Ndr protein kinase is regulated by phosphorylation on two conserved sequence motifs.

Ndr is a nuclear serine/threonine protein kinase that belongs to a subfamily of kinases implicated in the regulation of cell division and cell morphology. This subfamily includes the kinases LATS, Orb6, Cot-1, and Dbf2. We show here that Ndr is potently activated when intact cells are treated with okadaic acid, suggesting that Ndr is normally held in a state of low activity by protein phosphatase 2A. We mapped the regulatory phosphorylation sites of Ndr protein kinase and found that active Ndr is phosphorylated on Ser-281 and Thr-444. Mutation of either site to alanine strongly reduced both basal and okadaic acid-stimulated Ndr activity, while combined mutation abolished Ndr activity completely. Importantly, each of these sites (and also the surrounding sequences) are conserved in the kinase relatives of Ndr, suggesting a general mechanism of activation for kinases of this subfamily. Ser-281 and Thr-444 are also similar to the regulatory phosphorylation sites in several targets of the phosphoinositide-dependent protein kinase PDK1.(1) However, PDK1 does not appear to function as an upstream kinase for Ndr. Thus, Ndr and its close relatives may operate in a novel signaling pathway downstream of an as-yet-unidentified kinase with specificity similar to, but distinct from, PDK1.

Regulation of protein kinase cascades by protein phosphatase 2A.

Many protein kinases themselves are regulated by reversible phosphorylation. Upon cell stimulation, specific kinases are transiently phosphorylated and activated. Several of these protein kinases are substrates for protein phosphatase 2A (PP2A), and PP2A appears to be the major kinase phosphatase in eukaryotic cells that downregulates activated protein kinases. This idea is substantiated by the observation that some viral proteins and naturally occurring toxins target PP2A and modulate its activity. There is increasing evidence that PP2A activity is regulated by extracellular signals and during the cell cycle. Thus, PP2A is likely to play an important role in determining the activation kinetics of protein kinase cascades.

Calcium regulation of Ndr protein kinase mediated by S100 calcium-binding proteins.

Ndr is a nuclear serine/threonine protein kinase that belongs to a subfamily of kinases identified as being critical for the regulation of cell division and cell morphology. The regulatory mechanisms that control Ndr activity have not been characterized previously. In this paper, we present evidence that Ndr is regulated by EF-hand calcium-binding proteins of the S100 family, in response to changes in the intracellular calcium concentration. In vitro, S100B binds directly to and activates Ndr in a Ca2+-dependent manner. Moreover, Ndr is recovered from cell lysates in anti-S100B immunoprecipitates. The region of Ndr responsible for interaction with Ca2+/S100B is a basic/hydrophobic motif within the N-terminal regulatory domain of Ndr, and activation of Ndr by Ca2+/S100B is inhibited by a synthetic peptide derived from this region. In cultured cells, Ndr is rapidly activated following treatment with Ca2+ ionophore, and this activation is dependent upon the identified Ca2+/S100B-binding domain. Finally, Ndr activity is inhibited by W-7 in melanoma cells overexpressing S100B, but is unaffected by W-7 in melanoma cells that lack S100B. These results suggest that Ndr is regulated at least in part by changes in the intracellular calcium concentration, through binding of S100 proteins to its N-terminal regulatory domain.

Molecular cloning and characterization of a conserved nuclear serine(threonine) protein kinase.

Human, Drosophila melanogaster, and Caenorhabditis elegans cDNA clones encoding homologues of a serine(threonine) protein kinase (EC 2.7.1.37) (designated Ndr protein kinase) have been isolated and sequenced. The human and Drosophila cDNAs predict polypeptides of 54 kDa and 52 kDa, respectively, which share approximately 80% amino acid similarity. Northern analysis of human tissues revealed a ubiquitously expressed 3.9-kb transcript. Recombinant GST-Ndr underwent intramolecular autophosphorylation on serine and threonine residues in vitro but failed to transphosphorylate several standard protein kinase substrates. Transfection of the human cDNA into COS-1 cells resulted in the appearance of an intense nuclear staining in cells analyzed by indirect immunofluorescence; deletion mutagenesis identified a short basic peptide, KRKAETWKRNRR, responsible for the nuclear accumulation of Ndr. Thus, Ndr is a conserved and widely expressed nuclear protein kinase. The closest known relative of this previously uncharacterized kinase is Dbf2, a budding yeast protein kinase required for the completion of nuclear division.

The effect of sub-inhibitory concentrations of chlorhexidine on the proteolytic activity of Bacteroides gingivalis.

Bacteroides gingivalis was grown in the presence of chlorhexidine at concentrations lower than the minimum inhibitory concentration (1.25 mg/l). These sub-inhibitory concentrations were found to stimulate growth in terms of an increase in the number of viable cells, the greatest increase being at a concentration of 0.75 mg/l of chlorhexidine. The total proteolytic activity of the cultures (assayed by means of azocasein hydrolysis) grown in the presence of chlorhexidine and their specific activities (per 10(6) cells) were found to be less than those of the cultures grown in the absence of chlorhexidine. In the case of the trypsin-like activity of the microorganism, a different pattern was found. Thus, although the specific activities of the chlorhexidine-grown cultures were lower than those of the chlorhexidine-free cultures, the total activity in the chlorhexidine-grown cultures was greater.

The use of various handwashing agents to decontaminate gloved hands.

It has been suggested that gloved hands could be washed between patient treatments in the dental surgery and gloves re-used, provided they are undamaged. A series of experiments are described, which evaluate the effectiveness of four handwashing agents at removing defined bacterial inoculae from two types of latex rubber glove, a dedicated dental procedure glove (Regent Biogel D) which has a rough surface and a smooth-surfaced examination glove (Microtouch). The four agents tested were povidone iodine (Betadine), chlorhexidine (Hibiscrub), 60% iso-propyl alcohol, and a detergent triclosan preparation (Kleenex washcream). The Biogel D gloves required slightly shorter washing times to eradicate organisms than the Microtouch gloves. It was found that washing with water alone reduced the organisms on gloves by 300-1000-fold, but a minimum washing time of 20 seconds with povidone iodine or chlorhexidine was required to eradicate all the organisms inoculated, except bacterial spores, from both glove surfaces. Povidone-iodine and chlorhexidine were more effective washing agents than iso-propyl alcohol and triclosan soap.

The effect of chlorhexidine on Streptococcus sanguis biofilms.

The bactericidal effect of chlorhexidine on Streptococcus sanguis biofilms was investigated and compared with its effect on suspensions of the organism. The biofilms were found to be more resistant to the bactericidal effects of chlorhexidine than the bacterial suspensions in terms of the number of surviving organisms. The minimum inhibitory concentration (MIC) being 25 and 50 micrograms/ml, respectively, for the suspension and the 24 h biofilm. The age of the biofilm had a marked effect on the number of bacteria surviving. Older (72 h) biofilms tended to be more resistant to chlorhexidine than younger (24 h) ones, with the MIC being 200 and 50 micrograms/ml, respectively. Addition of blood to the test system greatly reduced the bactericidal effect of the chlorhexidine, increasing the MIC for the 24 h biofilm from 50 to 200 micrograms/ml.