Sp1 phosphorylation by Erk 2 stimulates DNA binding

The temperate mycobacteriophage L5 integrates site specifically into the genomes of Mycobacterium smegmatis, Mycobacterium tuberculosis, and Mycobacterium bovis bacillus Calmette-Guérin. This integrative recombination event occurs between the phage L5 attP site and the mycobacterial attB site and requires the phage-encoded integrase and mycobacterial-encoded integration host factor mIHF. Here we show that attP, Int-L5, and mIHF assemble into a recombinationally active complex, the intasome, which is capable of attB capture and formation of products. The arm-type integrase binding sites within attP play specialized roles in the formation of specific protein-DNA architectures; the intasome is constructed by the formation of intramolecular integrase bridges between one pair of sites, P4-P5, and the attP core, while an additional pair of sites, P1-P2, is required for interaction with attB.     

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.     

Sp1 and egr-1 have opposing effects on the regulation of the rat Pgp2/mdr1b gene

The promoter of the rat pgp2/mdr1b gene has a GC-rich region (pgp2GC) that is highly conserved in mdr genes and contains an consensus Sp1 site. Sp1's role in transactivation of the pgp2/mdr1b promoter was tested in Drosophila Schneider cells. The pgp2/mdr1b promoter was strongly activated by co-transfected wild type Sp1 but not mutant Sp1 and mutation of the Sp1 site abrogated Sp1-dependent transactivation. In gel shift assays, the same mutations abolished Sp1-DNA complex formation. Moreover, basal activity of the pgp2/mdr1b Sp1 mutant promoter was dramatically lower. Enforced ectopic overexpression of Sp1 in H35 rat hepatoma cells revealed that cell lines overexpressing Sp1 had increased endogenous pgp2/mdr1b mRNA, demonstrating that Sp1 activates the endogenous pgp2/mdr1b gene. Pgp2GC oligonucleotide also bound Egr-1 in gel shift assays and Egr-1 competitively displaced bound Sp1. In transient transfections of H35 cells (and human LS180 and HepG2 cells) Egr-1 potently and specifically suppressed pgp2/mdr1b promoter activity and mutations in the Egr-1 site decreased Egr-1 binding and correlated with pgp2/mdr1b up-regulation. Ectopic overexpression of Egr-1 in H35 cells decreased Pgp expression and selectively increased vinblastine sensitivity. In conclusion, Sp1 positively regulates while Egr-1 negatively regulates the rat pgp2/mdr1b gene. Moreover, competitive interactions between Sp1 and Egr-1 in all likelihood determine the constitutive expression of the pgp2/mdr1b gene in H35 cells.     

Regulatory roles of cyclin dependent kinase phosphorylation in cell cycle control

Cyclins and cyclin-dependent kinases (Cdks) are universal regulators of cell cycle progression in eukaryotic cells. Cdk activity is controlled by phosphorylation at three conserved sites, and many of the enzymes that act on these sites have now been identified. Although the biochemistry of Cdk phosphorylation is relatively well understood, the regulatory roles of such phosphorylation are, in many cases, obscure. Recent studies have uncovered new and unexpected potential roles, and prompted re-examination of previously assumed roles, of Cdk phosphorylation.     

Growth hormone regulation of SIRP and SHP-2 tyrosyl phosphorylation and association

SIRPs (signal-regulatory proteins) are a family of transmembrane glycoproteins that were identified by their association with the Src homology 2 domain-containing protein-tyrosine phosphatase SHP-2 in response to insulin. Here we examine whether SIRPalpha and SHP-2 are signaling molecules for the receptors for growth hormone (GH), leukemia inhibitory factor (LIF), or interferon-gamma (IFNgamma), cytokine receptor superfamily members that bind to and activate Janus kinase 2 (JAK2). In 3T3-F442A fibroblasts, GH rapidly stimulates tyrosyl phosphorylation of both SIRPalpha and SHP-2 and enhances association of SHP-2 with SIRPalpha. Consistent with JAK2 binding and phosphorylating SIRPalpha in response to GH, co-expression of SIRPalpha and JAK2 in COS cells results in tyrosyl phosphorylation of SIRPalpha and JAK2 association with SIRPalpha. LIF does not stimulate tyrosyl phosphorylation of SIRPalpha but stimulates greater tyrosyl phosphorylation of SHP-2 than GH. Additionally, LIF enhances association of SHP-2 with the gp130 subunit of the LIF receptor signaling complex. IFNgamma, which stimulates JAK2 to a greater extent than LIF, is ineffective at stimulating tyrosyl phosphorylation of SIRPalpha or SHP-2. These results suggest that SIRPalpha is a signaling molecule for GH but not for LIF or IFNgamma. Differential phosphorylation of SIRPalpha and SHP-2 may contribute to the distinct physiological effects of these ligands.     

Collaborative interactions between MEF-2 and Sp1 in muscle-specific gene regulation

We recently reported that previously activated T cells, irrespective of the nature of the first stimulus they encountered, are unable to respond to Staphylococcal enterotoxin B (SEB), nor to soluble anti-CD3 monoclonal antibody (mAb) presented by splenic antigen-presenting cells (APC). Such previously activated T cells are, however, fully capable of responding to plate-bound anti-CD3 plus splenic APC. These data suggest differential integration of the T-cell receptor (TCR) and co-stimulatory signalling pathways in naive versus antigen-experienced T cells. Consistent with this hypothesis, anti-CD28 mAb restores the proliferative capacity of resting ex vivo CD45RBlo CD4+ T cells (representing previously activated T cells) to both soluble anti-CD3 mAb and SEB. Interestingly, mAb-mediated engagement of cytotoxic T-lymphocyte antigen-4 (CTLA-4) completely negates the rescue effects mediated by anti-CD28 mAb in CD45RBlo cells. Nevertheless, the non-responsiveness of CD45RBlo CD4+ T cells cannot be reversed by anti-CTLA-4 Fab fragments, indicating that it is not related to negative regulatory effects of CTLA-4 engagement itself. Interestingly, the addition of interleukin-2 (IL-2) restores the proliferative capacity of CD45RBlo CD4+ T cells to SEB and soluble anti-CD3 mAb. Moreover, when rescued by IL-2, the cells are less susceptible to the negative regulatory effects of CTLA-4 engagement. Together, these findings suggest that the non-responsiveness of CD45RBlo CD4+ T cells to certain stimuli may be related to inadequate TCR signalling, primarily affecting IL-2 production.     

Negative regulation of Raf-1 by phosphorylation of serine 621

The elevation of cyclic AMP (cAMP) levels in the cell downregulates the activity of the Raf-1 kinase. It has been suggested that this effect is due to the activation of cAMP-dependent protein kinase (PKA), which can directly phosphorylate Raf-1 in vitro. In this study, we confirmed this hypothesis by coexpressing Raf-1 with the constitutively active catalytic subunit of PKA, which could fully reproduce the inhibition previously achieved by cAMP. PKA-phosphorylated Raf-1 exhibits a reduced affinity for GTP-loaded Ras as well as impaired catalytic activity. As the binding to GTP-loaded Ras induces Raf-1 activation in the cell, we examined which mechanism is required for PKA-mediated Raf-1 inhibition in vivo. A Raf-1 point mutant (RafR89L), which is unable to bind Ras, as well as the isolated Raf-1 kinase domain were still fully susceptible to inhibition by PKA, demonstrating that the phosphorylation of the Raf-1 kinase suffices for inhibition. By the use of mass spectroscopy and point mutants, PKA phosphorylation site was mapped to a single site in the Raf-1 kinase domain, serine 621. Replacement of serine 621 by alanine or cysteine or destruction of the PKA consensus motif by changing arginine 618 resulted in the loss of catalytic activity. Notably, a mutation of serine 619 to alanine did not significantly affect kinase activity or regulation by activators or PKA. Changing serine 621 to aspartic acid yielded a Raf-1 protein which, when expressed to high levels in Sf-9 insect cells, retained a very low inducible kinase activity that was resistant to PKA downregulation. The purified Raf-1 kinase domain displayed slow autophosphorylation of serine 621, which correlated with a decrease in catalytic function. The Raf-1 kinase domain activated by tyrosine phosphorylation could be downregulated by PKA. Specific removal of the phosphate residue at serine 621 reactivated the catalytic activity. These results are most consistent with a dual role of serine 621. On the one hand, serine 621 appears essential for catalytic activity; on the other hand, it serves as a phosphorylation site which confers negative regulation.     

Alterations in cell cycle regulation in mouse skin tumors

Using a modified tip of the atomic force microscope (AFM), we harvested several strands of genomic DNA from a nanometer region of mouse chromosomes. We have also co-developed a random PCR method to amplify the recovered genomic DNA, in which a single DNA molecule of several kilobasepairs could be amplified efficiently. A subsequent fluorescence in situ hybridization (FISH) indicated that the amplified DNA originally came from the tip-manipulated regions of mouse chromosomes. Several fragments containing unique sequences were identified using Southern hybridization after subcloning the PCR products into pUC18 plasmid. The present results showed a potential application of AFM to genomic analysis.     

Absence of p53-dependent cell cycle regulation in pluripotent mouse cell lines

We examined the expression of p53 in three lines of pluripotent embryonal carcinoma (EC) and ES cells. p53 mRNA and protein levels were constitutively high in two lines but absent from one. In the P19 line of EC cells neither p53 protein nor mRNA was detected. The first intron of the p53 gene in these cells had been invaded by a murine leukemia virus and there was extensive hypermethylation of the p53 gene accompanying its inactivation. In all three cell lines, irradiation resulted in arrest of the cells in the G2 but not in the G1 phase of the cell cycle despite the induction of p21cip1 in the cell lines expressing p53. Thus, the chromosomal stability of EC and ES cells appears to be not dependent on the p53 protein and we interpret our results to suggest that these cells may require the deletion of p53 dependent cell cycle regulation in order to become immortalized.     

Insulin regulation of the Ras activation/inactivation cycle

A theory is presented of the phase separation of supercoiled DNA into a nucleoid in a bacterial cell. The suspension consists of DNA interacting with globular proteins in excess salt. A cross virial between DNA and a protein is computed as well as the DNA self-energy arising from excluded volume. The cellular parameters of Escherichia coli would appear to be compatible with the thermodynamic equilibrium derived theoretically. The state of superhelical DNA in the nucleoid could be liquid crystalline and rippled.     

Proliferation, apoptosis and cell cycle regulation in prostatic carcinogenesis

Besides the MutLS-like system, Schizosaccharomyces pombe has an additional pathway of mismatch repair. This minor pathway, producing short excision tracts, repairs C/C and, with lower efficiency, other mismatches also. We investigated the involvement of the exo1+, msh2+ and pms1+ genes in the two pathways. The exo1+ gene encodes a 5' to 3' exonuclease, while msh2+ and pms1+ are homologs of Escherichia coli mutS and mutL, respectively. Intragenic two-factor crosses showed that exo1+, msh2+ and pms1+ are involved in the major, but not in the C/C-correcting, pathway. Post-meiotic segregation frequencies and mitotic mutation rates in single and double mutants supported this finding. Furthermore, msh2 delta was epistatic over exo1 delta, and the ExoI enzyme is likely to be redundant with other exonucleases.     

In vivo regulation of the early embryonic cell cycle in Xenopus

We report here the first extensive in vivo study of cell cycle regulation in the Xenopus embryo. Cyclin A1, B1, B2, and E1 levels, Cdc2 and Cdk2 kinase activity, and Cdc25C phosphorylation states were monitored during early Xenopus embryonic cell cycles. Cyclin B1 and B2 protein levels were high in the unfertilized egg, declined upon fertilization, and reaccumulated to the same level during the first cell cycle, a pattern repeated during each of the following 11 divisions. Cyclin A1 showed a similar pattern, except that its level was lower in the egg than in the cell cycles after fertilization. Cyclin B1/Cdc2 kinase activity oscillated, peaking before each cleavage, and Cdc25C alternated between a highly phosphorylated and a less phosphorylated form that correlated with high and low cyclin B1/Cdc2 kinase activity, respectively. Unlike the mitotic cyclins, the level of cyclin E1 did not oscillate during embryogenesis, although its associated Cdk2 kinase activity cycled twice for each oscillation of cyclin B1/Cdc2 activity, consistent with a role for cyclin E1 in both S-phase and mitosis. Although the length of the first embryonic cycle is regulated by both the level of cyclin B and the phosphorylation state of Cdc2, cyclin accumulation alone was rate-limiting for later cycles, since overexpression of a mitotic cyclin after the first cycle caused cell cycle acceleration. The activity of Cdc2 closely paralleled the accumulation of cyclin B2, but cell cycle acceleration caused by cyclin B overexpression was not associated with elevation of Cdc2 activity to higher than metaphase levels. Tyrosine phosphorylation of Cdc2, absent during cycles 2-12, reappeared at the midblastula transition coincident with the disappearance of cyclin E1. Cyclin A1 disappeared later, at the beginning of gastrulation. Our results suggest that the timing of the cell cycle in the Xenopus embryo evolves from regulation by accumulation of mitotic cyclins to mechanisms involving periodic G1 cyclin expression and inhibitory tyrosine phosphorylation of Cdc2.     

Transcriptional regulation of the rat poly(ADP-ribose) polymerase gene by Sp1

BACKGROUND: Exogenous surfactant therapy of lung donors improves the preservation of normal canine grafts. The current study was designed to determine whether exogenous surfactant can mitigate the damage in lung grafts induced by mechanical ventilation before procurement. METHODS AND RESULTS: Five donor dogs were subjected to 8 hours of mechanical ventilation (tidal volume 45 ml/kg). This produced a significant decrease in oxygen tension (p = 0.007) and significant increases in bronchoscopic lavage fluid neutrophil count (p = 0.05), protein concentration (p = 0.002), and the ratio of poorly functioning small surfactant aggregates to superiorly functioning large aggregates (p = 0.02). Five other animals given instilled bovine lipid extract surfactant and undergoing mechanical ventilation in the same manner demonstrated no significant change in oxygen tension values, lavage fluid protein concentration, or the ratio of small to large aggregates. All 10 lung grafts were then stored for 17 hours at 4 degrees C. Left lungs were transplanted and reperfused for 6 hours. After 6 hours of reperfusion the ratio of oxygen tension to inspired oxygen fraction was 307 +/- 63 mm Hg in lung grafts administered surfactant versus 73 +/- 14 mm Hg in untreated grafts (p = 0.007). Furthermore, peak inspired pressure was significantly (p < 0.05) lower in treated animals from 90 to 360 minutes of reperfusion. Analysis of lavage fluid of transplanted grafts after reperfusion revealed small to large aggregate ratios of 0.17 +/- 0.04 and 0.77 +/- 0.17 in treated versus untreated grafts, respectively (p = 0.009). CONCLUSIONS: Instillation of surfactant before mechanical ventilation reduced protein leak, maintained a low surfactant small to large aggregate ratio, and prevented a decrease of oxygen tension in donor animals. After transplantation, surfactant-treated grafts had superior oxygen tension values and a higher proportion of superiorly functioning surfactant aggregate forms in the air space than untreated grafts. Exogenous surfactant therapy can protect lung grafts from ventilation-induced injury and may offer a promising means to expand the donor pool.     

Calcium, protease action, and the regulation of the cell cycle

New layered matrix systems designed for zero-order sustained release are presented. The devices consist of a hydrophobic middle layer and press-coated hydrophillic and/or hydrophobic barrier layer(s). The systems overcome the inherent disadvantage of non-linear release associated with diffusion-controlled matrix devices by providing additional releasing surface with time to compensate for the decreasing release rate. The in vitro release of pseudoephedrine hydrochloride as a model compound from the matrices was examined. Modulation of release from a selected matrix containing aminophylline was evaluated in a crossover study using nine beagle dogs. Preliminary in vitro/in vivo correlation was also studied. The systems described in this paper can potentially be scaled up to commercial production.