New model of ErbB-2 over-expression in human mammary luminal epithelial cells.

The ErbB-2 receptor has been strongly implicated in the development of breast cancer. To establish a new model system to investigate the role of erbB-2 in tumorigenesis of the breast, the conditionally immortalised human mammary luminal epithelial cell line HB4a was transfected with erbB-2 cDNA. Biological and biochemical characterisation of the resulting cell lines demonstrated that high levels of ErbB-2 expression were sufficient to cause transformation in vitro but did not cause tumours in vivo. Transformation by overexpression of ErbB-2 correlated with ligand-independent tyrosine phosphorylation of ErbB-2 and the adaptor protein Shc. Over-expression of ErbB-2 also resulted in the ligand-independent constitutive association between Shc and another adaptor protein, Grb2, indicating that receptor activation was sufficient to activate downstream signalling pathways. Using the model described, it was found that elevation of ErbB-2 expression levels caused marked quantitative and qualitative alterations in responses to the ligands epidermal growth factor and heregulin. Data indicate a central role for ErbB-2 in mediating the responses induced by these ligands and suggest that these altered ligand-dependent responses play an important role in tumorigenesis in vivo.

Integrin-linked kinase regulates phosphorylation of serine 473 of protein kinase B by an indirect mechanism.

The serine threonine kinase protein kinase B regulates cellular activities as diverse as glycogen metabolism and apoptosis. Full activation of protein kinase B requires 3-phosphoinositides and dual phosphorylation on threonine-308 and serine-473. CaM-K kinase and 3-phosphoinositide dependent-kinase-1 phosphorylate threonine-308. Integrin-linked kinase reportedly phophorylates serine-473. Consistent with this, in a model COS cell system we show that expression of wild-type integrin-linked kinase promotes the wortmannin sensitive phosphorylation of serine-473 of protein kinase B and its downstream substrates, and inhibits C2-ceramide induced apoptosis. In contrast, integrin-linked kinase mutated in a lysine residue critical for function in protein kinases is inactive in these experiments, and furthermore, acts dominantly to block serine-473 phosphorylation induced by ErbB4. However, alignment of analogous sequences from different species demonstrates that integrin-linked kinase is not a typical protein kinase and identifies a conserved serine residue which potentially regulates kinase activity in a phosphorylation dependent manner. Mutation of this serine to aspartate or glutamate, but not alanine, in combination with the inactivating lysine mutation restores integrin-linked kinase dependent phosphorylation of serine-473 of protein kinase B. These data strongly suggest that integrin-linked kinase does not possess serine-473 kinase activity but functions as an adaptor to recruit a serine-473 kinase or phosphatase.

The induction of apoptosis in human mammary luminal epithelial cells by expression of activated c-neu and its abrogation by glucocorticoids.

The effects of expressing neu-T, a mutated constitutively activated form of c-neu, have been examined in the non-transformed conditionally immortalised human mammary luminal epithelial cell line, HB4a. A variant cell line, N4.1, which expressed neu-T, showed evidence of transformation, including partial loss of growth factor dependence and acquisition of anchorage-independent growth, but failed to give rise to tumours in nude mice, indicating that expression of neu-T alone was probably insufficient to cause tumorigenic progression to a full malignant phenotype. During characterisation of the N4.1 cell line, it was observed that under conditions of serum deprivation, it underwent apoptotic cell death, as demonstrated by light microscopy, flow cytometry and DNA gel electrophoresis. The induction of apoptotic cell death in the N4.1 cell line by serum deprivation was abrogated specifically by the addition of steroids with glucocorticoid activity but not any peptide growth factors studied. This study shows the induction of apoptosis by serum deprivation, and its abrogation by glucocorticoids occurring in human mammary luminal epithelial cells transformed by expression of neu-T, and implicates the involvement of receptor protein tyrosine kinases in an apoptotic signalling pathway in this cell type.

The GTPase dynamin binds to and is activated by a subset of SH3 domains.

Src homology 3 (SH3) domains have been implicated in mediating protein-protein interactions in receptor signaling processes; however, the precise role of this domain remains unclear. In this report, affinity purification techniques were used to identify the GTPase dynamin as an SH3 domain-binding protein. Selective binding to a subset of 15 different recombinant SH3 domains occurs through proline-rich sequence motifs similar to those that mediate the interaction of the SH3 domains of Grb2 and Abl proteins to the guanine nucleotide exchange protein, Sos, and to the 3BP1 protein, respectively. Dynamin GTPase activity is stimulated by several of the bound SH3 domains, suggesting that the function of the SH3 module is not restricted to protein-protein interactions but may also include the interactive regulation of GTP-binding proteins.

Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit.

Purified bovine brain phosphatidylinositol 3-kinase (Pl3-kinase) is composed of 85 kd and 110 kd subunits. The 85 kd subunit (p85 alpha) lacks Pl3-kinase activity and acts as an adaptor, coupling the 110 kd subunit (p110) to activated protein tyrosine kinases. Here the characterization of the p110 subunit is presented. cDNA cloning reveals p110 to be a 1068 aa protein related to Vps34p, a S. cerevisiae protein involved in the sorting of proteins to the vacuole. p110 expressed in insect cells possesses Pl3-kinase activity and associates with p85 alpha into an active p85 alpha-p110 complex that binds the activated colony-stimulating factor 1 receptor. p110 expressed in COS-1 cells is catalytically active only when complexed with p85 alpha.

Molecular characterization of the oligopeptide permease of Salmonella typhimurium.

The oligopeptide permease (Opp) of Salmonella typhimurium is a periplasmic binding protein-dependent transport system and handles any peptides containing from two to five amino acid residues. Opp plays an important nutritional role and is also required for the recycling of cell wall peptides. We have determined the nucleotide sequence of the opp operon. In addition to the four opp genes identified previously by genetic means (oppABCD) a fifth gene, oppF, is shown to be cotranscribed as part of the opp operon. Using reverse genetics, we show that oppF also encodes an essential component of the Opp transport system. The five proteins, OppABCDF, are shown to be the only proteins required for Opp function. Regulation of opp expression and of the differential expression of genes within the operon is investigated. We have devised a simple means of constructing lacZ gene fusions to any S. typhimurium chromosomal gene in vivo, using derivatives of bacteriophage Mu. Using this procedure, opp-lacZ gene fusions were selected. The resultant Opp-LacZ hybrid proteins were used to show that OppB, OppC and OppD are membrane-associated proteins. A detailed comparison of the Opp components with those of other binding protein-dependent transport systems provides insight into the mechanisms and evolution of these transport systems.

Stabilization of translationally active mRNA by prokaryotic REP sequences.

The REP sequence is a highly conserved inverted repeat that is present in about 25% of all E. coli transcription units. We show that the REP sequence can stabilize upstream RNA, independently of any other sequences, by protection from 3'-5' exonuclease attack. The REP sequence is frequently responsible for the differential stability of different segments of mRNA within an operon. We demonstrate that REP-stabilized mRNA can be translated in vivo and that cloning the REP sequence downstream of a gene can increase protein synthesis. This provides direct evidence that alterations in mRNA stability can play a role in determining bacterial gene expression. The implications of these findings for the mechanisms of mRNA degradation and for the role of RNA stability in the regulation of gene expression are discussed.

Peptide transport in Salmonella typhimurium: molecular cloning and characterization of the oligopeptide permease genes.

The oligopeptide permease is encoded by at least four genes which are transcribed as a single operon. We cloned and characterized this operon from Salmonella typhimurium, as well as the flanking genes, tonB, ana and a new gene, cwd, which affects cell wall synthesis. We correlated the physical map of opp DNA with a detailed genetic map of the opp operon and the individual opp genes were accurately located with respect to various restriction sites by Southern blotting. The region of the chromosome near opp was found to be highly unstable with deletions arising at a highly frequency. The operon also contains hot-spots for IS1 and IS5 insertions.

A family of related ATP-binding subunits coupled to many distinct biological processes in bacteria.

Many biological processes are coupled to ATP hydrolysis. We describe here a class of closely related ATP-binding proteins, from several bacterial species, which are associated with a variety of cellular functions including membrane transport, cell division, nodulation in Rhizobium and haemolysin export. These proteins comprise a family of structurally and functionally related subunits which share a common evolutionary origin, bind ATP and probably serve to couple ATP hydrolysis to each of these biological processes. This finding suggests a specific role for ATP in cell division, nodulation during nitrogen fixation and protein export, and allows us to assign a probable function to one of the protein components from each of these systems.

Peptide uptake by Salmonella typhimurium. The periplasmic oligopeptide-binding protein.

The uptake of most peptides, including many peptide antibiotics, by the oligopeptide permeases of Escherichia coli and Salmonella typhimurium requires the function of a specific peptide-binding protein (the OppA protein) located within the periplasm. The OppA protein is the largest and most abundant periplasmic substrate-binding protein known and has an unusually broad substrate-binding specificity. The OppA protein has been purified to homogeneity and anti-OppA antibodies have been raised. The sequence of the OppA protein has been deduced from the nucleotide sequence of the oppA gene. This protein is unrelated to any other known periplasmic substrate-binding protein, either immunologically or in its amino acid sequence. The role of this protein in peptide transport is discussed.

Nucleotide binding by membrane components of bacterial periplasmic binding protein-dependent transport systems.

Bacterial periplasmic binding protein-dependent transport systems require the function of a specific substrate-binding protein, located in the periplasm, and several membrane-bound components. We present evidence for a nucleotide-binding site on one of the membrane components from each of three independent transport systems, the hisP, malK and oppD proteins of the histidine, maltose and oligopeptide permeases, respectively. The amino acid sequence of the oppD protein has been determined and this protein is shown to share extensive homology with the hisP and malK proteins. Three lines of evidence lead us to propose the existence of a nucleotide-binding site on each of these proteins. A consensus nucleotide-binding sequence can be identified in the same relative position in each of the three proteins. The oppD protein binds to a Cibacron Blue affinity column and can be eluted by ATP but not by CTP or NADH. The oppD protein is labelled specifically by the nucleotide affinity analogue 5'-p-fluorosulphonylbenzoyladenosine. The identification of a nucleotide-binding site provides strong evidence that transport by periplasmic binding protein-dependent systems is energized directly by the hydrolysis of ATP or a closely related nucleotide. The hisP, malK and oppD proteins are thus responsible for energy-coupling to their respective transport systems.