We have identified in the Streptococcus pneumoniae genome sequence a two-component system (TCS13, Blp [bacteriocin-like peptide]) which is closely related to quorum-sensing systems regulating cell density-dependent phenotypes such as the development of genetic competence or the production of antimicrobial peptides in lactic acid bacteria. In this study we present evidence that TCS13 is a peptide-sensing system that controls a regulon including genes encoding Blps. Downstream of the Blp TCS (BlpH R) we identified open reading frames (blpAB) that have the potential to encode an ABC transporter that is homologous to the ComA/B export system for the competence-stimulating peptide ComC. The putative translation product of blpC, a small gene located downstream of blpAB, has a leader peptide with a Gly-Gly motif. This leader peptide is typical of precursors processed by this family of transporters. Microarray-based expression profiling showed that a synthetic oligopeptide corresponding to the processed form of BlpC (BlpC*) induces a distinct set of 16 genes. The changes in the expression profile elicited by synthetic BlpC* depend on BlpH since insertional inactivation of its corresponding gene abolishes differential gene induction. Comparison of the promoter regions of the blp genes disclosed a conserved sequence element formed by two imperfect direct repeats upstream of extended ؊10 promoter elements. We propose that BlpH is the sensor for BlpC* and the conserved sequence element is a recognition sequence for the BlpR response regulator.Signaling mechanisms controlling multicellular behavior of bacteria have attracted much attention in current research. In gram-negative bacteria, homoserine-lactone-based communication systems are prominent. Research in this area led to the term "quorum sensing" for phenomena that are controlled by cell density (12). In gram-positive bacteria, quorum sensing is accomplished by signaling systems that depend on the secretion and sensing of small peptides (11,19). At least two different mechanisms for sensing the presence of pheromone-like peptides are known (21). The first involves import of the peptide and interaction with an intracellular factor (22); the second involves binding to the extracellular portion of a membranebound histidine kinase. This leads to the autophosphorylation of the kinase and subsequent activation, e.g., phosphorylation of a cognate response regulator that mediates changes in gene expression. Quorum-sensing systems regulate a plethora of cellular functions. In Staphylococcus aureus, the AgrC-AgrAsystem is involved in the density-dependent regulation of virulence (18). In Lactobacillus strains, the production of bacteriocins is dependent on peptide-regulated two-component systems (TCS) (4, 10). In Streptococcus pneumoniae, the development of genetic competence (the natural ability to take up DNA) has been shown to be regulated by the comC-DE system (29).The com system of S. pneumoniae was the first quorumsensing system for which a biological function was defined. ...

Protein tyrosine kinases participate in the transduction and modulation of signals that regulate proliferation and differentiation of cells. Excessive or deregulated protein tyrosine kinase activity can cause malignant transformation. The catalytic activity of the T cell protein tyrosine kinase p56lck is normally suppressed by phosphorylation of a carboxyl‐terminal tyrosine, Tyr‐505, by another cellular protein tyrosine kinase. Here we characterize a human cytosolic 50 kDa protein tyrosine kinase, p50csk, which specifically phosphorylates Tyr‐505 of p56lck and a synthetic peptide containing this site. Phosphorylation of Tyr‐505 suppressed the catalytic activity of p56lck. We suggest that p50csk negatively regulates p56lck, and perhaps other cellular src family kinases.

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An Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL was engineered and has been successfully used to produce large quantities of the recombinant human protein-tyrosine kinase pSOcsk. The co-overproduction of the two chaperones with p5Ocsk results in increased solubility of the kinase and allows purification of milligram amounts of active enzyme. Analysis of the purified protein by SDS/polyacrylamide gel electrophoresis reveals a single band with an apparent molecular mass of 50 kDa, indicating that recombinant human p5Ocsk has been purified to near homogeneity. The purified enzyme displays tyrosine kinase activity as measured by both autophosphorylation and phosphorylation of exogenous substrates. Biochemical properties, including in vitro substrate specificity and enzymatic characteristics of the enzyme, have been assessed and compared with those of members of the Src family of protein-tyrosine kinases. Results indicate that p5Ocsk and p56kk have different substrate specificities and that p59csk and p60csrc have similar kinetic parameters. The

Abstract. Proteins which are inserted and anchored in the membrane of the ER by an uncleaved signalanchor sequence can assume two final orientations. Type I signal-anchor proteins translocate the NH2 terminus across the membrane while type 1I signalanchor proteins translocate the COOH terminus. We investigated the requirements for cytosolic protein components and nucleotides for the membrane targeting and insertion of single-spanning type I signalanchor proteins. Besides the ribosome, signal recognition particle (SRP), GTE and rough microsomes (RMs) no other components were found to be required. The GTP analogue GMPPNP could substitute for GTP in supporting the membrane insertion of IMC-CAT. By using a photocrosslinking assay we show that for secreted, type I and type II signalanchor proteins the presence of both GTP and RMs is required for the release of the nascent chain from the 54-kD subunit of SRP. For two of the proteins studied the release of the nascent chain from SRP54 was accompanied by a new interaction with components of the ER. We conclude that the GTP-dependent release of the nascent chain from SRP54 occurs in an identical manner for each of the proteins studied.