Cellular & molecular biology research最新文献

Platelet actin binding protein (ABP) as isolated from human platelets exists in at least four phosphorylated forms which we have designated ABP-0, ABP-1, ABP-2, and ABP-3 whose phosphate content ranges from 18 (ABP-0) to 40 (ABP-3) moles Pi/mole ABP. These forms differ in their resistance to calpain cleavage and ability to cross-link F-actin with ABP-3 being the best in each of these properties. Attempts to phosphorylate ABP-1, two or three with protein kinase C (PKC) were unsuccessful except if the proteins were pretreated with Escherichia coli alkaline phosphatase. All of the forms could be phosphorylated with cAMP-dependent kinase (PKA) and subsequent resistance to calpain cleavage conferred. Phosphorylation/dephosphorylation of ABP may be an important regulatory mechanism by which the cytoskeletal architecture is stabilized or transformed.

Basic Fibroblast Growth Factor-2 (FGF-2) promotes G1 to S transition of quiescent sparse adult bovine aortic endothelial cells. In addition to signal transduction through interaction with tyrosine kinase high affinity receptor, FGF-2 is translocated to the nucleus and accumulated into the nucleolus. These data suggest that FGF-2 functions directly in nuclear events. In vivo, correlations were established between the entrance of FGF-2 into the nucleus and an increase in rDNA transcription and in protein phosphorylation. In vitro, in experiments carried out with nuclei isolated from quiescent cells, addition of FGF-2 increases rDNA transcription by a factor of 5 and also increases protein phosphorylation. Nucleolin, a factor involved in control of rDNA transcription is preferentially phosphorylated. It has been shown that nucleolin and other factors implicated in rDNA transcription are substrates of protein kinase CKII. Using purified kinase CKII and nucleolin in an in vitro phosphorylation assay, we have shown that FGF-2 activates the protein kinase activity. These results suggest that FGF-2 could act as an activator of rDNA transcription through interactions with the protein kinase CKII.

Infection with the macaque strain of the simian immunodeficiency virus (SIVmac) induces simian immunodeficiency syndrome in rhesus macaques. This report describes the isolation and identification of antigenic variants of SIVmac in one of the infected monkeys (macaque #22803). Eight naive rhesus monkeys were inoculated with a titered viral stock of the molecularly cloned SIVmac239. Standard serological analysis revealed that all but two were seroconverted. Western blot analysis confirmed the seronegativity of macaque #22803. In addition, sera recovered from this monkey were not able to neutralize the parent SIVmac239. However, virus could be isolated from all of the infected animals, including macaque #22803. Sera recovered were reactive to the autologous virus. The results suggest that the virus from macaque #22803 may have undergone extensive antigenic shift in vivo. To test this hypothesis, a portion of the gag gene was amplified by the polymerase chain reaction (PCR), cloned, and sequenced. Sequence analysis revealed amino acid changes that were clustered between amino acids 200-245. Evaluation of the possible selective pressures contributing to the observed viral mutation revealed that in comparison with the other SIVmac239-infected monkeys, macaque #22803 produced an unusually high T cell proliferative response toward mitogen stimulation before infection, and continued to display a persistently high plasma viremia titer after infection.

Previous studies have shown that the expression of some human HOX genes can be induced by retinoic acid (RA) in cultured embryonal carcinoma (EC) cells. However, the mechanisms for the regulation of HOX gene expression by RA are still unclear. We have examined the effects of RA on the human MSX-1 (formerly named HOX-7) gene expression in cultured EC cells (NT2/D1). Furthermore, we have cloned and characterized the human MSX-1 promoter and analyzed the activities of the promoter in response to RA. Our results demonstrate that transcription of human MSX-1 is activated by RA in cultured EC cells. This activation is dose and time responsive. The MSX-1 promoter was shown to be TATA-box independent and able to promote transcription in RA-treated EC cells. DNase-I footprinting studies revealed protection of several GAGA factor binding sites and an NF-kappa B site upstream to the transcription start site by nuclear extracts prepared from EC cells. A downstream sequence was differentially protected by the nuclear extract from RA treated cells. This differential binding of the sequence with the nuclear extract was further confirmed by gel shift assays. This sequence confers to a heterologous promoter with the ability to respond to RA induction. Point mutation within this DNA fragment abolished the binding of the fragment to the nuclear extract and the response of this element in a heterologous promoter to RA induction. Deletion of this enhancer element together with the adjacent NF-kappa B and GAGA sites abolished the ability of the promoter to direct transcription in RA-treated EC cells. However, removal of a downstream DNA fragment from the promoter endowed the promoter with the ability to direct transcription in RA-untreated cells. Taken together, both positive and negative regulatory cis-elements are involved in the regulation of the MSX-1 promoter and coordinate to control the gene expression.

In enteric bacteria, the transcription of the Ntr regulon is regulated by a signal transduction system that measures and transmits information on the nitrogen status of the cell. Four of the components of this signal transduction apparatus have been previously identified, and the roles of these are known, to a first approximation, from studies with purified components. The sensor is a uridylyltransferase/uridylyl-removing enzyme (UTase/UR) that controls the uridylylation state of the PII protein. PII indirectly regulates the transcription of the Ntr regulon by acting through the kinase/phosphatase protein NRII. In the absence of unmodified PII, NRII autophosphorylates on a histidine residue, and these phosphoryl groups are transferred to the transcription factor NRI, resulting in the conversion of NRI to the form able to activate transcription. In the presence of PII and NRII, NRI approximately P is rapidly dephosphorylated, preventing the activation of Ntr transcription. This PII-dependent dephosphorylation of NRI approximately P is referred to as the regulated phosphatase activity. In this report, we describe improved methods for the purification of the UTase/UR and PII, and the crystallization of PII. We also present improved methods for the assay of the activities of the UTase/UR protein and PII. The results of our assays indicate that purified PII is effective in eliciting the regulated phosphatase activity, but does not affect the autophosphorylation of NRII or affect the transfer of phosphoryl groups from NRII approximately P to NRI. In addition, we demonstrate that the elicitation of the regulated phosphatase activity by PII is strongly dependent on the ratio of NRI approximately P to NRI, and that the isolated N-terminal domain of NRI, once phosphorylated, is dephosphorylated by the regulated phosphatase activity.

In the Gram-positive bacterium Bacillus subtilis, the developmental process of spore formation occurs in response to nutrient deprivation and requires the generation of two different cell types with distinct programs of gene expression. Entry into sporulation is regulated primarily by activation (phosphorylation) of the transcription factor encoded by spo0A. The phosphorylation state of Spo0A is controlled by a multi-component phospho-transfer pathway and by at least one phosphatase. Recent experiments indicate that several intracellular conditions that decrease the fidelity of chromosome transmission inhibit production of Spo0A approximately P. These conditions include inhibition of DNA replication, DNA damage, and some alterations in the chromosome partitioning and cell division machinery. Coupling accumulation of a critical level of Spo0A approximately P to these conditions seems to serve as a developmental checkpoint to ensure that cells do not attempt to sporulate unless they are able to provide an intact, undamaged chromosome for each of the two cell types needed for sporulation.

We have previously described a Chironomus tentans nuclear 42 kDa phosphoprotein preferentially associated with transcriptionally active chromatin. In an attempt to purify and identify the kinase responsible for the phosphorylation of the 42 kDa protein, a casein-phosvitin affinity chromatography was used. Unexpectedly, in the eluted kinase fraction, a novel 42 kDa casein kinase, designated protein kinase CK42, with a kinase activity similar, but not identical, to protein kinase CKII, could be identified. In other studies, a nuclear protein that comigrates with protein kinase CK42 in electrophoresis and is capable to bind different gene promoters in single-stranded forms in a sequence-selective manner was found. The observations that both protein kinase and ssDNA-binding activities could be ascribed to a 42 kDa protein raised the possibility that the ssDNA-binding 42 kDa phosphoprotein is a protein kinase. By specific ssDNA-binding affinity chromatography, using a biotinylated oligodeoxyribonucleotide promoter probe and Streptavidine-agarose matrix, evidence that both activities arise from the same protein molecules was obtained. The similarity in the enzyme activities between protein kinase CK42 and CKII raised the question of whether the former was an alpha subunit of the latter. To provide an answer to this issue, CKII, isolated and purified from an epithelial cell line of C. tentans, was characterized and compared with protein kinase CK42 purified from the same cell system. Like other purified CKII preparations, CKII from Chironomus is able to use ATP or GTP for phosphorylation of casein and phosvitin, and its activity is strongly inhibited by heparin and the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). However, the heparin and DRB sensitivities of protein kinase CKII were substantially higher than those of the protein kinase CK42. Due to their differential solubilities in NaCl and (NH4)2SO4 solutions, individual alpha and beta subunit pools of CKII could be detected. More than 80% of the nuclear alpha subunit was insoluble in 0.35 M NaCl, while all individual beta subunit were solubilized under the same conditions suggesting that a major portion of the nuclear CKII alpha subunit does not form heterooligomeric structures with the beta subunit, but binds tightly to nuclear components, probably to chromatin. The biochemical and immunological data taken together strongly suggest that CK42 is a novel DNA-binding protein kinase that is not the alpha subunit of CKII.

Polyamines have been reported to stimulate casein kinase 2 (CK2) in vitro. We have shown that the phosphorylation of different substrates is diversely stimulated by basic effectors and that the responsiveness of CK2 activity may be influenced by the overall conformation of the protein substrate but also by a specific interaction with the enzyme itself. Our data show that native hetero tetrameric CK2 is a spermine binding protein and a spermine binding site was identified in the N-terminal region of the beta subunit of CK2. We found that recombinant CK2 undergoes a progressive polymerization in low salt conditions, giving rise to three different polymer structures. A ring-like structure formed by the association of four protomers alpha 2 beta 2 was characterized by gel filtration, sucrose density gradient analysis and electron microscopy. Polyamines like spermine, when added under conditions where the enzyme preparation contains a mixture of various oligomers, could trigger their dissociation and their interconversion in the fully active ring structure. These results suggest that in the presence of positive effectors like polyamines, CK2 adopts a ring-like structural organization which may represent the active state of this kinase.