Identification of a cellular 110 000-Da protein substrate for the insulin-receptor kinase.

Addition of insulin to wheat-germ-lectin-purified glycoproteins derived from rat hepatocytes or rabbit brown adipose tissue results in the increased phosphorylation of a Mr-110 000 protein. This naturally occurring glycoprotein appears as a monomeric structure and is not part of the insulin receptor itself, since it is not immunoprecipitated by highly specific antibodies to insulin receptor. Phosphorylation of the Mr-110 000 protein and autophosphorylation of the receptor beta-subunit (Mr 95 000) are stimulated by insulin in a remarkably similar dose-dependent fasion, with half-maximal stimulation at 1 nM-insulin. Further, kinetic studies suggest that the phosphorylation of the Mr-110 000 protein occurs after autophosphorylation of the insulin-receptor kinase. In conclusion, the present identification of an endogenous substrate for the insulin-receptor kinase could suggest that some, if not all, effects of insulin may be mediated through activation of this kinase.     

Two different protein kinase activities are associated with the insulin receptor.

In intact rat hepatocytes insulin stimulates the phosphorylation of the beta-subunit of its receptor exclusively on serine residues, which are also phosphorylated in the absence of insulin. In contrast, in partially purified insulin receptors derived from these same cells and in highly purified insulin receptors obtained by immunoprecipitation with anti-receptor antibodies, the receptor beta-subunit is phosphorylated solely on tyrosine residues. For both cell-free systems, insulin's stimulatory action on receptor phosphorylation leads to an increase in phosphotyrosine. When partially purified receptors were used to phosphorylate two exogenous substrates, casein and histone, insulin was found to stimulate the phosphorylation of both tyrosine and serine. However, the basal and insulin-stimulated kinase activity of immunoprecipitated receptors was only tyrosine-specific. From these observations we propose that the insulin-receptor complex consists of two different insulin-stimulatable kinase activities: (1) a tyrosine-specific kinase, which is a constituent of the insulin-receptor structure and whose activation is likely to be the first post-binding event in insulin action; and (2) a serine-specific kinase, which is closely associated with the receptor in the cell membrane.     

Cloning, sequence analysis and expression of the F1F0-ATPase beta-subunit from wine lactic acid bacteria

The nucleotide sequences of the genes encoding the F1F0-ATPase beta-subunit from Oenococcus oeni, Leuconostoc mesenteroides subsp. mesenteroides, Pediococcus damnosus, Pediococcus parvulus, Lactobacillus brevis and Lactobacillus hilgardii were determined. Their deduced amino acid sequences showed homology values of 79-98%. Data from the alignment and ATPase tree indicated that O. oeni and L. mesenteroides subsp. mesenteroides formed a group well-separated from P. damnosus and P. parvulus and from the group comprises L. brevis and L. hilgardii. The N-terminus of the F1F0-ATPase beta-subunit of O. oeni contains a stretch of additional 38 amino acid residues. The catalytic site of the ATPase beta-subunit of the investigated strains is characterized by the two conserved motifs GGAGVGKT and GERTRE. The amplified atpD coding sequences were inserted into the pCRT7/CT-TOPO vector using TA-cloning strategy and transformed in Escherichia coli. SDS-PAGE and Western blot analyses confirmed that O. oeni has an ATPase beta-subunit protein which is larger in size than the corresponding molecules from the investigated strains.     

Pertussis toxin-sensitive G-proteins are not involved in activation of T-lymphocytes.

Multiple effects of pertussis toxin (PT) on Jurkat T-cells can be distinguished on the basis of their dose-response and their kinetics. High concentrations of PT deliver to cells an activating signal resulting in a rapid rise in [Ca2+]i followed by IL-2 synthesis. This activation is accompanied (within 2 h) by a down-regulation of the CD3/TCR complex from the cell surface. Cells then become refractory towards stimulation by CD3 mAb or PHA. All these effects, referred to as 'mitogenic effects', present the same dose-response curves with an EC50 of 0.5 micrograms/ml. Short term effects (PT-induced Ca2+ movements, down-regulation of CD3/TCR complex and inhibition of PHA and CD3-induced Ca2+ signal) are observed under conditions where no PT-induced ADP-ribosylation can be detected. In contrast, ADP-ribosylation of the 40,000 alpha-subunit of G-proteins requires a sustained (18 h) incubation of intact cells in the presence of low concentration (EC50 = 0.3 ng/ml) of PT. Dose-response curves for PT-dependent ADP-ribosylation and mitogenic effects are separated by three orders of magnitude. Covalent modification of G-protein has no effect on CD3-induced increase in [Ca2+]i and IL-2 synthesis induced by a combination of phorbol ester and either CD3 mAb, PHA or calcium ionophore. These data indicate that transduction of the mitogenic signal does not involve a PT-sensitive G-protein. Furthermore, inhibition of mitogenic signals following PT treatment results from a PT-induced activation leading to a down-regulation of the CD3/T cell receptor complex.     

Two types of receptor for insulin-like growth factors in mammalian brain.

Two types of receptor for insulin-like growth factors (IGFs) have been identified on adult rat and human brain plasma membranes by competitive binding assay, affinity labelling, receptor phosphorylation and interaction with antibodies to insulin receptors. The type I IGF receptor consists of two species of subunits: alpha-subunits (mol. wt. approximately 115 000), which bind IGF I and IGF II with almost equal affinity and beta-subunits (mol. wt. approximately 94 000), the phosphorylation of which is stimulated by IGFs. The alpha-subunits of type I IGF receptors in brain and other tissues differ significantly (mol. wt. approximately 115 000 versus 130 000), whereas the beta-subunits are identical (mol. wt. approximately 94 000). The type II IGF receptor in brain is a monomer (mol. wt. approximately 250 000) like that in other tissues. Two antibodies to insulin receptors, B2 and B9, interact with type I but not with type II IGF receptors. B2 is more potent than B9 in inhibiting IGF binding and in immunoprecipitating type I IGF receptors, in contrast to their almost equal effects on insulin receptors. This pattern is characteristic for IGF receptors in other cells. The presence of two types of IGF receptor in mammalian brain suggests a physiological role of IGFs in regulation of nerve cell function and growth. Since IGF II, but not IGF I, is present in human brain, we propose that IGF II interacts with both types of IGF receptor to induce its biological actions.     

Insulin receptors in the mammalian central nervous system: binding characteristics and subunit structure

Insulin receptors in rat and human central nervous system have been identified by binding of 125I-insulin on purified synaptic plasma membranes; affinity labelling of receptors by chemical cross-linking 125I-insulin; or phosphorylation of receptors with [gamma-32P]ATP. Brain insulin receptors showed significant differences in their binding characteristics and subunit structure when compared with receptors in other tissues like adipose and liver cells: absence of negatively cooperative interactions; a distinct binding specificity i.e. porcine proinsulin, coypu insulin and insulin-like growth factor I and II showed 2-5 times higher binding affinity in brain than in other cell types; a smaller molecular size of the brain receptor alpha-subunit than in other tissues (Mr approximately 115,000 instead of 130,000). In contrast, the size (Mr approximately 94,000) and function of the insulin receptor beta-subunit kinase was identical with that described in other cells. We conclude, that insulin receptors in mammalian brain represent a receptor subtype which may mediate growth rather than metabolic activity of insulin.     

Genome-wide MicroRNA Expression Profiles in COPD: Early Predictors for Cancer Development

Chronic obstructive pulmonary disease(COPD) significantly increases the risk of developing cancer. Biomarker studies frequently follow a case-control set-up in which patients diagnosed with a disease are compared to controls. Longitudinal cohort studies such as the COPD-centered German COPD and SYstemic consequences-COmorbidities NETwork(COSYCONET) study provide the patient and biomaterial base for discovering predictive molecular markers. We asked whether microRNA(miRNA) profiles in blood collected from COPD patients prior to a tumor diagnosis could support an early diagnosis of tumor development independent of the tumor type. From 2741 participants of COSYCONET diagnosed with COPD, we selected 534 individuals including 33 patients who developed cancer during the follow-up period of 54 months and 501 patients who did not develop cancer, but had similar age, gender and smoking history. Genome-wide miRNA profiles were generated and evaluated using machine learning techniques. For patients developing cancer we identified nine miRNAs with significantly decreased abundance(two-tailed unpaired t-test adjusted for multiple testing P 0.05), including members of the miR-320 family. The identified mi RNAs regulate different cancer-related pathways including the MAPK pathway(P = 2.3 x 10~(-5)). We also observed the impact of confounding factors on the generated miRNA profiles, underlining the value of our matched analysis. For selected miRNAs, qRT-PCR analysis was applied to validate the results. In conclusion, we identified several miRNAs in blood of COPD patients, which could serve as candidates for biomarkers to help identify COPD patients at risk of developing cancer.     

Activation of human T cells is associated with tyrosine phosphorylation of several cellular proteins

Human T lymphocytes are activated to proliferate after triggering the T Cell Antigen Receptor Complex. CD3-Ti, with either antigen, mitogenic lectins or monoclonal antibodies against its different subunits. Stimulation of Jurkat leukemic human T cells with anti-CD3 or anti-Ti monoclonal antibodies was found to induce, within 1 min, an increase in the phosphorylation of a set of cellular proteins that can be precipitated with anti-phosphotyrosine antibodies. Seven phosphotyrosine-containing proteins were separated with respective mol. wt of 21, 25, 38, 55, 70, 80 and 110 kDa, among which the 38 kDa species is predominant. Moreover, incubation of Jurkat T cells with sodium orthovanadate, a potent inhibitor of phosphotyrosine protein-phosphatases, was found to potentiate the effects of anti-CD3 mAb on tyrosine phosphorylation. In addition vanadate also induced IL-2 secretion in Jurkat cells when associated with the phorbol ester TPA, further demonstrating the importance of these phosphorylation reactions in the process of T cell activation. Our results therefore allow us to identify several protein substrates of a tyrosine kinase activity, whose stimulation appears to be an early event in human T cell activation through the antigen receptor pathway.