Y-27632, an inhibitor of Rho-associated kinases, prevents tyrosine phosphorylation of focal adhesion kinase and paxillin induced by bombesin: dissociation from tyrosine phosphorylation of p130(CAS).

A rapid increase in tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and Crk-associated substrate (CAS) are prominent early events triggered by many G protein-coupled receptors (GPCRs), but the mechanisms involved remain unclear. Here, we examined whether the Rho-associated protein serine/threonine kinase family (ROCK) is a critical Rho effector in the pathway that links GPCR activation to the tyrosine phosphorylation of FAK, CAS, and paxillin. Treatment of Swiss 3T3 cells with Y-27632, a preferential inhibitor of ROCK, dramatically inhibited the formation of actin stress fibers, the assembly of focal contacts, and the increase in tyrosine phosphorylation of FAK and paxillin induced by bombesin in these cells. Surprisingly, we found that treatment with Y-27632 did not produce any detectable effect on bombesin-elicited CAS tyrosine phosphorylation even at the highest concentrations of Y-27632 tested. HA-1077, a preferential inhibitor of ROCK activity structurally unrelated to Y-27632, also attenuated the increase in the tyrosine phosphorylation of FAK and paxillin but did not affect the tyrosine phosphorylation of CAS induced by bombesin in Swiss 3T3 cells. The results demonstrate that ROCK-dependent tyrosine phosphorylation of FAK and paxillin can be dissociated from a ROCK-independent pathway leading to tyrosine phosphorylation of CAS.

Calyculin-A induces focal adhesion assembly and tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin in Swiss 3T3 cells.

Treatment of intact Swiss 3T3 cells with calyculin-A, an inhibitor of myosin light chain (MLC) phosphatase, induces tyrosine phosphorylation of p125(Fak) in a sharply concentration- and time-dependent manner. Maximal stimulation was 4.2 +/- 2.1-fold (n = 14). The stimulatory effect of calyculin-A was observed at low nanomolar concentrations (<10 nM); at higher concentrations (>10 nM) tyrosine phosphorylation of p125(Fak) was strikingly decreased. Calyculin-A induced tyrosine phosphorylation of p125(Fak) through a protein kinase C- and Ca(2+)-independent pathway. Exposure to either cytochalasin-D or latrunculin-A, which disrupt actin organization by different mechanisms, abolished tyrosine phosphorylation of p125(Fak) in response to calyculin-A. Treatment with high concentrations of platelet-derived growth factor (20 ng/ml) which also disrupt actin stress fibers, completely inhibited tyrosine phosphorylation of p125(Fak) in response to calyculin-A. This agent also induced tyrosine phosphorylation of the focal adhesion-associated proteins p130(Cas) and paxillin. These tyrosine phosphorylation events were associated with a striking increase in the assembly of focal adhesions. The Rho kinase (ROK) inhibitor HA1077 that blocked focal adhesion formation by bombesin, had no effect on the focal adhesion assembly induced by calyculin-A. Thus, calyculin-A induces transient focal adhesion assembly and tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin, acting downstream of ROK.

Gbeta 5gamma 2 is a highly selective activator of phospholipid-dependent enzymes.

In this study, Gbeta specificity in the regulation of Gbetagamma-sensitive phosphoinositide 3-kinases (PI3Ks) and phospholipase Cbeta (PLCbeta) isozymes was examined. Recombinant mammalian Gbeta(1-3)gamma(2) complexes purified from Sf9 membranes stimulated PI3Kgamma lipid kinase activity with similar potency (10-30 nm) and efficacy, whereas transducin Gbetagamma was less potent. Functionally active Gbeta(5)gamma(2) dimers were purified from Sf9 cell membranes following coexpression of Gbeta(5) and Ggamma(2-His). This preparation as well as Gbeta(1)gamma(2-His) supported pertussis toxin-mediated ADP-ribosylation of Galpha(i1). Gbeta(1)gamma(2-His) stimulated PI3Kgamma lipid and protein kinase activities at nanomolar concentrations, whereas Gbeta(5)gamma(2-His) had no effect. Accordingly, Gbeta(1)gamma(2-His), but not Gbeta(5)gamma(2-His), significantly stimulated the lipid kinase activity of PI3Kbeta in the presence or absence of tyrosine-phosphorylated peptides derived from the p85-binding domain of the platelet derived-growth factor receptor. Conversely, both preparations were able to stimulate PLCbeta(2) and PLCbeta(1). However, Gbeta(1)gamma(2-His), but not Gbeta(5)gamma(2-His), activated PLCbeta(3). Experimental evidence suggests that the mechanism of Gbeta(5)-dependent effector selectivity may differ between PI3K and PLCbeta. In conclusion, these data indicate that Gbeta subunits are able to discriminate among effectors independently of Galpha due to selective protein-protein interaction.

Tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin does not require extracellular signal-regulated kinase activation in Swiss 3T3 cells stimulated by bombesin or platelet-derived growth factor.

The experiments presented here were designed to examine the contribution of the extracellular signal-regulated mitogen-activated protein kinases (ERKs) to the tyrosine phosphorylation of the focal adhesion proteins p125(Fak), p130(Cas), and paxillin induced by G protein-coupled receptors (GPCRs) and tyrosine kinase receptors in Swiss 3T3 cells. Stimulation of these cells with bombesin, lysophosphatidic acid (LPA), endothelin, and platelet-derived growth factor (PDGF) led to a marked increase in the tyrosine phosphorylation of these focal adhesion proteins and in ERK activation. Exposure of the cells to two structurally unrelated mitogen-activated protein kinase or ERK kinase (MEK) inhibitors, PD98059 and U0126, completely abrogated ERK activation but did not prevent tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin. Furthermore, different dose-response relationships were obtained for tyrosine phosphorylation of focal adhesion proteins and for ERK activation in response to PDGF. Putative upstream events in the activation of focal adhesion proteins including actin cytoskeletal reorganization and myosin light chain (MLC) phosphorylation were also not prevented by inhibition of ERK activation. Thus, our results demonstrate that the activation of the ERK pathway is not necessary for the increase of the tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin induced by either GPCRs or tyrosine kinase receptors in Swiss 3T3 cells.

Gbetagamma stimulates phosphoinositide 3-kinase-gamma by direct interaction with two domains of the catalytic p110 subunit.

Class I phosphoinositide 3-kinases (PI3Ks) regulate important cellular processes such as mitogenesis, apoptosis, and cytoskeletal functions. They include PI3Kalpha, -beta, and -delta isoforms coupled to receptor tyrosine kinases and a PI3Kgamma isoform activated by receptor-stimulated G proteins. This study examines the direct interaction of purified recombinant PI3Kgamma catalytic subunit (p110gamma) and Gbetagamma complexes. When phosphatidylinositol was used as a substrate, Gbetagamma stimulated p110gamma lipid kinase activity more than 60-fold (EC50, approximately 20 nM). Stimulation was inhibited by Galphao-GDP or wortmannin in a concentration-dependent fashion. Stoichiometric binding of a monoclonal antibody to the putative pleckstrin homology domain of p110gamma did not affect Gbetagamma-mediated enzymatic stimulation, whereas incubation of Gbetagamma with a synthetic peptide resembling a predicted Gbetagamma effector domain of type 2 adenylyl cyclase selectively inhibited activation of p110gamma. Gbetagamma complexes bound to N- as well as C-terminal deletion mutants of p110gamma. Correspondingly, these enzymatically inactive N- and C-terminal mutants inhibited Gbetagamma-mediated activation of wild type p110gamma. Our data suggest that (i) p110gamma directly interacts with Gbetagamma, (ii) the pleckstrin homology domain is not the only region important for Gbetagamma-mediated activation of the lipid kinase, and (iii) Gbetagamma binds to at least two contact sites of p110gamma, one of which is close to or within the catalytic core of the enzyme.

G proteins endogenously expressed in Sf 9 cells: interactions with mammalian histamine receptors.

Expression of functionally active mammalian histamine H1- and H2-receptors was recently demonstrated in Sf 9 cells. Either receptor elicited phosphoinositide degradation leading to an increased cytoplasmic calcium concentration. In the present study we focussed on identifying the Sf 9 guanine nucleotide-binding proteins (G proteins) involved. Immunodetection of Sf 9 membranes showed expression of G alpha isoforms belonging to all four G protein subfamilies. During prolonged baculovirus infection of Sf 9 cells, binding of guanosine 5'-o-(3-thiotriphosphate) as well as the intensities of G protein immunoreactivity, pertussis toxin-mediated ADP-ribosylation, GTP azidoanilide labelling of G alpha, and phosphate-labelling of G beta declined in cell membranes. Some 48 h after infection with mammalian histamine receptor-encoding viruses virtually no functional coupling of ligand-activated receptors to insect G proteins was observed despite a high level of expressed receptors. In contrast, Sf 9 cells infected only for 28 h allowed studies on histamine-induced G protein coupling. In membranes obtained from H1-receptor-expressing cells, histamine increased incorporation of GTP azidoanilide into Gq/11-like proteins whereas in membranes containing H2-receptors histamine enhanced GTP azidoanilide-labelling of Gq/11-like and G(S)-like proteins. In fura-loaded H1- and H2-receptor-expressing cells histamine induced the release of calcium from intracellular stores. This study shows firstly that Sf 9 G proteins couple to mammalian histamine receptors and secondly that H1-receptors activate only Gq/11, whereas H2-receptors activate Gq/11 and G(S), but neither receptor couples to Gi/o or G12. Finally, the time following baculovirus infection is critical for studying the functional coupling between recombinantly expressed and endogenous signal transduction components.

Direct activation of trpl cation channels by G alpha11 subunits.

G proteins of the Gq/11 subfamily functionally couple cell surface receptors to phospholipase C beta (PLC beta) isoforms. Stimulation of PLC beta induces Ca2+ elevation by inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release and store-dependent 'capacitative' Ca2+ entry through Ca(2+)-permeable channels. The Drosophila trp gene, as well as some human trp homologs, code for such store-operated channels. The related trp-like (trpl) gene product also forms a Ca(2+)-permeable cation channel, but is not activated by store depletion. Co-expression of the constitutively active Gq subfamily member G alpha 11 (G alpha 11) with trpl enhanced trpl currents 33-fold in comparison with co-expression of trpl with other G alpha isoforms or G beta gamma complexes. This activation could not be attributed to signals downstream of PLC beta. In particular, InsP3 infusion, modulation of protein kinase C activity or elevation of intracellular calcium concentration failed to induce trpl currents. In contrast, purified G alpha 11 (but not other G protein subunits) activated trpl channels in inside-out patches. We conclude that trpl is regulated by G11 proteins in a membrane-confined manner not involving cytosolic factors. Thus, G proteins of the Gq subfamily may induce Ca2+ entry not only indirectly via store-operated mechanisms but also by directly stimulating cation channels.

Distinct biochemical properties of the native members of the G12 G-protein subfamily. Characterization of G alpha 12 purified from rat brain.

G12 and G13 are insufficiently characterized pertussis toxin-insensitive G-proteins. Here, we describe the isolation of G alpha 12 from rat brain membranes. G alpha 12 was purified to apparent homogeneity by three steps of conventional chromatography, followed by two cycles of subunit-exchange chromatography on immobilized G subunits. Purified G alpha 12 bound guanosine 5'-[gamma-thio]triphosphate slowly and substoichiometrically. For isolation of functionally active G alpha 12, it was mandatory to use sucrose monolaurate as a detergent. Comparative studies of both rat-brain-derived members of the G12 subfamily revealed differences in the affinity of G alpha 12 and G alpha 13 for G beta gamma. G alpha 12 required a higher Mg2+ concentration for AlF4- -induced dissociation from immobilized G beta gamma than did G alpha 13. In addition, the G12 subfamily members differed in their sedimentation velocities, as determined by sucrose-density-gradient centrifugation. Analysis of sedimentation coefficients revealed a higher tendency of G12 to form supramolecular structures in comparison to G13 and other G-proteins. These G13 structures were stabilized by sucrose monolaurate, which in turn may explain the necessity for this detergent for purification of functionally active G alpha 12. Despite these distinct biochemical characteristics of G12 and G13, both purified G-proteins coupled to a recombinant thromboxane A2 (TXA2) receptor reconstituted into phospholipid vesicles. These data indicate, (1) significant differences in the biochemical properties of native members of the G12 subfamily, and (2) their specific coupling to TXA2 receptors.

[Experimental reproduction of the respiratory form of infection with the agent of epidemic late abortion of swine]. / Experimentelle Reproduktion der respiratorischen Verlaufsform der Infektion mit dem Erreger des Seuchenhaften Spätabortes der Schweine.

Porcine reproductive and respiratory syndrome has been observed in a large swine-farm. Diagnostic investigations were undertaken. Piglets infected with homogenate from the lung of a sick gilt developed biphasic fever up to 42 degrees C, inappetence and apathy. A few piglets showed red-blue discolored parts of the ears and of the skin in the abdominal region. A cytopathogenic agent has been isolated on cultivated alveolar-macrophages. Infection of another group of piglets with macrophages-propagated agent resulted in similar clinical signs too. Organs of euthanatized piglets have been examined anatomical, histological and with the fluorescent antibody technique. Specific fluorescence could be seen in tonsils, bronchial lymph nodes, spleen and in pathological-changed parts of the lungs. Antibodies against PRRS-virus were detected in blood samples of experimentally infected pigs.

Influenza virus A/swine-outbreaks in domestic pigs and antibody findings in human sera.

In the last years, the occurrence of influenza viruses A/H1N1 (Hsw1N1) in pig stocks of different countries has been increasingly reported. In general, the isolated viruses were related to the influenza virus A/New Jersey/8/76 H1N1 (Hsw1N1). Human infections were not reported in these outbreaks. Since March 1981, very limited influenza outbreaks in several pig stocks of the GDR with high morbidity and very low lethality have been observed. The illness took an uncomplicated path and generally subsided after 3 days. Some of the virus isolates were examined and typed as influenza virus A/swine/Potsdam/81/H1N1 (Hsw1N1). By serological examinations of convalescent pigs the aetiologic importance of the isolates was confirmed. Infection of the contact persons by the influenza virus A/swine/Potsdam/81 is to be regarded as likely according to the serological results.

[Transmissible gastroenteritis of swine as a model for infectious diarrhea]. / Die Transmissible Gastroenteritis der Schweine als Modell für infektionsbedingte Durchfallerkrankungen.

Transmissible gastro-enteritis is a virus-caused diarrhoeal disease of swine which may result in up to a total loss among newborn animals. Changes are, primarily, of pathologicoanatomic nature and take place in the small intestine. The epithelium of the intestinal villi is completely destroyed, and diarrhoea is the result. Deformation in blood composition is a secondary sequel. The diarrhoea proper causes dehydration and acidosis which, in conjunction with abnormal heart function due to hyperkalaemia, cause death of the piglets affected. Reference is made to the intrinsic immunological conditions in swine, and possibilities are discussed for immunoprophylactic control of transmissible gastro-enteritis by induction of effective lactogenic immunity. The approaches taken to vaccine application should largely correspond to the natural route of infection.