABP alpha and GABP beta 1) as shown by mobility shift assays and DNase I footprinting. Similar cis-elements in herpes simplex virus type 1 interact with rat liver GABP and the viral VP16 protein to mediate the induction of immediate early viral genes. A GC-rich region (-87 to -31) is identified by mobility shift assay, and a consensus sequence of an androgen response element is present at -396 to -382. The human aldose reductase promoter, thus, has regulatory response elements that may be important during early development and puberty. These regulatory elements may play a significant role in the development of certain diabetic complications.     

Malignant glaucoma and its management

Acetylcholine receptor-inducing activity (ARIA) is a glycoprotein initially purified from chick brain based on its ability to increase the synthesis of acetylcholine receptor (AChR) on cultured myotubes. cDNA encoding ARIA contains different domains and the functions of each domain in ARIA activity are not known. We used molecular genetic methods to construct a chimeric fusion protein, designated ARIA(S136-K205)-Fc, that contained the leader sequence, the EGF-like domain of chick ARIA (S136 to K205) and the Fc region of human immunoglobulin. The ARIA(S136-K205)-Fc cDNA was transfected into HEK 293 cells and stable cell lines secreting soluble ARIA(S136-K205)-Fc were obtained. The secreted ARIA(S136-K205)-Fc has a molecular mass of approximately 60 kDa and can be purified by protein G chromatography. The purified ARIA(S136-K205)-Fc retained its full biological activity of chick ARIA that included: (i) induction of tyrosine phosphorylation of erbB 3 receptor in C2C12 myotubes; and (ii) approximately 12-fold stimulation of AChR alpha-subunit mRNA synthesis when applied onto cultured chick myotubes. This Fc-tagged ARIA could be rapidly purified and provides a very useful ligand for identifying its true receptor(s) on muscle cell surface.